Exocytotic membrane fusion as studied in toxin-permeabilized cells.
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3,4-Dihydroxyphenylacetic Acid↗
Biomedical subjects
M Gratzl
Publications and source records attributed to M Gratzl.
Calculation and control of free divalent cations in solutions used for membrane fusion studies.
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Animals↗
Long-term survival of autologous adrenal medulla grafts in the great omentum of the rat.
The omentum, a rich source for trophic and angiogenic factors, was explored as a potential intermediate transplant site to facilitate long-term survival of chromaffin tissue. Autologous rat adrenal medullas were grafted into omental pockets. All grafts became densely vascularized. The grafted chromaffin tissue exhibited strong immunoreactivities for tyrosine hydroxylase, synaptophysin and chromogranin A throughout the observation period of 16 weeks. The expression of these markers implies that grafted chromaffin cells retained the key enzyme for catecholamine biosynthesis and the organelles required for catecholamine secretion. Moreover, intermediate transplant of chromaffin tissue to the omentum could provide a favourable conditioning microenvironment thus augmenting the potential for survival of functional chromaffin tissue.
Adrenal Medulla↗
Glucocorticoids rapidly inhibit oxytocin-stimulated adrenocorticotropin release from rat anterior pituitary cells, without modifying intracellular calcium transients.
Glucocorticoid hormones suppress the secretion of ACTH evoked by secretagogues such as CRF and arginine vasopressin. In this study, we investigated the effects of glucocorticoids on ACTH release induced by oxytocin (OT) and on intracellular free calcium ion levels in corticotropes prepared from the adenohypophyses of female Wistar rats. Pulsatile additions of physiological concentration of OT (10 nM) to superfused anterior pituitary cells caused pulsatile ACTH release about 4-fold above basal secretion with similar peak amounts of ACTH during subsequent OT pulses. Exposure of the cells to corticosterone (100 nM) or to a selective glucocorticoid receptor agonist RU 28362 (100 nM) for 30 min suppressed OT-stimulated but not basal ACTH release by approximately 60%. Inhibition gradually disappeared during subsequent pulses of OT in the absence of corticosterone. Pretreatment with the selective antagonist RU 38486 (1 microM) completely blocked the inhibitory effect of corticosterone on OT-induced ACTH secretion. Changes in free cytosolic calcium levels in single cultured pituitary cells were measured using the calcium indicator Fura-2. OT caused calcium transients in corticotropes, which were identified by immunocytochemistry. They responded in a similar manner to a second OT stimulus when preincubated for 30 min with corticosterone (1 microM) or with RU 28362 (1 microM). Our data indicate that glucocorticoids, via glucocorticoid receptors, rapidly inhibit OT-stimulated ACTH secretion by corticotropes without affecting intracellular calcium transients due to OT. Therefore, we conclude that rapid inhibition of ACTH release by glucocorticoids interferes with cellular signal transduction beyond the step of calcium mobilization.
Adrenocorticotropic Hormone↗
Neural cell adhesion molecules in rat endocrine tissues and tumor cells: distribution and molecular analysis.
The adhesive properties of neural cell adhesion molecules (NCAMs) can be modified by alternative splicing of the primary transcript or posttranslational modifications. In the present study, we describe distinct forms of alternative splicing and posttranslational modification of the extracellular domain of NCAM of various endocrine tissues and derived tumor cells of the rat. Using an antiserum detecting the immunoglobulin-like domains of NCAM as well as a monoclonal antibody recognizing the NCAM-specific polysialic acid (PSA), we observed a similar staining pattern in adrenals, pituitary, and neoplastic endocrine cells. In endocrine tumor cells [pheochromocytoma (PC12), insulinoma (RINA2), and pituitary tumor cells (GH3)], NCAM immunoreactivity was most intense at contact sites between the cells. The immunocytochemical data were substantiated by results of in situ hybridization histochemistry. Specifically, higher levels of NCAM mRNA were detected in the adrenal cortex than in the medulla. In the pituitary, NCAM mRNA was more abundant in the anterior and intermediate lobes than in the neural lobe. The sequence of NCAM mRNAs in endocrine cells was analyzed by polymerase chain reaction and S1 nuclease protection assays. We found that major exons 4-13 of the NCAM mRNA in endocrine tissues and related tumor cell lines were homologous to those in the brain. However, PC12, RINA2, and GH3 tumor cells; normal rat pituitaries; and adrenals contained different amounts of NCAM mRNA with an alternative extra exon, termed VASE (also called pi in mouse) between constitutive exons 7 and 8. In addition, in pituitaries, we detected an alternative exon in splice site a between the constitutive exons 12 and 13, termed a15, with or without an AAG triplett. These sites are thought to be important for the adhesive properties of NCAM. Therefore, these results suggest that modifications of NCAM may be important for adhesive interactions in normal and neoplastic endocrine cells.
Adrenal Glands↗
Concerted action of human chorionic gonadotropin and norepinephrine on intracellular-free calcium in human granulosa-lutein cells: evidence for the presence of a functional alpha-adrenergic receptor.
