Library

Notes: Abstract: Possible interactions between Met-enkephalin and cholecystokinin (CCK)-containing neurons in the rat sub-stantia nigra were investigated by looking for the effects of various opioid receptor ligands and inhibitors of enkephalin-degrading enzymes on the K+-evoked overflow of CCK-like material (CCKLM) from substantia nigra slices. The δ-opioid agonists d-Pen2,dPen5-enkephalin (50 μM) and Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET; 3 μM) enhanced, whereas the n-opioid agonists Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO; 10 μM) and MePhe3, D-Pro4-morphiceptin (PL 017; 10 μM) decreased, the K+-evoked release of CCKLM. By contrast, the δ-opioid agonist U-50488 H (5 μM) was inactive. The stimulatory effect of DTLET could be prevented by the 5 antagonist ICI-154129 (50 μM but not by the μ antagonist naloxone (1 μM). Conversely, the latter drug, but not ICI-154129, prevented the inhibitory effect of DAGO and PL 017. A significant increase in CCKLM overflow was observed upon tissue supervision with the peptidase inhibitors kelatorphan or bestatin plus thiorphan. This effect probably resulted from the stimulation of δ-opioid receptors by endogenous enkephalins protected from degradation, because it could be prevented by ICI-154129 (50 μM Furthermore, the peptidase inhibitors did not enhance CCKLM release further when S-opioid receptors were stimulated directly by DTLET (3 μM). These data indicate that opioids acting on d and n receptors may exert an opposite influence, i.e., excitatory and inhibitory, respectively, on CCK-containing neurons in the rat substantia nigra. Because CCK has anti-opioid properties, the δ-opioid control of CCKLM release might participate in the central mechanisms of opiate tolerance.

Notes: Abstract: The expression of the preproenkephalin A gene was investigated in adult rat dorsal root ganglia (DRG). A radioimmunoassayable Met-enkephalin (ME)-like material was detected in 0.1 M HCl extracts of rat DRG, representing ∼60 pg of ME equivalents/mg of protein. Chromatographic analyses indicated that the major component of the ME-like material coeluted with authentic ME. In northern blot experiments on total RNA extracted from DRG, a cDNA probe corresponding to the entire coding region of rat preproenkephalin A mRNA yielded a single band of the expected size for this mRNA, i.e., 1.5 kb. Polymerase chain reaction (PCR) experiments were carried out with DRG, striatum, and liver cDNAs using two primers flanking the 1,371–1,771 base region of the preproenkephalin A gene. Thirty PCR cycles performed on both striatum and DRG cDNAs generated a single band of 400 bp, as expected, whereas only trace amounts of this product were detectable using liver cDNAs. Nucleotide sequencing of the PCR product obtained with DRG cDNAs revealed a 100% homology with the 1,371–1,771 sequence of the preproenkephalin A gene. In situ hybridization with a cRNA probe showed that about 3.5% of DRG cells expressed the preproenkephalin A transcript. However, most of these cells probably did not process proenkephalin to enkephalins, as thorough immunohistochemical investigations with anti-ME antibodies allowed the detection of only one in ∼6,000 cells (in 30 sections of DRG) that exhibited ME-like immunoreactivity. Cells expressing preproenkephalin A mRNA were intermediate-sized neurons, suggesting that primary afferent ME-containing fibers belong to the A category and may participate in a local (spinal) inhibitory control of nociception.

Notes: Abstract: Biochemical mapping of five different peptide-like materials—calcitonin gene-related peptide (CGRP), substance P (SP), Met5-enkephalin (ME), cholecystokinin (CCK), and dynorphin A (1–8) (DYN)—was conducted in the dorsal and ventral zones of the spinal cord at the cervical, thoracic, and lumbar levels in 3-month-old rats 10 days after unilateral dorsal rhizotomy at the cervical level (C4-T2) or after neonatal administration of capsaicin (50 mg/kg s.c). In control rats, all peptide-like materials were more abundant in the dorsal than in the ventral zone all along the spinal cord. However, in both zones, absolute concentrations of CGRP, SP, ME, and CCK were significantly higher at the lumbar than at the cervical level. Rhizotomy-induced CGRP depletion (-85%) within the ipsilateral dorsal zone of the cervical cord was more pronounced than that due to neonatal capsaicin (-60%), a finding suggesting that this peptide is contained in both capsaicin-sensitive (mostly unmyelinated) and -insensitive (myelinated) primary afferent fibers. In contrast, similar depletions of SP (-50%) were observed after dorsal rhizotomy and neonatal capsaicin treatment, as expected from the presence of SP only in the capsaicin-sensitive small-diameter primary afferent fibers. Although the other three peptides remained unaffected all along the cord by either intervention, evidence for the existence of capsaicin-insensitive CCKergic primary afferent fibers could be inferred from the increased accumulation of CCK (together with SP and CGRP) in dorsal root ganglia ipsilateral to dorsal root sections.

