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During stress, the relationship between the central nervous system and the immune system is essential to maintain homeostasis. The main neuroendocrine system involved in this interaction is the hypothalamic-pituitary-adrenal axis (HPA), which via the synthesis of glucocorticoids will modulate the intensity of the inflammatory response. Anaesthetic agents could be interacting with the HPA axis during surgery. Although etomidate currently remains in the center of the discussions, it seems, at least experimentally, that most hypnotics have the capacity to modulate the synthesis of adrenal steroids. Nevertheless, with the large literature on this subject, etomidate seems to be the most deleterious hypnotic agent on the HPA axis function. Its use should be limited when HPA axis is already altered., (Copyright © 2014. Published by Elsevier SAS.)

The trout possesses two POMC genes as a result of duplication of its genome some 25-100 million years ago. One of the POMC molecules exhibits a unique C-terminal extension of 25 amino acid residues which is not found in any other POMC characterized so far. In order to isolate possible novel peptides derived from trout POMC-A, we have raised antibodies against two synthetic epitopes derived from the C-terminal region of the precursor. Two native decapeptides were isolated in pure form from an extract of trout pituitary. The primary structures of these peptides were established as Glu-Gln-Trp-Gly-Arg-Glu-Glu-Gly-Glu-Glu and Ala-Leu-Gly-Glu-Arg-Lys-Tyr-His-Phe-Gln-NH2. The structure of the trout POMC-A cDNA reveals that both peptides are flanked by pairs of basic amino acids or a glycine residue, indicating that they can actually be generated during post-translational processing of POMC-A., (Copyright 1997 Academic Press.)

A peptide related to urotensin II has been isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the peptide was established as Ala-Gly-Asn-Leu-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val and this sequence was confirmed by chemical synthesis. Frog urotensin II contains an additional amino acid residue compared with fish urotensin II peptides but the structure of the cyclic region of the molecule has been fully conserved. The data show that urotensin II is not confined to the caudal neurosecretory system of fish but is present in the central nervous system of a tetrapod.

Quantitative evaluation of octadecaneuropeptide-like immunoreactivity (ODN-Li) was carried out in hippocampus, cortex and cerebellum of group-reared or three month isolated male Wistar rats, whether muricidal or not. ODN-Li was lower in cortex and cerebellum of isolated animals, (mainly in muricidal) than of group-reared ones. A similar trend was apparent in the hippocampus. No significant differences were observed between muricidal and non-muricidal animals. The results are discussed in terms of the involvement of the endogenous ligands of the GABA-benzodiazepine receptor complex in the integrations related to mood and behaviour.

In order to study the morphological localization of the endogenous benzodiazepine ligand octadecaneuropeptide (ODN) in rat brain, we have developed antibodies against this peptide. Using a radioimmunoassay for ODN, we have observed that synthetic ODN and serial dilutions of several brain areas gave parallel displacement curves. By light microscope immunocytochemistry, ODN-immunoreactive material was only detected in glial and ependymal cells. Immunolabelled cells were found in high concentrations in the olfactory bulb, hypothalamus, hippocampus, periaqueductal gray, cerebral cortex and the circumventricular organs. In the cerebellar cortex, immunostaining was associated with Bergmann cells. The studies performed at the electron microscopic level confirmed the association of immunoreactive material with glial and ependymal cells. The present results suggest that ODN might play a role in the function of glial cells which have been shown to contain benzodiazepine receptors of the "peripheral type".

