Phorbol ester differentially regulates oxytocin receptor binding activity in hypothalamic cultured neurons and astrocytes.

Hypothalamic cultured neurons and astrocytes were used to investigate the cellular mechanisms underlying the oxytocin receptor-mediated downregulation through a possible involvement of protein kinase C (PKC). For this purpose, the effects of PKC activators, inhibitor and of OT on OT receptor binding activity were compared in both cultures. In neurons, phorbol-myristate-acetate (PMA), a potent PKC activator, increased the binding of an OT receptor antagonist whereas in astrocytes, a decrease was observed. Pre-treatment of the cells with bisindolylmaleimide (10(-4) M), a PKC inhibitor, prevented the PMA-induced up- and downregulation. In contrast, receptor downregulation resulting from treatment of both cells with OT (10(-9) M) was not affected by the PKC inhibitor. On the other hand, when PMA (10(-7) M) was tested along with OT (10(-9) M), a subsequent decrease in ligand binding was observed in astrocytes. In neurons, PMA attenuated the OT-induced downregulation. Structural analysis of neuron and astrocyte OT receptor mRNA by RT-PCR, subcloning and sequencing, demonstrated identical sequence to rat uterine receptor. In conclusion, these data suggest that activation of PKC has opposite effect on OT receptor binding activity in neurons and astrocytes but they do not support the involvement of PKC in the OT-induced downregulation.

Pharmacological characterization of oxytocin-binding sites in rat spinal cord membranes: comparison with embryonic cultured spinal cord neurones and astrocytes.

Detection and pharmacological characterization of OT-binding sites were performed on 12-day-old rat spinal cord membranes and on embryonic cultured spinal neurones and astrocytes after 12 days in culture. In neurone-enriched cultures, OT-binding sites were detected by autoradiography on cells morphologically comparable to neurone-specific enolase immunoreactive cells. In astrocyte cultures, as shown by combination of autoradiography and immunocytochemistry, OT-binding sites were detected on cells expressing the glial fibrillary acidic protein (a specific astrocytic marker). The pharmacological characterization was assessed by binding studies performed with a highly specific radioiodinated OT antagonist on postnatal rat spinal cord membranes and on embryonic cultured spinal cord neurones and astrocytes. The saturation studies suggested the presence of a single class of binding sites of high affinity for the OT antagonist on spinal membranes and cultured cells, as already described in the rat for central and peripheric OT receptors. The competition studies indicated that on spinal membranes, OT and AVP had the same high affinity, as classically described, whereas on cultured cells, AVP had a lower affinity, suggesting that culture conditions may influence the pharmacology of the spinal OT-binding sites. Involvement of NEM- and Gpp[NH]p-insensitive G-proteins in the coupling of the spinal OT-binding sites with the effector system was evidenced on 12-day-old rat spinal membrane preparations and on neurone and astrocyte cultures.

Downregulation of the oxytocin receptor on cultured astroglial cells.

Cultured astroglial cells obtained from rat fetal hypothalamus express oxytocin (OT) receptors, which have been previously characterized (Di Scala-Guenot and Strosser. Biochem. J. 284: 491-497, 1992), with a radioiodinated OT antagonist. In these cells, at steady-state binding at 37 degrees C, ice-cold acidic treatment released 10% of the bound ligand; with pronase treatment, 52% of the tracer was released. Because the binding was performed with an antagonist, one could assume that the radiolabeled ligand remains locked into the membrane in a state insensitive to the stripping agents rather than being internalized. Receptor downregulation induced by OT was concentration- and time-dependent, leading to a 72% loss of maximal binding capacity without changing the affinity of the receptor. On removal of OT the binding capacity recovered partially and the restoration process was blocked by monensin (20 microM) but not by cycloheximide (20 micrograms/ml), suggesting involvement of receptor recycling. Concerning the early mechanisms involved in the downregulation processes, uncoupling of the receptor from the G protein and the receptor phosphorylation by protein kinase C could be demonstrated. Treatment of the cells with the OT antagonist d(CH2)5OVT was shown to facilitate radioligand binding and to protect the receptor against OT-induced downregulation.

