Evaluation of the effectiveness of three sticky traps to monitor four species of cockroaches (Hexapoda: Blattaria) with simulated use tests.

Background: Cockroaches are the pest of major concern for the disinfestation programs of the sanitary system in Italy. Hygienic-sanitary interest is linked to the role of mechanical vectors of pathogens and to their allergological potential. Sticky traps are the best tool to monitor the presence of these insects and several types of them are available on the market. In most of the cases the traps are not indicated for a given species, but, instead, generically for cockroaches. Domestic cockroaches differ in morphology, size and habits. Consequently, the effectiveness of the trap can change in relation to the target species. Materials and methods: In this study three of the most employed traps in Italy were compared: the INDIA trap with and without its attractant tablet (hereafter mentioned as INDIA-A and INDIA-E, respectively), the ZAPI Simply trap and the CATCHMASTER Spider & Insect Glue trap. We chose the four most common species of cockroach (Blattodea) in Italy, Blatta orientalis (L.), Periplaneta americana (L.) (Blattidae), Blattella germanica (L.) and Supella longipalpa (F.) (Blattellidae). Each species of cockroach was tested separately inside arenas containing one of the traps. Each test (one species with one kind of trap) was replicated five times. Results and discussion: The INDIA-A trap collected more cockroaches of every species, followed by the INDIA-E. The ZAPI trap caught less specimens of each species in respect to the INDIA traps, with the only exception of B. orientalis, for which the ZAPI trap caught more than the INDIA-E. The CATCHMASTER trap performed significantly less for all the species. B. orientalis was the species most abundantly caught by all traps, followed by B. germanica, S. longipalpa and P. americana. No significant difference was observed in the catch according to the developmental stage. In general, there was no particular predisposition of any trap to catch a particular species. Conclusions: It is not possible to indicate a model of trap for each species of cockroach, but it is clear that different traps have different performances in terms of attractiveness and capture. Therefore, the choice of the trap affects the results of the monitoring, and as consequence, the evaluation of the infesting population of the pest.

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.

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.

Chromaffin cell cortical actin network dynamics control the size of the release-ready vesicle pool and the initial rate of exocytosis.

Morphological, biochemical, and membrane capacitance measurements were used to study the role of cortical filamentous actin (F-actin) in exocytosis. Fluorescence and electron microscopy of resting chromaffin cells revealed a cortical actin network that excluded secretory vesicles from the subplasmalemmal area. Phorbol ester (PMA) treatment disrupted cortical F-actin and increased both the number of vesicles within the 0-50 nm subplasmalemmal zone and the initial rate of stimulated catecholamine release. In PMA-pretreated cells, membrane capacitance studies showed an increased number of vesicles fusing with the plasmalemma during the first two depolarizations of a train. PMA did not affect voltage-dependent Ca2+ influx. The total number of vesicles fused with the plasma membrane correlated well with the number of vesicles occupying the 0-50 nm cortical zone. Therefore, cortical F-actin disassembly allows translocation of vesicles to the plasmalemma in preparation for exocytosis.

Serotonin, a neurotransmitter involved in the regulation of luteinizing hormone release.

The involvement of serotonin (5-HT) in the regulation of LH secretion is discussed on the basis of experimental and physiological models. The role of 5-HT on low amplitude pulsatile LH release in male rats is not yet clear, in spite of the fact that recent results suggest a weak permissive role. In ovariectomized rats, 5-HT expresses a negative influence on the increased rate of LH release, which is converted into a stimulatory effect by pretreatment of the animals with E2. Moreover, in castrated female rats there is a morning/afternoon oscillatory pattern in 5-HT metabolism of brain areas associated with the control of LH secretion including the hypothalamus. In this area, the fluctuation is modulated by E2 in such a way that the peak of 5-HT neural activity occurs simultaneously with the induced afternoon discharge of LH. On the other hand, P enhances the amplitude of the oscillation of hypothalamic 5-HT metabolism concomitantly with a potentiation of the induced LH surge. This facilitatory role of 5-HT upon phasic LH discharge is also evident in intact female rats. The preovulatory surge of LH is accompanied by an increased hypothalamic and, more precisely, ME 5-HT turnover. Furthermore, 5-HT stimulates in vitro LHRH release from the ME. Serotonergic nuclei located in the brain stem seem to mediate this effect.

Cytoskeleton dynamics during neurotransmitter release.

