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M L Vitale

Publications and source records attributed to M L Vitale.

36 records · Page 2Linked to original sources
PubMed1992

Protein kinase C activation by phorbol esters induces chromaffin cell cortical filamentous actin disassembly and increases the initial rate of exocytosis in response to nicotinic receptor stimulation.

Nicotinic stimulation and high K+ depolarization of bovine chromaffin cells cause disassembly of cortical filamentous actin networks. Previous work from our laboratory has demonstrated that disassembly of actin filaments is Ca(2+)-dependent, precedes exocytosis and occurs in cortical areas of low cytoplasmic viscosity which are the sites of exocytosis. It has also been suggested that protein kinase C is involved in catecholamine secretion from chromaffin cells. Therefore, the possibility that protein kinase C activation might be implicated in cortical filamentous actin disassembly was investigated. Here we report that phorbol myristate acetate, a protein kinase C activator, causes cortical filamentous actin disassembly. Short-term phorbol ester treatment does not alter the morphology of chromaffin cells; however, 1 h after phorbol ester exposure an increase in cell flattening and membrane ruffling is observed. Phorbol ester-induced cortical filamentous actin disassembly is inhibited by protein kinase C activity inhibitors, is independent of extracellular Ca2+ and has a slower time course than that induced by either nicotinic receptor stimulation or K(+)-depolarization. Phorbol ester effects are likely to be mediated by activation of protein kinase C and not by any changes in intracellular Ca2+ levels, as indicated by measurements of Ca2+ transients. Pretreatment of chromaffin cells with phorbol myristate acetate increases the initial rate of nicotine-evoked catecholamine release. Nicotine-induced cortical actin filament disassembly and catecholamine secretion are partially (29-40%) inhibited by pretreatment of cells with either calphostin C, staurosporine or sphingosine. The results suggest that protein kinase C may be involved in the reorganization of the cortical actin filament network priming the cells for release by removing a barrier to secretory granule mobility. However, its role in exocytosis is modulatory but not essential.

Actins↗
PubMed1992

Ca2+ and pH determine the interaction of chromaffin cell scinderin with phosphatidylserine and phosphatidylinositol 4,5,-biphosphate and its cellular distribution during nicotinic-receptor stimulation and protein kinase C activation.

Nicotinic stimulation and high K(+)-depolarization of chromaffin cells cause disassembly of cortical filamentous actin networks and redistribution of scinderin, a Ca(2+)-dependent actin filament-severing protein. These events which are Ca(2+)-dependent precede exocytosis. Activation of scinderin by Ca2+ may cause disassembly of actin filaments leaving cortical areas of low cytoplasmic viscosity which are the sites of exocytosis (Vitale, M. L., A. Rodríguez Del Castillo, L. Tchakarov, and J.-M. Trifaró. 1991. J. Cell. Biol. 113:1057-1067). It has been suggested that protein kinase C (PKC) regulates secretion. Therefore, the possibility that PKC activation might modulate scinderin redistribution was investigated. Here we report that PMA, a PKC activator, caused scinderin redistribution, although with a slower onset than that induced by nicotine. PMA effects were independent of either extra or intracellular Ca2+ as indicated by measurements of Ca2+ transients, and they were likely to be mediated through direct activation of PKC because inhibitors of the enzyme completely blocked the response to PMA. Scinderin was not phosphorylated by the kinase and further experiments using the Na+/H+ antiport inhibitors and intracellular pH determinations, demonstrated that PKC-mediated scinderin redistribution was a consequence of an increase in intracellular pH. Moreover, it was shown that scinderin binds to phosphatidylserine and phosphatidylinositol 4,5-biphosphate liposomes in a Ca(2+)-dependent manner, an effect which was modulated by the pH. The results suggest that under resting conditions, cortical scinderin is bound to plasma membrane phospholipids. The results also show that during nicotinic receptor stimulation both a rise in intracellular Ca2+ and pH are observed. The rise in intracellular pH might be the result of the translocation and activation of PKC produced by Ca2+ entry. This also would explain why scinderin redistribution induced by nicotine is partially (26-40%) inhibited by inhibitors of either PKC or the Na+/H+ antiport. In view of these findings, a model which can explain how scinderin redistribution and activity may be regulated by pH and Ca2+ in resting and stimulated conditions is proposed.