Increase of intracellular calcium induced by oxytocin in hypothalamic cultured astrocytes.

A recent study demonstrated oxytocin (OT) receptors on hypothalamic cultured astrocytes (Di Scala-Guenot and Strosser, 1992). The attempt in the present paper was to determine a possible intracellular calcium mobilization induced by OT receptor activation in these cells. Using the microspectrofluorimetric technique with fura-2, as calcium indicator, brief applications of OT on single astrocytes induced a transient and reversible dose-dependent increase of intracellular calcium concentration ([Ca2+]i) in most of the cells tested. In a few cells, OT application triggered intracellular calcium oscillations. Repetitive applications of OT generally produced a decreasing calcium signal, suggesting a desensitization of the receptor. OT-induced calcium release was prevented by a prior or simultaneous application of an OT antagonist. The origin of the calcium mobilization was assessed during conditions where no extracellular calcium was available. Neither removal of extracellular calcium nor addition of a calcium channel blocker, cadmium 100 microM, in the bathing solution, did affect the calcium response to OT, demonstrating that release of intracellular calcium is solely involved in the OT-induced [Ca2+]i increase. The OT-induced calcium mobilization was abolished after thapsigargin application (100 nM). This indicates that the calcium response to OT application was principally associated with activation of the IP3-sensitive calcium stores. Taken together these results demonstrate that OT receptors previously detected on hypothalamic cultured astrocytes are functional receptors which transduction pathways involve calcium mobilization from IP3-sensitive stores.

Oxytocin receptors on cultured astroglial cells. Kinetic and pharmacological characterization of oxytocin-binding sites on intact hypothalamic and hippocampic cells from foetal rat brain.

The ability of astroglial cells to exhibit oxytocin (OT)-binding sites has been investigated in embryonic hypothalamic and hippocampic astroglial cell cultures. The differential characteristics of binding of OT and [Arg8]vasopressin (AVP) agonists and antagonists to the OT-binding sites using the highly selective iodinated OT antagonist d(CH2)5-[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT ([125I]OTA) have been evaluated using intact cells maintained for 12 days in culture. The specific binding displayed features of reversibility. Computer analysis of the saturation studies using the LIGAND program indicated that, at 4 degrees C, the antagonist binds to a homogeneous population of sites with a Kd value of 0.02 nM and a low binding-site density of around 2 fmol/dish for hypothalamic cells and 6 fmol/dish for hippocampic cells. For hypothalamic cells, competition curves using unlabelled OT, AVP or V2 AVP agonist were characterized by a pseudo-Hill coefficient below unity (0.7), indicating possible heterogeneity among the binding sites. On the other hand, the dose-inhibition curves resulting from competition studies with hippocampic cells had a pseudo-Hill coefficient close to unity, except for OT. Computer analysis (LIGAND) indicated that the OT dose-inhibition curve was significantly better fitted to a two-site model, and this can be explained by two apparent forms of the receptor having high and low affinities for the displacing drug. The relative potencies of the peptides tested for binding to the high-affinity site were: AVP greater than OT greater than V1 AVP antagonist ([d(CH2)5-Tyr(Me)2]AVP) = V2 AVP agonist greater than AVP-Sar ([d(CH2)5-Sar7,Arg8]VP) in hypothalamic cultures, and OT = AVP greater than V1 AVP antagonist greater than V2 AVP agonist in hippocampic cultures. In addition, autoradiography allowed visualization of OT-binding sites, which are located on both soma and processes of astrocyte-like type of cells. In conclusion, these data provide evidence that glial cell cultures contain specific OT-binding sites which display pharmacological characteristics different from those already reported in neuronal cultures and in the adult rat brain.

Oxytocin receptors on cultured astroglial cells. Regulation by a guanine-nucleotide-binding protein and effect of Mg2+.

