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The melanotrope population of the frog intermediate lobe consists of two subtypes of cells, referred to as high-(HD) and low-density (LD) melanotrope cells, which differ markedly in their basal morphofunctional features as well as their in vitro response to hypothalamic factors, such as the stimulator thyrotropin-releasing hormone (TRH) and the inhibitor dopamine. In this study, we have investigated whether other major hypothalamic regulators of the release of alpha-melanocyte-stimulating hormone (alpha-MSH), such as gamma-aminobutyric acid (GABA) and neuropeptide Y (NPY), also differentially regulate frog melanotrope subpopulations. Our results show that in LD cells, both factors markedly inhibited proopiomelanocortin (POMC) mRNA accumulation and alpha-MSH secretion. In contrast, the secretory and biosynthetic activity of HD cells was not modified by GABA. NPY inhibited POMC transcript accumulation and tended to reduce alpha-MSH secretion in HD cells, yet these effects were less pronounced than those evoked in LD cells. In addition, GABA and NPY inhibited the KCl-induced rise in cytosolic free calcium levels in both subpopulations. Taken together, these results further indicate that frog melanotrope subpopulations are differentially regulated by the hypothalamus and strongly suggest that the intensity of such regulation is directly related to the activity of the cell subset. Thus, the LD subpopulation represents a highly responsive cell subset which is regulated by multiple neuroendocrine factors (TRH, dopamine, GABA and NPY), whereas the hormone storage HD subpopulation shows a moderate response to single stimulatory (TRH) and inhibitory (NPY) inputs.

Previous studies have shown that melanotrope cells of the pars intermedia of Rana ridibunda are inhibited by dopaminergic D2 agonists and stimulated by beta-adrenergic agonists. In the present study, we have examined the possible involvement of alpha-adrenoreceptors in the regulation of frog melanotrope cells. Reversed-phase HPLC analysis combined with electrochemical detection revealed the presence of both dopamine and noradrenaline in pars intermedia extracts (74.1 and 3.2 ng/mg protein, respectively), while adrenaline was undetectable. Administration of graded doses of noradrenaline and adrenaline (from 0.1 to 10 microM) to perifused frog neurointermediate lobes induced a dose-dependent inhibition of alpha-MSH release. The inhibitory effect of adrenaline was partially blocked by the D2-dopaminergic antagonist sulpiride and totally suppressed by concomitant administration of sulpiride and yohimbine (an alpha 2-adrenergic antagonist). Conversely, in the presence of sulpiride, noradrenaline provoked a strong stimulation of alpha-MSH secretion which was totally blocked by the beta-adrenergic antagonist propranolol. Taken together, our results indicate that endogenous catecholamines may exert a complex regulatory action on frog melanotrope cells through activation of dopaminergic D2, alpha 2- and beta-adrenergic receptors.

Previous reports have described the heterogeneity of different pituitary cell types on the basis of morphological and physiological criteria. In the present study, we investigated the possible existence of distinct subpopulations of melanotrope cells in the intermediate lobe of the pituitary of the frog, Rana ridibunda. Separation of dispersed pars intermedia cells in a Percoll density gradient made it possible to isolate two fractions of melanotrope cells whose morphological and functional properties were further characterized. Analysis of the relative volume and number of various cellular organelles showed that high-density cells had a larger number of secretory granules than low-density cells. Concurrently, radioimmunoassay quantification revealed that the concentration of alpha-melanocyte-stimulating hormone (alpha-MSH) was 2 times higher in the heavy cell population. The rate of secretion of alpha-MSH from cultured melanotrophs was significantly higher in low-density than in high-density cells. Thyrotropin-releasing hormone (TRH) was more potent in stimulating alpha-MSH release from the low-density than from the high-density cell subset. In contrast, the response to TRH persisted for a longer time in the high-density cell subpopulation. Taken together, these data demonstrate the existence of two subpopulations of melanotrope cells, and indicate that the low-density cells have a secretory rate substantially greater than high-density cells.

