P. Netchitailo

Direct radioimmunoassays for plasma corticosterone and aldosterone in frog. I. Validation of the methods and evidence for daily rhythms in a natural environment

Abstract Two radioimmunoassay techniques for direct measurement of frog plasma concentrations of corticosterone after prior ethanol extraction for deproteinization, and of aldosterone without prior extraction, are described. Specific antibodies against corticosterone 21-hemisuccinate and aldosterone 18,21-diacetate-3-carboxymethoxime derivatives conjugated to bovine serum albumin are raised in rabbits. The sensitivity threshold of the assays allows the assessment of corticosterone in 10-μl and aldosterone in 5-μl samples of plasma. Sephadex LH-20 chromatography demonstrates the validity of both methods. The intra- and interassay reproducibilities and the accuracy of each assay have been studied. The conditions making it possible to reduce aldosterone fluctuations during blood taking have been ascertained. Using these techniques corticosterone and aldosterone concentrations have been assessed in the plasma of 214 frogs caught in their natural habitat at 4-hr intervals during a 40-hr period in mid-June. The existence of synchronous and reproducible 24-hr rhythms of corticosterone and aldosterone plasma levels has been demonstrated. High concentrations of both corticosteroids are recorded during the night and low concentrations are recorded during daylight. The amplitude of corticosterone fluctuations is 3.5-fold greater than that of aldosterone fluctuations. Corticosteroid rhythms are compared to activity phases of frogs during the day at this period of the year.

Atrial natriuretic factor-like immunoreactivity in the central nervous system of the frog

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.

Localization and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium

The distribution of ANF was studied in the heart of the frog (Rana ridibunda) using indirect immunofluorescence. ANF-like immunoreactivity was localized mainly in the right and left atrium, most of cardiocytes being intensively labelled. At the electron microscopic level, all secretory granules present in atrial cardiocytes contained ANF immunoreactive material. Using a specific radioimmunoassay, we found higher concentrations of ANF in the left atrium (208 +/- 25 ng/mg protein) than in the right atrium (120 +/- 16 ng/mg protein) whilst in the rat, the right atrium contains the highest ANF concentration. The concentration of ANF in the ventricle was 10 times lower than in the whole atrium (32 +/- 4 ng/mg protein). Sephadex G-50 gel filtration of atrial extracts showed that ANF-like immunoreactivity eluted in three peaks. Most of the immunoreactivity corresponded to high molecular weight material eluting at the void volume while 20% of the material co-eluted with synthetic (Arg 101-Tyr 126) ANF. These results indicate that frog cardiocytes synthetize a peptide which is immunologically and biochemically related to mammalian ANF.

Localization and identification of immunoreactive atrial natriuretic factor (ANF) in the frog ventricle

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.

The amino acid sequences of frog heart atrial natriuretic-like peptide and mammalian ANF are closely related.

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.

Action of vasoactive intestinal peptide (VIP) on amphibian adrenocortical function, in vitro

Vasoactive intestinal peptide (VIP) is located in chromaffin cells of the frog adrenal gland and is able to stimulate corticosteroid secretion in amphibians. In the present study we have investigated the possible involvement of prostaglandins, microfilaments and calcium in the mechanism of action of VIP on frog adrenocortical tissue. Rana ridibunda interrenal dice were perifused with amphibian culture medium for more than 10 hours. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. In the presence of indomethacin (5 microM), a specific blocker of prostaglandin biosynthesis, the spontaneous secretion of corticosteroids was markedly reduced (80%) but the stimulatory effect of VIP was not altered. The administration of the microfilament disrupting agent cytochalasin B (50 microM) inhibited both spontaneous and VIP-induced corticosteroid secretion. In the absence of calcium, the spontaneous level of corticosteroid was reduced to about 60% but VIP was still able to stimulate corticosteroid secretion. From these data we conclude that the integrity of the cytoskeleton is required for the secretory response of adrenocortical cells to VIP, whereas neither prostaglandins nor calcium are involved in VIP-induced adrenocortical stimulation.

In vitro study of frog (Rana ridibunda pallas) neurointermediate lobe secretion by use of a simplified perifusion system: I. Effect of TRH analogs upon α-MSH release☆

Abstract We have recently shown that the tripeptide l -pyroglutamyl- l histidyl- l -proline-amide (mammalian TRH) stimulates α-MSH secretion in amphibia. Using a perifusion system technique, we have compared the stereochemical requirements for hormone-receptor interaction of frog melanotrophs with mammalian thyrotrophs and mammatrophs. Of all the analogs tested, only the TRH analog l - N -(2-oxopiperidine-6-yl-carbonyl)-histidyl-thiazolidine-4-carboxamide (MK-771) was equipotent with TRH. All analogs which were known to be TRH agonists in mammals (e.g., [Pic] 3 -TRH, [Pro-hydrazide] 3 -TRH) were also relatively active on α-MSH release. Seven analogs were totally inactive on both mammalian pars distalis and frog pars intermedia . The discrepancies concerned only two TRH analogs in which the histidine moiety has been altered [Tyr] 2 -TRH and [Lys] 2 -TRH). The biological potencies of these analogs were 17 and 8% on α-MSH release whereas both molecules were devoid of activity in mammals.

In vitro effect of prostaglandins on corticosterone and aldosterone production by frog interrenal gland

Summary In order to elucidate the role of prostaglandins of the E and F series on adrenal steroidogenesis, we have studied corticosterone and aldosterone production by frog interrenal. Rana ridibunda interrenal dice were perifused with amphibian culture medium for ten hours. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. Perifusion of interrenal fragments with increasing concentrations of PGE 1 and PGE 2 (ranging from 8.8 nM to 2.8 μM) led to a dose-related increase in both corticosterone and aldosterone biosynthesis, the magnitude of the stimulation being 1.3 fold higher for aldosterone than for corticosterone. High concentrations of PGF 2α (2.8 μM) were only responsible for a slight increase in corticosteroid biosynthesis while PGF 1α was almost inactive. Indomethacin an inhibitor of prostaglandin biosynthesis caused a marked decrease of spontaneous production of corticosterone (−84%) and aldosterone (−75%) but did not alter the stimulation of steroidogenesis induced by ACTH. From these data, it was concluded that 1) exogenous prostaglandins control corticosteroid production in amphibia ; 2) endogenous prostaglandins are required for spontaneous biosynthesis of corticosteroids ; 3) endogenous prostaglandins are not involved in ACTH-induced steroidogenesis.

Neuronal and paracrine regulation of adrenal steroidogenesis: interactions between acetylcholine, serotonin and vasoactive intestinal peptide (VIP) on corticosteroid production by frog interrenal tissue

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)

II. Atrial natriuretic factor (ANF) stimulates the release of α-MSH from frog neuro- intermediate lobes in vitro. Interaction with dopamine, GABA and neuropeptide Y.

Abstract The action of atrial natriuretic factor (ANF) on α-MSH release from frog neurointermediate lobe was studied in vitro using a perifusion technique. Synthetic ANF Arg101Tyr126, at concentrations ranging from 10 −7 to 10 −5 M, caused a dose-related stimulation of α-MSH release. In addition, dopamine, GABA and NPY, three neuroendocrine factors which inhibit α-MSH secretion totally suppressed the action of ANF on α-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.