Biomedical subjects

H Vaudry

Publications and source records attributed to H Vaudry.

At least 325 records · Page 18Linked to original sources

PubMed1993

Effects of modifications of extracellular and intracellular calcium concentrations on the bioelectrical activity of the isolated frog semicircular canal.

In the inner ear, calcium appears to play a major role in different processes including mechanoelectrical transduction, adaptation during prolonged stimulation and electrical resonance. The aim of the present study was to investigate the effect of an increase of the cytosolic calcium content and a reduction of the perilymphatic concentration of calcium, on the bioelectrical activity of the isolated frog semicircular canal. Under resting conditions, the spontaneous activity of the afferent fibers and the difference of potential between the endolymphatic and perilymphatic compartments, called endolymphatic potential, were recorded. When the sensory epithelium was mechanically stimulated three additional parameters were investigated: the variations of the endolymphatic potential (ampullar direct current), the variations of the ampullar nerve potential (nerve direct current) and the frequency of the evoked afferent spikes. Increase of the intracellular calcium concentration by administration of the calcium ionophore A23187 (3 x 10(-6) M, 20 min) into the perilymphatic compartment, caused a biphasic effect on the spontaneous activity of the ampullar nerve which increased rapidly, reaching a maximum within 15 min, and then gradually declined to stabilize at 74% of the control 1 h after withdrawal of A23187. A23187 did not induce any modifications of the endolymphatic potential, the ampullar direct current or the frequency of the evoked afferent spikes. In contrast, A23187 induced a significant reduction of the nerve direct current which decreased by 31% of the control 1 h after withdrawal of the ionophore. Gradual reduction of the perilymphatic concentration of calcium (from 2 to 1 mM) induced a dose-dependent increase of the spontaneous activity of the ampullar nerve and the frequency of the evoked afferent spikes. Reduction of the perilymphatic calcium concentration from 1.6 to 1.2 mM caused a transient increase of the endolymphatic potential, while 1 mM Ca2+ induced a decrease to 88% of the control. The nerve direct current slightly increased for calcium concentrations ranging from 1.8 to 1.4 mM and decreased in the presence of 1.2 mM CaCl2. These data suggest that an increase of calcium into the cytosol induces an alteration of the mechanisms responsible for the spontaneous release of the afferent neurotransmitter and the electrogenic spreading of the postsynaptic potentials. In contrast, an excess of calcium does not impair the mechanisms involved in the generation of the action potentials. Our results also suggest that reduction of the perilymphatic calcium concentration may lead to modifications of the physical and electrical properties of the cell membranes of the labyrinthine epithelium and/or the ampullar afferent fibers.

Animals↗

PubMed1993

Immunocytochemical localization of atrial natriuretic factor (ANF)-like peptides in the brain and heart of the treefrog Hyla japonica: effect of weightlessness on the distribution of immunoreactive neurons and cardiocytes.

The localization of atrial-natriuretic factor (ANF)-like immunoreactivity was investigated in the brain and heart of the treefrog Hyla japonica by the indirect immunofluorescence technique. Concurrently, the effect of weightlessness on the distribution of ANF-containing neurons and cardiocytes was studied in frogs that were sent into space for 9 days on the space station "MIR." In control animals, the amygdala contained the most prominent group of ANF-immunoreactive cells and fibers. ANF-positive neurons and nerve processes were also detected in other areas of the telencephalon such as the nucleus olfactorius, the pallium mediale, and the striatum. In "space frogs," the intensity of labeling of the amygdala and nucleus olfactorius was similar to that seen in control animals. In contrast, the pallium and the striatum of "space frogs" were totally devoid of positive cell bodies. In the diencephalon, of all animals, numerous ANF-immunoreactive perikarya and fibers were seen in the hypothalamus, the anterior thalamus, the infundibulum, and the median eminence. ANF-positive cell bodies were also noted in the lateral forebrain bundle of control frogs but were absent in "space frogs." The major difference between control and "space frogs" was observed in the posterior nuclei of the thalamus. In "space frogs," the nucleus posterocentralis thalami and the nucleus posterolateralis thalami exhibited large ANF-immunoreactive perikarya, while, in control frogs, these nuclei only contained scarce positive nerve fibers. In the mesencephalon, ANF-positive cell bodies and nerve processes were seen in the nucleus tegmenti mesencephali, the interpeduncular nucleus, and the nucleus cerebelli of all animals. However, stained perikarya were only observed in the nucleus reticularis isthmi of control frogs. In the heart, atrial cardiocytes exhibited intense ANF-like immunoreactivity. ANF-positive myocytes were also detected in the subpericardial region of the ventricle. The density and distribution of the staining were identical in the heart of control and "space frogs." These data support the concept that prolonged exposure to microgravity affects biosynthesis and/or release of ANF-related peptides in discrete regions of the amphibian brain.

