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The diastereoselective Michael alkylation of alpha-substituted and alpha,alpha'-disubstituted cyclohexanone benzylimines with ethylidenemalonate diesters was carried out for mechanistic and synthetic purposes. In the first case, an inverse regioselectivity occurred in comparison with what is generally observed since the Michael adducts resulted from alkylation of the non substituted enamine tautomer. With alpha,alpha'-disubstituted imines, in all cases, the stereochemistry of the major diastereomer was the one anticipated from a mechanism including a chairlike complex approach with a preferred exo position for the beta-methyl group of the ethylidenemalonic acid diesters. Furthermore, diphenyl 2-ethylidenemalonate 4 was found to be a highly electrophilic synthetic equivalent of crotonic esters.

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To investigate a possible direct action of glucocorticoids on adrenal steroidogenesis, the effect of corticosterone on the conversion of pregnenolone into various metabolites by frog adrenal tissue was examined. Frog interrenal slices were incubated with [3H]pregnenolone (1 mCi/ml) and the various labelled metabolites analysed by reverse-phase high-performance liquid chromatography. With the methanol gradient used, five identified steroids were resolved: progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone and aldosterone. Corticosterone (10 micrograms/ml) induced a 45-80% decrease in all steroids synthesized from [3H]pregnenolone. In contrast, the glucocorticoid agonist dexamethasone did not reduce the rate of conversion of pregnenolone into its metabolites. In addition, the inhibitory effect of corticosterone was not reversed by the specific glucocorticoid antagonist RU 43044. These results show that corticosterone exerts a direct inhibitory effect on adrenal steroid secretion. In addition, our data indicate that the ultra-short regulation induced by corticosterone is not mediated through glucocorticoid receptors.

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The localization of the N-terminal fragment of the atrial natriuretic factor (ANF) precursor in the heart of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques using an antiserum directed against synthetic rat ANF (Asp11-Ala37). At the optic level, positive material was found in most atrial myocytes. Staining of consecutive sections of frog heart with antibodies against N-terminal and C-terminal regions of the proANF molecule showed that both peptides are contained in the same cardiocytes. In the rat atrium, antibodies against the N-terminal ANF region induced a more intense labeling than in the frog atrium. Electron microscopic studies indicated that all secretory granules present in frog atrial cardiocytes contain N-terminal ANF-like immunoreactive material. The positive material localized in frog atrium was characterized by gel filtration and radioimmunological detection. Serial dilutions of frog atrial extracts exhibited displacement curves which were parallel to that obtained with synthetic human ANF (Asn1-Asp30). Sephadex G-50 gel chromatography of the immunoreactive material showed that the N-terminal ANF-like immunoreactivity eluted in a single peak corresponding to high molecular weight material. These results indicate that the N-terminal fragment of frog proANF is immunologically and biochemically related to the homologous mammalian peptide.

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The distribution of atrial natriuretic factor (ANF) binding sites was investigated in the central nervous system of the frog Rana ridibunda using the technique of in vitro receptor autoradiography by means of [125I]-labeled ANF-28. The anatomic distribution of ANF recognition sites was determined on Kodak ARX films apposed onto tissue sections, and their distribution was examined in greater detail by analysis of autoradiograms generated by using emulsion-coated sections. The highest levels of ANF binding sites were found in the olfactory bulb, the dorsal pallium, the septum, the habenular nucleus, the dorsal infundibular nucleus, the interpeduncular nucleus, and in the tectum. Moderate levels of ANF binding sites were observed in the thalamus and throughout the mesencephalon, whereas low levels were detected in the lateral and medial pallium, the medial forebrain bundle, and the nucleus rotondus. In the pituitary gland, the neural and distal lobes were densely loaded with ANF binding sites, whereas no autoradiographic labeling was observed in the pars intermedia. In general, there was a good correlation between the location of ANF receptors and the distribution of ANF-containing neurons, as previously determined by immunocytochemistry. Together these results support the view that ANF may act as a neurotransmitter or a neuromodulator in various regions of the frog brain.