Specific binding sites for the radio-iodinated oxytocin (OT) antagonist d(CH2)5-[Tyr(Me)2,Thr4, Tyr-NH2(9)]OVT ([125I]OTA) have been characterized on cultured hypothalamic astroglial cell membranes. The rate of association of the ligand to OT-binding sites was identical in the presence and the absence of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p, 0.1 mM), whereas the monophasic dissociation reaction became biphasic in the presence of Gpp[NH]p. Scatchard analysis of equilibrium binding of [125I]OTA resulted in a linear plot with a single class of binding sites (Kd 0.06 nM) which were insensitive to the addition of Gpp[NH]p. Unlabelled OT and [Arg8]vasopressin (AVP) bound to high- (H) and low- (L) affinity states with a dissociation constant ratio (KL/KH) of 100 for both hormones. Binding with both high and low affinity required the presence of Mg2+ in the incubation buffer, and the addition of Gpp[NH]p decreased the KL/KH ratio to 10 and increased the percentage of low-affinity binding sites. On the other hand, neither omission of Mg2+ from the buffer nor the addition of Gpp[NH]p altered the binding of either OT or V1 AVP antagonists to OT receptors. In the presence of a G-protein inactivator (N-ethylmaleimide; 3 mM) during OT competition studies the affinities of the two OT-binding sites were unchanged, but 90% of the high-affinity binding sites were converted into the low-affinity state. These results obtained with cultured hypothalamic astroglial cells provide further evidence for a coupling of OT receptors with a guanine-nucleotide-binding protein, with a requirement for Mg2+.

Are chronic alcohol-induced central vasopressin changes determinant in strain dependency of tolerance in mice?

As vasopressin (VP) has been related to tolerance, we were interested in following central VP levels after chronic alcohol exposure of two selected mouse lines (C57Bl and Balb/c). Strongly elevated VP and VP mRNA levels have been noted, in particular in the hypothalamus. The phenomenon is much more marked in Balb/c mice than in C57Bl; in extrahypothalamic areas in the changes in VP noted in septum and amygdala are only apparent in Balb/c mice. Hypothalamic norepinephrine and serotonin, known to partly control VP release, also reacts in a strain dependent manner to alcohol. This study provides neurochemical evidence that long term ethanol intoxication selectively activates central vasopressinergic and aminergic neurons in mice. Such activation appears to be strain dependent; therefore it may be related to the unequal capacities of these strains to adapt to chronic alcohol intoxication. Such phenomena may partly account for differences between individuals in tolerance to chronic alcohol in men.

Evidence for the presence of guanylate cyclase-coupled receptors for atrial natriuretic peptide on pituicytes of the neurohypophysis.

The aims of the present study were to determine whether natriuretic peptide receptors coupled to guanylate cyclase are present in the neural lobe (NL) of the pituitary and eventually localized on pituicytes and/or on nerve fibers and whether cyclic GMP may be involved in the regulation of vasopressin secretion. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) enhanced cyclic GMP content of NLs in a dose-related fashion, with ED(50) values of about 5 x 10(-8)M, while CNP failed to significantly elevate guanylate cyclase activity. ANP stimulated cyclic GMP accumulation in NLs lacking functional nerve fibers, while it was without significant effect on isolated nerve terminals. In the brain, ANP-enhanced cyclic GMP production was similarly expressed in glial and not in neuronal cultures, although intracellular guanylate cyclase activity (stimulated by sodium nitroprusside) was present in both cell types. Finally, the cell permeant S-bromoguanosine 3':5'-monophosphate GMP failed to change either basal or isoproterenol-stimulated vasopressin secretion from incubated NLs. We conclude that in the NL, as well as in brain tissue cultures, the guanylate cyclase-NP receptor complex (most probably the ANP-A subtype) is localized on pituicytes/filial cells rather than on nerve fibers/cells and that cyclic GMP may not be directly involved in the regulation of vasopressin output from the NL.

Expression of vasopressin and opiates but not of oxytocin genes studied by in situ hybridization in embryonic rat brain primary cultures.