Animals↗

PubMed1993

In vitro study of catecholamine release from perifused frog adrenal slices.

The adrenal gland of amphibians is composed of a mixed population of adrenochromaffin and corticosteroid-secreting cells. It has previously been shown that chromaffin cells synthesize several bioactive substances (including biogenic amines and neurotransmitters) which may act locally to regulate corticosteroid secretion. In the present report, we have studied the secretory activity of adrenochromaffin cells in Rana ridibunda. Frozen sections of adrenal gland were immunolabeled with antisera against tyrosine hydroxylase or phenylethanolamine-N-methyltransferase. Comparison of homologous fields on consecutive sections indicated that 77% of catecholaminergic cells produce adrenaline. The concentrations of catecholamines were measured by means of high performance liquid chromatography analysis coupled to electrochemical detection. The concentrations of dopamine, noradrenaline, and adrenaline in fresh adrenal tissue were 24 +/- 4, 763 +/- 68, and 1032 +/- 118 ng/mg wet weight, respectively. After a 12-hr perifusion period, the concentration of adrenaline in the tissue was reduced by 62%, whereas noradrenaline only decreased by 22%. The secretion rates of adrenaline and noradrenaline from perifused adrenal slices significantly diminished during the first 7 hr of the experiment and then remained relatively stable for about 10 hr. Exposure of adrenal tissue to a depolarizing concentration of potassium (55 mM) induced an immediate and substantial rise of adrenaline and noradrenaline release and a delayed increase in corticosterone output. Acetylcholine, which stimulates corticosterone secretion from frog adrenocortical cells, induced a slight but not significant increase of adrenaline and noradrenaline release. Similarly, the selective cholinergic agonists muscarine and nicotine did not significantly affect catecholamine release, while muscarine mimicked the stimulatory action of acetylcholine on corticosterone secretion. This study validates the use of the perifusion model to investigate the mechanism of control of catecholamine release from frog adrenochromaffin tissue. The results presented herein indicate that, in contrast to mammals, the secretion of catecholamines from the amphibian adrenal gland is not regulated by cholinergic inputs.

Adrenal Cortex Hormones↗

PubMed1993

Ontogeny of diazepam-binding inhibitor-related peptides (endozepines) in the rat brain.