Using adjacent serial brain sections, a morphometric method has been developed for analysing the coexistence of the neurophysial hormones, vasopressin (VP) and oxytocin (OT), with their specific neurophysins (N). A significative correlation was found between the immunoreactive areas stained with (1) anti-VP and anti-N-VP sera, and between the immunoreactive areas detected with anti-OT and anti-N-OT antibodies. Besides, the immunoreactive areas stained with (1) anti-VP and anti-OT antibodies, (2) anti-N-OT and anti-VP antibodies, (3) anti-N-OT and anti-N-VP antibodies or (4) anti-OT and anti-N-VP antibodies were totally independent. A different method projecting the microscope images on a reference grid with a camera lucida permitted to quantify the coexistence of an ovine corticotropin-releasing factor-related-peptide (41-CRF) with OT in the paraventricular neurons of the Brattloro rat brain. In these animals, the same method applied after total hypophysectomy demonstrated that the neurons synthezising simultaneously 41-CRF and OT projected their axons to the neurohypophysis; the same operation increased the relative number of neurons containing 41-CRF only; it can be supposed that they originated the infundibular terminals.

Recent studies have shown that biologically active peptides and monoaminergic neurotransmitters coexist in certain neuronal cell populations. Using the immunofluorescence technique, we have examined the localization of enkephalins, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase in the adrenal gland of the frog Rana ridibunda. Most chromaffin cells which stained for tyrosine hydroxylase contained VIP-like immunoreactivity, whereas methionine- (Met-) and leucine- (Leu-) enkephalin-like immunoreactivity was detected in about 40% of the cells revealed by the anti-tyrosine hydroxylase serum. No VIP- or enkephalin-like immunoreactive nerve fibres were observed. Since in the frog, the chromaffin cells are in close contact with the adrenocortical (interrenal) tissue, a possible action of VIP and opiates on corticosteroidogenesis has been investigated. At doses 10(-6) and 10(-5) M, 20-min infusions of synthetic porcine or chicken VIP elicited a significant increase in corticosterone and aldosterone production by perifused frog adrenals, in a dose-dependent manner. As compared to ACTH, VIP was several orders of magnitude less effective in stimulating corticosteroid production. Morphine, Met- and Leu-enkephalins (10(-5) M) had no effect on spontaneous secretion of corticosteroids. In addition, Met- and Leu-enkephalins (10(-5) M) did not alter the production of corticosterone induced by ACTH. THese results suggest that VIP contained in the chromaffin cells of the frog adrenal gland may exert a local action in stimulating corticosteroid production by the interrenal tissue.

A neuropeptide Y (NPY)-like peptide has been localized in the frog brain by the direct immunofluorescence method. Subcellular localization of NPY has been studied by PAP immunocytochemistry and the immunogold technique. Using a specific radioimmunoassay technique, NPY-like peptide has been quantified in various brain areas and the immunoreactive material was characterized by Sephadex G-50 gel filtration and high performance liquid chromatography. The results indicate that frog NPY is closely related to the porcine molecule and largely distributed in the frog brain. This peptide may play both neurotransmitter and neuroendocrine functions in amphibia.

To investigate the possible direct effect of two aldosterone antagonists (Canrenoate-K and Prorenoate-K) upon mineralocorticoid biosynthesis a perifusion system technique has been developed. Frog interrenal tissue was selected for its ability to secrete huge amounts of aldosterone (twice as much as corticosterone in resting conditions). Throughout the experiment, secretion of aldosterone was measured every ten minutes by means of a sensitive and highly specific radioimmunoassay method. Increasing concentrations of both Canrenoate-K and Prorenoate-K (ranging from 10(-4)M to 10(-3)M) caused a dose-related inhibition of aldosterone output. At a dose of 3.16 x 10(-4)M, Prorenoate-K appeared to be somewhat more potent (57.8% inhibition) than Canrenoate-K (47.8% inhibition). Infusion of both Canrenoate-K and Prorenoate-K at a dose of 5 x 10(-4)M during 1 or 2 hours induced a similar sharp decrease in mineralocorticoid secretion. Thus, it appears that Canrenoate-K and Prorenoate-K beside their well known effects at renal tubular receptor sites do also inhibit aldosterone biosynthesis. These results indicate that in vivo administration of aldosterone antagonists may first involve a transient decrease in aldosterone secretion. Furthermore, they suggest that mineralocorticoid biosynthesis might be regulated by a short loop feedback mechanism.