Adrenal Medulla↗
PubMed1992

Loss and Ca(2+)-dependent retention of scinderin in digitonin-permeabilized chromaffin cells: correlation with Ca(2+)-evoked catecholamine release.

Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.

Adrenal Medulla↗
PubMed1991

Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin.

Immunofluorescence and cytochemical studies have demonstrated that filamentous actin is mainly localized in the cortical surface of the chromaffin cell. It has been suggested that these actin filament networks act as a barrier to the secretory granules, impeding their contact with the plasma membrane. Stimulation of chromaffin cells produces a disassembly of actin filament networks, implying the removal of the barrier. The presence of gelsolin and scinderin, two Ca(2+)-dependent actin filament severing proteins, in the cortical surface of the chromaffin cells, suggests the possibility that cell stimulation brings about activation of one or more actin filament severing proteins with the consequent disruption of actin networks. Therefore, biochemical studies and fluorescence microscopy experiments with scinderin and gelsolin antibodies and rhodamine-phalloidin, a probe for filamentous actin, were performed in cultured chromaffin cells to study the distribution of scinderin, gelsolin, and filamentous actin during cell stimulation and to correlate the possible changes with catecholamine secretion. Here we report that during nicotinic stimulation or K(+)-evoked depolarization, subcortical scinderin but not gelsolin is redistributed and that this redistribution precedes catecholamine secretion. The rearrangement of scinderin in patches is mediated by nicotinic receptors. Cell stimulation produces similar patterns of distribution of scinderin and filamentous actin. However, after the removal of the stimulus, the recovery of scinderin cortical pattern of distribution is faster than F-actin reassembly, suggesting that scinderin is bound in the cortical region of the cell to a component other than F-actin. We also demonstrate that peripheral actin filament disassembly and subplasmalemmal scinderin redistribution are calcium-dependent events. Moreover, experiments with an antibody against dopamine-beta-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.

Actins↗
PubMed1991

Suckling-induced changes of vasoactive intestinal peptide concentrations in hypothalamic areas implicated in the control of prolactin release.

The present study was designed to investigate whether the vasoactive intestinal peptide (VIP) concentration in hypothalamic nuclei, dorsal raphe nucleus (DR) and pituitary lobes of lactating rats changes in physiological situations when prolactin (PRL) secretion is stimulated (suckling) or inhibited (pup separation). In addition VIP levels in blood plasma were determined in both situations. Acute suckling induced changes in VIP concentration only in the rostral part of the anterior hypothalamic (rAHN) and the paraventricular (PVN) nuclei of all the brain areas examined. VIP concentration in the rAHN increased at 5 min from 3.52 +/- 0.30 (mean +/- SEM) to 8.67 +/- 1.91 ng/mg protein (p less than 0.05) but fell to baseline values after 30 min suckling (p less than 0.05; 5 vs. 30 min). Although changes in VIP concentration in the suprachiasmatic nucleus (SCN) did not attain statistical significance, they followed the same trends as the changes of VIP in the rAHN. The opposite pattern of changes was observed in the PVN with a decrease in VIP concentration following 5 min suckling (p less than 0.01). At 30 min the VIP values showed a trend towards 0-min values. Pup removal did not affect VIP concentrations in the rAHN, PVN, SCN, median eminence, supraoptic nucleus and DR. VIP values were not detectable in the arcuate nucleus in any of the experimental situations examined. Lactation increased VIP concentration only in the rAHN and PVN when lactating rats with their pups were compared with virgin female diestrous rats. VIP concentration in the anterior lobe of the pituitary from lactating rats did not change with pup separation or suckling.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Expression of scinderin, an actin filament-severing protein, in different tissues.

Scinderin is a calcium-dependent actin filament-severing protein recently discovered in the chromaffin cells of adrenal medulla. In view of the wide tissue distribution of gelsolin, another actin filament-severing protein, experiments were performed to determine the tissue expression of scinderin. Extracts prepared from different bovine tissues were tested by actin-DNase I Sepharose 4B-binding procedure and immunoprecipitation followed by immunoblotting with scinderin and gelsolin antibodies. Among the tissues tested, scinderin was found to be present in the adrenal medulla, brain, anterior and posterior pituitaries, kidney, salivary gland and testis. Scinderin was not found in liver, plasma, skeletal and heart muscles. Gelsolin was expressed in all of the above tissues. The results suggest that scinderin seems to be restricted to tissues with high secretory activity.