Benzodiazepine receptors are expressed very early in the brain during embryonic life, suggesting that endogenous ligands for these receptors may play an important role during ontogenesis in the central nervous system. In the present study, the distribution and characterization of diazepam-binding inhibitor-related peptides (endozepines) in the rat brain was investigated during embryonic and postnatal development using an antibody raised against the biologically active region of the precursor molecule. Immunohistochemical labelling showed that, in newborn rats, endozepine-like immunoreactivity was present in ependymal cells of the hypothalamus. Although the number of positive cells increased by day 5, the intensity of the immunoreaction in each cell diminished. In 15-day-old rats, both the number of endozepine positive cells and the intensity of the immunoreaction increased in the ependymal layer. At day 40, a dense accumulation of immunoreactive tanycytes and glial cells was observed in the median eminence and the arcuate nucleus. Endozepines were detected by radioimmunoassay in all regions of the brain as early as embryonic day 18. The concentration of endozepine-related peptides increased in the hypothalamus and olfactory bulb during late gestation. Between birth and postnatal day 5, the levels of endozepines decreased two- to four-fold in all brain regions studied. Thereafter, endozepine concentration increased gradually until day 25. Reversed-phase high-performance liquid chromatography analysis of tissue extracts revealed that the olfactory bulb, pituitary, hypothalamus and cerebellum contained only one immunoreactive peak eluting at 39 min (peak C). In the telencephalon two peaks were observed: peak C and a second one eluting at 34 min (peak B). Peak B was present as early as embryonic day 20 and the ratio peak B/peak C gradually increased until day 25. At day 25 peak B was also detected in hippocampus, medulla oblongata, cortex and striatum extracts. In any brain region, no immunoreactivity co-eluting with the octadecaneuropeptide was observed. Sephadex G-50 gel filtration of hypothalamus extracts of 25-day-old animals, confirmed the existence of only one immunoreactive compound with an apparent molecular weight of 10,000. In the telencephalon two major species were resolved, with apparent molecular weights of 10,000 and 8800, and a minor one of 6500 mol. wt. In conclusion, the present study shows that endozepines are expressed in the rat brain as early as embryonic day 18 and the amount of endozepine-like material increases rapidly during the two days preceding birth. The results also indicate that diazepam-binding inhibitor is processed to different molecular forms depending on the brain region.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Localization and characterization of PACAP receptors in the rat cerebellum during development: evidence for a stimulatory effect of PACAP on immature cerebellar granule cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are abundant in the brain and particularly in the cerebellum of adult rats. In contrast, the occurrence of PACAP binding sites has not been investigated during ontogenesis. The aim of the present study was to determine the distribution and biochemical characteristics of PACAP binding sites in the rat cerebellum during postnatal development, and to examine the effect of PACAP on immature cerebellar granule cells. Autoradiographic studies revealed that PACAP binding sites are transiently expressed in a germinative matrix of the cerebellar cortex, the external granule cell layer, and in the medulla, from postnatal days 8 to 25. A population of PACAP binding sites persisted in the internal granule cell layer in the mature cerebellum. Emulsion-coated cytoautoradiography, performed on cultured immature granule cells from eight-day-old rat cerebellum, demonstrated that transient PACAP binding sites are expressed by cerebellar immature granule cells. Biochemical characterization of binding revealed the occurrence of two classes of PACAP recognition sites exhibiting, respectively, high (Kd = 0.39 +/- 0.08 nM) and low (Kd = 21.2 +/- 9.4 nM) affinity for PACAP27. The two naturally occurring forms PACAP38 and PACAP27 were equipotent in competing for [125I]PACAP27 binding. In contrast, the [Des-His1]PACAP38 analog was eight times less efficient and vasoactive intestinal polypeptide only induced weak displacement of the binding. Exposure of cultured immature granule cells to PACAP27 resulted in a dose-dependent stimulation of the production of cAMP, indicating that PACAP binding sites represent authentic receptors positively coupled to adenylate cyclase. These results show that PACAP receptors are actively expressed in the cerebellum of rats during postnatal development. The presence of functional PACAP receptors in the external granule cell layer suggests that PACAP may play a role in the control of proliferation and/or differentiation of granule cells.

Adenylyl Cyclases↗

PubMed1993

Localization of somatostatin receptors in subcortical visual centres of the rat during development. Effect of neonatal enucleation on the expression of somatostatin receptors.