The ionic conductance mechanisms underlying the action potential behaviour of frog melanotrophs in primary culture were studied by using the patch-clamp technique in whole-cell configuration. The action potentials spontaneously generated by these cells were predominantly sodium spikes with a calcium component. Voltage-dependent sodium, calcium, potassium and calcium-activated potassium currents were identified and analysed separately. The voltage-dependent sodium current was characterized by its fast kinetic, its low-threshold activation, its voltage-dependent inactivation and a tetrodotoxin sensitivity. Calcium currents were identified on the basis of their ionic selectivity to divalent cations (Ba2+, Ca2+, Co2+) and their time course. Only two of the three well-documented calcium currents could be detected in frog melanotrophs. A sustained calcium current (ICaS) and an inactivating calcium current (ICaN) were elicited by step depolarizations up to -20 mV. ICaN inactivated for membrane potentials more positive than -50 mV; its inactivation appeared to be both voltage- and calcium-dependent. Transient calcium current (ICaT) has never been observed. Two types of potassium currents were identified: voltage-dependent potassium (IKV) and calcium-activated potassium currents, (IK[Ca]). They were both suppressed by tetraethylammonium chloride, whereas only IK(Ca) was blocked by cobalt. These major ionic currents underlying spontaneous electrical activity are assumed to be involved in the process of alpha-melanocyte-stimulating hormone release. The present study provides the ground for future investigations regarding the relationships between the electrical and secretory activities in amphibian pars intermedia cells.

The localization of CRF-41 related peptide was studied in the brain and posterior pituitary of the homozygous rats for the inherited diabetes insipidus (Brattleboro strain, DI) and of the Long-Evans rats (LE) as control. It was compared to the distribution of vasopressin (AVP), oxytocin (OXY) and OXY-neurophysin (N I). In both strains, CRF-41 was identified in two morphologically distinct systems: one was a hypothalamoneurohypophysial system simultaneously containing CRF-41, OXY and N I; the other was a hypothalamoinfundibular system carrying CRF-41 only. CRF containing neurons were located in the periventricular area of the anterior hypothalamus, in the retrochiasmatic part of the supraoptic nuclei (SON) and, for some of them, in the antechiasmatic part of SON. CRF immunostainings were enhanced by colchicine treatment in LE rats and by DDAVP therapy in DI rats.

Comparative ultrastructural localization of corticotropin-releasing factor (CRF) and oxytocin was performed in the rat median eminence of Long Evans and Brattleboro rats. The peroxidase-antiperoxidase technique used on serial ultrathin sections revealed CRF and oxytocin neurosecretory granule colocalization in the same fibers of the internal layer running towards the posterior pituitary. It is probable that both these peptides coexist in the same granules. In the Brattleboro rats, while genetically lacking vasopressin, CRF was nevertheless shown to be present. In these rats, as was demonstrated in the Long Evans rats, CRF distribution paralleled that of oxytocin only in the internal zone of the median eminence.

The distribution of immunoreactive corticotropin-releasing hormone (CRF) in the forebrain and pituitary of the frog Rana ridibunda was studied by means of specific radioimmunoassay and immunohistochemistry using the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. Relatively high concentrations of CRF-like material were found in both chiasmatic and infundibular regions of the hypothalamus (352 +/- 11 and 422 +/- 36 pg, respectively). Large amounts of CRF were also found in neurointermediate lobe extracts. Standard curves of synthetic CRF and the dilution curves for hypothalamic or neurointermediate lobe extracts were parallel. After Sephadex G-75 gel filtration, CRF-like immunoreactivity eluted in a single peak, in the same position as synthetic ovine CRF. Reversed-phase high-performance liquid chromatography of the material purified on Sephadex G-75 revealed 5 components with CRF-like immunoreactivity. The major peak had a retention time of 22 min as compared to 25.4 min for ovine CRF and 36 min for rat CRF. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the frogs. The great majority of the CRF-positive perikarya were seen in the ventral region of the preoptic nucleus. A few scattered perikarya were also observed in the dorsal preoptic nucleus and in the retrochiasmatic region. Immunoreactive fibers were found in the infundibular nucleus and in various extrahypothalamic zones. CRF-containing neurons were apparently distinct from mesotocinergic and vasotocinergic neurons. A large number of immunoreactive nerve fibers were observed in the median eminence in close contact with the capillaries of the pituitary portal plexus and in the neural lobe. A few CRF-positive fibers were detected in the intermediate lobe, whereas the distal lobe was totally negative. These results show that the diencephalon and pars intermedia-nervosa of the frog contain a peptide immunologically related to mammalian CRF.

