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Notes: Abstract: The src family protein tyrosine kinases (PTKs) are nonreceptor kinases. Some PTKs of this family are ubiquitously expressed, whereas others have a more restricted expression, as in neurons. Lymphoid cell kinase (lck) p56lck is highly expressed in tissues of lymphoid origin and believed to be specific for hematopoietic cells. Reports suggesting that CD4 is expressed in neurons prompted us to analyze the possibility that p56lck is also expressed in these cells. By western blot and immunoprecipitations using anti-lck antibody, an lck-like protein was detected in lysates from primary cultures of rat cerebellar granular neurons. This 56-kDa phosphoprotein was autophosphorylated in vitro and also phosphorylated enolase, similarly to p56lck. It was shown to be located actually in the neurons by immunocytofluorescence. Partial proteolysis mapping showed that the 56-kDa phosphoprotein had a peptide pattern very similar to the p56lck protein. Retrotranscription-PCR allowed the detection of an lck RNA in the neurons. The lck kinase domain was completely identical to the lymphocyte lck kinase domain, but the 5′ end was modified in the neurons. These results show that p56lck is not lymphoid specific as is widely believed; its expression in neurons might underlie the toxicity of the HIV glycoprotein gp120 to neurons.

Notes: Abstract: Adenosine can influence dopaminergic neuro-transmission in the basal ganglia via postsynaptic inter-action between adenosine A2A and dopamine D2 receptors. We have used a human neuroblastoma cell line (SH-SY5Y) that was found to express constitutively moderate levels of adenosine A1 and A2A receptors (∼100 fmol/mg of protein) to investigate the interactions of A2A/D2 receptors, at a cellular level. After transfection with human D2L receptor cDNA, SH-SY5Y cells expressed between 500 and 1,100 fmol of D2 receptors/mg of protein. In membrane preparations, stimulation of adenosine A2A receptors decreased the affinity of dopamine D2 receptors for dopamine. In intact cells, the calcium concentration elevation induced by KCl treatment was moderate, and dopamine had no effect on either resting intracellular free Ca2+ concentration ([Ca2+]i) or KCl-induced responses. In contrast, pretreatment with adenosine deaminase for 2 days dramatically increased the elevation of [Ca2+]i evoked by KCl, which then was totally reversed by dopamine. The effects induced by 48-h adenosine inactivation were mimicked by application of adenosine A1 antagonists and could not be further reversed by acute activation of either A1 or A2A receptors. Acute application of the selective A2 receptor agonist CGS-21680 counteracted the D2 receptor-induced [Ca2+]i responses. The present study shows that SH-SY5Y cells are endowed with functional adenosine A2A and A1 receptors and that A2A receptors exert an antagonistic acute effect on dopamine D2 receptor-mediated functions. In contrast, A1 receptors induce a tonic modulatory role on these dopamine functions.

Notes: Abstract: The time course of D2 receptor expression assessed by the levels of the corresponding binding sites and mRNA was studied in rat striatum during ontogenesis and in primary cultures of cells taken at embryonic day (E) 17 and postnatal day (P) 4. In the two experimental situations, the amount of D2 receptor mRNA and number of binding sites increased regularly from E16 to P15, indicating that expression of D2 receptors in striatal neurons occurs independently from a dopaminergic input. Incubation of striatal primary cultures with 10−5M retinoic acid significantly increased the level of D2 receptor mRNA, whereas thyroid hormone, vitamin D3, and steroid hormones (estradiol, testosterone, and corticosterone) had no effect. The transcriptional activity of the rat D2 receptor gene promoter region, which bears a retinoic acid-responsive element, was increased by retinoic acid in transfected C6 glioma cells but not in transfected MMQ prolactin cells. Thyroid hormone and vitamin D3 were not effective in either cell line. Finally, mutations of the putative retinoic acid-responsive element inhibited the transcriptional effect of retinoic acid. These results suggest that retinoic acid is a key factor in regulation of the embryonic onset of the dopaminergic D2 receptor.