The structure of alpha-melanocyte-stimulating hormone (alpha-MSH) has been determined in the pars intermedia of the frog Rana ridibunda. Pulse-chase labeling of frog neurointermediate lobes with selective amino acids revealed that the composition of frog alpha-MSH is similar to that of alpha-MSH from all mammalian species yet studied. Tryptic mapping of nexly synthetized alpha-MSH generated two fragments with the following amino acid composition: (T1) Trp, Pro, Lys, Gly, Val and (T2) Tyr, Arg, Phe, His, Ser, Glu. Concurrently, alpha-MSH was purified from 100 neurointermediate lobes to apparent homogeneity by reverse-phase HPLC. The sequence of the peptide determined by automated Edman degradation was Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. The structure of frog alpha-MSH is thus identical to mammalian des-N alpha-acetyl alpha-MSH and differs from the sequence of toad (Xenopus laevis) alpha-MSH only by the first residue (Ser instead of Ala). These results confirm that the sequence of alpha-MSH has been highly preserved during evolution.

The action of atrial natriuretic factor (ANF) on alpha-MSH release from frog neurointermediate lobe was studied in vitro using a perifusion technique. Synthetic ANF Arg101-Tyr 126, at concentrations ranging from 10(-7) to 10(-5)M, caused a dose-related stimulation of alpha-MSH release. In addition, dopamine, GABA and NPY, three neuroendocrine factors which inhibit alpha-MSH secretion totally suppressed the action of ANF on alpha-MSH production. The neural lobe of the amphibian pituitary contains numerous ANF immunoreactive fibers, and this regulatory peptide may diffuse from nerves terminating in the pars nervosa to the pars intermedia. Thus, our results suggest that ANF of hypothalamo-neurohypophysial origin may be involved in the multineuronal regulation of amphibian melanotrophs.

The presence of neuropeptide tyrosine (NPY) in the intermediate lobe of the frog pituitary was demonstrated using indirect immunofluorescence, the immunogold technique and a specific radioimmunoassay combined with high pressure liquid chromatography (HPLC). A high density of NPY-containing fibers, was found among the parenchymal cells of the intermediate lobe. These fibers originated from the ventral infundibular nucleus, travelled via the median eminence to the pars intermedia. At the electron microscopic level, NPY-like material was found exclusively in nerve fibers where the product of the immunoreaction was associated to dense-core vesicles. High concentrations of NPY-like peptide were found in neurointermediate lobe extracts. After Sephadex G-50 gel filtration the major peak of immunoreactive material appeared to co-elute with synthetic porcine NPY. Conversely, HPLC analysis revealed that the NPY-like peptide of the frog pituitary had a retention time shorter than the porcine NPY. The localization of NPY-like material in the pars intermedia suggested a possible role of NPY in the regulation of melanotropic cell secretion. In fact, graded concentrations of synthetic NPY induced a dose-dependent inhibition of alpha-melanotropin (alpha-MSH) release in vitro. The lack of effect of a dopaminergic antagonist on NPY-induced alpha-MSH release inhibition demonstrated that the local dopaminergic system could not account for the NPY action. These results indicate that NPY located in the hypothalamo-hypophyseal system of the frog may act as a melanotropin-release inhibiting factor.

The action of the central-type benzodiazepine-receptor agonist clonazepam on alpha-MSH release has been studied in vitro using perifused frog neurointermediate lobes. High concentrations of clonazepam (3.16 X 10(-5) and 10(-4) M) caused an inhibition of alpha-MSH release and this effect was reversed by the central-type benzodiazepine-receptor antagonist Ro 15-1788. High doses of GABA (10(-5) and 10(-4) M) induced a biphasic effect on pars intermedia cells: a brief stimulation followed by a sustained inhibition of alpha-MSH secretion. Administration of clonazepam (10(-5) M) in the presence of various concentrations of GABA (10(-6) to 10(-4) M) led to a potentiation of both stimulatory and inhibitory phases of alpha-MSH secretion induced by GABA. Ro 15-1788 completely abolished the potentiating effect of clonazepam. Thus our results indicate that endogenous benzodiazepine receptors may modulate the effects of GABA on alpha-MSH secretion.