Previous studies have shown that both somatostatin and somatostatin receptors are expressed in the visual system of the rat early during ontogenesis, suggesting that somatostatin plays a role in the development of visual functions. In the present study, we have examined the localization of somatostatin receptors in the lateral geniculate nucleus and the superior colliculus of the developing rat by autoradiography, and we have investigated the effect of neonatal enucleation on the density of receptors in young and adult animals. In the dorsal part of the lateral geniculate nucleus, somatostatin receptors were transiently expressed during the first two postnatal weeks, while in the ventral part, receptors were detected during the whole developmental period. In the superior colliculus, a high density of somatostatin receptors was measured before eye opening; thereafter the concentration of receptors decreased markedly in the whole structure to reach the adult level. Uni- or bilateral enucleation one day after birth only induced minor modifications of the expression of somatostatin receptors in the lateral geniculate nucleus and colliculus. Conversely, bilateral enucleation caused a significant reduction of the density of receptors in the inter-geniculate leaflet and the medial zone of the ventral lateral geniculate nucleus. Taken together, these results indicate that, in the relay nuclei, somatostatin receptors are not borne by retinal afferents. The presence of high densities of receptors in the lateral geniculate nucleus and superior colliculus during development suggests that somatostatin exerts trophic activities in these structures. In adult rat, somatostatin is probably involved in the modulation of visual information in the superior colliculus, but not in the dorsal lateral geniculate nucleus.

Amino Acid Sequence↗

PubMed1993

Neuroanatomical and physiological evidence for the involvement of pituitary adenylate cyclase-activating polypeptide in the regulation of the distal lobe of the frog pituitary.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38 amino-acid peptide which belongs to the glucagon/secretin/vasoactive intestinal peptide superfamily. The sequence of PACAP is identical in all mammalian species studied so far but frog PACAP differs by one amino-acid from mammalian PACAP. The aim of the present study was to investigate the presence of PACAP in the hypothalamo-pituitary complex of the frog Rana ribibunda and to determine the biological activity of frog PACAP on homologous pituitary cells. The distribution of PACAP-containing neurons and fibers was examined by the indirect immunofluorescence method using an antiserum raised against the N-terminal region of the peptide. In the hypothalamus, PACAP-immunoreactive perikarya were localized in the preoptic nucleus and the dorsal and ventral infundibular nuclei. Beaded nerve fibers were observed coursing from the ventral infundibular nucleus to the external vascular layer of the median eminence. A dense network of immunoreactive axons terminated in the vicinity of the capillaries of the hypophysial portal system. The neurointermediate lobe and the distal lobe of the pituitary were devoid of immunoreactive elements. The amount of PACAP-like immunoreactive material in hypothalamus extracts was measured by radioimmunoassay; the apparent concentration of PACAP was 4.5 ng/mg protein. Synthetic frog PACAP38 and PACAP27 induced a similar dose-dependent stimulation of cAMP production in isolated frog distal lobe pituitary fragments (ED50 = 2 x 10(-8) M). At the maximum dose tested (5 x 10(-6) M), both frog PACAP38 and PACAP27 produced a 4-fold increase in cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Two molecular forms of gonadotropin-releasing hormone from the brain of the frog, Rana ribibunda: purification, characterization, and distribution.

The molecular forms of GnRH in amphibia have not been characterized structurally. An antiserum to mammalian GnRH that shows appreciable cross-reactivity with chicken GnRH-I ([Gln8]GnRH), chicken GnRH-II ([His5,Trp7,Tyr8]GnRH), and salmon GnRH ([Trp7, Leu8]GnRH) was used in a RIA to detect GnRH-related peptides in an extract of the brain of the European green frog, Rana ridibunda. Two peptides were purified to apparent homogeneity, and determination of their primary structures showed that they are identical to mammalian GnRH and chicken GnRH-II. Salmon GnRH and lamprey GnRH-I ([Tyr3,Leu5,Glu6,Trp7,Lys8]GnRH) were not identified in this species of frog. Immunocytochemical analysis using specific antisera has identified regions of the frog diencephalon and telencephalon in which chicken GnRH-II and mammalian GnRH are localized to different populations of neurons. This differential distribution suggests distinct physiological roles for the two forms of GnRH in amphibia.

Animals↗

PubMed1993

Immunohistochemical distribution, biochemical characterization, and biological action of tachykinins in the frog adrenal gland.