Notes: Abstract: Ca2+ ions trigger the release of hormones and neurotransmitters and contribute to making the secretory vesicles competent for fusion. Here, we present evidence for the involvement of the GTP-binding protein Rab3a in the sensitivity of the exocytotic process to internal [Ca2+]. The secretory activity of bovine adrenal chromaffin cells was elicited by Ca2+ dialysis through a patch-clamp pipette and assayed by monitoring changes in cell membrane capacitance. Microinjection of antisense oligonucleotides directed to rab3a mRNA increased the secretory activity observed at low (0.2–4 µM) [Ca2+], but did not change the maximal activity observed at 10 µM free [Ca2+]. Moreover, after a train of depolarizing stimuli, the secretory activity of antisense-injected cells dialyzed with 10 µM [Ca2+] was increased significantly compared with that of control cells. This result suggests that the activity of either Rab3a or its partners might change upon stimulation. We conclude that Rab3a, together with its partners, participates in the Ca2+ dependence of exocytosis and that its activity is modulated further in a stimulus-dependent manner. These findings should provide some clues to elucidate the role of Rab3a in synaptic plasticity.

Notes: Abstract: We report the isolation of a full-length eel tyrosine hydroxylase (TH) cDNA that is characterized by a long 3′ untranslated region and by a diversity restricted to the 3′ end owing to the differential use of three polyadenylation signals. The longest eel TH mRNA was distinctive in the presence of four pentameric elements (AUUUA) in the AU-rich 3′ noncoding region. Such a diversity could provide the basis of posttranscriptional or translational regulation of eel TH gene expression. Comparison of the eel TH sequence with those of other aromatic amino acid hydroxylases (TH, tryptophan hydroxylase, and phenylalanine hydroxylase) and phylogenetic analysis confirmed that the N-terminal regulatory domain is highly divergent, contrasting with the conservation of the catalytic core of the enzyme. Molecular phylogenies including the available sequences of the three hydroxylase genes suggested that the duplication of their common ancestor occurred before the emergence of arthropods. The regional expression of the eel TH mRNA was studied by semiquantitative PCR, northern blots, and in situ hybridization and compared with the immunocytochemical localization of TH protein. The data showed that TH mRNA is mostly expressed in the olfactory and hypothalamic areas, whereas sparse TH-expressing cell bodies are present in the telencephalic region and brainstem. No labeling was detected in the mesencephalic area, in striking contrast with that found in amphibians and amniotes.

Notes: Lactotrophs from lactating rats were separated by unit gravity sedimentation on a continuous density gradient of bovine serum albumin and were identified in two populations located in the light fractions (fractions 3–5) and in the heavy fractions (fractions 7–9) of the gradient. After 7 days in vitro, the effects on prolactin release of thyrotropin-releasing hormone (TRH) and dopamine before and after pretreatment with 17β-estradiol were studied by a continuous perifusion system and reverse hemolytic plaque assay. Light fraction lactotrophs spontaneously released large quantities of prolactin (22 ng/ml/2 min/106 cells) and this basal release was markedly elevated (51 ng/ml/2 min/106 cells) by pretreatment with 17β-estradiol (10−8 M, 48 h), while the amount of intracellular prolactin remained stable. Mean hemolytic plaque area was increased in the same manner by 17β-estradiol pretreatment but the number of cells and the percentage of plaque-forming cells were not changed. Perifusion of dopamine-containing medium (10−7 M) almost completely blocked basal prolactin release from light fraction cells and this inhibition was markedly reduced by 17β-estradiol pretreatment. TRH-containing medium (10−7 M) weakly stimulated basal prolactin release (about 190% from basal) and this response was significantly enhanced (to about 300% of basal release) by 17β-estradiol pretreatment. Both dopaminergic inhibition and TRH-stimulatory effects were dose-dependent and their half maximal effect values were not changed by 17β-estradiol pretreatment. Secretion of prolactin evaluated at the single cell level by the reverse hemolytic plaque assay corroborated the results obtained from perifusion experiments. Lactotrophs from heavy fractions released small amounts of prolactin (12 ng/ml/2 min/106 cells) and neither this basal release nor the amount of intracellular prolactin were markedly modified by 17β-estradiol pretreatment. As opposed to the light fraction cells, lactotrophs found in heavy fractions were very sensitive to TRH (10−7 M) stimulation with maximal stimulation reaching ten times basal release, but were less sensitive to dopamine (10−7 M), with an inhibition of only 40% basal prolactin liberation. Pretreatment of heavy fraction lactotrophs with 17β-estradiol induced similar effects to those observed after pretreatment of light fraction cells: the stimulation by TRH was increased (from 11 times to 16 times) whereas the inhibition by dopamine was diminished (from 35% to 60%), but cell number and the percentage of prolactin-secreting cells remained unchanged. From the above results, we suggest that: 1) lactotrophs in the lactating rat pituitary can be divided into two major subpopulations with regard to cellular size and density, prolactin production and responsiveness to TRH and dopamine; 2) 17β-estradiol pretreatment increases basal prolactin release from light fraction cells but does not affect basal prolactin release from heavy fraction cells in this way; 3) pretreatment with 17β-estradiol enhances TRH stimulation and reduces dopaminergic inhibition of prolactin release from lactotrophs.