Expression of arginine-vasopressin (AVP), oxytocin (OT), dynorphin and enkephalin genes was studied with the in situ hybridization technique in embryonic rat brain serum-free cultures. Neurones were prepared from hypothalamus and extrahypothalamic structures of 16-day-old rat embryos. After 7 days in culture, AVP gene expression occurred in hypothalamic cultures only, whereas ProOT mRNAs were undetectable. By contrast, prodynorphin and proenkephalin mRNAs could be detected in both hypothalamic and extrahypothalamic cultures, however, with a higher number of cells containing proenkephalin mRNAs. These observations demonstrated that AVP, dynorphin and enkephalin, but not OT genes, can be expressed in cultures prepared from embryonic rat brain as young as 16 days old. This is the first report of an early expression of opioid peptide genes within the central nervous system suggesting that opioids could be involved in the early phases of nervous system development.

Characterization of oxytocin-binding sites in primary rat brain cell cultures.

Detection and characterization of oxytocin-binding sites in dissociated brain cell cultures were performed, using a highly selective iodinated oxytocin antagonist [( 125I]OTA). Dissociated cells derived from hypothalamus and extrahypothalamic forebrain of 16-day-old fetal rats were maintained in chemically defined medium in order to enrich the cultures in neuronal cells. Specific binding of [125I]OTA, demonstrated in both hypothalamic and forebrain cell cultures, was temperature- and time-dependent; maximal binding was obtained by incubating the iodinated ligand for 60 min at 37 degrees C. The binding parameter were shown to be identical in both cell type cultures. The Scatchard plot analysis suggested the presence of a single class of binding sites of high affinity (Kd about 0.06 nM) and low binding capacity (Bmax about 4 fmol/dish). The specificity of these binding sites tested in competition experiments revealed that the unlabelled OT antagonist was the most potent in inhibiting specific [125I]OTA binding (Ki = 0.1 nM). A lower affinity, of the nM range was demonstrated for oxytocin (OT), arginine-vasopressin (AVP) and the V1 antagonist, whereas the V2 AVP agonist poorly competed for [125I]OTA binding sites (Ki about 250 nM). In conclusion, the [125I]OTA binding characteristics, identical in both hypothalamic and forebrain cultures, fulfil the classical conditions required for the existence of receptor sites since the binding was reversible, saturable and specific. As these characteristics were similar to those already described in the adult rat, at the central level in hippocampus, and at the periphery in the mammary gland, it could be postulated that [125I]OTA binds to an OT receptor site.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of neurophysin-containing neurons in primary cultures of rat hypothalami is related to the age of the embryo: morphological study and comparison of in vivo and in vitro neurophysins, oxytocin, and vasopressin content.

The morphological development of immunocytochemically identified neurophysin neurons and the evolution of neuropeptide content (neurophysins, vasopressin, and oxytocin) were studied in primary cultures of hypothalami obtained from 15- to 19-day-old embryos. According to their perikaryal surface, two populations of neurons were distinguished: large and small cells. Full development (defined by the perikaryal surface) of these neurons was reached at day 21 only in cultures from 15- or 16-day-old embryos. These two types of neurons may correspond to the magnocellular and parvocellular neurons described in vivo. Total neurophysins, vasopressin, and oxytocin content were measured by specific radioimmunoassays. Ontogeny of neurophysins and vasopressin showed a good correlation between cells cultured from 15- to 16-day-old embryos and hypothalami from age-matched rats. However, oxytocin was never detected in any of the cultures whatever the age of the embryos. Under our experimental conditions, hypothalamic primary cultures from 15- to 16-day-old embryos therefore appeared to be suitable for studying the differentiation and regulation of neurophysin- and vasopressin-containing neurons.

Variations of somatostatin-like immunoreactivity in the circumoesophageal ganglia, the hepatopancreas, the mantle edge, and the hemolymph of shell-repairing snails (Helix aspersa).