The distribution of tachykinin-like immunoreactivity (LI) was studied in the adrenal gland of the frog Rana ridibunda using the immunofluorescence technique. A dense network of varicose fibers immunoreactive to both substance-P (SP) and neurokinin-A (NKA) was found in the adrenal tissue. In contrast, no positive fibers could be detected using antineurokinin-B (NKB) antibodies. At the electron microscope level, the immunogold technique revealed that tachykinin-LI was sequestered in dense core vesicles of 50-70 nm. Bilateral transection of either splanchnic or vagus nerves or total lesion of celiac sympathetic ganglion did not suppress tachykinin-LI. A combination of HPLC analysis and RIA detection was used to characterize tachykinin-LI in frog adrenal extracts. Two major peaks were resolved, which coeluted, respectively, with synthetic ranakinin, a novel tachykinin previously isolated from the frog brain, and [Leu3,Ile7]NKA previously isolated from the frog gut. No NKB could be detected in the extracts. The effects of various synthetic tachykinins on corticosteroid secretion were studied using perifused frog adrenal slices. For concentrations ranging from 10(-8)-10(-4) M, SP induced a dose-dependent stimulation of corticosterone and aldosterone release. A desensitization phenomenon was observed when iterative or prolonged infusions of SP were administered to the tissue. All mammalian or amphibian tachykinin-related peptides tested in our model also enhanced corticosteroid production. The effectiveness of the tachykinins tested was: [Pro7] NKB > NKA > ranakinin > [Pro9]SP > SP > kassinin > physalaemin > NKB > [Leu3,Ile7]NKA. SP also enhanced prostaglandin E2 and prostacyclin release in the effluent perifusate and the response preceded by 10-15 min the increase in corticosteroid output. Indomethacin (5 x 10(-6) M), a specific blocker of cyclooxygenase activity, totally suppressed SP-evoked steroid secretion. These data indicate that tachykinin-induced stimulation of steroidogenesis was mediated through activation of the arachidonic acid cascade. Taken together, our results show that the frog adrenal gland is innervated by a dense network of peptidergic fibers containing both ranakinin and [Leu3,Ile7]NKA, which, in vitro, stimulates corticosteroid secretion by adrenocortical cells through a prostaglandin-dependent mechanism. The present results support the view that tachykinins released by nerve fibers exert a neuroendocrine control on corticosteroid release in amphibians.

6-Ketoprostaglandin F1 alpha↗

PubMed1993

Effect of cytochalasin-B on the metabolism of polyphosphoinositides in andrenocortical cells.

We have previously shown that microfilament-disrupting agents inhibit steroid secretion by frog adrenocortical cells. To determine the role of microfilaments in the process of corticosteroid production, we studied the effects of cytochalasin-B and chaetoglobosin-C on polyphosphoinositide metabolism in myo-[3H]inositol-prelabeled frog interrenal (adrenal) slices. Immunocytochemical labeling of adrenocortical cells in primary culture with actin antiserum showed that cytochalasin-B (5 x 10(-5) M) induced a complete and reversible disruption of microfilaments, whereas chaetoglobosin-C, a cytochalasin analog that cannot interact with actin, did not modify the organization of the microfilament network. Cytochalasin-B caused a dramatic inhibition of corticosteroid release from perifused frog interrenal slices, whereas chaetoglobosin-C did not affect steroid secretion. Analysis of labeled inositol phosphates and phosphoinositides revealed that cytochalasin-B, but not chaetoglobosin-C, caused a significant increase in tritiated inositol content (+38%) and concurrently inhibited the formation of polyphosphoinositides (-48%). Cytochalasin-B reduced the production of phosphatidylinositol (-63%), phosphatidylinositol monophosphate (-46%), phosphatidylinositol bisphosphate (-46%), and lyso-phosphatidylinositol (-66%). Cytochalasin-B also blocked the stimulatory effect of angiotensin-II on the breakdown of phosphatidylinositol, phosphatidylinositol monophosphate, and phosphatidylinositol bisphosphate and the formation of lyso-phosphatidylinositol and inositol phosphates. The present results provide evidence of a role for microfilaments in polyphosphoinositide metabolism in adrenocortical cells. These data indicate that microfilaments are required for the incorporation of inositol into membrane phospholipids and are necessary for angiotensin-II-induced phospholipase activation.

