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Notes: Objective To study the relation between various perinatal factors and the sequelae of very preterm birth, applying logistic regression analysis.Design In a nationwide collaborative study in the Netherlands, perinatal and follow up data were collected on 899 liveborn singleton nonmalformed infants with gestational age less than 32 weeks or birthweight less than 1500 g born in 1983.Main outcome measures Neonatal mortality rate and total handicap rates (minor and major) in surviving children at two years and five years of age.Results Comparing breech with vertex presentation, the odds ratio for neonatal mortality (adjusted for duration of pregnancy, birthweight, maternal hypertension and prolonged rupture of membranes) is 1.6 (P〈0.05). Comparing abdominal versus vaginal delivery, the odds ratio indicates equal risks. When breech and vertex presentation are analysed separately it appears that breech presenting infants have a significantly lower mortality risk when born by caesarean section compared with vaginal delivery. However, comparing abdominal versus vaginal delivery in breech presentation, the odds ratio for handicap at five years (0.9) is not significantly different from 1.Conclusion The data presented suggest a reduced neonatal mortality rate in breech presenting infants born by caesarean section but because of the observational design of the study the statistical analysis described only identifies a possible trend and cannot prove the issue.

Notes: Objective To determine the effect of early labour, maternal analgesia and fetal hypoxia on circulating fetal oxytocin concentrations.Design Prospective observational study.Setting Delivery suite in a District General Hospital.Subjects Fifty women at term who did not require oxytocin administration or more than one form of analgesia. Study groups: vaginal delivery with (1) no analgesia, (2) pethidine, or (3) epidural analgesia. Caesarean section under regional analgesia (4) prior to, and (5) after the onset of labour.Interventions Samples of blood were collected from the umbilical artery (UA) and umbilical vein (UV) immediately after fetal delivery prior to placental separation or oxytocic administration.Main outcome measures Plasma oxytocin (OT) concentration, umbilical vein pH, cystine aminopeptidase activity.Results The geometric mean UA–OT was significantly greater than UV–OT in all groups and was not altered by pethidine; however, epidural administration increased the UA–UV difference. The UA–UV difference at caesarean section was not significantly altered by the onset of labour. There was no correlation between UV pH and UA–UV plasma oxytocin. Cystine aminopeptidase activity was not detectable in UA and UV plasma.Conclusions Fetal OT production is increased by epidural but not by pethidine analgesia. It is not influenced by the onset of labour or fetal hypoxia.

Notes: Objective To establish the plasma evolution of prothrombin fragments 1+2 (F1+2), thrombin–antithrombin III complexes (TAT), fibrin fragment D-Dimers (DD), von Willebrand factor antigen (vWf), Type 1 plasminogen activator inhibitor antigen (PAI) and blood platelet count during normal pregnancy and to compare these values with those obtained in hypertensive or pre-eclamptic pregnancies.Design Cross-sectional study.Subjects Forty-seven healthy pregnant women with gestational age ranging between 5 and 40 weeks, and fourteen women with gestational age ranging between 25 and 38 weeks presenting with either gestational hypertension (n= 4) or pre-eclampsia (n= 10). Numbers of nulliparous women in the control, hypertension and pre-eclampsia groups were 13/47 (28%), 1/4 (25%) and 9/10 (90%), respectively.Results All six markers increased with gestational age in normal pregnant women (P〈0.01). Using the upper limit of 95% prediction interval obtained from regression curves as normality threshold, TAT showed the best sensitivity (71%vs 〈30% for F 1+2, DD, vWf, PAI and platelet count).Conclusion TAT appears to be an interesting marker for detecting haemostatic system alterations in pregnancies complicated by hypertension or pre-eclampsia. A large prospective study to determine its clinical usefulness for such complicated pregnancies is currently in progress.

Notes: Objective To determine if pulse oximetry could detect any changes in fetal arteriolar oxygen saturation resulting from maternal administration of oxygen.Design A prospective study comparing study comparing the fetal pulse oximetry reading before and after giving 27% and 100% oxygen to the mother. The data were collected using an experimental pulse oximeter and a sensor specifically adapted to cope with the problems of fetal pulse oximetry.Setting Labour ward, St. James's University Hospital, Leeds University, UK.Subjects Twelve fetuses presenting by the vertex in normal uncomplicated labour.Main outcome measures The change in fetal arteriolar oxygen saturation recorded by the pulse oximeter in response to oxygen administration to the mother.Results Twenty-seven percent oxygen increased the average fetal arteriolar oxygen saturation by 7.5%, the effect being reversed when the oxygen was withdrawn. One hundred percent oxygen increased fetal arteriolar oxygen saturation by 11% and when the oxygen was withdrawn oxygen saturation dropped by 10%. One hundred percent inspired maternal oxygen was more effective than 27%. The gradient of the fetal oxygen regression slope is steeper with 100% oxygen than 27% and it is steeper when oxygen is given compared to when it is withdrawn. This suggests that the fetus responds to the new placental oxygen gradient by accepting oxygen more rapidly than it gives it up. Using a quadratic regression model, it took 9 min for fetal oxygen saturation to reach its maximum value after giving the mother oxygen.Conclusion This study confirms that a pulse oximeter is able to measure an increase in fetal arteriolar oxygen saturation when oxygen is administered to the mother.

Notes: Objectives 1. To compare the ultrasound biparietal diameter and crown-rump length of fetuses with and without Down's syndrome in the first half of pregnancy; 2. To investigate the effect of estimation of gestational age using either measure on the detection rate of serum screening for Down's syndrome.Design Matched case-control study. Cases were singleton Down's syndrome pregnancies with a biparietal diameter or a crown-rump length recorded. Five controls were matched to each case on: medical centre; the date of the ultrasound scan examination (within two years); gestational age measured as the number of days since the first day of the last menstrual period; and the ultrasound measure used (ie the biparietal diameter (the measure of choice), or the crown-rump length otherwise). If a woman had a serum screening test for Down's syndrome, the biparietal diameter or crown-rump length measurement had to be taken prior to the screening test so that the result of the test could not influence whether a scan was performed.Setting Ten antenatal screening centres in seven countries in Europe and North America.Subjects Two hundred and one women with singleton Down's syndrome pregnancies and 1005 women with unaffected singleton pregnancies.Results The median biparietal diameter of fetuses with Down's syndrome was identical to that among the controls (median difference 0.0mm, 95% confidence intervals (CI)–0.5 to 0.5mm). The estimates of gestational age based on biparietal diameter yielded a median gestational age less than that based on the women's last menstrual period: three days less for cases and two days less for controls; small but statistically significant differences probably reflected a minor systematic difference in the conversion of a biparietal diameter to a gestational age estimate. The median crown-rump length of fetuses with Down's syndrome was also identical to that among controls (median difference 0.0mm, 95% CI–1.5 to 2.0 mm). There was no significant difference between the median gestational age estimate based on crown-rump length and that based on the women's last menstrual period.Conclusion In antenatal screening for Down's syndrome the routine use of an ultrasound biparietal diameter or crown-rump length measurement to estimate gestational age will not adversely affect the detection rate. To avoid differences in gestational age estimates using the last menstrual period and the biparietal diameter influencing screening performance, separate medians should be derived for each serum marker using the two methods of estimating gestational age. The appropriate set of medians can then be used to calculate the multiple of the median value for each woman screened depending on the method used to estimate her gestational age.

Notes: Objective To examine microvascular endothelial cell function in vivo in pre-eclampsia.Design Iontophoresis of acetylcholine (Ach), which gives rise to endothelial cell dependent vasodilatation, and of sodium nitroprusside (SNP), which elicits vasodilatation independently of functioning vascular endothelium.Setting Södersjukhuset, Stockholm, Sweden.Subjects Ten pre-eclamptic patients, ten healthy pregnant women and ten healthy nonpregnant women were examined.Main outcome measures The degree of vasodilatation following iontophoretic administration of Ach compared with SNP was recorded with a laser Doppler technique, the data being analysed on a personal computer.Results Both Ach and SNP administration resulted in marked vasodilatation; the magnitude of the vasodilatation was similar in the three groups of women.Conclusion Following iontophoretic administration of endothelial cell dependent or independent vasodilatators, laser Doppler measurement of blood flow demonstrated no microvascular endothelial cell dysfunction in pre-eclamptic women.

Notes: Objective To investigate the relationship between preterm delivery and maternal serum insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein-1 (IGFBP-1) levels.Design A study over a 12 month period in which all samples were collected according to a pre-set protocol.Setting St. Bartholomew's Hospital, London.Subjects Thirty-eight nonpregnant adult females, 456 pregnant women at various gestational ages, 84 women with average-for-gestational-age babies at term delivery, and 49 pregnant women with preterm delivery (44 with singleton pregnancy and five with twin pregnancy).Main outcome measures Serum IGF-I and IGFBP-1 levels were determined by radioimmunoassay.Results Serum IGF-I concentrations increased as pregnancy progressed. In the third trimester, serum IGF-I levels in singleton preterm deliveries were lower than those in normal pregnancies, and IGFBP-1 concentrations were higher than those in normal pregnancies. This phenomenon was not obvious in the second trimester. Maternal circulating IGFBP-1 levels were correlated inversely with birthweight in women with singleton preterm delivery.Conclusions Neither IGF-I nor IGFBP-1 appears to play a significant role in preterm delivery since maternal serum IGF-I and IGFBP-1 levels are similar in preterm and term deliveries.

Notes: Objective To determine the concentrations of the metabolites of prostaglandin E2 (PGEM) and of prosta-glandin F2α (PGFM) prior to the onset of labour and during spontaneous labour, and to correlate the changes in concentrations of these metabolites with labour outcome.Design Longitudinal study throughout labour.Setting Labour ward of a large maternity unit.Subjects Seven primigravid and 11 parous women in the late third trimester with no signs of labour, and 17 primigravid and 11 parous women in spontaneous labour.Interventions Six of the primigravid women required augmentation with oxytocin because of dysfunctional labour.Results Before labour, parous women had significantly higher concentrations of both PGEM (P〈0.007) and PGFM (P〈0.006) compared with primigravid women. During labour, PGFM concentrations were significantly higher in both primigravid (P〈0.0002) and parous (P〈0.0001) women compared with the concentrations of these metabolites in women not in labour; the same was true for PGEM in primigravid (P〈0.003) but not in parous (P= 0.1) women. There was a small but significant increase (P〈0.02) in PGEM as labour progressed in both the normal groups. Amniotomy was associated with a significant increase in PGFM in primigravid and parous women (P〈0.002 and P〈0.009, respectively). The concentration of PGFM one hour following amniotomy correlated inversely with the amniotomy to delivery interval in both the normal primigravid (r=−0.624; P= 0.04) and the parous (r= 0.745; P= 0.021) groups. Women with dysfunctional labour showed no significant rise in PGEM or PGFM. Their PGFM concentrations were significantly lower than those seen in normal labour (P〈0.05). The concentration of PGFM in cord blood was significantly higher (P〈0.0001) in the parous women who laboured than in women delivered by elective caesarean section. There was no difference in the corresponding concentrations of PGEM (P= 0.9).Conclusions These data show that spontaneous labour is associated with increased concentrations of prostaglandin metabolites in the maternal plasma, and are consistent with PGF2α being an important stimulator of uterine contractility, with a relative deficiency of PGF2α being associated with dysfunctional labour.

Notes: Objective To assess the feasibility of discharging selected patients home within 72 h of vaginal hysterectomy.Design Women for whom vaginal hysterectomy was planned and who were invited to take part. Those who accepted were visited at home by a staff nurse experienced in gynaecology to assess home conditions and family support. If these were suitable, the general practitioner was informed and invited to comment. If the operation was uneventful women were discharged on the third post operative day and visited at home by the nurse until the seventh day.Setting Leeds Western Health Authority.Main outcome measures The number of minor complications requiring treatment by the general practitioner, the readmission rate and the acceptability to the woman and her family.Results For 11 out of 61, home conditions proved unsuitable for early discharge. In six of the remaining 50, abdominal hysterectomy proved preferable to vaginal. Thirty women were discharged home on the third day after vaginal hysterectomy, five on the fourth and seven on the fifth. Two developed urinary tract infections, treated by their general practitioners. Two required readmission to hospital. Twenty-eight of 30 discharged on the third post operative day and nine of 12 discharged on the fourth or fifth post operative day were enthusiastic about the scheme.Conclusions Early discharge following uncomplicated vaginal hysterectomy in selected patients appears to be a safe procedure, appreciated by the majority of women. Its adoption as a routine procedure would enable the surgical throughput in a unit to be maintained on a smaller bed complement.

