M.T. Strosser

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.

Inhibitory effect and mode of action of somatostatin on lipolysis in chicken adipocytes.

The effect of somatostatin on lipolysis was investigated utilizing isolated chicken adipocytes. Somatostatin-14 and -28 inhibited basal lipolysis. This ability to suppress glycerol release (used as an index of lipolysis) was emphasized in presence of stimulated lipolysis. Concentration of 1 ng/ml somatostatin-14 (0.625 nM) and somatostatin-28 (0.312 nM) was found to inhibit completely the glycerol release induced by concentrations of glucagon up to 2 ng/ml (0.58 nM). The percentage of inhibition was dose-dependent. The antilipolytic effect of somatostatin-14 was also observed during ACTH and aminophylline-stimulated lipolysis. Among the mechanisms which could account for the inhibition, a possible competitive effect of somatostatin-14 with 125I-labelled glucagon binding to adipocyte membranes was excluded. The small inhibiting effect of somatostatin-14 on glycerol release prompted by dibutyryl cyclic AMP, together with the significant inhibiting effect on aminophylline-stimulated lipolysis argued for a reduction of cyclic AMP accumulation. The increase of cyclic AMP levels induced by glucagon was substantially reduced in presence of somatostatin-14. It was concluded that in chicken adipocytes somatostatin inhibited the rate of lipolysis and that reduction on cyclic AMP could be responsible, at least in part, for the antilipolytic effect.

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)

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.

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).