Actin Cytoskeleton↗

PubMed1993

Vasopressin stimulates cortisol secretion from human adrenocortical tissue through activation of V1 receptors.

It has previously been shown that arginine vasopressin (AVP) exerts a direct stimulatory action on rat adrenocortical cells. In the present study, we have investigated the possible effect of AVP on cortisol secretion by normal human adrenocortical tissue. The occurrence of endogenous AVP in the human adrenal gland has been studied by means of the indirect immunofluorescence technique. The presence of AVP-containing cells was observed in both cortex and medulla. The action of AVP on corticosteroidogenesis has been investigated in vitro using a perifusion system technique coupled to a specific RIA for cortisol. Graded doses of AVP (from 10(-11)-10(-9) M) increased cortisol secretion in a dose-dependent manner (ED50, 4.5 x 10(-11) M). AVP also induced a significant stimulation of cortisol release from acutely dispersed adrenocortical cells. Prolonged administration of AVP (3 h) induced a rapid and transient increase in cortisol output, followed by a gradual decline in cortisol secretion. Repeated pulses of AVP, given at 90-min intervals, resulted in reproducible stimulations of cortisol output. Selective agonists and antagonists have been used to determine the type of receptor involved in the response of adrenocortical cells to AVP. Oxytocin at doses up to 10(-7) M had virtually no effect on cortisol secretion. The stimulatory effect of AVP was blocked by the V1 antagonist [beta-mercapto-beta,beta-cyclopentamethylene propionyl 1, OMe-Tyr2, Arg8]AVP. In contrast, the V2 antagonist [d(CH2)5D-Phe2,Ile4,Ala9-NH2]AVP did not affect the response of the adrenal gland to AVP. The selective V2 agonist [deamino-Cys1,D-Arg8]AVP did not mimic the stimulatory effect of AVP on cortisol secretion. Taken together, these results suggest that AVP, locally released by intracortical cells, may act as a paracrine factor to stimulate adrenal steroidogenesis in man. The effect of AVP on cortisol secretion appears to be mediated through activation of typical V1 receptors.

Adrenal Cortex↗

PubMed1993

Effect of the serotonin-4 receptor agonist zacopride on aldosterone secretion from the human adrenal cortex: in vivo and in vitro studies.

We have recently shown that serotonin (5-HT) stimulates cortisol secretion from human adrenocortical tissue in vitro through activation of 5-HT4 receptors. The aim of the present study was to investigate the effect of the 5-HT4 agonist racemic zacopride on aldosterone secretion from the human adrenal gland in vivo and in vitro. In vivo studies were conducted on 28 healthy volunteers pretreated with dexamethasone. The subjects received a single oral dose of placebo, 10 micrograms zacopride, or 400 micrograms zacopride. Plasma aldosterone levels increased significantly within 90 min after the administration of 400 micrograms zacopride, remained elevated for 60 min, and gradually returned to the baseline within 180 min. In contrast, the administration of 10 micrograms zacopride or placebo did not modify the aldosterone concentration. No significant changes were observed in renin, ACTH, or cortisol levels. In vitro studies were conducted on perifused human adrenocortical slices. Administration of 20-min pulses of zacopride (from 10(-11) - 10(-6) mol/L) induced a dose-dependent increase in aldosterone secretion. The minimal effective dose was 10(-10) mol/L, and half-maximal stimulation was obtained with a dose of 7 x 10(-8) mol/L. Zacopride was 100 times more potent in stimulating aldosterone than cortisol release. Taken together, the present data suggest that 5-HT-evoked aldosterone secretion involves the activation of 5-HT4 receptors.

Adolescent↗

PubMed1993

Effect of dopamine on adenylate cyclase activity, polyphosphoinositide metabolism and cytosolic calcium concentrations in frog pituitary melanotrophs.