Notes: Continuous cell perifusion and reverse hemolytic plaque assay have been used to show a regulatory action of 17 β-estradiol on lactotroph responsiveness to thyrotropin-releasing hormone (TRH) or dopamine (DA) in vitro. Lactotroph-enriched cell cultures were obtained from adult male rats after trypsinization and mechanical dissociation followed by separation on a continuous bovine serum albumin gradient at unit gravity. After 7 days in culture, perifusion experiments showed that prolactin was continuously released and this release was increased by TRH and decreased by DA. Both TRH-induced secretion and DA-induced inhibition of prolactin release were dose-dependent with a half maximal effect obtained at 7 × 10−9 M for TRH and at 10−9 M for DA. It was shown by reverse hemolytic plaque assay that about 55% of the cells were plaque-forming (lysis of red blood cells) and were thus identified as prolactin-secreting cells. This was similar to a previous result obtained by immunofluorescent staining. Heterogeneity among lactotrophs with regard to the quantity of prolactin released was clearly shown by the varying plaque areas in all preparations. In order to make a quantitative analysis of the effect of 17 β-estradiol on TRH-stimulation and DAergic inhibition in these heterogeneous prolactin cells, they were divided into two groups: large plaques (≥ 3 × 103μ m2) constituted about 35% of all plaque-forming cells, and small plaques (〈 3 × 103μ m2), about 65%. Pretreatment with 17β-estradiol (10−8 M) either for 10 h or 48 h markedly increased TRH-stimulated prolactin release and decreased the inhibitory effect of DA both in perifusion and reverse hemolytic plaque assay experiments. However, these pretreatments did not change the values of half maximum dose for TRH and DA. TRH transformed about 7% of the small plaques into large plaques and this proportion was increased to 25% after 17β-estradiol treatment. On the contrary, DA and its more stable analogue bromocriptine increased the percentage of small plaques by 10% to 15% but this effect was decreased after 17β-estradiol treatment. We conclude that: 1) Normal rat pituitary lactotrophs show heterogeneity with respect to their spontaneous release and responsiveness to TRH and DA; 2) pretreatment with 17β-estradiol increases the response to TRH and decreases the response to DA without altering the doses at which they have half maximal effect; 3) there is no significant difference between the effect of 17β-estradiol obtained after 10 h and after 48 h pretreatment.