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Despite few studies conducted in non-mammalian species, there has been a number of reports pertaining to the occurrence of a natriuretic-like substance in lower organisms. Thus, an immunoreactive substance reacting with antibodies directed against mammalian atrial natriuretic factor has previously been detected both in heart atria and ventricles of a chordate, the frog. This substance was isolated and purified from frog heart atria and its amino acid sequence established. The sequence, Ala-Pro-Arg-Ser-Ser-Asp-Cys-Phe-Gly-Ser-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln- Ser-Gly - Met-Gly-Cys-Gly-Arg-(Phe), is highly homologous to known mammalian ANF sequences. However, when aligned with the complete mammalian ANF precursor sequence at positions 121 to 151, it exhibits a single amino acid insertion at position 129 and other substitutions at positions 121, 125, 133, 135, 144, 147 and 148. Some evidence is also presented concerning the occurrence of uncleaved frog pronatriodilatin, the precursor form of ANF. This study represents the first report pertaining to the structure of a non-mammalian ANF and its precursor.

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In order to determine the role of the cytoskeleton in adrenal steroidogenesis, we have studied the effect of cytochalasin B (a microfilament-disrupting agent) and vinblastine (an antimicrotubular drug) on corticosteroid secretion by frog interrenal tissue in vitro. Perifusion of interrenal fragments with cytochalasin B (50 mumol/l) induced a marked inhibition of basal corticosteroid output. In addition, stimulation of corticosteroidogenesis by all corticotrophic factors was also inhibited by cytochalasin B. Using an immunohistochemical technique and specific anti-tubulin antiserum, we verified that vinblastine (10 mumol/l) was responsible for the disappearance of the microtubular network in adrenocortical cells. Administration of vinblastine (10 mumol/l) did not affect the spontaneous secretion of corticosterone and aldosterone and had no effect on the steroidogenic response of interrenal glands to angiotensin II and acetylcholine. In contrast, vinblastine was responsible for a marked decrease in serotonin-induced stimulation of corticosteroid production. On the other hand, data from high-performance liquid chromatography showed that infusion of cytochalasin B or vinblastine was not associated with the production of any new steroid which could interfere in the radioimmunoassays. Taken together, these data suggest that microfilaments are involved in a late and common step of corticosteroidogenesis while microtubules are only required for the coupling of the secretory response to certain corticotrophic factors such as ACTH and serotonin.

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In order to investigate a possible role of atrial natriuretic factor (ANF) in the control of corticosteroid biosynthesis in amphibians, we have examined the effect of synthetic ANF (Arg 101-Tyr 126) on perifused frog interrenal slices. ANF did not affect the spontaneous secretion of corticosterone and aldosterone. In contrast, ANF (10(-6) M) inhibited ACTH-and angiotensin II-stimulated corticosteroid production. ANF was more potent in suppressing aldosterone than corticosterone secretion. Immunocytochemical studies using a specific ANF antiserum revealed the presence of ANF-like immunoreactive fibers in the vicinity of interrenal cells. It is thus proposed that, in amphibians, both "hormonal" ANF secreted by myocytes and "neurohormonal" ANF delivered by peptidergic nerve terminals coursing among interrenal cells may partake in the regulation of corticosteroidogenesis.