Notes: Objectives To analyse the incidence and factors associated with the ovarian hyper-stimulation syndrome (OHS) in our IVF/GIFT programme before and after the introduction of a strategy to cryopreserve all embryos from women judged to be at risk.Design Two hundred forty-one consecutive IVF/GIFT cycles from January to December 1989.Setting Specialist fertility unit, Manchester, UK.Interventions Pituitary suppression was effected by a daily subcutaneous injection of buserelin (500 μg) beginning 7 days before the expected menses. The ovarian stimulation was with variable amounts of human menopausal gonadotrophin. Ovulation was induced with 10 000 i.u. human chorionic gonadotrophin (hCG). From January to May (period A), gametes/embryos were replaced and 2000 i.u. hCG given, irrespective of the serum oestradiol (E2) concentration. From June to December (period B), all the embryos from women with an E2〉3500 pg/ml on the day of ovulatory trigger were electively cryopreserved.Main outcome measures Serum E2, features of moderate or severe OHS, clinical pregnancies.Results The OHS occurred in 10/105 (9.5%) and 12/136 (8.8%) cycles in periods A and B, respectively. Fewer women (6% versus 60%, P〈0.05) who had their embryos cryopreserved developed severe OHS compared with women with an E2 〉3500 pg/ml who became pregnant after gamete/embryo transfer in period A. The main factors associated with the development of OHS were serum E2 concentrations 〉3500 pg/ml, whether gamete/embryos were replaced and the additional hCG given, the occurrence of a pregnancy and the presence of polycystic ovary disease.Conclusion The elective cryopreservation of all embryos from women with high E2 levels reduced the severity, but not the incidence of symptomatic OHS.

Notes: Objectives To compare the outcome of two methods of maternity care during the antenatal period and at delivery. One was to be midwife-led for both antenatal care and delivery, the latter taking place in rooms similar to those in one's own home to simulate home confinement. The other would be consultant-led with the mothers labouring in the delivery suite rooms with resuscitation equipment for both mother and baby in evidence, monitors present and a delivery bed on which both anaesthetic and obstetric procedures could be easily and safely carried out.Design Randomised controlled trial.Setting Leicester Royal Infirmary Maternity Hospital.Subjects Of 3510 women who were randomised, 2304 were assigned to the midwife-led scheme and 1206 were assigned to the consultant-led scheme.Main outcome measures Complications in the antenatal, intrapartum and postpartum periods were compared as was maternal morbidity and fetal mortality and morbidity. Satisfaction of the women with care over different periods of the pregnancy and birth were assessed.Results There were few significant differences in antepartum, intrapartum and postpartum events between the two groups. There was no difference in the percentage of mothers and babies discharged home alive and well. Generally higher levels of satisfaction with care antenatally and during labour and delivery were shown in those women allocated to midwife care.

Notes: Objective To examine indications for the induction of labour and variations in the current policy of induction at different levels of obstetric specialisation and to compare the outcome of induced and spontaneous labour.Design A prospective 1 year birth cohort.Setting Maternity hospitals in the two northernmost administrative provinces of Finland, including one university hospital and three central hospitals, three local hospitals and five health centres.Subjects Eight thousand six hundred and six singleton pregnancies, including 1679 with induced labour.Main outcome measure Data collection on age, parity, social factors and education at antenatal clinic. Data on labour collected from the hospital records after delivery.Results Labour was induced significantly more often at units of the lowest level of specialisation, the health centres (29.4%) than at the local hospitals (23.6%, P〈0.003) or in the most specialised central hospitals (17.7%, P〈0.0001). Cases of induced labour accumulated on working days. Indicative reasons, such as maternal or fetal conditions, comprised 45.0% of the indications for induction, the most common causes being elective reasons, e.g. timing of labour (51.3%). The risk of elective induction was 2.6 times greater at the primary care level than at the central hospitals (95% confidence limit, CL 2.0.3.2). The corresponding risk ratio for local hospitals was 1.8 (CL 1.5–2.1). The risk of caesarean section was 1.5 times greater in the elective induction group than in the spontaneous group (CL 1.1–1.9) and 2.9 times greater in the indicative induction group. The most common indication for caesarean section was dysfunctional, arrested labour; causes such as fetal asphyxia or antenatal haemorrhage were not seen in excess.Conclusion The practice of induction of labour are not consistent in different hospitals. The opinions of individual practitioners and staff routines influence the induction policy nearly as much as do medical reasons. Despite the safety of induction, a liberal induction policy leads to an increase in operative deliveries creating potential risks for the mother and child and greater expense.

Notes: Objective To perform a clinical analysis of newborn babies with a fractured clavicle and investigate the possible role of relative calcium deficiency in the aetiology.Design Prospective descriptive study (clinical aspects); case controlled study (calcium, phosphate, alkaline phosphatase).Setting The Nazareth Hospital, Israel.Subjects All babies with a fractured clavicle detected in the newborn period and their mothers delivered between 23 August 1987 and 22 June 1989. The majority of the population were Arab.Interventions Serum calcium, phosphate and alkaline phosphatase were measured on the third postpartum day in 42 affected babies (with uncomplicated deliveries), their mothers, and a matched group of babies and mothers as controls.Main outcome measures Clinical associations of fractured clavicle of the newborn; statistical comparison of affected group with controls (calcium study).Results The incidence of fractured clavicle was 18.7 per 1000 singleton vaginal births; 38 in the posterior clavicle at delivery, 27 in the anterior, position unascertained in nine. Four (5.2%) occurred in instrumental deliveries, three (3.9%) in assisted breech delivery; shoulder dystocia was noted in 13. The male to female ratio of 51:26 was significantly different but not due to birth weight. Affected babies were significantly heavier than the unaffected population. The incidence was higher in parous mothers older than 25 years of age but there was no evidence of increased incidence with increasing parity. No significant results were obtained in the calcium study between affected babies and their mothers, when compared with controls.Conclusions Fractured clavicle of the newborn is a benign form of birth trauma from which heavier babies are at greater risk. It occurs in 1 to 2% of deliveries, most of these being uncomplicated vaginal births, and is often undetected. There is no evidence for relative calcium deficiency.

Notes: Objective To investigate the endocrine changes associated with spontaneous miscarriage after fetal heart activity has been demonstrated.Design Prospective study during the first trimester of pregnancy comparing the circulating levels of human chorionic gonadotrophin (hCG), Schwangerschaft protein 1 (SP-1), pregnancy-associated plasma protein A (PAPP-A), oestradiol (E2), and progesterone (P), and fetal growth (crown-rump length [CRL] and gestational sac volume [GSV]) in women who miscarried after the identification of fetal heart activity with those of normal singleton and twin pregnancies achieved following in vitro fertilisation (IVF) and embryo transfer (ET).Setting The Assisted Conception Unit of King's College Hospital, London.Subjects Nine women who miscarried after demonstration of fetal heart activity, 52 normal singleton and 22 normal twin pregnancies.Interventions Weekly blood tests and ultrasound assessments of CRL and GSV.Results Four fetuses (all singleton) died between 9 and 12 weeks gestation (Group 1), and seven (three singleton and two twin) died between 16 and 20 weeks gestation (Group 2). In Group 1, both fetal growth and placental function, as assessed by serial measurements of CRL and GSV, and of serum levels of PAPP-A, SP-1 and hCG respectively, were reduced before fetal death. In Group 2, while fetal growth was maintained in all but one case, placental function was reduced in 4 of 5 women.Conclusion These findings suggest that there may be a relationship between trophoblast dysfunction and some forms of miscarriage. Furthermore, the pattern of the reduction in the circulating levels of the placental proteins in later miscarriages suggests that the function of specific cell types may be impaired.

Notes: Objective To compare the outcome of in vitro fertilisation (IVF) and gamete intrafallopian transfer (GIFT) cycles in women with or without ultrasound features of polycystic ovary syndrome (PCOS).Design A consecutive series from January to December 1989.Subjects Twenty-five women with PCOS scheduled for assisted conception. The controls were 139 women with normal ovaries.Setting A single centre specialist fertility unit, Manchester, UK.Interventions Pituitary desensitisation was with buserelin. In the PCOS group ovarian stimulation was with 1 ampoule (75 iu FSH) of hMG/day in 12 women (Group I) and two ampoules/day in 13 (Group II). The controls (Group III) were given two ampoules of hMG daily. Human chorionic gonadotrophin (hCG; 10 000 iu) was given when three follicles measured 〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:14700328:BJO365:ges" location="ges.gif"/〉20 mm diameter.Main outcome measures Serum oestradiol (E2) concentrations, number of follicles, clinical pregnancies, features of the ovarian hyperstimulation syndrome (OHS).Results Women with PCOS (Groups I or II) had more follicles 〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:14700328:BJO365:ges" location="ges.gif"/〉14 mm diameter on the day of the hCG injection (P〈0.005), higher serum E2 concentrations on the day after the hCG (P〈0.05) and more oocytes retrieved (P〈0.05) than the controls. The OHS was more prevalent in those with PCOS (32% versus 6.5%; P〈0.05). The clinical pregnancy rate per embryo transfer (27% versus 22%) or gamete transfer (25% versus 39%) and the rate of spontaneous miscarriage (33% versus 12%) were not statistically different.Conclusions The pregnancy rate and outcome of pregnancy following IVF or GIFT in women with or without PCOS are similar. Women with PCOS are at a higher risk of developing OHS.

Notes: Objective To point out the association between infertility treatments and the increasing number of multiple deliveriesDesign Comparison over time of the incidence of multiple deliveries, the proportion of deliveries resulting from assisted conception (AC), and ovulation inductor sales.Data Use of existing statistics: vital statistics, surveys of AC centres and ovulation inductor sales.Results Between 1972 and 1989 the incidence of twin deliveries rose from 8.8/1000 to 11.2/1000, and the incidence of triplet deliveries from 0.9/10 000 to 4.4/10 000. This upward trend was particularly remarkable among women aged 30 to 39. Since 1978, the triplet delivery rates and the sales of Human Menopausal Gonadotrophin have been rising similarly. Between 1985 and 1989, 26 per cent of the triplet deliveries followed assisted conception and nearly 50 per cent were estimated to be due to ovulation inductor agents.Conclusions The results suggest a strong influence of infertility treatments and especially ovulation inductor agents in the dramatic increase of triplet deliveries.

Notes: Objective To ascertain the number and type of obstetric computer systems (OCS) in Great Britain, and to ascertain user satisfaction with these systems.Design A postal questionnaire was circulated to every consultant obstetrician in Great Britain at the beginning of 1992.Main outcome measures Information was sought on the hardware, software and uses of obstetric computer systems. Satisfaction with, benefits and problems of the system were also assessed.Results There was an 87.5% response rate. Of the 264 units questioned, 100 units reported that they had a computer system. Sixty-five units used terminals connected to a mainframe or minicomputer and 17 used stand-alone personal computers (PCs). Local area networks (LANs) were used in 19 units and wide area network (WANs) in 22 units. Software varied from commercial turnkey systems to in-house systems. The quoted annual running cost ranged from £50 to £48 000. Most units were satisfied with their system. Problems included slow operating times, unreliability, user unfriendliness, deficiencies in training and inadequate customer support services.Conclusions Obstetric computer systems are now coming into widespread use. Despite problems, the use of such systems is likely to increase. This survey establishes a database for those units who are considering acquiring or changing their computer system for the purpose of audit or research.

Notes: Objective To determine the distribution of platelet volumes and numbers through pregnancy, and to compare these to changes in platelet volumes and numbers in women with pre-eclampsia.Subjects Four hundred twenty-eight women with normal pregnancy from whom four or more platelet measurements were available were identified. 74 women with pre-eclampsia (blood pressure 〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:14700328:BJO216:ges" location="ges.gif"/〉 140/90 mmHg, at least 0.5g protein/24 h urine collection) from whom platelet measurements were available between 27 and 30 weeks of gestation were identified.Results Mean platelet volume and platelet number remained constant in normal pregnancies between the first trimester and the end of pregnancy. A persistent increase of 〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:14700328:BJO216:ges" location="ges.gif"/〉 0.8 fl (〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:14700328:BJO216:ges" location="ges.gif"/〉 90th centile) in mean platelet volume was found in 14 out of 15 pre-eclamptic patients between 24 weeks and 38 weeks of gestation and in only 13 of 428 normal pregnant individuals. Platelet numbers were decreased by 〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:14700328:BJO216:ges" location="ges.gif"/〉 50 × 109/l (i.e. to less than the 10th centile) in 12 of the 15 patients with pre-eclampsia. 10% of the normal pregnant population showed a similar decline in platelet numbers showing that changes in platelet numbers may be a less accurate assessment of the development of pre-eclampsia.Conclusion We suggest that longitudinal determination of platelet volumes may be of use in identifying those women at risk of pre-eclampsia.