It has become apparent in recent years that the cytoskeleton and its associated proteins play a major role in secretion. This review summarizes recent findings on the cytoskeleton organization and the molecular topology of its regulatory proteins, as well as the dynamic changes that occur in this organelle during secretion from neurons and secretory cells. Although two apparently different ultrastructures and molecular organizations of the cytoskeleton seem to be involved in neuronal and secretory cell secretion, there are similarities between the two systems. In both neurons and secretory cells, Ca2+ plays a pivotal role in the control of cytoskeleton dynamics, especially in the changes in actin filament networks observed during secretion.

Biphasic Effect of Basic Fibroblast Growth Factor on Anterior Pituitary Folliculostellate TtT/GF Cell Coupling, and Connexin 43 Expression and Phosphorylation.

Basic fibroblast growth factor (bFGF) is a mitogenic and differentiating cytokine. In the anterior pituitary, folliculostellate (FS) cells constitute the major source of bFGF. bFGF affects endocrine cell proliferation and secretion in the anterior pituitary. In addition, bFGF increases its own expression by acting directly on FS cells. FS cell Cx43-mediated gap junction intercellular communication allows the establishment of an intrapituitary network for the transmission of information. In the present study, we assessed how bFGF regulates FS cell coupling. Time course studies were carried out on the FS cell line TtT/GF. Short-term bFGF treatment induced a transient cell uncoupling and the phosphorylation in Ser368 of membrane-bound Cx43 without modifying Cx43 levels. We demonstrated the involvement of the protein kinase C (PKC) isoform α in the phosphorylation of Cx43 in S368. Moreover, we showed that bFGF induced PKCα activation by stimulating its expression, phosphorylation and association with the plasma membrane. The long-term incubation with bFGF increased TtT/GF cell coupling, total Cx43 levels and Cx43 accumulation at the cell membrane of cytoplasmic projections. The Cx43 level increase was a result of the stimulation of Cx43 gene transcription as mediated by the extracellular-regulated kinase 1/2 signalling pathway. Taken together, the data show that bFGF modulates TtT/GF cell coupling by activating different pathways that lead to opposite effects on Cx43 phosphorylation and expression depending on the duration of the exposure of the cells to bFGF. A short-term bFGF exposure reduces cell-to-cell communication as a mean of desynchronising FS cells. By contrast, long-term exposure to bFGF enhances cell-to-cell communication and facilitates coordination among FS cells.

The cortical actin cytoskeleton of lactotropes as an intracellular target for the control of prolactin secretion.

We investigated the role of cortical actin filaments (F-actin) in the regulation of PRL secretion in cultured normal anterior pituitary cells. F-actin dynamics were evaluated by fluorescence microscopy, and PRL secretion from attached cells was measured by the reverse hemolytic plaque assay. F-actin localized to the periphery of lactotropes. PRL-releasing factors such as TRH, vasoactive intestinal peptide (VIP), and forskolin, or removal of the PRL-inhibiting factor dopamine (DA) from cultures chronically exposed to DA, caused fragmentation, i.e. focal disassembly of cortical F-actin. Basal, VIP-, and DA withdrawal-induced cortical F-actin disassembly were dependent on extracellular Ca2+ whereas TRH- and forskolin-induced disassembly were not. Short-term (5 min) treatment of cells with the F-actin-disrupting agent cytochalasin D (CD) enhanced basal PRL secretion but did not further stimulate TRH- or VIP-induced PRL secretion. The results support the existence of a causal link between F-actin disassembly and increased PRL secretion. On the other hand, exposure of cultures to DA decreased the percentage of cells showing cortical F-actin disassembly within minutes. Longer treatments (2-4 h) caused stabilization of cortical actin filaments as revealed by the protection vis-a-vis the depolymerizing effect of CD. The protective effect was specific for lactotropes and was evident with DA concentrations as low as 50 nM. Chronic exposure of the cells to DA blocked CD- and TRH-evoked actin disassembly and PRL secretion while VIP-induced effects were partially inhibited. Stabilization of F-actin with the marine sponge venom, jasplakinolide, also decreased basal and stimulated PRL secretion. In conclusion, our results suggest that, first, the cortical actin cytoskeleton of lactotropes is an integrator of the multiple factors regulating PRL secretion directly on the lactotrope, and second, the tonic inhibition of PRL secretion is mediated, at least in part, by DA-induced stabilization of cortical F-actin.

Intracellular mechanisms involved in dopamine-induced actin cytoskeleton organization and maintenance of a round phenotype in cultured rat lactotrope cells.