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The adrenocortical cells of frog interrenal (adrenal) tissue are controlled by multiple factors. Recently, we have shown that corticosteroidogenesis is stimulated by acetylcholine released from splanchnic nerve terminals as well as by serotonin and vasoactive intestinal peptide (VIP) which are both contained in chromaffin cells. Since these 3 putative neuroregulators are known to interact with each other on various target organs, we have investigated possible coordinate actions of acetylcholine, serotonin and VIP on adrenal steroid production, using a perifusion system technique for frog interrenal tissue. Simultaneous infusion of submaximal doses of VIP (10(-5) M) and acetylcholine (5 X 10(-5) M) induced stimulations of corticosteroids (corticosterone and aldosterone) which were strictly additive. When VIP (10(-5) M) and serotonin (5 X 10(-6) M) were infused together, a potentiation of the individual responses was observed. In contrast, concomitant infusion of acetylcholine (5 X 10(-5) M) and serotonin (5 X 10(-6) M) caused a total blockage of the stimulatory effect of serotonin. Muscarine (10(-5) M) caused a similar blockade of the response of adrenocortical cells to serotonin while nicotine (5 X 10(-5) M) did not alter the stimulatory effect of serotonin. The inhibitory effect of acetylcholine on serotonin-induced steroidogenesis was antagonized by atropine (10(-5) M). Thus, acetylcholine appears to block the corticotropic action of serotonin by interacting with typical muscarinic receptors. Taken together our results indicate that 3 of the neuroregulators which participate in the control of adrenal steroidogenesis, namely acetylcholine, serotonin and VIP, may interact on their target cell to modulate the activity of their congeners.(ABSTRACT TRUNCATED AT 250 WORDS)

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The localization of atrial natriuretic factor (ANF)-like immunoreactivity in the central nervous system of the frog Rana ridibunda was examined by the indirect immunofluorescence technique, using an antiserum against synthetic ANF (Arg101-Tyr126). Immunoreactive cell bodies were principally found in the dorsal and medial pallium, the medial septal nucleus, the ventrolateral and anteroventral areas of the thalamus, the lateral forebrain bundle, the posterolateral thalamic nuclei, the preoptic nucleus, the dorsal infundibular nucleus, and the anteroventral tegmentum nucleus of the mesencephalon. Numerous cell bodies and a very dense fiber bundle were visualized in the interpeduncular nucleus. All the areas mentioned above contained a high density of immunoreactive fibers. In addition, the amygdala, the infundibular nucleus, the median eminence, and most of the areas of the mesencephalon contained a moderate number of ANF-positive nerve processes. In the frog pituitary, fibers and nerve terminals were found in the peripheral zone of the neural lobe. The intermediate and anterior lobes of the frog pituitary were totally devoid of ANF immunoreactivity. These results indicate that ANF-like material is widely distributed in the frog brain and that ANF may be involved in various brain functions including neuroendocrine regulations.

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During long-term fasting in birds and mammals, protein utilization initially decreases (phase I), is thereafter maintained at a low value (phase II), and then further increases (phase III). To delineate hormonal and biochemical changes responsible for these modifications, the effect of food deprivation for 50 days was studied in 6 male king penguins captured at the beginning of their natural breeding fast. During phase II, both rate of mass loss and plasma uric acid concentration remained at low levels, whereas plasma beta-hydroxybutyrate concentration increased. In phase III there was by contrast a 2.5-fold increase in the rate of mass loss, an eightfold increase in plasma uric acid, and an 80% drop in plasma beta-hydroxybutyrate. Plasma corticosterone was low and steady in phase II and increased three times in phase III. During the overall fast, there were no significant variations in plasma insulin, but there was a fourfold increase in plasma glucagon and a decrease in plasma thyroxine and triiodothyronine. These findings suggest that protein sparing (phase II) requires low levels of corticosterone, insulin, and thyroid hormones, whereas the further increase in protein utilization (phase III) is due to an increase in plasma corticosterone. The high plasma glucagon concentration in phase III is presumably responsible for a transient increase in plasma glucose observed at this stage; such increase in glucagon could enhance gluconeogenesis from amino acids.