Notes: Objective To investigate the effect of subcutaneous oestradiol and testosterone on the proportion of type III collagen in the skin of postmenopausal women.Design A cross sectional comparison.Setting Dulwich Hospital menopause clinic.Subjects Fourteen untreated women and 11 women who had received subcutaneous oestradiol and testosterone for a median 8.0 years (range 3–14). Ten of the untreated women received subcutaneous hormone implants and the effect on skin collagen was studied prospectively.Measurements The proportion of type III collagen in skin biopsies taken from the lateral aspect of the thigh.Results The median type III collagen content in the skin of the women who had received hormone replacement therapy (25.4%, range 21.4–30.2) was significantly higher (P〈0.01) than in the untreated women (19.6%, range 18.2–28.8). The proportion of type III collagen in the skin of 10 untreated women increased significantly (P〈0.01) from a median of 19.9% (range 18.2–23.9) to 22.4% (range 20.5–31.5) following 6 months of treatment with hormone implants.Conclusion This study indicates an increase in the proportion of type III collagen in women receiving hormone replacement therapy.

Notes: Objective To compare the Charing Cross Hospital scoring system with FIGO staging for gestational trophoblastic disease.Design A retrospective analysis of patients referred to Charing Cross during the period 1979–1989.Setting National referral centre.Subjects Two hundred and seven women with gestational trophoblastic disease presenting between 1979–1989.Main outcome measures The women were classified according to the Charing Cross Hospital scoring system and FIGO clinical staging and the results of treatment compared.Results Of the 207 women studied, there were 102 low risk, 39 medium risk and 66 high risk according to the Charing Cross system and 26 stage 0,83 stage 1,23 stage 2,51 stage 3 and 24 stage 4 according to the FIGO system. If the FIGO system had been used to determine therapy, 17 women would have been at risk of under-treatment and nine of overtreatment. The main prognostic factors contributing to a higher Charing Cross score and thus more intensive chemotherapy from the outset were: HCG 〉 105, interval from antecedent pregnancy 〉12 months and term delivery.Conclusions The Charing Cross scoring system incorporates factors predictive of the presence of drug resistant tumour and thus allows more appropriate use of chemotherapy from the start of treatment than is possible with the anatomically based FIGO staging. Moreover, the system is easy to apply relying only on history and clinical examination in addition to a reliable quantitative HCG assay.

Notes: Objective To test the effect of peritoneal fluid from infertile women with minimal or mild endometriosis on sperm movement characteristics in comparison with fertile and infertile women with no endometriosis.Design A prospective observer-blind trial.Setting Academic infertility department.Subjects 57 women undergoing diagnostic laparoscopy or laparoscopic sterilisation.Main outcome measures Changes in sperm movement characteristics in semen samples provided during routine infertility investigation or from sperm donors. Computer assisted semen analysis (CASA) performed using a Celltrack-S system.Results Significant reductions in linearity (P 〈0.05), amplitude of lateral head displacement (P 〈0.01), straight line velocity (P 〈0.01), and curvilinear velocity (P 〈0.01) (but not percentage motility) were observed.Conclusions Peritoneal fluid from women with minimal or mild endometriosis adversely effects sperm movement characteristics in comparison to fertile women.

Notes: Objective To determine the long term efficacy of laparoscopic ovarian diathermy in the management of refractory anovulatory infertility in women with the polycystic ovary syndrome.Design Open study of 50 women treated over a period of 3 years and 3 months, with follow up until 18 months after the last woman was treated.Setting Teaching Hospital.Subjects Fifty consecutive women with refractory anovulatory infertility (mean duration 5.3 (SD 3.5) years). All had been treated unsuccessfully with anti-oestrogens and more than half with gonadotrophins.Interventions Laparoscopic ovarian diathermy.Main outcome measures Ovulatory cycles and pregnancies.Results Forty-three women (86%) ovulated following ovarian diathermy; the mean time to ovulation was 23 (SD 6.2) days. Three nonresponders ovulated following anti-oestrogen treatment to which they were previously resistant. Thirty-three women have conceived 58 pregnancies; 22 had no treatment other than ovarian diathermy prior to their first post-operative conception; in seven an anti-oestrogen was given because of lengthening cycles; two were treated elsewhere with gonadotrophins without prior postdiathermy anti-oestrogen therapy and conceived; four had the operation repeated and two of these conceived. Twenty-six women conceived within the first 8 post-operative months. Forty-two pregnancies ended in the birth of normal live healthy babies, eight are ongoing and eight miscarried. Of the 22 women who had no pelvic abnormality other than polycystic ovaries, 19 (86%) have had one or more successful pregnancies.Conclusion Laparoscopic ovarian diathermy is a very effective treatment for anti-oestrogen resistant anovulatory infertility in women with the polycystic ovary syndrome and should be considered as the next step in those who fail to respond to anti-oestrogen treatment.

Notes: Objective To study the physiological responses to noninvasive cardiovascular autonomic function tests in normal pregnancy.Design Cardiovascular autonomic responses in 60 women at 22 to 29 weeks gestation and 62 nonpregnant women were investigated using the Valsalva manoeuvre as well as orthostatic, quiet breathing, deep breathing, and isometric handgrip tests.Results Compared with nonpregnant women, those who were pregnant showed significantly lower heart rate variability during normal breathing and a blunted tachycardic reaction to blowing during the Valsalva manoeuvre. The vagally controlled biphasic heart rate response to standing was also attenuated in the pregnant group.Conclusions The cardiovascular responses were blunted in mid-pregnancy indicating a decrease in parasympathetic cardiovascular control.

Notes: Objective To study cardiovascular responses to noradrenaline during early pregnancy.Design Administration of incremental intravenous infusions of noradrenaline under basal conditions.Setting University Hospital, Nottingham.Subjects Nineteen women admitted for termination of pregnancy in first or second trimester and 18 nonpregnant women as control subjects.Interventions Recordings of blood pressure and heart rate responses during the infusion of noradrenaline. Blood samples taken before, during and after the infusion.Main outcome measures Evoked responses of systolic and diastolic blood pressure and heart rate at steady state during the noradrenaline infusion. Plasma catecholamine concentrations measured by high performance liquid chromatography.Results There were no significant differences between the evoked pressor responses to noradrenaline in pregnancy compared to the nonpregnant state, but there was a lesser bradycardia in response to a standardised change in blood pressure. Neither basal plasma catecholamine concentrations nor those achieved during infusion of noradrenaline differed between pregnant and nonpregnant women. Thirty minutes after discontinuance of the infusion there was a persistent elevation of heart rate in all women, although plasma catecholamine concentrations had returned to basal levels. In pregnant women, systolic and diastolic blood pressures were also elevated at this time, compared to preinfusion levels.Conclusion Cardiovascular regulatory mechanisms are altered in pregnancy with a diminution in the bradycardic response to a pressor challenge evoked by noradrenaline and a delayed recovery from that challenge.

Notes: The cause of neuronal death in amyotrophic lateral sclerosis (ALS) is unknown. Recently, it was found that some patients with autosomal-dominant familial ALS (FALS) have point mutations in the gene that encodes Cu/Zn superoxide dismutase (SOD1). In this study of postmortem brain tissue, we examined SOD activity and quantified protein carbonyl groups, a marker of oxidative damage, in samples of frontal cortex (Brodmann area 6) from 10 control patients, three FALS patients with known SOD1 mutations (FALS-1), one autosomal-dominant FALS patient with no identifiable SOD1 mutations (FALS-0), and 11 sporadic ALS (SALS) patients. Also, we determined the activities of components of the electron transport chain (complexes I, II-III, and IV) in these samples. The cytosolic SOD activity, which is primarily SOD1 activity, was reduced by 38.8% (p 〈 0.05) in the FALS-1 patients and not significantly altered in the SALS patients or the FALS-0 patient relative to the control patients. The mitochondrial SOD activity, which is primarily SOD2 activity, was not significantly altered in the FALS-1, FALS-0, or SALS patients. The protein carbonyl content was elevated by 84.8% (p 〈 0.01) in the SALS patients relative to the control patients. Finally, the complex I activity was increased by 55.3% (p 〈 0.001) in the FALS-1 patients relative to the control patients. These results from cortical tissue demonstrate that SOD1 activity is reduced and complex I activity is increased in FALS-1 patients and that oxidative damage to proteins is increased in SALS patients.

Notes: Abstract— Chronic administration of ethanol results in the development of tolerance and dependence. The molecular mechanism underlying these behavioral actions of ethanol is poorly understood. Several lines of evidence have suggested that some of the pharmacological actions of ethanol are mediated via a potentiation of GABAergic transmission. Chronic ethanol administration results in a reduction in the GABAA receptor-mediated 36Cl− uptake in cortical synaptoneurosomes and primary cultured neurons. We and others have shown that it also results in a 40-50% reduction in GABAA receptor α-subunit mRNA levels in the rat cerebral cortex. In the present study, we investigated the expression of α1, α2, and α3 subunits of the GABAA receptor in the cerebral cortex and the α1 subunit in the cerebellum by immunoblotting using polyclonal antibodies raised against α1-, α2-, and α3-subunit polypeptides following chronic ethanol treatment. These results reveal that chronic ethanol administration to rats results in a 61 ± 4% reduction in level of the GABAA receptor α1subunit (51 kDa), 47 ± 8% reduction in level of the α2subunit (53 kDa), and 30 ± 7% reduction in level of the α3subunit (59 kDa) in the cerebral cortex and a 56 ± 5% reduction in content of the α1 subunit in the cerebellum. In summary, this ethanol-induced reduction in content of the GABAA receptor α subunits may underlie alterations in the GABAA receptor function and could be related to cellular adaptation to the functional disturbance caused by ethanol.

Notes: [125I]RTI-55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [125I]RTI-55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [125I]RTI-55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [125I]RTI-55 binding sites in human brain using homogenized membrane preparations and quantitative autoradtography. Analysis of the association, dissociation, and saturation data favored two-phase processes. A curve-fitting analysis of the data derived in saturation experiments found a high-affinity site with KD= 66 ± 35 pM and Smax= 13.2 ± 10.1 pmol/g of tissue and a low-affinity site with KD= 1.52 ± 0.55 nM and Bmax of 47.5 ± 11-2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI-55 〉 GBR-12909 〉 mazindol 〉 WIN 35428 〉 = methylphenidate 〉 (−)-cocaine 〉 buproprion 〉 (±)-amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with KD= 370 ± 84 pM. It appears that [125I]RTI-55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.

Notes: Protein zero (P0), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P0 (P0-ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N-linked glycosylation. The x-ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 Å, 50 Å (native), and 90 Å between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x-ray diffraction data on the orientation of P0-ED, and we propose how this immunoglobulin-like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that β-strand predictions for P0-ED are virtually superimposable with those of the VH domain of the phosphocholine-binding immunoglobulin M603 of mouse, which has a similar number of residues as P0-ED and a structure that has been solved crystallographically. The dimensions of P0-ED from the space-filling model, developed using PC- based molecular modeling software, were found to be 44 Å× 25 Å× 23 Å. On the assumption that neither the shape nor the orientation of P0-ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P0-ED from apposed membranes if the molecules were oriented so that the β- strands were approximately perpendicular to the membrane surface. The apposed P0-EDs would fully overlap at the closest apposition of the membranes, partially overlap in the native state, and align end to end in the incompletely swollen state. The P0-ED regions analogous to the complementarity-determining regions of immunoglobulins can account for the recognition of P0-ED from apposed membranes in the incompletely swollen state. Two of the faces of P0-ED that show charge complementarity could account for the homophilic interactions of P0-ED from apposed membranes in the native state. This association can be stabilized further by hydrophobic interactions. The N- linked nonasaccharide after energy minimization fit into a cavity, which was at the base of P0-ED and which was lined with three positively charged residues. Thus, the carbohydrate may help to maintain the orientation of P0 at the membrane surface. Our model shows how the single immunoglobulin-like domain of P0 can account for distinct structural states of myelin membrane packing by homophilic interactions.

Notes: The possible existence of a dopamine D2 receptor-mediated regulation of dopamine release was investigated in the goldfish retina. Isolated retinas were preloaded with [3H]dopamine and superfused with D2 dopamine receptor agonists or antagonists to determine if there was an effect on [3H]dopamine release. The D2 receptor antagonist sulpiride increased both baseline [3H]- dopamine release and [3H]dopamine release induced by an increase in extracellular potassium concentration. The D2 receptor agonists LY-171555 and RU-24213 did not reduce baseline [3H]dopamine release but completely inhibited [3H]dopamine release induced by an increase in [K±]o. This action of the D2 agonists was blocked by sulpiride. These studies demonstrate the existence of D2 receptor, possibly autoreceptor, regulation of dopamine release in the teleost retina.