The participation of the actin cytoskeleton in the control of PRL secretion by dopamine (DA) is not yet fully understood. Recently, we demonstrated that DA induces cortical actin assembly and stabilization in anterior pituitary PRL-secreting cells (lactotropes) that can be linked to DA-induced inhibition of PRL secretion. Here we show that DA prevents cell flattening and the formation of cytoplasmic actin cables in cultured rat lactotropes. The effects of DA were reversible, mediated by D2 receptors, exclusive to lactotropes, and independent of other anterior pituitary cells present in the cultures. Because cAMP and Ca2+ mediate DA-induced inhibition of PRL secretion and synthesis, we investigated whether morphological responses to DA were dependent on these second messengers. Either inhibition of protein kinase A activity with the specific inhibitor KT5720 or blockade of Ca2+ channels with nifedipine inhibited cell flattening and induced cytoplasmic actin filament breakdown. Nifedipine was as effective as DA, but KT5720 was less effective than DA. Increased intracellular cAMP levels provoked cell flattening, which was blocked by nifedipine and KT5720, but not by DA. The results suggest that Ca2+-dependent pathways control cell shape in most lactotropes; however, in a subpopulation of lactotropes, cAMP-dependent pathways may also contribute to DA morphological responses. Next, we studied the participation of the Rho family of guanosine triphosphatases, which is known to regulate the dynamics of actin filaments. Inactivation of Rho by C3 exoenzyme induced cytoplasmic actin cable disassembly and lactotrope rounding up. No additive effects were observed among Rho-, cAMP-, and Ca2+-dependent pathways. However, C3-induced morphological responses were blocked by increased cAMP levels, suggesting that Rho-dependent steps are upstream cAMP-dependent steps. DA-induced actin cytoskeleton reorganization in lactotropes may involve modifications in the expression and localization of actin-binding proteins. DA increased expression of the actin anchoring proteins talin and alpha-actinin, but not of vinculin. DA enhanced association of talin to cell membranes. Increased talin-membrane interaction may be implicated in DA-induced maintenance of a round phenotype in lactotrope cells.

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.

Dynamic changes in chromaffin cell cytoskeleton as prelude to exocytosis.

Earlier work by us as well as others has demonstrated that filamentous actin is mainly localized in the cortical surface of chromaffin cell. This F-actin network acts as a barrier to the chromaffin granules, impeding their contact with the plasma membrane. Chromaffin granules contain alpha-actinin, an anchorage protein that mediates F-actin association with these vesicles. Consequently, chromaffin granules crosslink and stabilize F-actin networks. Stimulation of chromaffin cell produces disassembly of F-actin and removal of the barrier. This interpretation is based on: (1) Cytochemical experiments with rhodamine-labeled phalloidin indicated that in resting chromaffin cells, the F-actin network is visualized as a strong cortical fluorescent ring; (2) Nicotinic receptor stimulation produced fragmentation of this fluorescent ring, leaving chromaffin cell cortical areas devoid of fluorescence; and (3) These changes are accompanied by a decrease in F-actin, a concomitant increase in G-actin, and a decrease in the F-actin associated with the chromaffin cell cytoskeleton (DNAse I assay). We also have demonstrated the presence in chromaffin cells of gelsolin and scinderin, two Ca(2+)-dependent actin filament-severing proteins, and suggested that chromaffin cell stimulation activates scinderin with the consequent disruption of F-actin networks. Scinderin, a protein recently isolated in our laboratory, is restricted to secretory cells and is present mainly in the cortical chromaffin cell cytoplasm. Scinderin, which is structurally different from gelsolin (different pIs, amino acid composition, peptide maps, and so on), decreases the viscosity of actin gels as a result of its F-actin-severing properties, as demonstrated by electron microscopy. Stimulation of chromaffin cells either by nicotine (10 microM) or high K+ (56 mM) produces a redistribution of subplasmalemmal scinderin and actin disassembly, which preceded exocytosis. The redistribution of scinderin and exocytosis is Ca(2+)-dependent and is not mediated by muscarinic receptors. Furthermore, our cytochemical experiments demonstrate that chromaffin cell stimulation produces a concomitant and similar redistribution of scinderin (fluorescein-labeled antibody) and F-actin (rhodamine phalloidin fluorescence), suggesting a functional interaction between these two proteins. Stimulation-induced redistribution of scinderin and F-actin disassembly would produce subplasmalemmal areas of decreased cytoplasmic viscosity and increased mobility for chromaffin granules. Exocytosis sites, evaluated by antidopamine-beta-hydroxylase (anti-D beta H) surface staining, are preferentially localized in plasma membrane areas devoid of F-actin.

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.

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.

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.

Comparison of vesicular volume and quantal size in bovine chromaffin cells.