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The localization of ANF-like immunoreactivity in the ventricle of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques, using an antiserum against synthetic ANF (Arg 101-Tyr 126). At the optic level, an appreciable number of positive cardiocytes was observed in the frog ventricle. Electron microscopic studies showed that all secretory granules present in ventricular cardiocytes contain immunoreactive ANF. The immunoreactive material has been characterized by Sephadex G-50 gel chromatography and reverse phase high performance liquid chromatography (RP-HPLC). After gel filtration, ANF-like immunoreactivity eluted in 3 peaks. The major immunoreactive peak corresponded to high molecular weight material, while one peak co-eluted with synthetic ANF (Arg 101-Tyr 126). Further analysis of frog ventricular extracts by RP-HPLC revealed that the low molecular weight material has the same retention time as synthetic ANF, suggesting a high degree of sequence homology between amphibian and mammalian ANF. These results indicate that in amphibians, ventricular cardiocytes synthesize a peptide immunologically and chemically related to mammalian ANF.

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The possible role of arachidonic acid metabolites of the lipoxygenase pathway in the regulation of steroidogenesis was studied in vitro using perifused frog interrenal (adrenal) glands. Graded doses of arachidonic acid (10(-6)-10(-4)M) increased the production of corticosterone and aldosterone in a dose-dependent manner. In the presence of indomethacin (5 X 10(-6)M), the effect of arachidonic acid on steroid secretion was totally abolished. Nordihydroguaiaretic acid (NDGA: 10(-6)M), a lipoxygenase inhibitor, did not alter the spontaneous secretion of corticosteroids and did not impair the stimulatory effect of arachidonic acid. In the presence of NDGA, both ACTH and angiotensin II were still able to stimulate corticosteroid production. Our data support the view that arachidonic acid metabolites play an important role in the regulation of amphibian steroidogenesis. Moreover, the results show that the lipoxygenase pathway is not involved in the spontaneous secretion of corticosteroids and in angiotensin II- or ACTH-induced steroidogenesis.

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In order to determine the role of intermediate filaments in adrenal steroidogenesis, we have studied the effect of IDPN (beta-beta'iminodipropionitrile), an intermediate filaments perturbing agent, on corticosteroid secretion by frog interrenal glands in vitro. A 6-h administration of IDPN (10(-3) M) did not affect the spontaneous release of corticosterone and aldosterone. While IDPN did not alter the response of adrenal fragments to ACTH, the drug caused a marked decrease in angiotensin II-induced stimulation of corticosterone and aldosterone production. These results indicate that, in contrast to microfilaments, which play an important role in spontaneous steroidogenesis, intermediate filaments are not required for basal corticosteroid secretion but are involved in the mechanism of action of angiotensin in frog adrenocortical cells.

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In order to determine the role of microfilaments in adrenal steroidogenesis, we have studied the effect of cytochalasin B, a microfilament-disrupting agent, on the kinetics of [3H] pregnenolone conversion to labelled metabolites by frog interrenal tissue in vitro. Cytochalasin B (5 x 10(-5)M) induced a 50 to 70% decrease in corticosterone, 18-hydroxycorticosterone and aldosterone biosynthesis while the formation of progesterone and 11-desoxycorticosterone was not affected. These results suggest that microfilaments interfere in the conversion of 11-desoxycorticosterone to corticosterone probably by controlling the movement of 11-desoxycorticosterone from the reticulum to the mitochondria.

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In order to determine the role of the microtubular network in adrenal steroidogenesis, we have studied the effect of vinblastine, a potent antimicrotubular agent on corticosteroid secretion by frog adrenocortical fragments in perifusion. Administration of vinblastine (10(-5) M) did not alter the spontaneous secretion of corticosteroids and had no effect on the steroidogenic response to angiotensin II and prostaglandin E1. In contrast, vinblastine induced a marked decrease in ACTH-induced stimulation of corticosteroidogenesis. To investigate further the site of action of the microtubular system in ACTH-induced steroidogenesis we have studied the effect of vinblastine on the different steps of the stimulation of the adenylate cyclase system. Our results show that vinblastine does not alter the stimulatory action of cAMP, the second messenger of ACTH. In addition, the corticosteroidogenic effect of forskolin and NaF (which respectively stimulate the adenylate-cyclase subunit and the guanyl nucleotide regularly protein) was not affected by vinblastine. These results indicate that the microtubular network interferes in the coupling of the secretory response of the adrenal glands to ACTH, either at the level of the binding of ACTH to its receptor or in the coupling of the receptor to the guanyl nucleotide regulatory protein.