Notes: To date three β subunits of the GABAA receptor have been identified in rat brain as a result of cDNA library screening. The β2 subunit has been reported to have a wide distribution in rat brain based on in situ hybridization studies quantifying β2 mRNA. To study the β2 subunit more directly, we have raised a polyclonal antibody to a synthetic peptide representing residues 315–334 of the intracellular loop of the β2 subunit. The antibody, which had been affinity-purified, recognized the β2 peptide but did not immunolabel homologous β1 and β3 subunit peptides, indicating that this antibody is specific for the β2 subunit of the receptor. In western blots of the purified receptor, the antibody recognized a major diffuse band of 54–58 kDa arid exhibited minor labeling of lower-molecular-mass polypeptides. In western blots of cortex homogenate, the antibody exhibited nervous system-specific labeling of a 55-kDa band that comigrated with the 55-kDa band of the purified receptor. Quantitative immunolabeling of this 55-kDa polypeptide permitted direct determination of the relative amounts of the β2 subunit in different brain regions. The brainstem contained the highest relative specific activity of the β2 subunit, followed by the inferior colliculus, olfactory lobe, and cerebellum. Lower levels of immunolabeling were seen in hypothalamus, hippocampus, thalamus, and cortex.

Notes: Abstract— The mRNA levels of secretogranin II, chromo-granin B, and VGF were compared in brains of control and AF64A-treated rats. This toxin induces specific lesions of the septohippocampal cholinergic pathway. As a consequence of this treatment, the Chromogranin B message was elevated in the dentate gyrus granule cells of the hippocampus. In the paraventricular nucleus of the hypothalamus, a concomitant elevation of the messages of secretogranin II and corticotropin-releasing factor occurred in the parvocellular neurons, and an increase of those of secretogranin II and VGF occurred in a subgroup of magnocellular neurons. Further increases for secretogranin II were seen in the amygdaloid nuclei and the reticular thalamic nuclei and increases for Chromogranin B in the temporal cortex, substantia nigra compacta, and ventral tegmental area. These results indicate that the toxin-induced lesion of the cholinergic pathway innervating the hippocampus apparently leads to the stimulation of several defined groups of neurons that react with an increase in the mRNA levels of their secretory peptides. We suggest that changes in mRNA expression of these peptides are useful parameters for defining neurons under chronic stimulation. Key Words: Secretory peptides—Large dense core vesicles—Corticotropin releasing factor—Septohippocampal cholinergic system—Hippocampus—AF64A.

Notes: Abstract— Glutathione levels in neurons and gllal cells were investigated in a neuronal-glial coculture and in separate cultures. Brain cell suspensions obtained from cerebral hemispheres of fetal rats were cultured, and after 5 days the glutathione content of this cell population, consisting mainly of neurons and astroglial cells, was 23.0 nmol/mg of cell protein, with a significantly high content in glial cells (28.0 nmol/mg of protein) in comparison with neurons (18.8 nmol/mg of protein). When the neurons and glial cells were separated and recultured in fresh medium, neu-ronal glutathione rapidly decreased, whereas glial glutathione remained unchanged. Cysteine is a rate-limiting precursor for glutathione synthesis, and its level was also decreased in neurons, but not in glial cells. Cysteine was taken up rapidly by both neurons and glial cells, but cys-tine was taken up only by glial cells. This accounts for the rapid decrease of glutathione in the cultured neurons, because the culture medium contains cystine, but not cys-teine. It was also found that the cultured glial cells released cysteine into the medium. These results suggest that neurons maintain their glutathione level by taking up cysteine provided by glial cells.

Notes: Using a controlled cortical impact model of traumatic brain injury (TBI) coupled with tissue microdialysis, interstitial concentrations of aspartate and glutamate (together with serine and glutamine) were assessed in rat frontal cortex. Histological analysis indicated that the severity of injury following severe TBI (depth of deformation = 3.5 mm) was approximately twice that occurring following moderate TBI (depth of deformation = 1.5 mm). Both groups demonstrated significant postinjury maximal increases in excitatory amino acid (EAA) concentration, which were proportional to the severity of injury. The mean ± SEM fold increase in dialysate concentrations of aspartate was 38 ± 13 (n = 5) for moderate TBI and 74 ± 12 (n = 5) for severe TBI. Fold increases in glutamate concentrations were 81 ± 26 and 144 ± 23 for moderate and severe TBI, respectively. Although these increases normalized within 20–30 min following moderate TBI, concentrations of aspartate and glutamate took 〉60 min to normalize after severe TBI. Changes in levels of nontransmitter amino acids were much smaller. Fold increases for serine concentrations were 4.6 ± 0.6 and 7.6 ± 1.7 in moderate and severe TBI, respectively; glutamine concentrations had similar small fold increases (2.6 ± 0.2 and 4.1 ± 0.6, respectively). Calculation of interstitial concentrations following severe TBI indicated that aspartate and glutamate maximally increased to 123 ± 20 and 414 ± 66 μM, respectively. To determine the extent to which such tissue concentrations of EAAs could contribute to the injury seen in TBI, the EAA receptor agonists N-methyl-d- aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid were slowly injected into rat cortex. Remarkably similar histological injuries were produced by this procedure, supporting the notion that TBI is an excitotoxic injury.

Notes: Abstract— Regulations of the increase in intracellular Ca2+concentration ([Ca2+]i) and inositol 1, 4, 5-trisphosphate (IP3) production by increasing intracellular cyclic AMP (cAMP) levels or activating protein kinase C (PKC) were studied in rat frontocortical cultured neurons. Amitriptyline (AMI; 1 mM), a trìcyclic antidepressant, and bradykinin (BK; 1 μM) stimulated IP3 production and caused transient [Ca2+]i increases. Pretreatment with forskolin (100mkUM, 15 min) decreased the AMI-and BK-induced [Ca2+]i increases by 33 and 48%, respectively. However, this treatment had no effect on the AMI-and BK-induced IP3 productions. Dibutyryl-cAMP (2 mM, 15 min) also decreased the AMI-and BK-induced [Ca2+]i increases by 23 and 47%, respectively. H-8 (30 μM), an inhibitor of protein kinase A (PKA), attenuated the ability of forskolin to inhibit the AMI-and BK-induced [Ca2+]i increases, suggesting that the activation of cAMP/PKA was involved in these inhibitory effects of forskolin. On the other hand, forskolin treatment had no effect on 20 mM caffeine-, 10 μM glutamate-, or 50 mM K+-induced [Ca2+]i increases. Pretreatment with phorbol 12-myristate 13-acetate (PMA; 100 nM, 90 min) decreased both the AMI-induced [Ca2+]i increases and the IP3 production by 31 and 25%, respectively. H-7 (200 μM), an inhibitor of PKC, inhibited the ability of PMA to attenuate the [Ca2+]i increases. PMA also inhibited the BK-induced IP3 production and the [Ca2+]i increases. Taken together, these results suggest that activation of cAMP/ PKA may inhibit the IP3-mediated Ca2+ release from internal stores; on the other hand, activation of PKC may inhibit the phosphatidylinositol 4,5-bisphosphate breakdown and consequently reduce the [Ca2+]i increases or inhibit independently both responses. PKA and PKC may differently regulate the phosphatidylinositol-Ca2+ signaling in rat frontocortical cultured neurons.

Notes: Abstract— In this study we demonstrate that a 51-kDa phosphoprotein, previously identified as morphine regulated and showing different basal levels among rat strains, is glial fibrillary acidic protein (GFAP). Chronic morphine increased levels of GFAP immunoreactivity by 〉70% in the ventral tegmental area (VTA) of outbred Sprague-Dawley rats. This increase in GFAP content was not observed in rats that were treated concomitantly with morphine and naltrexone, an opiate receptor antagonist, and did not occur in response to a single acute injection with morphine. No alterations in GFAP levels were observed in response to chronic morphine in several other regions of the CNS studied, including the substantia nigra, locus coeruleus, cerebral cortex, and spinal cord. There were also inherent differences in levels of GFAP immunoreactivity in the VTA of drug-naive Fischer 344 and Lewis rats, two inbred rat strains that differ in their relative preference for morphine and other drugs of abuse. The VTA of drug-naive Lewis rats contained more than twofold higher levels of GFAP compared with drug-naive Fischer rats. This strain difference was also apparent in the locus coeruleus but not in several other brain regions or in spinal cord. Because the mesolimbic dopamine system is thought to play a critical role in mediating the reinforcing properties of opiates and other drugs of abuse, it is possible that the opiate induction of GFAP and inherent Lewis versus Fischer strain differences in GFAP levels in the VTA may be related to the reinforcing and/or addictive properties of opiates mediated by this brain region, as well as to genetic differences in drug preference.

Notes: Abstract: Analyses of samples of articles in the Journal of Neurochemistry between 1956 (the year of its foundation) and 1990 were used to obtain numerical indices of the history of neurochemistry. Data suggest that the acceleration of neurochemical research did not merely reflect the increase of biochemical research in general and that it involved progressive decreases and increases of interest in major constituents and transmitters, respectively, as indicated by both numbers and citations of papers. Papers on all classes of transmitters increased steadily and in the order of amines 〉 amino acids, acetylcholine 〉 peptides. Within the field of brain metabolism, papers on energy metabolism decreased markedly. Use of techniques other than those of biochemistry/neurochemistry altered strikingly with decreases of histological, electrophysiological, and pharmacological methods and increases of chemical, immunological, and tissue culture methods. Citations by neuroscience core journals between 1975 and 1988 suggest that the relative prominence of neurochemistry within neuroscience has remained constant. Analyses indicate that the influence of the U.S.A. relative to that of other regions has remained fairly steady between 1956 and 1990, but that number of papers from the U.K. has declined, whereas the influences of Western Europe and other areas appear to have recently increased substantially. Sociological changes have been the virtual disappearance of single-author papers, an increase of multiauthorship (〉3), and a recent striking increase of assertive sentence titles.

Notes: Abstract: Neuromelanin is a poorly understood pigment that accumulates in catecholaminergic neurons during normal aging. Electron paramagnetic resonance spectroscopy, an especially effective technique for investigating melanins, is used in the present study to show unambiguously that neuromelanin is a melanin; however, it is not well modeled by synthetic dopamine melanin and thus is an atypical melanin. Some of the unusual features of neuromelanin can be explained by postulating two distinct sources for its free radicals, the dominant one possibly derived from a precursor containing sulfur. Examination of human substantia nigra by electron paramagnetic resonance spectroscopy during the purification of neuromelanin also demonstrates, contrary to some other studies, that a portion of the paramagnetic metal ions in this tissue are bound to the pigment in situ. Combined with previous histochemical data, these observations have implications for the mechanism through which neuromelanin accumulates in vivo and are consistent with its having a cytoprotective function under normal conditions, but a cytotoxic role at advanced ages and in patients with Parkinson's disease. Other results of this study show that homogenizing tissues during the purification of any natural pigment may cause contamination of the pigment by extraneous metal ions and that subsequent incubation in hot acid, though most effective in removing metal ions and hydrolyzing proteins, leads to degradation of melanin. A purification procedure using incubation in acid at room temperature, however, is well suited for identifying and characterizing unknown natural pigments by electron paramagnetic resonance spectroscopy.

Notes: Abstract: To clarify the regulatory mechanism of the N-methyl-d-aspartate (NMDA) receptor/channel by several protein kinases, we examined the effects of purified type II of protein kinase C (PKC-II), endogenous Ca2+/calmodulin-dependent protein kinase II (CaMK-II), and purified cyclic AMP-dependent protein kinase on NMDA receptor/ channel activity in the postsynaptic density (PSD) of rat brain. Purified PKC-II and endogenous CaMK-II catalyzed the phosphorylation of 80–200-kDa proteins in the PSD and l-glutamate-(or NMDA)-induced increase of (+)-5-[3H]methyl-10, 11-dihydro-5H-dibenzo[a, d]cyclohepten-5, 10-imine maleate ([3H]MK-801; open channel blocker for NMDA receptor/channel) binding activity was significantly enhanced. However, the pretreatment of PKC-II-and CaMK-II-catalyzed phosphorylation did not change the binding activity of l-[3H]glutamate, cis-4-[3H](phospho-nomethyl)piperidine-2-carboxylate ([3H]CGS-19755; competitive NMDA receptor antagonist), [3H]glycine, α-[3H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, or [3H]-kainate in the PSD. Pretreatment with PKC-II-and CaMK-II-catalyzed phosphorylation enhanced l-glutamate-induced increase of [3H]MK-801 binding additionally, although purified cyclic AMP-dependent protein kinase did not change l-glutamate-induced [3H]MK-801 binding. From these results, it is suggested that PKC-II and/or CaMK-II appears to induce the phosphorylation of the channel domain of the NMDA receptor/channel in the PSD and then cause an enhancement of Ca2+ influx through the channel.