Immunocytology and radioimmunoassays demonstrate the presence of immunoreactive somatostatin-like (IR-SOM-LI) material in the tissues of young or adult snails Helix aspersa bred under short or long day in controlled artificial conditions. Neurosecretory cells in the circumoesophageal ganglia and in the digestive gland, small fibers in the hepatopancreas, and small granules in the mantle epithelial cells are immunoreactive toward antisomatostatin. In all experimented animals shell fractures induce variations of the IR-SOM-LI concentrations in all assayed tissues whatever the lighting conditions were underwent. These findings support the hypothesis that in Helix one or more substances related to the vertebrate tetradecapeptide is (are) involved in the repair processes but that the storage and the metabolism may be different during the biological cycle of the snails. These results are compared with those previously published using different gastropods and different methods.

The ultrastructure of A, B and D pancreatic cells in normal and in diabetic ducks.

Ultrastructurally and immunocytochemically identified A, B and D cells are highly concentrated in the splenic bulb of the duck pancreas. Ultrastructural features of normal A, B and D cells are similar in the duck and in other species so far studied. However, normal D cells present a striking characteristic, i.e. apical accumulation of dense bodies, which seem to derive from multivesicular bodies and are probably involved in a catabolic regulatory process. Subtotal pancreatectomy in the duck, leaving the splenic bulb and inducing transient diabetes, produces strong secretory stimulation of A and B cells, as indicated by the development of the rough endoplasmic reticulum and the Golgi apparatus and transient degranulation, more marked in B cells. Numerous B cells with degenerative aspects, observed after 12 days, seem to be exhausted following prolonged hyperstimulation: this could explain why diabetes reappears in some cases. In contrast, in D cells, functional inhibition after surgery is suggested by a dramatic increase in the number and size of the dense bodies, associated with a marked decrease in secretory vesicle storage. The morphological data correlate well with the previously reported evolution of plasma and pancreatic hormone concentration after surgery, and suggest that the normal inhibitory control of glucagon and insulin secretion by the local release of somatostatin might be reduced or suppressed during transient diabetes in subtotally depancreatized ducks.

Genotypic differences in central neurotransmitter responses to aging in mice.

Dopamine, serotonin, cholinergic and somatostatin responses to aging have been followed in striatum and hippocampus of two inbred strains of mice (C57BL and BALB/c). A striking strain dependency is noted. Dopaminergic mechanisms in BALB/c mice become particularly defective in striatum where both dopamine release and dopamine turnover are affected. Also, striatal cholinergic activity and somatostatin levels are more disturbed in BALB/c than in C57BL mice. For cholinergic neurotransmission and somatostatin levels, similar results are noted in hippocampus. Conversely, C57BL mice react to aging by increased serotonin turnover in hippocampus and decreased 5HIAA levels in both structures studied, whereas the BALB/c strain remains unaffected. Also, structure dependency is observed: in C57BL mice serotonin turnover appears to be unchanged in striatum and increased in hippocampus; in the BALB/c the slowing down of dopamine activity in striatum is not observed in hippocampus. This unequal capacity of central neurotransmitters and neuromodulators to adapt to aging processes, depending upon the genotype, the nervous structure and the neurotransmitter considered may be involved in man in specific pathologies in aged individuals.

Electrical stimulations of perifused magnocellular nuclei in vitro elicit Ca2+-dependent, tetrodotoxin-insensitive release of oxytocin and vasopressin.

Isolated rat paraventricular (PVN) and supraoptic (SON) nuclei were perifused in vitro and oxytocin and vasopressin releases were measured by radioimmunoassay during rest and during electrical stimulation. Stimulations at a frequency of 10 Hz (10-s bursts, every 10 s for 5 min) and an intensity of 4 mA, induced significant hormone release only with long duration pulses (10 ms). Short pulses (1 ms) applied at various frequencies (10, 20, 40 or 80 Hz) and intensities (4, 5, 10 or 20 mA) had no effect. The electrically evoked release of both hormones was not affected by tetrodotoxin (TTX), a sodium channel blocker, but was blocked in low-calcium medium or in the presence of gallopamil hydrochloride (D-600), a calcium channel blocker. These results suggest that, following electrical stimulation, oxytocin and vasopressin are released locally within the magnocellular nuclei even when blocking action potentials. The possibility of dendritic release is discussed.

Influence of mouse genotype on responses on central neurotransmitters to long-term alcohol exposure in striatum and hippocampus.