Notes: Abstract— The incubation of brain stem slices from adult rats in a K+-enriched medium containing a 5-HT uptake inhibitor (fluoxetine) significantly increased their capacity to synthesize 5-HT from tryptophan. The K+-induced stimulation of 5-HT synthesis was at least partly dependent on the depletion of the indoleamine in tissues since: (1) a good correlation was found between the respective changes in 5-HT release and synthesis evoked by high K+ concentrations in the presence of various 5-HT uptake inhibitors; (2) the modifications in endogenous 5-HT levels produced by in vim treatments with drugs (reserpine, pargyline) or by incubating slices with 5-HT altered the stimulating effect of high K+ concentrations and fluoxetine on 5-HT synthesis; (3) the replacement of Ca2+ by Co2+ (4 mM) or EGTA (0.1 mM) in the incubating medium completely prevented the increased 5-HT release and synthesis evoked by high K+ concentrations and fluoxetine.The extraction of tryptophan hydroxylase from incubated tissues revealed that the increased 5-HT synthesis occurring in K+-enriched medium was associated with an activation of this enzyme. Kinetic analyses indicated that this activation resulted from an increase in the Vmax of tryptophan hydroxylase, its apparent affinities for both tryptophan and 6-MPH4 being not significantly affected. In contrast to the tryptophan hydroxylase from tissues incubated in normal physiological medium, the activated enzyme from tissues depolarized by K+ was hardly stimulated by Ca2+-mediated phosphorylating conditions. This led to the proposition of a hypothetical model by which the Ca2+ influx produced by the neuronal depolarization would trigger the activity of a Ca2+-dependent protein kinase capable of activating tryptophan hydroxylase. Although this sequence is still largely speculative it must be emphasized that, as expected from such a model, the regional differences in the K+-evoked activation of tryptophan hydroxylase in slices (cerebral cortex 〉 brain stem 〉 spinal cord) were parallel to those of the Ca2+-dependent protein phosphorylation (r= 0.92) and those of the activating effect of phosphorylating conditions on soluble tryptophan hydroxylase (r= 0.96).

Notes: Although the serotoninergic innervation is immature in the brains of young rats, the 5-HIAA content is similar to that found in adults. As indicated by the ratio of 5-HIAA to 5-HT levels in the brain stem and the forebrain, the catabolism of the indolamine was more rapid during the first 3 postnatal weeks than in adults. This was contirmed by measuring the total formation of [3H]5-HIAA from [3H]5-HT newly synthesized from L-[3H]tryptophan in brain stem slices of young and adult rats.Electrolytic lesions of midbrain raphe nuclei (B7 and B8) performed on the 5th postnatal day resulted in parallel decreases in brain 5-HT and 5-HIAA levels; this ruled out the possibility that 5-HIAA might be formed from 5-HT synthesized outside serotoninergic neurons, using peripheral 5-hydroxytryptophan. Inhibition of 5-HT storage by reserpine pretreatment did not alter the higher capacity of newborn tissues to catabolize exogenous [3H]5-HT. Therefore, possible differences in 5-HT binding in serotoninergic neurons between newborn and adult rats were not likely to account for the differences in 5-HT catabolism. Estimation of the rate of 5-HIAA efflux from the brain after MAO inhibition did not reveal marked changes with age.The activity of MAO type A, the enzyme involved in 5-HT catabolism, was higher during early life than later on. This could be shown by using 5-HT as substrate and clorgyline as a selective inhibitor. An opposite pattern of development was seen for MAO B, measured with benzylamine as substrate and deprenyl as selective inhibitor.These results suggest that the high 5-HIAA levels found in the brains of young rats can be attributed mainly to the presence of high MAO A activity during early life.