Notes: Abstract: Exposure to manganese compounds often occurs as the result of industrial production or mining. Although manganese appears in traces in animal and human tissue and is essential to certain biological processes, it is also toxic. In humans and animals, toxicity is mainly associated with the nervous system. The mechanism underlying behavioral and biochemical alterations observed after manganese intoxication is not fully understood. We have shown that the manganese present in serum after exposure to manganese oxide is bound to transferrin as trivalent manganic ion. In this study of manganese uptake and storage we used a clone of human neuroblastoma cells (SHSY5Y). These cells differentiate and express catechol-aminergic properties. Saturation binding analysis of the transferrin-manganese complex to the cells revealed a single class of binding sites, with an apparent KD of 13 ± 1 nM and a density of 11, 000 ± 2, 000 binding sites per cell. The complex was internalized in a temperature-dependent way and reached saturation after 2 h when ∼2% of the added manganese had been internalized. About 80% of the internalized manganese was found in ferritin after 24 h of exposure. The results demonstrate that the transferrin receptor on SHSY5Y cells can bind and internalize a manganese-transferrin complex as efficiently as an iron-transferrin complex, although a saturation of the manganese uptake was achieved.

Notes: Abstract: Ischemia-induced changes in 31P NMR relaxation were examined in 16 piglets. NMR spectra were acquired under control conditions and during complete cerebral ischemia induced via cardiac arrest. Changes in T1 were assessed directly in six animals during control conditions and after 30–45 min of complete ischemia when changes in brain P1 levels had reached a plateau. The T1 for P1 did not change, i.e., 2.3 ± 0.5 s during control conditions versus 2.4 ± 1.0 s during ischemia. To evaluate phosphocreatine and ATP, two types of spectra, with a long (25-s) or short (1-s) interpulse delay time, were collected during the first 10 min of ischemia (n = 10). Both types of spectra showed the same time course of changes in phosphocreatine and ATP levels, implying that the T1 relaxation times do not change during ischemia. There were no changes in the linewidths of phosphocreatine, ATP, or P1 during ischemia, implying that the T*2 values remain constant. Our results suggest that the 31P T1 and T*2 for phosphocreatine, Pi, and ATP do not change during ischemia, and therefore changes in 31P NMR peak intensity accurately reflect changes in metabolite concentrations.

Notes: Abstract: The present study reports the ion dependency of 2β-carbomethoxy-3β-(4-fluorophenyl)[3H]tropane ([3H]- CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [3H]CFT binding to synaptosomal P2 membranes requires low concentrations of Na+ (peak binding between 20 and 50 mM Na+), is stimulated by phosphate anion or l-, but is unaffected or only slightly affected by F-, Cl-, Br-, NO3-, or SO42-, Concentrations of Na+ of 〉50 mM become inhibitory except in the presence of l-, which shifts peak binding levels toward higher Na+ concentrations and also elevates the peak binding level. K+ strongly decreased [3H]CFT binding with a shallow inhibition curve, and Na+ could not overcome this effect. Saturation analysis of [3H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 mM; 1 mM plus 49 mM NaCIversus 10 mM plus 40 mM NaCI; or 1 mM plus 129 mM Nal versus 10 mM plus 120 mM Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.

Notes: Abstract: Monoamine oxidases (MAOs) A and B play important roles in the metabolism of neuroactive, vasoactive amines. Human platelets contain only MAO B, often used as an indicator of brain MAO B. The validity of this model remained to be evaluated. This report describes the molecular cloning of human MAO B from frontal cortex and platelets. Two overlapping PCR-amplified clones of human platelet MAO B and four PCR-amplified clones of human frontal cortex MAO B covering the entire coding region were sequenced using five internal oligomers and M13 reverse and forward primers. The nucleotide sequences of human MAO B cDNA from platelet and frontal cortex were identical to that of human liver MAO B except for three nucleotides that differed in frontal cortex: nucleotides 440 A → G, 794 C → T, and 825 C → T. Whether or not these differences are artifactual, all three represent silent mutations, which would not alter the amino acid of the encoded polypeptides. Thus, the deduced amino acid sequences of MAO B from frontal cortex, platelet, and liver are identical. These findings indicate the validity of using platelet MAO B mRNA as a marker for brain MAO B and provide a new approach to study the role of brain MAO B in humans.

Notes: Abstract: Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving β-and α1-adrenergic receptors. The effects of N-monomethyl-l-arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited β-adrenergic stimulation of cGMP formation as well as α1-adrenergic potentiation of β-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of l-arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.

Notes: Abstract: We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxy-genase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [3H]-arachidonic acid, formed products that eluted in four major groups designated as R17–30, R42–50, R51–82, and R83–90 based on their retention times in reverse-phase HPLC. These fractions were further segregated into as many as 13 peaks by normal-phase HPLC and a second reverse-phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact-mass spectrometry. Based on HPLC retention times and gas chromatography/electron impactmass spectrometry analysis, the more polar fractions (R17–30) contained prostaglandin D2 as the major cyclooxygenase product. Minor products included 6-keto prostaglandin F1α, prostaglandin E2, prostaglandin F2α, and thromboxane B2. Fractions R42–50, R51–82. and R83–90 contained epoxygenase and lipoxygenase-like products. The major metabolite in fractions R83–90 was 5, 6-epoxyeicosatrienoic acid (EET). Fractions R51–82 contained 14, 15-and 8, 9-EETs, 12-and 5-hydroxyeicosatetraenoic acids, and 8, 9-and 5, 6-dihydroxyeicosatrienoic acids (DHETs). In fractions R42–50, 14, 15-DHET was the major product. When radiolabeled [3H]14, 15-EET was incubated with astrocyte homogenate, it was rapidly metabolized to [3H]14, 15-DHET. The metabolism was inhibited by submicromolar concentration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxyalcohols from 14, 15-DHET was not observed. In conclusion, astro-cytes readily metabolize arachidonic acid to 14, 15-EET, 5, 6-EET, and their vicinal-diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.

Notes: Abstract: Intrastriatally infused ouabain (200 or 1,000 μM) markedly increased the extracellular levels of striatal spermidine and spermine in dialysis experiments in halothane-anesthetized rats. The effects of ouabain (1 mM) on sper- midine release were rapid and unaffected by local infusion of the competitive N-methyl-d-aspartate (NMDA) antagonist 3-(2-carboxypiperazin-4-yl)propyl-1 -phosphonic acid (CPP; 100 μM) or by systemically administered MK-801 (0.3 mg/kg i.p.), both of which treatments markedly inhibit the effects of intrastriatally administered NMDA. The peak effects of ouabain (1 mM) on spermine release were delayed with respect to those on spermidine release, or to the effects of NMDA, and were also insensitive to locally administered CPP (100 μM). However, systemically administered MK-801 (0.3 mg/kg i.p., 30 min before the striatal infusion of drugs), which totally inhibits the effects of NMDA, or CPP (10 mg/kg i.p.; 30 min before the striatal infusion of drugs) partially inhibited the effects of ouabain on spermine release, suggesting partial mediation of the delayed effects of ouabain on spermine release by indirect NMDA-receptor activation. Despite partial sensitivity of ouabain-induced spermine release to systemically administered NMDA antagonists, both spermidine and spermine can be released in vivo by sodium-pump inhibition, independently of NMDA-receptor activation.

Notes: Abstract: The characteristics of adenosine and inosine outflow evoked by 5 min of ischemia-like conditions in vitro (superfusion with glucose-free Krebs solution gassed with 95% N2/5% CO2) were investigated on rat hippocampal slices. The viability of the slices after “ischemia” was evaluated by extracellular recording of the evoked synaptic responses in the CA1 region. The evoked dendritic field potentials were abolished after 5 min of superfusion under “ischemia” but a complete recovery occurred after 5 min of reperfusion with normal oxygenated Krebs solution. No recovery took place after 10 min of “ischemia.” The addition of the adenosine A, receptor antagonist 8-phenylthe- ophylline to the superfusate antagonized the depression of the evoked field potentials caused by 5 min of “ischemia.” Five minutes of “ischemia” brought about a six- and fivefold increase in adenosine and inosine outflow, respectively, within 10 min. Tetrodotoxin reduced the outflow of adenosine and inosine by 42 and 33%, respectively, whereas the removal of Ca2+ caused a further increase. The NMDA receptor antagonist d(-)-2-amino-7- phoshonoheptanoic acid and the non-NMDA antagonist 6,7-dinitroquinoxaline-2,3-dione brought about small, not statistically significant decreases of adenosine and inosine outflow. The glutamate uptake inhibitor dihydrokainate did not affect the outflow of adenosine and inosine. Inhibition of ecto-5′-nucleotidase by α, β-methylene ADP and GMP did not affect basal adenosine outflow but potentiated “ischemia”-evoked adenosine outflow. It is concluded that ischemia-like conditions in vitro evoke a Ca2+-independent adenosine and inosine outflow, through a mechanism that partly depends on propagated nervous activity but does not involve excitatory amino acids. The efflux of adenosine is probably responsible for the depression of the evoked synaptic electrical activity during “ischemia” in the hippocampal slices.

Notes: Abstract: 5-Hydroxytryptamine (5-HT) specifically and reversibly inhibits nicotine-induced currents and catecholamine release in bovine adrenal chromaffin cells in culture. Pharmacological analysis indicates that the inhibition is not mediated by known 5-HT receptor subtypes. The inhibition is noncompetitive over a range of nicotine concentrations between 1 and 100 μM. Preincubation with either 5-HT or substance P significantly protects the response from nicotine-induced desensitization. It is concluded that 5-HT inhibits nicotinic acetylcholine receptors on bovine adrenal chromaffin cells, probably by binding to a noncompetitive site on the receptor itself. Because both blood and the chromaffin cells contain 5-HT, the inhibition provides an opportunity for negative control of catecholamine secretion from the adrenals.

Notes: Abstract: The light/dark cycle influences the rhythmic production of melatonin by the trout pineal organ through a modulation of the serotonin N-acetyltransferase (NAT) activity. In static organ culture, cyclic AMP (cAMP) levels (in darkness) and NAT activity (in darkness or light) were stimulated in the presence of forskolin, isobutylmethylxanthine, or theophylline. Analogues of cAMP, but not of cyclic GMP, induced an increase in NAT activity. Light, applied after dark adaptation, inhibited NAT activity. This inhibitory effect was partially prevented in the presence of drugs stimulating cAMP accumulation. In addition, cAMP accumulation and NAT activity increase, induced by forskolin, were temperature dependent. Finally, melatonin release, determined in superfused organs under normal conditions of illumination, was stimulated during the light period of a light/dark cycle by adding an analogue of cAMP or a phosphodiesterase inhibitor. However, no further increase in melatonin release was observed during the dark phase of this cycle in the presence of the drugs. This report shows for the first time that cAMP is a candidate as intracellular second messenger participating in the control of NAT activity and melatonin production by light and temperature.

Notes: Abstract: Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na+]i., [K+]i, and [Ca2+]i, and pH under conditions when [H+]i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K+ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 μM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin-stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion-selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca2+], and [Na+]., The increase in [Na+], in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin-induced loss of K+ and subsequent depolarization lead to an increase in Na+ influx and stimulation of the Na+/K+ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H+] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K+], depresses both oxidative phosphorylation and glycolysis; and that the nigericin-induced alterations in ion levels and activities of energy-producing pathways can explain some of the deleterious effects of ischemia and hypoxia.

Notes: Abstract: Formation of 6-hydroxydopamine (6-OHDA) from dopamine has been hypothesized to mediate neuro-degeneration induced by some psychostimulants. Although the emergence of a 6-OHDA-like substance was reported in the striatum of methamphetamine-treated rats, this substance has not been identified by a direct approach. We used mass fragmentography to search for 6-OHDA in the rat frontal cortex and striatum after the administration of a number of drugs including 3,4-dihy-droxyphenyl-L-alanine, methamphetamine, amphetamine, and cocaine, all of which increase synaptic dopamine. No 6-OHDA was detected after the acute systemic administration of these agents. Intraventricular administration of 6-OHDA (10 μg/rat.) produced measurable concentrations of 6-OHDA that were higher in the striatum than in the frontal cortex. Intraventricular administration of 2,4,5-trihydroxy-phenyl-D,L-alanine (6-OHDOPA; 10 μg/rat) produced similar concentrations of 6-OHDA in both regions. Pargyline, but not carbidopa (α-methyldopahydrazine), enhanced the effect of intraperitoneal 6-OHDOPA administration (80 mg/kg). We conclude that (1) 6-OHDOPA can cross the blood-brain barrier and is converted to 6-OHDA in the brain, (2) 6-OHDA is a substrate for monoamine oxidase(s) and therefore a search for its purported deaminated metabolite is warranted, and (3) acute treatment with the above stimulants either does not lead to the formation of 6-OHDA or produces concentrations below the detection limit of the assay (〈34 pg/mg of protein).