It has previously been shown that dopamine plays a pivotal role in the regulation of alpha-melanocyte-stimulating hormone (alpha-MSH) secretion from the intermediate lobe of the pituitary. In the present study, we have investigated the various intracellular mechanisms that are associated with the action of dopamine on frog pituitary melanotrophs. Dopamine reduced forskolin-stimulated cyclic adenosine monophosphate (cAMP) production and the inhibitory effect of dopamine was blocked by the dopaminergic D2 receptor antagonist sulpiride. The D2 receptor agonist apomorphine inhibited incorporation of [3H]inositol into membrane phospholipids. Dopamine also inhibited the formation of inositol trisphosphate and provoked accumulation of phosphatidylinositol bisphosphate. The inhibitory effect of dopamine on inositol trisphosphate production was mimicked by D2 receptor agonists and blocked by sulpiride. Using a double-wavelength microfluorimetric approach, we found that dopamine caused a rapid and transient decrease in K(+)-evoked stimulation of intracellular calcium concentration. The time-courses of the responses of the various intracellular messengers indicate that blockage of voltage-dependent calcium channels is the primary event associated with activation of dopamine D2 receptors, while inhibition of polyphosphoinositide breakdown, related to blockage of voltage-dependent calcium channels, and reduction of cAMP production are secondary events which may contribute to the sustained inhibitory effect of dopamine on alpha-MSH release.

Adenylyl Cyclases↗

PubMed1993

Localization, characterization and activity of pituitary adenylate cyclase-activating polypeptide in the frog adrenal gland.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been isolated from the frog brain and the sequence of the peptide appears to be strikingly similar to that of mammalian PACAP. In the present study, we have investigated the localization of PACAP in the frog interrenal (adrenal) gland by immunocytochemistry using antisera directed against PACAP 38 or PACAP 27. Two types of PACAP-immunoreactive fibres were observed: thick varicose fibres coursing between adrenal cells and thin processes located in the walls of blood vessels irrigating the gland. Bilateral transection of the splanchnic nerves did not affect the intensity and distribution of PACAP immunoreactivity. The mean +/- S.E.M. concentration of PACAP, measured by radioimmunoassay in crude adrenal extracts, was 0.65 +/- 0.16 nmol/g wet tissue. Two molecular forms of PACAP in the adrenal gland were characterized by reversed phase high-performance liquid chromatography combined with radioimmunoassay quantification. The elution profiles revealed the existence of two peaks exhibiting the same retention times as synthetic frog PACAP 38 (fPACAP 38) and PACAP 27, the predominant form being PACAP 38. The possible involvement of PACAP in the regulation of adrenal steroidogenesis was investigated in vitro using a perifusion system for frog adrenal slices. Graded doses of fPACAP 38 (0.1-10 mumol/l) increased the secretion of both corticosterone and aldosterone in a dose-dependent manner. Administration of repeated pulses of fPACAP 38 (1 mumol/l), at 120-min intervals, led to a reproducible stimulation of corticosteroid secretion without any tachyphylaxis. Prolonged infusion (2 h) of the peptide induced a rapid increase in corticosterone and aldosterone output, followed by a gradual decline in the secretion rate, suggesting the occurrence of a desensitization phenomenon. Synthetic porcine vasoactive intestinal peptide, which is structurally related to PACAP, was about ten times less potent than fPACAP 38 in stimulating steroidogenesis while the [Des-His1]-fPACAP 38 analogue was 100 times less effective. These results demonstrate that a peptide closely related to fPACAP 38 is present in fibres innervating the frog adrenal gland and could participate in the regulation of corticosteroid secretion, particularly during neurogenic stress.

Adrenal Glands↗

PubMed1992

Neuropeptide Y inhibits alpha-MSH release from rat hypothalamic slices through a pertussis toxin-sensitive G protein.