Animals↗
PubMed1990

Chromaffin cell scinderin, a novel calcium-dependent actin filament-severing protein.

Scinderin, a novel Ca2+-activated actin filament-severing protein, has been purified to homogeneity from bovine adrenal medulla using a combination of several chromatographic procedures. The protein has an apparent mol. wt of 79,600 +/- 450 daltons, three isoforms (pIs 6.0, 6.1 and 6.2) and two Ca2+ binding sites (Kd 5.85 x 10(-7) M, Bmax 0.81 mol Ca2+/mol protein and Kd 2.85 x 10(-6) M, Bmax 1.87 mol Ca2+/mol protein). Scinderin interacts with F-actin in the presence of Ca2+ and produces a decrease in the viscosity of actin gels as a result of F-actin filament severing as demonstrated by electron microscopy. Scinderin is a structurally different protein from chromaffin cell gelsolin, another actin filament-severing protein described. Scinderin and gelsolin have different mol. wts, isoelectric points, amino acid composition and yield different peptide maps after limited proteolytic digestion by either Staphylococcus V8 protease or chymotrypsin. Moreover, scinderin antibodies do not cross-react with gelsolin and gelsolin antibodies fail to recognize scinderin. Immunofluorescence with anti-scinderin demonstrated that this protein is mainly localized in the subplasmalemma region of the chromaffin cell. Immunoblotting tests with the same antibodies indicated that scinderin is also expressed in brain and anterior as well as posterior pituitary. Presence of scinderin and gelsolin, two Ca2+-dependent actin filament-severing proteins in the same tissue, suggests the possibility of synergistic functions by the two proteins in the control of cellular actin filament networks. Alternatively, the actin filament-severing activity of the two proteins might be under the control of different transduction and modulating influences.

Actins↗
PubMed1990

Serotonergic innervation of the rat testis.

The presence of 5-hydroxytryptamine (5-HT) was determined by h.p.l.c. in perchloric extracts of each isolated compartment of the adult rat testis. The testicular capsule, interstitial cells and interstitial fluid contained 5-HT, but 5-HT was not detected in the tubular compartment. In a group of adult rats, one testis was unilaterally denervated, and the contralateral testis used as control. The superior spermatic nerve, arising from the renal plexus, was excised and 1 week after surgery 5-HT content was measured in the capsule and interstitial fluid of both testes. Denervation caused a significant fall (34%) in 5-HT content. These results indicate that at least part of the testicular 5-HT derives from a serotonergic innervation of the gonad.

Animals↗
PubMed1988

Dorsal raphe serotoninergic branching neurons projecting both to the lateral geniculate body and superior colliculus: a combined retrograde tracing-immunohistochemical study in the rat.

Injections of HRP into the superior colliculus labelled cells in the lateral cell groups of the dorsal raphe nucleus. The cytoarchitectural features and location of these cells showed remarkable similarities with those known to project to the lateral geniculate body, and, therefore, the possible existence of branching neurons in the dorsal raphe nucleus projecting to these two visual structures was tested. Injections into the lateral geniculate body and the superior colliculus of several fluorescent tracers--namely, Fast Blue, Fluoro-Gold, propidium iodide, rhodamine-B-isothiocyanate, and Diamidino Yellow, used in different combinations, showed single- and double-labelled neurons in the lateral wings of the dorsal raphe nucleus. In order to verify the chemical nature of these cells, the tissue was processed for immunofluorescence with serotonin antibodies. The results obtained showed several triple-labelled cells exhibiting two fluorescent tracers as well as 5-hydroxytryptamine-like immunoreactivity. Some immunonegative tracer-positive cells were also observed, suggesting their nonserotoninergic nature. Finally, electrolytic lesions of the lateral wings of the dorsal raphe nucleus caused a gradual disappearance of serotonin-immunoreactive fibers in these visual areas following different survival times. This correlated well with a decrease in the serotonin content studied by high-pressure liquid chromatography. These results support a role of the serotoninergic dorsal raphe projection to the lateral geniculate body and to the superior colliculus in the processing of visual information, and they suggest that serotonin may have a coordinating influence on primary visual centers.