A striking strain dependency in response of central neuromediators (dopamine, serotonin, acetylcholine activities and somatostatin) to various periods of alcohol treatment has been noted in striatum and hippocampus in mice. For biogenic amines the C57Bl strain loses tolerance to prolonged alcohol injury earlier than the Balb/c strain. At the opposite the Balb/c strain exhibits a remarkable long lasting tolerance for cholinergic activity. The unequal capacity to adapt, also appears to depend on the nervous structure and the neurotransmitter considered. Such discrepancies may underly differences in behavioral changes observed in alcoholics.

[Evaluation of perfusion technics of the magnocellular nucleus of the hypothalamus in vitro and in vivo]. / Evaluation des techniques de périfusion des noyaux magnocellulaires de l'hypothalamus in vitro et in vivo.

In suckled rats, OT necessary for the occurrence of the neurosecretory bursts on OT cells, is probably released inside the magnocellular nuclei. In order to demonstrate this in vivo release and to precise the mechanism involved, perifusions were realized in vivo on lactating rats and in vitro with isolated magnocellular nuclei. In vivo, the push-pull perifusion of a single supraoptic nucleus (SON) was realized simultaneously with the recording of the electrical activity of OT cells in the contralateral nucleus. This would allow to determine the possible relationships between the amount of OT released in the SON and the electrical activity of OT cells (bursting activation during suckling or continuous activation during an hyperosmotic stimulation). Results obtained showed 1) that OT was released in vivo inside the SON, 2) that this release was specifically increased during the milk ejection reflex and 3) that this increase was only detectable inside the SON. However, this technique did not permit to determine either the mechanism of OT release or the neuron elements (perikaryon, dendrites, axon collaterals) responsible for this release. That is for why, in vitro perifusions were undertaken with isolated magnocellular nuclei. The first stage was to determine the stimulus able to induce a reproducible increase of OT release. Among the chemical stimuli (neurotransmitters, K+) only K+ at a 56 mM concentration increased OT release by SON but this increase was small and non significant. Repetitive electrical stimulations with short pulses (0.2 to 5 msec) were ineffective even if the pulse intensity was raised up to 10 mA and the frequency up to 80 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucagon and somatostatin secretion from the perfused splenic bulb of duck pancreas.

Glucagon, somatostatin and insulin secretions were evaluated in a new type of perfusion preparation: the naturally A and D cell rich splenic bulb of duck pancreas. Stable basal levels were observed with 11 mM glucose, corresponding to normoglycaemia, and all secretions were stimulated by 1 mM 3-isobutyl-1-methyl-xanthine and by 10 mM arginine, demonstrating the technique's validity. In the absence of aminoacids in the perfusion medium, A cell blindness to glucose was corrected by physiological levels of insulin (2 ng/ml); insulinodependency of A cells, and unresponsiveness of D cells to glucose, probably not ruled by insulin, were observed. However, in the presence of aminoacids, glucagon was inhibited and somatostatin secretion stimulated by glucose (33 mM), independently of insulin (2 ng/ml). Aminoacids greatly influenced pancreatic hormone release.

Genotypic differences in age and chronic alcohol exposure effects on somatostatin levels in hippocampus and striatum in mice.

Somatostatin contents of striatum, hippocampus, and pons medulla have been followed in two inbred strains of mice (C57B1/6J and Balb/cJ) with aging and long term alcohol exposure (over a 25 month period of intoxication). Marked strain dependent differences in basic levels of somatostatin and genotypic variations in reactivity of this neuropeptide to aging processes and chronic alcohol exposure were demonstrated. The Balb/c strain exhibits a significant age dependent decrease in striatal and hippocampal somatostatin levels whereas the C57B1 mice remain unchanged until the 27 month. Moreover, only the Balb/c strain reacts to chronic alcohol exposure, showing a significant increase in somatostatin levels in those structures affected by the aging process. Such genotypic differences may be involved in man in specific pathologies in aged individuals and in alcohol induced behavioral alterations in alcoholics.