Notes: The envelope glycoprotein gp120 of the human immunodeficiency virus HIV-1 has been proposed to cause neuron death in developing murine hippocampal cultures and rat retinal ganglion cells. In the present study, cultured human embryonic cerebral and spinal neurons from 8- to 10-week-old embryos were used to study the neurotoxic effect of gp120 and gp160. Electrophysiological properties as well as N-methyl-d-aspartate (NMDA)-induced currents were recorded from neurons maintained in culture for 10–30 days. Neither voltage-activated sodium or calcium currents nor NMDA-induced currents were affected by exposure of neurons to 250 pM gp120 or gp160. In contrast, when neurons were subjected to photometric measurements using the calcium dye indo-1 to monitor the intracellular free Ca2+ concentration ([Ca2+]i), gp120 and gp160 (20–250 pM) potentiated the large rises in [Ca2+]i induced by 50 μM NMDA. The potentiation of NMDA-induced Ca2+ responses required the presence of Ca2+ in the medium, and was abolished by the NMDA antagonist d-2-amino-5-phosphonovalerate (AP5) and the voltage-gated Ca2+ channel inhibitor nifedipine. Moreover, exposure of a subpopulation of spinal neurons (25% of the cells tested) to 20–250 pM gp120 or gp160 resulted in an increase in [Ca2+]i that followed three patterns: fluctuations not affected by AP5, a single peak, and the progressive and irreversible rise of [Ca2+]i. The neurotoxicity of picomolar doses of gp120 and gp160 cultures was estimated by immuno-fluorescence and colorimetric assay. Treatment of cultures with AP5 or nifedipine reduced gp120-induced toxicity by 70 and 100% respectively.

Notes: The Raf kinases play an important and specific role in the activation of extracellular signal-regulated kinases (ERK) cascade. Beside its role in the control of proliferation and differentiation, the ERK cascade has also been implicated in neuron-specific functions. In order to gain clues on the function of Raf kinases in the adult central nervous system (CNS), we performed a comparative analysis of the distribution and subcellular localization of the different Raf kinases in rat brain with antibodies specific for the different Raf kinases. We show that B-Raf and Raf-1 proteins are present in most brain areas, whereas A-Raf is not detected. Interestingly, the two Raf proteins have an approximately similar pattern of distribution with a rostro-caudal decreasing gradient of expression. These two kinases are colocalized in neurons but they are differentially located in subcellular compartments. Raf-1 is localized mainly in the cytosolic fraction around the nucleus, whereas B-Raf is widely distributed in the cell bodies and in the neuritic processes. In addition, we demonstrated that numerous B-Raf isoforms are present in the brain. These isoforms have a differential pattern of distribution, some of them being ubiquitously expressed whereas others are localized to specific brain areas. These isoforms also have a clear differential subcellular localization, specially in Triton-insoluble fractions, but also in synaptosomal, membrane and cytosolic compartments. Altogether these results suggest that each Raf protein could have a distinct signalling regulatory function in the brain with regard to its subcellular localization.

Notes: Post-transcriptional modification of the neural cell adhesion molecule (NCAM) by polysialic acid significantly decreases NCAM adhesiveness and more generally modifies cell-cell interactions. Polysialic acid-NCAM (PSA-NCAM) is mainly expressed in the developing nervous system. In the adult, its expression is restricted to regions that retain morphological plasticity, such as the hypothalamo-neurohypophysial system during lactation in rats. Since cell-cell interactions and synaptic contacts in the hypothalamo-neurohypophysial system are greatly increased during lactation, we examined whether PSA-NCAM expression is modified during this period. lmmunohistochemistry and immunoblotting showed that, compared with virgin rats, PSA-NCAM dramatically decreased during lactation in both the supraoptic nuclei and the neurohypophysis, and returned to its initial level only after weaning. This decrease was progressive and became significant only at the end of the first week of lactation. By contrast, modifications in the level of NCAM protein or changes in the splicing pattern of NCAM mRNAs could not be detected. The decline in polysialic acid on the NCAM molecule could strengthen membrane appositions, thereby stabilizing the newly established synapses and neurohaemal contacts in the hypothalamo-neurohypophysial system that accompany the increased neuronal activity that occurs during lactation. We also studied the regulation of the phosphorylated microtubule-associated protein-1B (MAP1B-P), whose distribution pattern largely overlaps with that of PSA-NCAM in the adult brain. Expression of MAP1B-P was greatly increased during lactation in the hypothalamic axons projecting into the neurohypophysis. Thus, the expression patterns of both PSA-NCAM and MAP1B-P may reflect the permanent structural plasticity characterizing the hypothalamo-neurohypophysial system in the adult.