Animals↗
PubMed1987

Dorsal raphe serotonergic projection to the retina. A combined peroxidase tracing-neurochemical/high-performance liquid chromatography study in the rat.

The existence of retinopetal neurons in the rat was verified using a morphological and neurochemical approach. Horseradish peroxidase injected into the posterior chamber of the eye labeled polygonal, ovoid, fusiform and small multipolar neurons in the lateral cell groups of the dorsal raphe nucleus. Very small electrolytic lesions of this region produced after several days of survival a significant decrease in the serotonin content of the retina. These results demonstrate the existence of a centrifugal projection to the retina from the lateral cell groups of the dorsal raphe nucleus and show its probable serotonergic nature. Besides, they also provide a new possibility to explain the presence of serotonin in the retina.

Animals↗
PubMed1987

Dorsal raphe lesion alters the estrous cycle and the preovulatory gonadotropin release.

The purpose of this study was to examine the role that the dorsal raphe (DR)-median eminence (ME) serotonergic projection may have in the proestrous gonadotropin and prolactin (PRL) release. DR electrolytic lesions were performed in cycling rats during the first day of diestrus. The effect of DR lesions after 48-72 h of survival (short-term lesioned animals) or after 35-40 days of survival (long-term lesioned animals) on estrous cyclicity, preovulatory gonadotropin and PRL releasing pattern, ovulation and serotonin (5-HT) content of the ME were studied. Following DR lesions the estrous cycle became irregular, remaining in the diestrus phase for several days. Preovulatory gonadotropin release in short-term lesioned animals was increased; on the contrary, in long-term lesioned rats a delay in the surge of these two hormones and a decrease in LH secretion were detected. Long-term lesioned animals also showed a diminished secretion of PRL. The number of ova did not differ between control and lesioned animals. DR lesions in both short- and long-term lesioned rats reduced 5-HT levels in the ME by about 50% and nullified the normal 5-HT decline during the afternoon of proestrus. Our results suggest that the DR exerts a stimulatory influence on the preovulatory gonadotropin release by means of its 5-HT projection to the ME. As the pattern of hormonal secretion in lesioned animals remains similar to that of controls, it may be suggested that this pathway acts as a fine modulator of the mechanisms involved in the regulation of LH and FSH release in cycling rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Serotonergic terminals in the anterior hypothalamic nucleus involved in the prolactin release during suckling.

The present studies were designed to localize within the hypothalamus and neighboring areas the serotonergic terminals which are implicated in suckling-induced PRL release. The initial experiments were performed to characterize the circulating hormone profile induced by suckling in lactating rats, previously separated from their pups. Five minutes of suckling induced an increase in serum PRL only. During these 5 min, 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid concentrations were determined in the pars nervosa of the pituitary gland, hypothalamic nuclei, dorsal, and median raphe nuclei. An increase by 80% (P less than 0.01) in 5-HT concentration was found only in the rostral part of the anterior hypothalamic nucleus (rNHA). In order to investigate causal effect between the altered 5-HT neuronal activity in the rNHA and the suckling-induced PRL release, serotonergic neurotoxin was bilaterally injected in the rNHA on day 1 of lactation. Litters were adjusted to eight pups each and weighed daily to determine litter growth rates. On day 8 of lactation, litters were separated from their mothers for 4 h and allowed to suckle for 5 or 15 min after which the mothers were decapitated. Litters from lesioned animals grew at a lower rate (P less than 0.0001) than control and sham-operated animals. Serum PRL increased with suckling in animals bearing the correct rNHA lesions, but the values were lower than in control and sham-operated animals after 5 (P less than 0.05) and 15 (P less than 0.01) min. Therefore we postulate that the rNHA is the site of termination of a stimulatory serotonergic pathway on PRL release induced by suckling.

5,7-Dihydroxytryptamine↗
PubMed1986

Serotonin induces gonadotrophin release through stimulation of LH-releasing hormone release from the median eminence.