Notes: Abstract: Cholinergic neurons in PNS and CNS are identified by the presence of choline acetyltransferase and the accumulation of choline by a high-affinity, sodium-coupled choline transporter to be used for acetylcholine synthesis. It appears that expression of choline acetyltransferase can be altered by several physiological conditions, including hormones and trophic factors, but little is known about control of expression of the sodium-coupled choline carrier or whether these two phenotypic markers are regulated similarly. In the present study, the cholinergic human neuroblastoma LA-N-2 was used to investigate regulation of expression of choline acetyltransferase and choline up take activity associated with differentiation and neurite extension. Cells grown in serum-containing basal medium maintained a relatively undifferentiated morphology, expressed low levels of choline acetyltransferase activity, and accumulated choline by a sodium-dependent process followed by conversion to acetylcholine. Transfer of cells to an enriched, serum-free defined medium resulted in morphological and neurochemical differentiation, with an enhancement of cholinergic phenotype. Hemicholinium-sensitive choline uptake activity was increased about sixfold over a 4-day period, with no change in choline acetyltransferase or acetylcholinesterase specific activity. Acetylcholine synthesis was increased in parallel with the changes in choline accumulation; choline metabolism in the differentiated cells differed significantly from that observed in the undifferentiated cells, with proportionally less converted to phosphorylcholine and proportionally more remaining as unmetabolized choline and converted to acetylcholine. The enhanced choline accumulation appeared to be mediated by an increased number of choline carriers, demonstrated by increased binding of the affinity ligand [3H]-choline mustard to the transporter and by an increased Vmax for the uptake process. The increased expression of the transport function appeared to be under transcriptional control. as the enhancement of uptake was blocked by the RNA polymerase II inhibitor α-amanitin as well as by the protein synthesis inhibitor cycloheximide. These results show that expression of sodium-coupled choline carriers and choline acetyltransferase may be regulated separately in the differentiating neuroblastoma LA-N-2 and that neurotransmitter synthesis is controlled by provision of precursor rather than at the level of the biosynthetic enzyme.

Notes: Abstract: Using stable cell lines containing a series of deletions of the myelin basic protein (MBP) promoter directing the bacterial chloramphenicol acetyltransferase gene in a peripheral neurinoma cell line, we have studied the sequences in the MBP promoter needed for induction by cyclic AMP. Stimulation of expression from the MBP promoter by cyclic AMP is not a rapid response. Expression begins after 24 h and reaches a maximum at ˜72 h. The results from the stable transformants indicate at least one region that appears to be essential to the induction of transcription directed by the MBP promoter. The region that is necessary for induction does not contain a consensus cyclic AMP response element. A specific binding site involved in the induction by cyclic AMP was localized to an NFI binding site.

Notes: Abstract: Mice carrying a mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) have recently been produced to provide an animal model for Lesch-Nyhan disease. The current-studies were conducted to characterize the consequences of the mutation on the expression of HPRT and to characterize potential changes in brain purine content in these mutants. Our results indicate that the mutant animals have no detectable HPRT-immunoreactive material on western blots and no detectable HPRT enzyme activity in brain tissue homogenates, confirming that they are completely HPRT deficient (HPRT-). Despite the absence of HPRT-mediated purine salvage, the animals have apparently normal brain purine content. However, de novo purine synthesis, as measured by [14C]formate incorporation into brain purines, is accelerated four- to fivefold in the mutant animals. This increase in the synthesis of purines may protect the HPRT- mice from potential depletion of brain purines despite complete impairment of HPRT-mediated purine salvage.

Notes: Abstract: Muscarinic cholinergic and α1-adrenoceptor-mediated stimulation of phosphoinositide hydrolysis in rat cerebral cortex were compared by measuring carbachol- and noradrenaline-induced accumulation of various intermediates of the phosphoinositide cycle. Unlike carbachol, noradrenaline in the presence of guanosine 5′-O-(3-thiotriphosphate) did not stimulate phospholipase C activity in brain cortical membranes. In cortical slices, the efficacy of noradrenaline to stimulate accumulation of 3H-inositol phosphates and [32P]phosphatidic acid was 2.5 to threefold that of carbachol. However, noradrenaline was less effective than carbachol in stimulating accumulation of [3H]CDP-diacylglycerol and resynthesis of phosphatidylinositol. This was not due to calcium inhibition of CTP:phosphatidate cytidyltransferase or to different lithium requirements for carbachol- and noradrenaline-stimulated accumulation of [3H]CDP-diacylglycerol. The noradrenaline-induced unbalance of the phosphoinositide cycle, which was most apparent at relatively high concentrations of calcium (2.5 mM) in the incubation buffer, was qualitatively reproduced with ionomycin. The use of the α1a-subtype-selective adrenoceptor antagonists WB4101 and 5-methylurapidil revealed a single α1a-like component mediating the effects of noradrenaline. Our results suggest that the primary mechanism for phospholipase C activation by brain α1 adrenoceptors involves an increase in intracellular calcium concentration.

Notes: Abstract: There is evidence of abnormalities in the brain-stem monoamine-containing neurons in infants with sudden infant death syndrome (SIDS). By taking advantage of the rich innervation of the human basal ganglia by monoam-inergic afferents from cell bodies in the brainstem, we studied the synaptic chemistry of catecholamine and associated neurons of the putamen obtained postmortem from 14 SIDS infants, eight age-matched control infants, and older control subjects of various ages. We found significantly lower concentrations of dopamine and higher homovanillic acid/DA ratios in samples from SIDS infants compared with age-matched control infants. Noradrenaline and 5-hydroxytryptamine were lower in SIDS compared with control subjects, but the difference did not reach statistical significance. There was no clear evidence that dihydroxyphe-nylacetic acid and 5-hydroxyindoleacetic acid were altered. Immunoblot analysis of striatal tissue showed that samples from infants with SIDS, which exhibited lower DA, also had lower tyrosine hydroxylase protein. Other transmitter-specific neuronal markers were also assessed, including enzymes associated with cholinergic and GABA-containing neurons. We found significantly decreased choline acetyltransferase activities. However, GABA, glutamate, or somatostatin concentrations or monoamine oxidase activities were unchanged in SIDS. We also noted age-dependent changes in brain weights and some synaptic markers by comparing the age-matched infants with older control subjects. Analysis of variance revealed that homovanillic acid, dihydroxyphenylacetic acid, and monoamine oxidase B activities were increased with age. DA and choline acetyltransferase were also found to be positively correlated in putamen. Our findings suggest developmental changes in some transmitter-specific neurons in SIDS that may result from apneic episodes or chronic hypoxia induced before death.

Notes: Abstract: The present study was initiated to examine the effects of ATP on acetylcholine (ACh) synthesis. The exposure of superior cervical ganglia to ATP increased ACh stores by 25%, but this effect was also evident with ADP, AMP, and adenosine, but not with βγ-methylene ATP, a nonhydrolyzable analogue of ATP, or with inosine, the deaminated product of adenosine. Thus, we attribute the enhanced ACh content caused by ATP to the presence of adenosine derived from its hydrolysis by 5′-nucleotidase. The adenosine-induced increase of tissue ACh was not the consequence of an adenosine-induced decrease of ACh release. The extra ACh remained in the tissue for more than 15 min after the removal of adenosine, but it was not apparent when ganglia were exposed to adenosine in a Ca2+-free medium. Incorporation of radiolabelled choline into [3H]ACh was also enhanced in the presence of adenosine, suggesting an extracellular source of precursor. Moreover, the synthesis of radiolabelled forms of phosphorylcholine and phospholipid was not reduced in adenosine's presence, suggesting that the extra ACh was not likely derived from choline destined for phospholipid synthesis. Aminophylline did not prevent the adenosine effect to increase ACh content; this effect was blocked by dipyridamole, but not by nitrobenzylthioinosine (NBTI). In addition, two benzodiazepine stereoisomers known to inhibit stereoselectively the NBTI-resistant nucleoside transporter displayed a similar stereoselective ability to block the effect of adenosine. Together, these results argue that adenosine is transported through an NBTI-resistant nucleoside transporter to exert an effect on ACh synthesis. The extra ACh accumulated as a result of adenosine's action was releasable during subsequent preganglionic nerve stimulation, but not in the presence of vesamicol, a vesicular ACh transporter inhibitor. We conclude that the mobilization of ACh is enhanced as a result of adenosine pretreatment.

Notes: Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [14C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC50, 0.33 μM), the potent 5-HT3 receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [14C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT3 receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [14C]guanidinium uptake, on the one hand, and to displace [3H]zacopride specifically bound to 5-HT3 receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [14C]guanidinium uptake was directly controlled by 5-HT3 receptors. Various compounds such as inorganic cations (La3+, Mn2+, Ba2+, Ni2+, and Zn2+), D-tubocurarine, and memantine inhibited [14C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT3 receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT3 receptor stimulation, prevented the effects of 5-HT plus SP on [14C]guanidinium uptake. The cooperative effect of SP on this 5-HT3-evoked response resulted neither from an interaction of the peptide with the 5-HT3 receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro9]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [14C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [14C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT3 receptors in NG 108-15 cells.

Notes: Abstract: Several lines of evidence indicate that cyclic AMP (cAMP) induces oligodendrocyte differentiation. However, the mechanism(s) of this stimulation remains unknown. Because in several cell types the transcriptional activity of various cAMP-responsive genes is regulated through a cisacting DNA sequence known as cAMP response element (CRE), we investigated the possible presence of a CRE binding (CREB) protein in myelinating oligodendrocytes. A double-stranded oligonucleotide containing a tandem repeat of the CRE sequence was labeled with T4 kinase in the presence of [32P]ATP and then incubated with a nuclear protein extract from 14-day-old rat brain oligodendrocytes. The reaction mixture was then electrophoresed on nondenaturing polyacrylamide gels. The results indicated the presence of a protein that specifically binds to the CRE sequence. The results were supported by southwestern blotting assays in which the CRE probe bound to a ˜45-kDa protein species. In separate experiments, it was shown that the 45-kDa protein can be phosphorylated in vitro by the catalytic subunit of protein kinase A. Developmental analysis of CREB protein expression indicated a peak at 14 days of age, preceding the peak of myelinogenesis.

Notes: Abstract: Abstract: [3H]Adenosine transport was characterized in cerebral cortical synaptoneurosomes prepared from postmortem human brain using an inhibitor-stop/centrifugation method. The adenosine transport inhibitors dipyridamole and dilazep completely and rapidly blocked transmembrane fluxes of [3H]adenosine. For 5-s incubations, two kinetically distinguishable processes were identified, i.e., a high-affinity adenosine transport system with Kt and Vmax values of 89 μM and 0.98 nmol/min/mg of protein, respectively, and a low-affinity adenosine transport system that did not appear to be saturable. For incubations with 1 μM [3H]adenosine as substrate, intrasynaptoneurosomal concentrations of [3H]adenosine were 0.26 μM at 5 s and 1 μM at 600 s. Metabolism of accumulated [3H]adenosine to adenine nucleotides was 15% for 5-s, 23% for 15-s, 34% for 30-s, 43% for 60-s, and 80% for 600-s incubations. The concentrations (μM) of total accumulated 3H-purines ([3H]-adenosine plus metabolites) at these times were 0.3, 0.5, 1.0, 1.3 and 5.6, respectively. These results indicate that in the presence of extensive metabolism, the intrasynaptoneurosomal accumulation of 3H-purines was higher than the initial concentration of 1 μM [3H]adenosine in the reaction medium. For 5-, 15-, 30-, 60-, and 600-s incubations in the presence of the adenosine deaminase inhibitor EHNA and the adenosine kinase inhibitor 5′-iodotubercidin, metabolism of the transported [3H]adenosine was 14, 14, 16, 14, and 38%, respectively. During these times, total 3H-purine accumulation was 0.3, 0.5, 0.5, 0.7, and 1.8 μM, respectively. Thus, the apparently “concentrative'’accumulation of 3H-purines can be prevented by inhibition of adenosine metabolism and, taken together, these results suggest that adenosine transport in at least synaptoneurosomes prepared from postmortem human brain is via a nonconcentrative and equilibrative system.