Electrochemical measurements of vesicular content released were compared with the morphometric measurements of vesicular size in bovine chromaffin cells. Cross-sectional vesicular diameters were determined from electron micrographs. Two methods were used to determine the frequency histograms of "true" vesicular diameters (i.e. diameters of the vesicles in the equatorial plane): (i) "peeling off" method [Coupland R. E. (1968), Nature 217, 384-388], and (ii) summation of individual probabilities of "true" vesicular diameters. Quantal size was estimated from the area under the spontaneous current spike detected electrochemically. The frequency histograms of "true" vesicular diameters are found to be skewed (thus not well described by a Gaussian function) irrespective of the method used to calculate them, as are the frequency histograms of the cube roots of the quantal sizes. Furthermore, we also find that the frequency histograms of electrochemical measurements (the cube roots of quantal sizes) have lower skews and coefficients of variation than those of morphometric measurements ("true" vesicular diameters), with discrepancy being especially pronounced for noradrenaline-secreting cells. Such a difference in both coefficients of variation and skews suggests that the intravesicular catecholamine concentration is not uniform, but that it is lower for vesicles of larger size. In conclusion a variety of factors--vesicular volume, vesicular surface area to volume ratio, binding capacity of chromogranin and/or ATP, likely determines the amount of catecholamine stored in the vesicle.

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.

Filipin vs enzymatic localization of cholesterol in guinea pig, mink, and mallard duck testicular cells.

To test the validity of filipin cytochemistry for localization of cholesterol in testicular cells, we compared the results obtained by this technique with those obtained by a two-step enzymatic method involving cholesterol esterase and cholesterol oxidase. In all the animals models tested (guinea pig, mink, and mallard duck) the disappearance of subsurface filaments along Sertoli cell junctional membranes was accompanied by a significant increase in the number of filipin-cholesterol complexes/microns 2 in these membranes. Enzyme histochemistry allowed localization of free cholesterol in the limiting membrane of multivesicular bodies, in membranes within lysosomes, in mitochondrial membranes, and in junctional membranes, with or without subsurface filaments. The method also permitted selective visualization of cholesterol esters in lipid droplets. We conclude that filipin mapping of cholesterol induces false-negative cytochemical results. The enzymatic method is superior to filipin because it allows localization of free cholesterol in junctional membranes and of cholesterol esters in lipid droplets. This compartmentalization of the compounds may represent the basis of a system that helps to maintain constant free cholesterol levels in the testis.

Human platelets contain scinderin, a Ca(2+)-dependent actin filament-severing protein.

A large body of biochemical and morphological evidence suggests that actin polymerizes in response to various stimuli which activate platelets. Previous work has shown the presence in platelets of gelsolin, a Ca(2+)-dependent regulator of actin filament length. This present work demonstrates that human platelets contain scinderin, another Ca(2+)-dependent actin filament-severing protein recently discovered in our laboratory. Extracts prepared from platelets were subjected to DNase-I-Sepharose 4B affinity chromatography. EGTA eluates from the affinity columns contained scinderin as demonstrated by mono and two-dimensional polyacrylamide gel electrophoresis and immunoblotting with scinderin antibodies. The concentration of scinderin in platelets was 75 fmol/mg total protein. This might represent 11% of the total actin filament-severing activity if both proteins are equally potent, on a molar basis, in severing actin filaments. Double staining immunocytochemical studies with antibodies against scinderin and rhodamine phalloidin, a probe for F-actin, also demonstrated the presence of scinderin in platelets. These findings suggest that scinderin may participate in the regulation of platelet actin networks.

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)

Scinderin and chromaffin cell actin network dynamics during neurotransmitter release.

It has been demonstrated that filamentous actin (F-A) is mainly localized in the cortical surface of the chromaffin cell. This F-A network acts as a barrier to the chromaffin granules impeding their contact with the plasma membrane. Stimulation of chromaffin cells with either nicotine or a depolarizing concentration of K+ induces the disassembly of cortical F-A in focal areas underneath the plasma membrane. Sites of exocytosis are localised to these areas with low concentration of F-A. The cortical surface of the chromaffin cell also contains scinderin, a Ca(2+)-dependent actin filament-severing protein recently isolated in our laboratory. Nicotine and high K+ stimulation also induce redistribution of cortical scinderin. Both nicotine and high K(+)-induced scinderin redistribution and F-A disassembly are Ca(2+)-dependent events which seem to precede neurotransmitter secretion. A possible target for protein kinase C in the modulation of secretion is the cortical F-A network. Treatment of chromaffin cells with phorbol esters prior to secretion induced scinderin redistribution, F-A disassembly and enhanced the initial rate of subsequent nicotine-evoked catecholamine release. The present results strongly indicate the involvement of the cortical cytoskeleton in the regulation of neurotransmitter release.