The cells of the frog pars intermedia synthesize a 36 000 (36K) protein called proopiomelanocortin (POMC). After [3H]glucosamine incorporation, separation of newly synthesized products by SDS-polyacrylamide gel electrophoresis showed that this 36K protein was glycosylated. Tryptic mapping revealed only one site of glycosylation and showed that the carbohydrate side-chain was located in the N-terminal region of POMC. The 36K protein was not released by the melanotrophs, but it generated, through specific intracellular proteolytic cleavage, a number of smaller peptides which were subsequently released. These peptides were identified by various methods including selective amino-acid incorporation, HPLC purification, acid-urea gel electrophoresis, tryptic and chymotryptic mapping, assay of melanotropic activity, radioimmunoassays and immunoprecipitations. Some of the newly synthesized N-terminal (18K) fragment of the POMC was secreted intact while a portion of it was further processed, via an intermediate peptide, to give mature gamma-MSH. All three of these peptides were glycosylated. In addition, the mature peptide (gamma-MSH) exhibited a low but significant melanotropic activity. The C-terminal portion of the prohormone was very rapidly processed to give des N alpha-acetyl alpha-MSH, corticotropin-like-intermediate lobe peptide (CLIP) and beta-endorphin. Authentic alpha-MSH was always absent in cellular extracts: acetylation to give rise to alpha-MSH was a late enzymatic process strictly linked to hormonal release. Since acetylation of alpha-MSH is required for full biological activity of this peptide, it is possible to conceive that this later step could be under neuroendocrine control. Using the perifusion technique we have been able to show the complexity of the control mechanisms regulating amphibian melanotrophs. It is generally accepted that the aminergic innervation of the intermediate lobe of the pituitary is involved in the hypothalamic control of melanotropin release. We have demonstrated that, in amphibians, dopamine inhibits alpha-MSH secretion through D2-type dopaminergic receptors whereas norepinephrine and (or) epinephrine stimulate alpha-MSH secretion via beta-adrenergic receptors. The existence of peptidergic fibers within parenchymal cells of the pars intermedia has been demonstrated. Evidence for TRH-containing fibers has been obtained by immunohistochemistry. Using a specific radioimmunoassay for TRH, we have confirmed the presence of TRH in the neurointermediate lobe of the frog. We have shown that TRH is a powerful MSH-releasing factor in these animals.(ABSTRACT TRUNCATED AT 400 WORDS)

We have recently shown that the release of alpha-MSH by the intermediate lobe of the frog pituitary is inhibited by neuropeptide Y (NPY). Using the perifusion technique, we have compared in the present study, the alpha-MSH release inhibiting activities of NPY, various NPY short chain analogues and two other members of the pancreatic polypeptide family, peptide YY (PYY) and avian pancreatic polypeptide (APP). The order of biological potency was NPY greater than NPY[2-36] greater than NPY[16-36] greater than NPY[25-36] greater than NPY[1-15]. Among the two pancreatic polypeptides tested, PYY appeared to be almost as potent as NPY while APP was 6 times less active than NPY. Neither NPY[1-15] nor NPY[16-36] could antagonize the inhibitory effect of NPY on alpha-MSH release. The structure-activity relationship study suggests that the bioactive determinant of NPY is located in the C-terminal part of the molecule.

In amphibians, alpha-MSH secretion is stimulated by thyrotropin-releasing hormone (TRH). In the present study we show that extracellular calcium influences directly the spontaneous secretion of alpha-MSH and that it participates in the mechanism of action of TRH on frog melanotrophs.