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L. Desrues, M. C. Tonon, and H. Vaudry


Previous studies have demonstrated that TRH is a potent stimulator of α-MSH secretion from frog pituitary melanotrophs. In order to determine the intracellular events responsible for TRH-evoked α-MSH release, we have investigated the effect of TRH on polyphosphoinositide breakdown in frog pars intermedia. Neurointermediate lobes were labelled to isotopic equilibrium with myo-[3H]inositol.

TRH stimulated the rate of incorporation of [3H]inositol into the phospholipid fraction. The effect of TRH was concentration-dependent; half-maximal stimulation of α-MSH release and inositol incorporation occurred at 12 and 28 nmol TRH/1 respectively. In prelabelled neurointermediate lobes, lithium (10 mmol/l) enhanced the radioactivity in inositol monophosphate, bisphosphate (IP2) and trisphosphate (IP3). LiCl (10 mmol/l) induced a 38% inhibition of α-MSH release from perifused neurointermediate lobes but did not impair TRH-induced α-MSH secretion. In the presence of LiCl, TRH (1 μmol/l) induced a transient increase of the radioactivity in IP3, which was evident by 30 s and maximal by 1 min (+ 100%). TRH treatment also increased the radioactivity in IP2, which reached a plateau after 5 min (+ 100%). The increase in radioactivity in IP3 induced by TRH was closely paralleled by a rapid loss of [3H]phosphatidylinositol bisphosphate (PIP2), which was maximal by 1 min (−70%).

These results indicate that, in frog pars intermedia, TRH-evoked α-MSH secretion is coupled to breakdown of PIP2. The data suggest that, in amphibian melanotrophs, as previously shown in GH3 tumour cells and in rat pituitary mammotrophs, TRH causes rapid stimulation of polyphosphoinositide-hydrolysing phospholipase C.

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E. Louiset, L. Cazin, M. Lamacz, M.-C. Tonon, and H. Vaudry


Modulation of the activity of K+ channels by TRH and the possible involvement of this modulation in TRH-induced release of α-MSH were studied in cultured frog melanotrophs, using patch-clamp and perifusion techniques. Pars intermedia cells were enzymatically dispersed and cultured in Leibovitz medium. In order to test the viability of cultured cells, the amount of α-MSH released into the medium was measured by radioimmunoassay every day for 1 week of culture. The total amount of α-MSH released during the first 4 days of culture was 8·6 times higher than the intracellular content of α-MSH on day 1. Melanotrophs were identified by an indirect immunofluorescence technique using a specific antiserum to α-MSH. Recordings obtained in whole-cell, cell-attached and excised patch-clamp configurations showed that TRH induced a transient polarization concomitant with an increase in the probability of opening of Ca2+-activated K+ channels. This transient response was followed by a depolarization accompanied by an enhanced frequency of action potential discharge. TRH also induced a decrease in voltage-dependent K+ conductance. Application of tetraethylammonium, a K+ channel blocker, depolarized the cells and increased the basal secretory level without noticeable changes in TRH-evoked α-MSH release.

These results demonstrate that the neuropeptide TRH both stimulates Ca2+-sensitive K+ channels and inhibits voltage-dependent K+ current in pituitary melanotrophs. Our data indicate that TRH-induced secretion of α-MSH is not a direct consequence of the lowering of K+ conductance. It thus appears that basal and TRH-induced α-MSH release occur through distinct pathways; the spontaneous release of α-MSH is probably linked to membrane potential, while modulation of the electrical activity is not directly involved in TRH-induced activation of the secretory process.

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L Desrues, H Vaudry, M Lamacz, and M C Tonon


We have previously demonstrated that γ-aminobutyric acid (GABA) is a potent regulator of secretory and electrical activity in melanotrophs of the frog pituitary. The aim of the present study was to investigate the intracellular events which mediate the response of melanotrophs to GABA.