Notes: —The effects of Ca2+ ions on the metabolism of [3H]serotonin and [3H]-labelled catecholamines have been examined in hippocampal slices or synaptosomes. The formation of [3H]-5 hydroxyindoles ([3H]serotonin + [3H]-5 hydroxyindoleacetic acid) from [3H]tryptophan and that of [3H]-labelled catecholamines from [3H]tyrosine were increased when Ca2+ was omitted from the incubating medium. However, the total synthesis of 5-HT from tryptophan and that of catecholamines from tyrosine did not seem to be significantly changed. Altered formation of tritiated amines were due to changes in the specific activities of respective precursor amino acids. This reflected altered sizes of the free amino acid pools caused by Ca2+-dependent in vitro proteolysis. This must be taken into consideration when studying in vitro Ca2+ dependency of neutrotransmitter metabolism.

Notes: —The relationships between plasma tryptophan and 5-HT metabolism in the CNS were studied in newborn rats and compared with adults. Both the concentration of free tryptophan in plasma and that of the amino-acid in brain were much higher immediately after birth than later on. Drugs such as salicylate and chlordiazepoxide, which increased brain tryptophan concentrations in adults by displacing the plasma amino acid bound to serum albumin, were ineffective in newborn rats: most of the amino acid being already free in their plasma. The study of 5-HT metabolism in brain stem slices revealed that the affinity of the uptake process for tryptophan was higher in newborn than in adult animals, whereas the reverse situation was observed for the enzyme complex involved in 5-HT synthesis (lower apparent Km in adults). In addition, the catabolism of newly synthesized 5-HT was more rapid in newborn than in adult tissues. Finally, the free state of tryptophan in plasma of newborn animals induced in brain both a high amino acid concentration and, in contrast to the situation observed in adults, a synthesis rate of 5-HT very near its maximal value.

Notes: The effects of changes in intraneuronal levels of 5-HT induced by monoamine oxidase inhibitors (MAOI) given in vivo or exogenous 5-HT added in vitro on 5-HT synthesis in striatal slices of the rat have been examined. The synthesis of 5-HT was estimated by the measurement of the total formation of [3H]5-HT and [3H]5-hydroxyindole acetic acid from [3H]tryptophan and by calculation of the conversion index (CI) of tryptophan into 5-HT. The small formation of [3H]tryptamine and [3H]indole acetic acid from [3H]tryptophan was taken into account in the estimation of 5-HT synthesis. Both MAOI pretreament (180 min) and 5-HT (2·8 μM) inhibited synthesis. The latter effect persisted in catecholamine depleted tissues and was related to intraneuronal changes in 5-HT levels, since it could be prevented by chlorimipramine. The inhibition of 5-HT synthesis was related to the decreased conversion of tryptophan into 5-hydroxytryptophan and could not be prevented by p-chloro-phenylalanine pretreatment which depleted 5-HT levels or by dibutyryl cyclic AMP which normally stimulated 5-HT synthesis. Tryptophan uptake in slices was not affected by exogenous 5-HT. The various mechanisms possibly involved in the end product regulation process of 5-HT synthesis are discussed.

Notes: —Tryptophan hydroxylase form pig brain has been purified using a method which involved sonic disintegration of a whole homogenate, ammonium sulphate fractionation, hydroxylapatite fractionation, column chromatography on Sephadex G-100 or G-200 and finally electrophoresis on poly-acrylamide gel. The enzyme was stabilized during purification by tryptophan and dithiothreitol. The partially purified enzyme has a molecular weight of 55,000-60,000 as measured by gel-filtration. The Km of the soluble partially purified enzyme was 0-4 mm, which differed significantly from that of the particulate enzyme (0·02mm). Enzyme activity was not stimulated by ferrous ion. However, it was inhibited by the chelating agents 8-hydroxyquinoline, O-phenanthroline and EDTA. In contrast to dopamine, high concentration of tryptophan (10 mm), 5-hydroxytryptamine, tryptamine and tyramine at 0-5 mm concentration did not inhibit the enzyme in the presence of dimethyltetrahydropterin (DMPH4). A number of monoamine oxidase inhibitors, phenelzine, pheniprazine and chlorgyline at 1 mm strongly inhibit the formation of 5-hydroxytryptamine. Evidence is presented for the presence of an endogenous inhibitor of tryptophan hydroxylase.