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The distribution of atrial natriuretic factor-like immunoreactivity in the central nervous system of the frog Rana ridibunda was investigated by indirect immunofluorescence and the immunogold technique, using an antiserum generated in rabbits against synthetic atrial natriuretic factor (Arg 101-Tyr 126). A stereotaxic atlas of neurons containing atrial natriuretic factor-like material was prepared to show the widespread distribution of atrial natriuretic factor-positive cell bodies and fibres in the brain. Appreciable numbers of immunoreactive perikarya were observed in the dorsal and medial pallium, the medial septal nucleus, the anteroventral and ventrolateral areas of the thalamus, the lateral forebrain bundle, the posterocentral and posterolateral thalamic nuclei, the preoptic nucleus, the dorsal infundibular nucleus and the anteroventral tegmental nucleus of the mesencephalon. A heavy accumulation of atrial natriuretic factor-like immunoreactive cell bodies and very dense fibre bundles were noted in the interpeduncular nucleus of the mesencephalon. Fibres were generally seen where cell bodies were observed, particularly in all regions of the pallium and septum nuclei, in the ventral thalamus, the infundibular nucleus and the tegmental area. Moderate numbers of fibres were also noted in several regions where cell bodies were absent, mainly in the amygdala and the infundibular nucleus, the median eminence and most mesencephalic regions. At the electron microscopic level, the immunoreactivity was restricted to dense core vesicles and absent in clear vesicles. These results provide the first evidence for the presence of atrial natriuretic factor in the brain of a non-mammalian chordate. The localization of atrial natriuretic factor-positive material in the frog central nervous system suggests that this peptide may act as a neuromodulator or a neurotransmitter in amphibians.

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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.

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The presence of atrial natriuretic factor (ANF)-like material in the hypothalamo-pituitary complex of the frog (Rana ridibunda) was studied by means of indirect immunofluorescence and immunogold techniques. In the hypothalamus, numerous ANF-positive cell bodies were located in the preoptic nucleus, the lateral forebrain bundle and the dorsal infundibular nucleus. The ventral infundibular area and the median eminence contained only ANF-immunoreactive fibers. A dense network of fibers and nerve terminals was observed in the neural lobe of the pituitary. Scarce nerve endings were also noted in the intermediate lobe whereas ANF-like immunoreactivity was totally absent in the anterior lobe of the pituitary. At the ultrastructural level, positive material appeared restricted to dense core vesicles (about 100 nm in diameter). These results demonstrate that an ANF-like substance is contained in nerve terminals of the frog pars nervosa, and support the hypothesis that ANF may exert a physiological role in the control of release of neurohypophyseal peptides.

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The role of prostaglandins (PGs) in calcium-induced corticosteroid secretion by frog adrenal (interrenal) gland has been examined in vitro using a perifusion technique. Increasing concentrations of CaCl2 (4-10 mM) stimulated in a dose-dependent manner aldosterone, PGE2 and 6-keto-PGF1 alpha production, whereas TXB2 was not affected. The kinetics of the adrenal response to CaCl2 indicated that the increase in PG output always preceded that of steroid. Administration of cobalt (4 mM), a calcium-channel inhibitor, blocked the calcium-induced stimulation of PGs and corticosteroids. Infusion of indomethacin (5 X 10(-6) M), a specific cyclooxygenase inhibitor, significantly decreased the basal production of PGs and steroids, and prevented the stimulatory effect of CaCl2 (6 mM). Infusion of the calcium ionophore A 23187 (10(-6) M), for 20 min, induced a marked stimulation of PG and steroid production. Taken together, these data support the notion that biosynthesis of prostaglandins is associated with calcium-induced corticosteroid secretion in frog adrenal cells.

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