The possible existence of extrapituitary melanocyte stimulating hormone (MSH) in various regions of the rat brain has been studied in intact and hypophysectomized rats. Using a sensitive and specific radioimmunoassay (RIA), alphaMSH has been found in a number of brain regions in intact rats. The standard curves of synthetic alphaMSH and the dilution curves for pars intermedia nervosa (PIN), pars distalis (PD), hypothalamus and thalamus extracts were strictly parallel. The alphaMSH concentrations were measured in PIN (6,225 +/- 962 ng/mg wet tissue); PD (12.5 +/- 1.41 ng/mg); pineal (380 +/- 29 ng/g wet tissue); hypothalamus (645 +/- 161 ng/g) and thalamus (33.3 +/- 5.26 ng/g). In rats hypophysectomized for 1 or 2 months, the highest concentrations of immunoreactive alphaMSH were found in pineal (353 +/- 140 ng/g wet tissue), hypothalamus (85.8 +/- 14.1 ng/g) and thalamus (39.8 +/- 13.9 ng/g). Hypophysectomy significantly reduced hypothalamic MSH content and concentration but did not alter MSH concentration in pineal and thalamus. From these results, we conclude that hypothalamic alphaMSH is, in part, of hypophyseal origin while pineal and thalamus alphaMSH does not originate from the pituitary. After Sephadex G-25 gel filtration, synthetic alphaMSH and PIN extracts showed a single peak of both bioactive and immunoreactive alphaMSH. In the same conditions, extracts from the 5 brain regions studied in hypophysectomized rats chromatographed as a single peak of immunoreactive MSH but as 2 peaks of apparent bioactive MSH, 1 concident with synthetic alphaMSH and the other far after the salt volume. We conclude that alphaMSH is found in a number of brain areas and its presence after hypophysectomy would indicate synthesis within the central nervous system.

While mammalian dopamine receptors have been extensively characterized, very little attention has been given to these receptors in lower vertebrates. Dopamine is thought to be a physiologically important melanotropin release-inhibiting factor in amphibians. By administering selective dopamine receptor agonists and antagonists to superfused neurointermediate lobes of Xenopus laevis and monitoring their effect on the release of melanophore-stimulating hormone (MSH), we have examined the characteristics of the receptors involved in the dopamine-induced inhibition of MSH secretion. The results show that the receptor system involved has characteristics of both classical D-2 receptors and alpha-adrenergic receptors. This is concluded from: the agonistic effect of both the D-2 receptor agonists LY-171555 and apomorphine; the antagonism of this effect, not only by the D-2 receptor antagonists sulpiride, domperidon and haloperidol, but also by the alpha-adrenergic receptor antagonist phentolamine, and the observation that the effect of adrenaline, which is extremely potent in inhibiting MSH release, could be fully blocked by D-2 receptor antagonists. Both the fact that the dopamine-induced inhibition of MSH secretion could not be blocked by haloperidol, domperidon and phentolamine, and that adrenaline-induced inhibition was hardly blocked by phentolamine, led us to suggest the possible presence of multiple receptors or receptor sites. We find no evidence for the involvement of dopamine D-1 receptors in the regulation of MSH release. The effects of dopamine agonists and antagonists were also studied in vivo by monitoring changes in pigment dispersion of dermal melanophores. The results are consistent with our in vitro findings and indicate, moreover, that in the living animals there must also be a non-catecholaminergic system involved in the inhibition of MSH release from the pars intermedia.

We determined the corticotropin-releasing factor(CRF)-like immunoreactivity by radioimmunoassay in intermediate and posterior pituitary lobes of stalk-sectioned and sham-operated control rats. The antigenic determinant read by the CRF antibody used was contained within the region of amino acids 26 (Gln) to 37 (Leu) of the molecule. Intermediate and posterior lobes of control rats contained similar amounts of CRF (591 +/- 78 and 487 +/- 34 pg/mg protein, respectively). The section of the pituitary stalk produced a marked decrease in CRF-like immunoreactivity in both structures. The CRF content on the intermediate lobe after stalk section was 62 +/- 17 pg/mg protein, a decrease of 90%, and that of the posterior lobe was 90 +/- 13 pg/mg protein, an 83% decrease. Our results suggest that most of the CRF-like immunoreactivity in the rat intermediate and posterior pituitary lobes is contained in nerve fibers of brain origin and support the hypothesis of a role of CRF in the release of intermediate and posterior lobe peptides.

Corticotropin-releasing factor-like immunoreactivity (CRF-LI), arginine vasopressin-like immunoreactivity (AVP-LI) and ACTH-releasing bioactivity of hypothalamic tissue from 63- to 143-day-old ovine fetuses and from 7- and 150-day-old lambs have been assessed. CRF-LI and AVP-LI contents and concentrations increased steadily between 63 and 138 days of gestation, decreased at day 143 then rose again in postpartum animals. In terms of concentration, the AVP-LI/CRF-LI ratio remained close to 5 between 63 and 123 days of intrauterine life, decreased steadily in late gestation down to 1.2 at day 143 increased again in lambs. The total hypothalamic ACTH-releasing bioactivity increased 26-fold between 63 and 138 days of gestation and remained constant until day 143. The highest values were observed in 150-day-old-lambs. These data support the view that AVP and oCRF1-41 might be important hypothalamic factors involved in the regulation of ACTH release by the pituitary gland of the sheep fetus. In addition, they suggest that AVP is more important in young fetuses and lambs than in prepartum animals.