We first observed that GABA (1–100 μm inhibited both basal and forskolin-stimulated cyclic AMP (cAMP) formation. The inhibitory effect of GABA on cAMP levels was mimicked by the GABAB receptor agonist baclofen (100 μm) and totally abolished by a 4-h pretreatment with pertussis toxin (01 μg/ml). In contrast, the specific GABAA agonist 3-aminopropane sulphonic acid (3APS) did not affect cAMP production. Both GABA and 3APS (100 μm each) induced a biphasic effect on α-MSH release from perifused frog neurointermediate lobes, i.e. a transient stimulation followed by an inhibition of α-MSH secretion. Administration of forskolin (10 μm) prolonged the stimulatory phase and attenuated the inhibitory phase evoked by GABA and 3APS, indicating that cAMP modulates the response of melanotrophs to GABAA agonists. Ejection of 3APS (1 μm) in the vicinity of cultured melanotrophs caused a massive increase in intracellular calcium concentration ([Ca2+]i). The stimulatory effect of 3APS on [Ca2+]i was abolished when the cells were incubated in a chloride-free medium. The formation of inositol trisphosphate was not affected by 3APS, suggesting that the increase in [Ca2+]i cannot be ascribed to mobilization of intracellular calcium stores. ω-Conotoxin did not alter the secretory response of frog neurointermediate lobes to 3APS, while nifedipine blocked the stimulation of α-MSH secretion induced by 3APS.

In conclusion, the present data indicate that, in frog pituitary melanotrophs, (i) the stimulatory phase evoked by GABAA agonists can be accounted for by an influx of calcium through L-type calcium channels, (ii) the inhibitory effect evoked by GABAB agonists can be ascribed to inhibition of adenylate cyclase activity and (iii) cAMP attenuates the inhibitory phase evoked by GABAA agonists. Taken together, these data suggest that activation of GABAB receptors may modulate GABAA receptor function.

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M. Lamacz, M. C. Tonon, E. Louiset, L. Desrues, L. Cazin, J. Guy, G. Pelletier, and H. Vaudry


The effect of modifications of extracellular calcium concentrations on α-MSH release has been studied using perifused frog neurointermediate lobes. Increasing concentrations of calcium (from 2 to 10 mmol/l) gave rise to a dose-related stimulation of α-MSH secretion, whereas reduction of Ca2+ from 2 to 15 mmol/l partially inhibited α-MSH release. The direct effect of extracellular Ca2+ on α-MSH secretion was confirmed by the dose-dependent stimulation of α-MSH release induced by the calcium ionophore A23187. Perifusion with a calcium-free medium or blockade of Ca2+ channels by 4 mmol Co2+/l both resulted in an inhibition of spontaneous and TRH-induced α-MSH release. Conversely, administration of verapamil or methoxy-verapamil (10 μmol/l each) did not alter basal secretion and had no effect on the response of the glands to TRH. Nifedipine (10 μmol/l), which was able to block KCl (20 mmol/l)-evoked α-MSH release, induced a slight inhibition of basal α-MSH secretion, indicating that extracellular Ca2+ levels may regulate α-MSH release in part by Ca2+ influx through voltage-dependent Ca2+ channels. In contrast TRH-induced α-MSH release was not affected by nifedipine or dantrolene (10 μmol/l), and BAY-K-8644 (1 μmol/l) did not significantly modify the response of neurointermediate lobes to TRH. Taken together, these results suggest that TRH-induced α-MSH secretion is associated with calcium influx across the plasma membrane and that calcium entry caused by TRH may occur through nifedipine/verapamil-insensitive Ca2+ channels.

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V Compère, D Lanfray, H Castel, F Morin, J Leprince, B Dureuil, H Vaudry, G Pelletier, and M C Tonon

In the central nervous system of mammals, the gene encoding diazepam-binding inhibitor (DBI) is exclusively expressed in glial cells. Previous studies have shown that central administration of a DBI processing product, the octadecaneuropeptide ODN, causes a marked inhibition of food consumption in rodents. Paradoxically, however, the effect of food restriction on DBI gene expression has never been investigated. Here, we show that in mice, acute fasting dramatically reduces DBI mRNA levels in the hypothalamus and the ependyma bordering the third and lateral ventricles. I.p. injection of insulin, but not of leptin, selectively stimulated DBI expression in the lateral ventricle area. These data support the notion that glial cells, through the production of endozepines, may relay peripheral signals to neurons involved in the central regulation of energy homeostasis.