Notes: Abstract: Dopamine (DA) stimulated K+ efflux (assessed as 86Rb+ efflux) in retinal suspensions of posthatched chicken. This effect was dose dependent (EC50= 22 μM), was mimicked by the D1-selective antagonist SKF-38393, and reversed by the D1-selective antagonist SCH-23390, indicating an involvement of D1 receptors. Analogues of cyclic AMP (CAMP) did not mimic the DA action. Moreover, DA failed to affect cAMP levels, suggesting that adenylyl cyclase (AC) was not involved. In contrast, forskolin (FSK) stimulated both K+ efflux and cAMP accumulation in the retina (EC50 of 10 μM for both effects). The FSK-elicited K+ efflux was not mimicked by 1,9-dideoxy-FSK (an analogue of FSK that does not activate AC), suggesting that FSK stimulated K+ efflux through the activation of AC. Both DA and FSK inhibited Na+,K+-ATPase activity in the retina. However, the DA-elicited K* efflux was independent of this inhibition, whereas the FSK effect on K+ efflux was largely due to the inhibitory action of the diterpene of the ion pump. A possible role of protein kinase C (PKC) in the DA action was explored. The PKC activator 4β-phorbol 12-myristate 13-acetate (4β-PMA) potently (EC50= 4 nM) stimulated K+ efflux. This action was not mimicked by the inactive isomer 4α-PMA. When added together, DA and 4β-PMA behaved in an additive manner, suggesting separate mechanisms of action for these two drugs. Moreover, DA failed to stimulate retinal phosphoinositide hydrolysis, a well-known pathway leading to PKC activation. These data suggest that DA acting through D1 receptors and independently of AC can modulate its target cell excitability in the chick retina by stimulating K+ efflux pathways. The mechanism of the DA action remains to be clarified.

Notes: Abstract: The acute effects of serum on sodium-potassium (Na+-K+) pump activity and glucose uptake in cultured rat skeletal muscle were studied. Addition of serum to myo-tubes in phosphate-buffered saline caused Na+-K+ pump activity (as measured by changes in the ouabain-sensitive component of both membrane potential and 86Rb uptake) to increase, with peak effects obtained after 30 min. The effect was blocked completely by treatment with amiloride, but not by tetrodotoxin, which blocks voltage-dependent Na+ channels. On transfer of myotubes to Na+-free, choline buffer, resting Na+-K+ pump activity decreased to about 10% of that in phosphate-buffered saline. Addition of regular serum, but not Na+-free serum, caused Na+-K+ pump activity to increase slightly. Similar results were obtained with serum on glucose uptake, the peak effect being reached within 15 min. Stimulation of glucose uptake by serum was partially reduced by amiloride and was not altered by tetrodotoxin. Removal of external Na+ also eliminated serum effects on glucose uptake. The results demonstrate that there are similar signals involving Na+-H+ exchange for serum-induced increases in Na+-K+ pump activity and glucose transport. The lack of complete blockade of serum-induced elevation of glucose transport suggests an additional, as yet undefined, intracellular signal for stimulation of this transport system.

Notes: Abstract: D,L-(E)-2-Amino-4-propyl-5-phosphono-3-pen-tenoic acid (CGP 39653). a new, high-affinity, selective NMDA receptor antagonist, interacts with rat cortical membranes in a saturable way and apparently to a single binding site, with a KD of 10.7 nM and a receptor density of 2.6 pmol/mg of protein. Displacement analysis of [3H]CGP 39653 binding shows a pharmacological profile similar to that reported for another NMDA antagonist, 3-[(±)-2-carboxypiperazin-4-yI]propyl-1-phosphonic acid (CPP). Glycine, however, is able to discriminate between the two ligands; in fact, it does not affect [3H]CPP binding but inhibits [3H]CGP 39653 binding in a biphasic way. D-Serine, another agonist at the strychnine-insensitive glycine binding site of the NMDA receptor complex, inhibits [3H]CGP 39653 binding in the same way as glycine, with a potency that correlates with its binding affinity at the glycine site. In addition, 7-chlorokynurenic acid, an antagonist at the glycine site, is able to reverse the displacement of [3H]CGP 39653 by glycine in a dose-dependent manner. Furthermore, the dissociation rate constant of [3H]CGP 39653 is enhanced in the presence of glycine, whereas the presence of NMDA receptor ligands does not modify the rate of dissociation of [3H]CGP 39653 from the receptor. These results indicate that part of the binding of the NMDA antagonist CGP 39653 can be potently modified by glycine through an allosteric mechanism, and suggest the existence of two antagonist preferring NMDA receptor subtypes that are differentially modulated through the glycine binding site.

Notes: Abstract: Heterogeneity of binding affinities for a variety of ligands was observed for γ-aminobutyric acid type A (GABAA) receptors in the rat CNS, at both GABA and ben-zodiazepine recognition sites. Photoaffinity labeling by [3H]flunitrazepam and [3H]muscimol to affinity column-purified receptor proteins was examined by gel electropho-resis in sodium dodecyl sulfate. Anesthetic barbiturates (pentobarbital) and steroids (alphaxalone) both differentially stimulated the incorporation of [3H]flunitrazepam more so into the 51-kDa α1 subunit than into the 53-kDa aL2 polypeptide, and incorporation of [3H]muscimol into the 55-kDa β2 subunit more so than the 58-kDaβ3 polypeptide. Binding to these polypeptides was also affected differentially by other allosteric modulators and competitive inhibitors, including the benzodiazepine “type 1” selective ligand CL218.872. Heterogeneity in affinity of this drug for the single 51-kDa α1 polypeptide strongly suggests that type I receptors, like type II, are heterogeneous. In brain sections, the extent of enhancement of [3H]muscimol binding showed significant regional variation, similar for both steroids and barbiturates, and the GABA analogues THlP and taurine inhibited muscimol binding with regional variations in affinity that were almost opposites of each other. Modulation of [3H]flunitrazepam binding by steroids, barbiturates, and THlP significantly varied with regions. Taken together, ligand binding heterogeneity exhibited by photoaffinity labeling and autoradiography demonstrate the existence of multiple pharmacological-binding subtypes resulting from the combination of multiple polypeptide gene products into several oligomeric isoreceptors. Comparison of the regional distribution of binding subtypes with that of different subunit gene products allows the following conclusions about possible subunit compositions of native pharmacological receptor subtypes present in the brain: Benzodiazepine pharmacology of the oligomeric receptor isofotms is dependent on the nature of α and subunits other than α, GABA-benzodiazepine coupling is dependent on the nature of the α subunits, GABA site pharmacology is dependent on the nature of the β sub-units, and several subunits including α and β contribute to the degree of sensitivity to steroids and barbiturates. Finally, the presence of discrete subunits may be necessary but is not sufficient to postulate a defined pharmacological property.

Notes: Abstract: Specific antisera were raised against the A and B chains of glycoprotein III. lmmunoblotting revealed that in adrenal medulla both chains migrate very closely together in two-dimensional electrophoresis. Both chains with slightly differing molecular sizes are found in several endocrine tissues and in brain, kidney, liver, and serum. The mRNA has an analogous widespread distribution. In primary cultures of chromaffin cells the level of message becomes significantly increased by treatment with hista-mine or 12-O-tetradecanoylphorbol 13-acetate/forskoIin. However, the increase is small when compared with that of secretogranin II. The subcellular localization of glycoprotein III in endocrine organs and in the posterior pituitary was investigated by subcellular fractionation and immuno-electron microscopy. Glycoprotein III was found to be confined to the large densecore vesicles of these organs. For a discussion of the function of glycoprotein III, its localization in these organelles has to be taken into account.

Notes: Abstract: Astrocytes exposed to hypoosmotic stress swell and subsequently reduce their size to almost their original volume, a phenomenon called regulatory volume decrease (RVD). We found that during hypoosmotic swelling there was a twofold increase in phosphatidylinositol (PI) hydrolysis. This increase was inhibited by the phosphdipase C inhibitor, U-73122 (10 μM). Inhibition of PI hydrolysis resulted in blockage of RVD. We also examined whether agents that stimulate PI hydrolysis would enhance RVD. These agents significantly accelerated RVD. The rank order of potency was endothelin (20 nM) ≥ norepinephrine (100 μM) 〉 endothelin-3 (7 nM) 〉 thrombin (1 U/ml) ≥ ATP (500 μM) 〉 bradykinin (20 μM) ≥ carbachol (500 μM), as indicated by RVD rate constants. The extent of PI hydrolysis induced by these agents at the beginning of RVD exhibited a logarithmic relationship with the magnitude of RVD enhancement. Also, there was a linear relationship between the rate of PI hydrolysis and RVD rate constants. Our results suggest that stimulated PI hydrolysis is involved in the regulation of cell volume in astrocytes.

Notes: Abstract: Adenosine A2a receptors have been localized to GABAergic striatopallidal neurons, but their functional role is unknown. To address this question, the modulation of endogenous GABA release by adenosine A2a receptors was examined in slices of rat globus pallidus. The selective adenosine A2a receptor agonist CGS-21680 (3.0–10 nM) significantly increased electrically stimulated release (overflow) of GABA, with 10 nM CGS-21680 resulting in a 44% increase compared with the control. Both the nonselective adenosine receptor antagonist 8-phenyltheophylline (10 μM) and the selective A2a receptor antagonist KF-17837 (100 nM) abolished the CGS-21680-induced increase in GABA overflow. Higher concentrations of CGS-21680 (0.10–1.0 μM) decreased GABA overflow by ˜25%.8-Phenyltheophylline (10 μM) antagonized these effects, whereas KF-17837 (100 nM) did not, suggesting actions of CGS-21680 on other adenosine receptors at these concentrations. These results demonstrate that activation of adenosine A2a receptors augments electrically stimulated release of GABA from globus pallidus slices and suggest a mechanism by which adenosine may modulate GABAergic output from the striatopallidal efferent system.

Notes: Abstract: The transforming growth factors-β (TGFs-β) are multifunctional peptide-growth factors that have been localized in neuronal and glial cells of the C'NS of mice. rats, and chick embryos. We tested the TGF-β isoforms 1. 2. and 3 for their protective ctlects against neuronal degeneration caused by cytotoxic hypoxia or by the excitatory amino acid i -glutamate. A cytotoxic hypoxia was induced in cultured chick embryo telencephalic neurons by adding I m.l/ sodium cyanide to the culture medium tor a period of 30min. I reatment with TGF-81 (1-30 ng/ml) led to a statistically significant increase in cell viability, neuronal ATP levels. and protein content of the cultures assessed 72 h after the toxic insult. TGF-33 was able to reduce the cyanide-induced neuronal damage at concentrations of 0.3 and 1 ng/ ml. whereas TGF-33, only showed neuroprotective activity at concentrations of 30 and 50 ng/ml. Both pre- and posttreatment with TGF-31, also prevented the degeneration of cultured chick embryo telencephalic neurons that had been exposed to I mM L-glutamate in a buffered salt solution for a period of 60 min. Furthermore, TGF-β1 (0.3-3 ng/ml). and to a lesser extent TGF-β3 (0.1-1 ng/ml). significantly reduced excitotoxic injury of cultured neurons from rat cerebral cortex that had been exposed to serum-free culture medium supplemented with 1 m.M L-glutamate. These results demonstrate that the TGFs-β are able to prevent the degeneration of primary neuronal cultures, which was caused by energy depletion and activation of glutamate receptors, in an isoform-specific manner.

Notes: Abstract: The effects of the selective dopamine D2 receptor antagonists YM-09151-2 and l-sulpiride on the in vivo release of dopamine (DA), l-3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in rat striatum were investigated. The drugs were injected into the striatum through a microinjection needle attached to a dialysis probe. YM-09151-2 (0.1 or 1.0 μg/0.5 μl) injected into the striatum produced a dramatic rapid-onset transient increase in striatal DA release in a dose-dependent manner. However, the DA increase induced by l-sulpiride (15 or 75 ng/0.5 μl) was small and of slower onset. An increase of DOPAC levels by YM-09151-2 was biphasic: The first peak occurred at 40 min, followed by a delayed-onset gradual increase. Slower-onset gradual increases were also found in DOPAC levels after l-sulpiride injection and in HVA levels after injections of both YM-09151-2 and l-sulpiride. The infusion of tetrodotoxin (TTX; 2 μM) revealed two different types of DA release mechanisms: The rapid-onset transient DA release induced by YM-09151-2 was TTX insensitive, whereas the slower-onset DA release induced by l-sulpiride was TTX sensitive. Moreover, the rapid-onset transient DA release was Ca2+ independent and was not affected by pre-treatment with l-sulpiride or nomifensine. Therefore, it is concluded that YM-09151-2 injected into the striatum produced a transient striatal DA release that is independent of D2 receptors and the action potential.

Notes: Abstract: Casein kinase II (CKII) is a protein kinase acting in the intracellular cascade of reactions activated by growth factor receptors, and that has a profound influence on cell proliferation and survival. In this investigation, we studied the changes in the activity and levels of CKII in the rat brain exposed to 10. 15 and 20 min of transient forebrain ischemia followed by variable periods of reperfusion. The cytosolic CKII activity decreased during reperfusion by ∼ 30 and ∼ 50% in the selectively vulnerable areas, striatum and the CA1 region of the hippocampus, respectively. In the resistant CA3 region of hippocampus and neocortex, the activity increased by ∼ 20 and ∼ 60%, respectively. The postischemic changes in CKII activity were dependent on the duration of the ischemic insult. The levels of CKII did not change after ischemia, suggesting that the enzyme is modulated by covalent modification or is interacting with an endogenous inhibitor/activator. Treatment of the cytosolic fraction from cortex of rats exposed to ischemia and 1 h of reperfusion with agarose-bound phosphatase decreased the activity of CKII to control levels, suggesting that CKII activation after ischemia involves a phosphorylation of the enzyme. The correlation between postischemic CKII activity and neuronal survival implies that preservation or activation of CKII activity may be important for neuronal survival after cerebral ischemia.

