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ABSTRACT

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.

ABSTRACT

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.

ABSTRACT

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 GABA_B receptor agonist baclofen (100 μm) and totally abolished by a 4-h pretreatment with pertussis toxin (01 μg/ml). In contrast, the specific GABA_A 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 GABA_A 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 GABA_A agonists can be accounted for by an influx of calcium through L-type calcium channels, (ii) the inhibitory effect evoked by GABA_B agonists can be ascribed to inhibition of adenylate cyclase activity and (iii) cAMP attenuates the inhibitory phase evoked by GABA_A agonists. Taken together, these data suggest that activation of GABA_B receptors may modulate GABA_A receptor function.

ABSTRACT

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.