The arcuate nucleus of the hypothalamus contains various types of peptidergic neurons. In particular, two distinct populations of neurosecretory neurons containing neuropeptide Y (NPY)- and alpha-melanocyte-stimulating hormone (alpha-MSH)-like immunoreactivity have been identified in the arcuate nucleus. Double-labeling immunocytochemical data have recently shown that NPY-containing fibers make synaptic contacts with proopiomelanocortin (POMC) immunoreactive neurons. We have thus investigated the possible effect of NPY on the release of alpha-MSH from rat hypothalamic slices in vitro, using the perifusion technique. NPY significantly inhibited KCl-stimulated alpha-MSH release in a dose-dependent manner. The inhibitory effect of NPY was mimicked by the Y2 agonist, NPY-(13-36), while the Y1 agonist, [Leu31,Pro34]NPY, was devoid of effect. Pretreatment of hypothalamic slices with pertussis toxin (PTX) blocked the inhibitory effect of NPY, suggesting that the action of NPY on POMC neurons is mediated through a PTX-sensitive G protein. These results support the notion that NPY may play a physiological role in the regulation of alpha-MSH release from hypothalamic neurons.

Animals↗

PubMed1992

Primary structure and receptor-binding properties of a neurokinin A-related peptide from frog gut.

A tachykinin peptide was isolated from an extract of the intestine of the European green frog, Rana ridibunda, and its primary structure was established as: His-Lys-Leu-Asp-Ser-Phe-Ile-Gly-Leu-Met.CONH2. This sequence was confirmed by chemical synthesis and shows two amino acid substitutions (leucine for threonine at position 3 and isoleucine for valine at position 7) compared with neurokinin A. Binding parameters for synthetic [Leu3,Ile7]neurokinin A and mammalian tachykinins were compared using receptor-selective radioligands and crude membranes from tissues enriched in the NK1, NK2 and NK3 receptors. [Leu3,Ile7]Neurokinin A was approx. 3-fold less potent than substance P in inhibiting the binding of 125I-labelled [Sar9,Met(O2)11]substance P (labelled with Bolton-Hunter reagent) to rat submandibular gland (NK1 receptor), 8-fold less potent than neurokinin A in inhibiting the binding of [2-[125I]iodohistidine1]neurokinin A to rat stomach fundus (NK2 receptor) and 6-fold less potent than neurokinin B in inhibiting the binding of 125I-Bolton-Hunter-labelled scyliorhinin II to rat brain (NK3 receptor). Thus the frog neurokinin A-related peptide shows moderate affinity but lack of selectivity for all three tachykinin-binding sites in rat tissues. This non-selectivity is similar to that displayed by the molluscan tachykinin, eledoisin, which also contains an isoleucine residue in the corresponding position in the molecule.

Amino Acid Sequence↗

PubMed1992

Isolation and primary structure of urotensin II from the brain of a tetrapod, the frog Rana ridibunda.

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.

Amino Acid Sequence↗

PubMed1992

Immunohistochemical distribution and biological activity of pituitary adenylate cyclase-activating polypeptide (PACAP) in the central nervous system of the frog Rana ridibunda.

The primary structure of frog pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been determined and the results show that the sequence of PACAP has been highly conserved during evolution. In particular, the structure of the 1-27 fragment of PACAP is identical in frog and mammals. Using an antiserum raised against PACAP27, we have investigated the distribution of PACAP-containing neurons in the central nervous system of the frog Rana ridibunda by the immunofluorescence technique. The main populations of immunoreactive perikarya were located in the medial and ventral diencephalon, i.e., the preoptic nucleus, the ventral and dorsal infundibular nuclei, the nucleus posterocentralis thalami, and the ventral and ventrolateral areas of the thalamus. In the telencephalon, sparse cell bodies were found in the nucleus accumbens septi, the amygdala, the pallial commissure, and the bed nucleus of the pallial commissure. In the hindbrain, the torus semicircularis, the nucleus profundus and the nucleus anteroventralis tegmenti of the mesencephalon also contained populations of PACAP-immunoreactive perikarya. Beaded nerve fibers were observed throughout the brain. Occasionally they formed bundles, e.g., from the ventral infundibulum to the external vascular layer of the median eminence, from the central thalamus to the optic tectum, and rostrocaudally, from the nucleus accumbens septi to the nucleus entopeduncularis. Other areas, such as the interpeduncular nucleus, the nucleus isthmi and the roots of cranial nerves V and VIII in the medulla oblongata, were also densely innervated. The adenylate cyclase-stimulating activity of PACAP was tested by using a static incubation technique for hypothalamic slices.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