The effects of serotonin (5-HT) on the release of gonadotrophins and LH-releasing hormone (LHRH) were examined in an in-vitro perifusion system using median eminences and/or anterior pituitaries obtained from male or pro-oestrous female rats. Animals were killed by decapitation between 12.00 and 13.00 h. A serial double-chamber perifusion system was employed. Three types of experiments were performed. In the first, median eminences were placed in the first chamber and one anterior pituitary in the second chamber. In the second group, only the anterior pituitary was perifused. In the third group, only five median eminences were perifused. In the first and second experiments, LH, FSH and prolactin were determined in the perifusion efflux by radio-immunoassay (RIA). In the third experiment, LHRH was determined by RIA. Addition of 5-HT (final concentrations 0.06, 0.6 and 6.0 mumol/l) into the first chamber containing the median eminences stimulated the release of LH and FSH from the pituitary, but did not affect the levels of prolactin in the effluent in the same experiment (pro-oestrous rats). The stimulatory effect of 5-HT was blocked by the addition of cyproheptadine (l mumol/l) in the perifusion fluid. The introduction of 5-HT (0.6 mumol/l) into the tube connecting the first and second chambers did not modify the release of LH, nor did 5-HT added to the pituitaries perifused alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Serotonin stimulates gonadotrophin release by acting directly on the median eminence.

The study examined the effect of serotonin (5-HT) on gonadotrophin release and its tissue site of action using a sequential double chamber perifusion system. Cycling female rats were killed between 12.00 and 13.00 h on the day of proestrus. Median eminences and anterior pituitaries were removed and transferred to perifusion chambers. Two types of experiments were performed: a) five median eminences (ME) were placed in the first chamber and one anterior pituitary in the second chamber. b) In the second group, only the anterior pituitary was perifused. The effluent from the first chamber perifused the second chamber. The effluent from the second chamber was collected for hormone assays. Addition of serotonin (final concentration 0.06, 0.6 and 6.0 uM) stimulated the release of LH and FSH into the perifusion fluid draining the pituitary in series with the median eminences. Pretreatment with the 5-HT receptor blocker cyproheptadine (1 uM) completely inhibited the stimulatory effect of 5-HT. Serotonin was ineffective in stimulating gonadotrophin release when injected into the tube connecting the first and the second chambers (50 ul of 0.6 uM solution) or when the anterior pituitary was perifused alone. Serotonin did not affect the prolactin release in any of the experimental conditions studied. These observations demonstrate that serotonin stimulated the gonadotrophin release by acting on serotoninergic receptors at the level of the median eminence.

Animals↗
PubMed1984

Median eminence serotonin involved in the proestrus gonadotropin release.

Numerous studies have suggested that serotonin (5-HT) is involved in the regulation of anterior pituitary hormone release. In the present study, the 5-HT concentrations of the median eminence and anterior pituitary lobe were measured during the estrous cycle and lactation in order to correlate changes in 5-HT levels with changes in serum luteinizing hormone, follicle-stimulating hormone, and prolactin. On the day of proestrus, median eminence 5-HT concentrations declined significantly between 14.00 and 16.30 h at the beginning of the gonadotropin and prolactin surges. No changes in 5-HT concentrations were found between the morning and afternoon on other days of the cycle. In the anterior pituitary, the levels of 5-HT did not change during the estrous cycle. 5-HT turnover rates were also estimated in the median eminence on proestrus and diestrus 1. The median eminence 5-HT synthesis rate increased in the afternoon of proestrus at 16.30 h. 5-HT was also measured in the anterior pituitary and the median eminence of lactating rats in four experimental situations: mothers with their litter until decapitation, mothers separated from their pups 4 h earlier, and mothers separated from their pups 4 h earlier, after which the pups were allowed to suckle for 5 or 30 min. In spite of the acute changes in circulating prolactin, 5-HT levels in the median eminence were not affected in any situation studied. These results suggest that 5-HT in the median eminence is involved in the control of gonadotropin release. The data further suggest that 5-HT does not act directly on the anterior pituitary to modulate gonadotropin or prolactin release.

Animals↗
PubMedDate not supplied

Catecholamine distribution in adult rat testis.

Catecholamine distribution in the adult rat testis was examined using a sensitive radioenzymatic method. Norepinephrine was present in the capsule and the interstitial fluid, in higher concentrations than dopamine, while in the interstitial cell preparations only norepinephrine was found. Epinephrine was undetectable in all testicular compartments investigated. No catecholamines were found in the seminiferous tubules. Testicular denervation caused a significant decrease in capsular catecholamines, confirming the neural origin of these amines.

Animals↗