Notes: Abstract: “Synaptic-like microvesicles” are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS. a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation. we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine β-hydroxylase and cytochrome b561. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic-like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [3H]-Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin: however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of ∼ 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine β-hydroxylase. Immunoelectron microscopy of synaptic-like microvesicle fractions showed an intense labeling for synaptophysin on 60-90-nm organelles. Whereas abundant gold labeling for cytochrome b561 was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic-like microvesicles of the chromaffin cell.

Notes: Abstract: The cellular localization of transforming growth factor-α (TGFa) mRNA in juvenile and adult rat forebrain was examined using in situ hybridization with a 35S-labeled cRNA probe. TGFα cRNA-labeled neuronal perikarya were distributed across many forebrain regions including the olfactory bulb, caudate-putamen, nucleus accumbens, olfactory tubercle, ventral pallidum, amygdala, hippocam-pal stratum granulosum and CA3 stratum pyramidale, and piriform, entorhinal, and retrosplenial cortices. TGFα cRNA-hybridizing cells were also localized to several thalamic nuclei and to the suprachiasmatic, dorsomedial, and ventromedial nuclei of the hypothalamus. In addition, labeled cells were present in regions of white matter including the corpus callosum, anterior commissure, internal and external capsules, optic tract, and lateral olfactory tract. Thus, both neurons and glia appear to synthesize TGFα in normal brain. Hybridization densities were greater in neuronal fields at 2 weeks of age compared with the adult, suggesting a role for TGFα in the development of several forebrain systems. Our results demonstrating the prominent and widespread expression of TGFα mRNA in forebrain, combined with the extremely low abundance of epidermal growth factor mRNA in brain, support the argument that TGFα is the principal endogenous ligand for the epidermal growth factor receptor in normal brain.

Notes: Abstract: Apoptotic cell death plays a critical role in the development of the nervous system. The death of mature nondividing neurons that fail to receive appropriate input from the target field has been extensively studied. However, the mechanisms mediating the extensive cell death occurring in areas of the developing brain where proliferating neuroblasts differentiate into mature nondividing neurons have not been analyzed. We show here that the cell cycle arrest of a proliferating cell of neuronal origin by removal of serum results in either apoptotic cell death or differentiation to a mature nondividing neuronal cell. The proportion of cells undergoing death or differentiation is influenced in opposite directions by treatment of the cells with cyclic AMP and retinoic acid. This suggests that following the withdrawal of signals stimulating neuroblast cell division, neuronal cells either can cease to suppress a constitutive suicide pathway and hence die by apoptosis or, alternatively, can differentiate into a mature neuronal cell. Regulation of the balance between apoptosis and neuronal differentiation could therefore play a critical role in controlling the numbers of mature neurons that form.

Notes: Abstract: To examine the possibility that NaF enhances phosphoinositide-specific phospholipase C (PIC) activity in neural tissues by a mechanism independent of a guanine nucleotide binding protein (Gp), we have evaluated the contribution of Gp activation to NaF-stimulated phosphoinositide hydrolysis in human SK-N-SH neuroblastoma cells. Addition of NaF to intact cells resulted in an increase in the release of inositol phosphates (450% of control values; EC50 of ∼ 8 mM). Inclusion of U-73122, an aminosteroid inhibitor of guanine nucleotide-regulated PIC activity in these cells, resulted in a dose-dependent inhibition of NaF-stimulated inositol lipid hydrolysis (IC50 of ∼ 3.5 μM). When added to digitonin-permeabilized cells, NaF or guanosine-5′-O-thiotriphosphate (GTPγS) resulted in a three- and sevenfold enhancement, respectively, of inositol phosphate release. In the combined presence of optimal concentrations of NaF and GTPγS, inositol phosphate release was less than additive, indicative of a common site of action. Inclusion of 2–5 mM concentrations of guanosine-5′-O-(2-thiodiphosphate) (GDPβS) fully blocked phosphoinositide hydrolysis elicited by GTPγS, whereas that induced by NaF was partially inhibited (65%). However, preincubation of the cells with GDPβS resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTPγS and NaF-stimulated inositol phosphate release were inhibited by inclusion of 10 μM U-73122 (54–71%). The presence of either NaF or GTPγS also resulted in a marked lowering of the Ca2+ requirement for activation of PIC in permeabilized cells. These results indicate that in SK-N-SH cells, little evidence exists for direct stimulation of PIC by NaF and that the majority of inositol phosphate release that occurs in the presence of NaF can be attributed to activation of Gp.

Notes: Abstract: Although guanine nucleotide binding proteins (G proteins) are one of the critical components of signal transduction units for various membrane receptor-mediated responses, little information is available regarding their status in brain of patients with neurodegenerative illnesses. We measured the immunoreactivity of G protein subunits (Gsα, Giα, Goα, Gq/11α, and Gβ) in autopsied cerebellar and cerebral cortices of 10 end-stage patients with dominantly inherited olivopontocerebellar atrophy (OPCA) who all had severe loss of Purkinje cell neurons and climbing fiber afferents in cerebellar cortex. Compared with the controls, the long-form Gsα (52-kDa species) immunoreactivity was significantly elevated by 52% (p 〈 0.01) in the cerebellar cortex of the OPCA patients, whereas the Gi1α concentration was reduced by 35% (p 〈 0.02). No statistically significant differences were observed for Goα, Gi2α, Gβ1, Gβ2, or Gq/11α in cerebellar cortex or for any G protein subunit in the two examined cerebral cortical subdivisions (frontal and occipital). The cerebellar Gsα elevation could represent a compensatory response (e.g., sprouting, reactive synaptogenesis) by the remaining cerebellar neurons (granule cells?) to neuronal damage but also might contribute to the degenerative process, as suggested by the ability of Gsα, in some experimental preparations, to promote calcium flux. Further studies will be required to determine the actual functional consequences of the G protein changes in OPCA and whether the elevated Gsα is specific to OPCA cerebellum, because of its unique cellular pattern of morphological damage, or is found in brain of patients with other progressive neurodegenerative disorders.

Notes: Abstract: Regulation of the expression of the nerve growth factor (NGF) gene has been reported previously to be mediated via the protooncogene c-fos. Activation of the protein kinase C pathway and 1,25-dihydroxyvitamin D3[1,25(OH)2D3] has also been reported to increase the pool of NGF transcripts in L929 fibroblasts. Here we show that activation of the cyclic AMP second messenger pathway antagonized the effect of phorbol 12-myristate 13-acetate (PMA) or serum on NGF synthesis, whereas it enhanced that of 1,25(OH)2D3. A positive effect was also observed when serum, PMA, and 1,25(OH)2D3 were added together, but dexamethasone reduced this enhancement. There was no close correlation between the increase in c-fos mRNA and that in NGF mRNA, suggesting that expression of the c-fos protooncogene is not necessarily followed by induction of the NGF gene. Rather, these two genes are simultaneously, and not sequentially, induced after forskolin treatment. It appears that regulation of the NGF gene depends on a repertoire of multiple regulatory AP-1 complexes arising from activation of the second messenger pathways. This suggests that NGF gene expression is under the control of a complex interplay among second messenger pathways, protooncogenes, and steroid hormones such as 1,25(OH)2D3 and glucocorticoids.

Notes: Abstract: Phospholipase D (PLD) is activated by many neuro-transmitters in a novel signal transduction pathway. In the present work, PLD activity was studied comparatively in hippocampal slices of newborn and adult rats. Basal PLD activity in adult rats was almost three times higher than in newborn rats. In newborn rats, L-glutamate and 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) time- and concentrationdependently enhanced the formation of [3H]phosphatidylpropanol ([3H]PP) and of [3H]phosphatidic acid in the presence of 2% propanol. N-MethylD-aspartate and kainate (both 1 mM) caused small, but significant increases (∼50%). whereas α-amino-3-hydroxy-5-methylisoxazole-4-propionate (100 μM) was ineffective. Maximally effective concentrations of glutamate (1 mM) and of 1S,3R-ACPD (300 μM) increased the PLD activity to almost 300% of basal activity; the EC50 values were 199 and 47 μM, respectively. Glutamate receptor antagonists, such as DL-2-amino-3-phosphonopropionic acid (AP3). DL-2-aminc-5-phosphonovalenic acid, and kynurenate (all 1 mM) did not inhibit the glutamate-evoked increase of PP formation. In slices of adult rats, the response to 1S,3R-ACPD was significant, but small, whereas glutamate was effective only in the presence of the glutamate uptake inhibitor L-aspartate-β-hydroxarnate. It is concluded that glutamate activates PLD in rat hippocampus through an AP3-resistant metabotropic receptor. This effect is subject to ontogenetic development, with one important factor being glutamate uptake.

Notes: Abstract: Gliosis is commonly observed in the CNS following tissue damage, and it also occurs in aging and in many neurodegen-erative diseases. Glial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. An inhibition or delay in GFAP synthesis could mitigate scar formation and thus reduce the formation of a physical barrier. The consequence of this would be to allow neurons and oligodendrocytes to reestablish a functional environment. (—)-Deprenyl, a specific monoamine oxidase (MAO) B inhibitor, has been used as an effective antipar-kinsonian drug, and it has been reported to possess neuroprotective and neurorescue properties. Using northern and slot blots to detect GFAP mRNA in C6 glioma cells, we have demonstrated that (—)-deprenyl decreases the abundance of GFAP mRNA in a time- and dose-dependent manner. The effect seems to be specific to MA0 B inhibitors because (+)-deprenyl and clorgyline exhibit no effect. This study indicates therefore that (-)-deprenyl may be useful for regulating astrogliosis following CNS injury as well as in some neurodegenerative diseases.

Notes: Abstract: Using a radioligand binding assay, we examined ionic modulation and G protein coupling of neuropeptide FF(NPFF) receptors in membranes of rat brain and spinal cord. We found that NaCl (but not KCl or LiCl) and MgCl2 increased specific 125I-YLFQPQRFamide (125I-Y8Fa) binding to NPFF receptors in both tissues in a dose-dependent manner, with optimal conditions being 60 mM NaCl and 1 mM MgCl2. Guanine nucleotides dose-dependently inhibited specific 125I-Y8Fa binding to rat brain and spinal cord membranes with maximal effects of 64 ± 6 and 71 ± 2%, respectively. The order of potency was nonhydrolyzable GTP analogues 〉 GTP 〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:00223042:JNC1894:ges" location="ges.gif"/〉 GDP 〉 GMP, ATP. The guanine nucleotide inhibition was observed in the absence and presence of NaCl and MgCl2. The mechanism of inhibition in spinal cord membranes appeared to be a reduction in the number of NPFF receptors; in one experiment, control KD and Bmax values were 0.068 nM and 7.2 fmol/mg of protein, respectively, and with 0.1 μM guanylylimidodiphosphate the respective values were 0.081 nM and 4.9 fmol/mg, a 32% reduction in receptor number. Similar results were obtained with guanosine 5′-0-(3-thiotriphosphate). Our data suggest that 125I-Y8Fa binding sites in rat CNS are G protein-coupled NPFF receptors regulated by GTP and cations.

Notes: Abstract: We have studied the effect of long-term treatment with hydrocortisone on the expression of acetylcholine receptors (AChRs) at the neuromuscular junctions of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord motoneurons. Hydrocortisone increased accumulation of junctional AChRs in a dose-and time-dependent fashion. This increase was due to both decreased degradation and increased synthesis of AChRs. Other glucocorticoids, dexamethasone and prednisolone, exerted similar effects. Our study demonstrates a novel action of glucocorticoids on human junctional AChRs.

Notes: Abstract: The type 1 angiotensin II (All) receptor (AT1-R) has been implicated in the physiological actions mediated by All in the brain. In view of the reported hyperactivity of the brain All system in the spontaneously hypertensive rat (SHR), we compared the expression of AT,-R mRNAs in the brains of normotensive [Wistar Kyoto (WKY)] and SHR animals. Northern blot analysis showed about three- and ∼20-fold increases in the levels of AT1-R mRNAs from the hypothalamus and brainstem areas, respectively, of the SHR compared with the WKY rat brain. This was attributable to greater levels of both AT,1A- and AT,1B-R mRNA subtypes in these areas from the SHR. These observations suggest that increased All receptor levels in SHR brain may, in part, be a result of increased expression of the AT1-R gene.