Luteal cells are known to possess receptors for LH/hCG and receptors of the beta-adrenergic type. Interactions of specific agonists with either receptor lead to the activation of adenylate cyclase and subsequently to an increase of cAMP. Since in the human there is also evidence for the presence of alpha-adrenergic receptors, we have investigated whether activation of these receptors is linked to calcium as a second messenger and performed measurement of intracellular free calcium (Ca2+) with Fura-2 in single human granulosa-lutein cells. Addition of either hCG (100, 1,000, 25,000 IU/L) or norepinephrine (NE; known to interact with both alpha- and beta-adrenergic receptors), beta-adrenergic receptor agonist isoproterenol (ISO), or alpha-adrenergic receptor agonist phenylephrine (PHE; all at 10 and 100 mumol/L) did not increase free intracellular Ca2+. However, the addition of combinations of NE/hCG, PHE/hCG, but not the combination ISO/hCG, induced a transient increase in cytosolic free Ca2+. The NE/hCG-evoked calcium signal was not abolished in the presence of the beta-adrenergic receptor antagonist propranolol and was not affected by removal of extracellular Ca2+. Furthermore, we tested whether catecholamines affected the release of progesterone in the presence or absence of hCG. As expected, hCG (10,000 IU/L) stimulated progesterone release by cultured granulosa-lutein cells. When these cells were incubated with NE, PHE, or ISO (at 10 mumol/L), production of progesterone by these cells was not affected. However, the combinations of NE and PHE with hCG abolished the hCG-induced progesterone accumulation, but ISO coincubated with hCG did not. Taken together, our results indicate: 1) the presence of functional alpha-adrenergic receptors on human granulosa-lutein cells; 2) simultaneous activation of two different receptors (for hCG and alpha-agonists) are able to evoke intracellular Ca2+ elevation, implicating postreceptor interactions in human granulosa lutein cells; 3) this process occurs even in the absence of extracellular Ca2+, indicating the involvement of intracellular Ca2+ stores, most likely due to activation of phosphoinositide pathway; 4) catecholamines most likely acting via alpha-adrenergic receptors, inhibit the LH/hCG-induced release of progesterone.
Calcium↗
Effect of oxytocin on free intracellular Ca2+ levels and progesterone release by human granulosa-lutein cells.
Oxytocin and its receptor are found in the corpus luteum in a variety of species, including the human. In the present study we used fura-2 microfluorimetry to investigate whether activation of the oxytocin receptor of cultured human granulosa-lutein cells causes intracellular calcium (Ca2+) signals and affects progesterone release. Although after 1 day in culture, cells were not responsive to oxytocin, the number of responsive cells increased steadily during the first 3 days in culture, reaching a maximum on days 4 and 5 (59-66%) and then declined again until day 8. Effective oxytocin concentrations were apparently independent of the culture day, and concentrations as low as 10 nmol/L increased intracellular free Ca2+ levels from 70-140 nmol/L (basal levels) to maximal peak levels of 800 nmol/L. The oxytocin-induced Ca2+ signal was not affected by removal of extracellular Ca2+ with EGTA. Moreover, depletion of intracellular Ca2+ stores by ionomycin treatment rendered the cells unresponsive to oxytocin, pointing also at the intracellular source of the oxytocin-inducible Ca2+ signal. Interestingly, after one single stimulation with oxytocin, cells became refractory to additional stimuli, and only extremely high concentrations of oxytocin induced a second increase in intracellular free Ca2+. To examine the possible effects of oxytocin on progesterone release by cultured cells, we incubated cells on culture day 2 (20% responsive cells in the fura measurements) and culture day 5 (66% responsive cells in the fura measurements) for 24 h with oxytocin (10 nmol/L) and hCG (10,000 IU/L). Although hCG significantly stimulated progesterone release, oxytocin alone was without a stimulatory effect on either day. However, a significant augmentation of the effect of hCG on progesterone release was found in incubations of cells on day 5. Interestingly, the effects of hCG also included stimulation of oxytocin release by cultured granulosa-lutein cells into the culture medium, as determined by RIA. In summary, our data indicate the presence of a functional oxytocin receptor on human granulosa-lutein cells that is linked to Ca2+ as a second messenger released from intracellular Ca2+ stores. The number of oxytocin-responsive cells increases during differentiation in culture. Moreover, oxytocin release induced by hCG and a stimulatory effect of oxytocin on the hCG-induced progesterone production during the period of maximal responsiveness of cultured cells were found. We, therefore, propose that oxytocin may have autocrine and/or paracrine functions in human granulosa-lutein cells, including fine-tuning of progesterone release.
Calcium↗
Noradrenaline release from permeabilized synaptosomes is inhibited by the light chain of tetanus toxin.
Noradrenaline release from rat brain cortical synaptosomes permeabilized with streptolysin O can be triggered by microM concentrations of free Ca2+. This process was inhibited within minutes by tetanus toxin and its isolated light chain, but not by its heavy chain. The data demonstrate that the effect of tetanus toxin on NA release from purified synaptosomes is caused by the intraterminal action of its light chain.
Animals↗
Basic fibroblast growth factor (bFGF) in rodent testis. Presence of bFGF mRNA and of a 30 kDa bFGF protein in pachytene spermatocytes.
We have previously described a 30 kDa basic fibroblast growth factor (bFGF)-like protein in rodent testicular homogenates and have shown that pachytene spermatocytes are the sites of predominant immunoreactivity for this bFGF-like protein (Mayerhofer, A., Russell, L.D., Grothe, C., Rudolf, M. and Gratzl, M. (1991) Endocrinology 129, 921-924). We have now addressed the question whether this 30 kDa bFGF-like protein is a large bFGF form and whether it is produced by pachytene spermatocytes. We detected bFGF mRNA in homogenates of isolated mouse spermatocytes (which consisted mainly of pachytene spermatocytes) using S1 nuclease protection assays. As shown by Western blot analyses, the bFGF mRNA in mouse spermatocytes is translated into bFGF of an approximate molecular weight of 30 kDa. Neither bFGF mRNA, nor bFGF itself, was observed in isolated mouse Leydig cells. These results indicate that the immunoreactive bFGF-like protein observed previously in germ cells of the murine testis is identical to bFGF. Thus, germ cells of the testis produce bFGF, which may exert regulatory function in the process of spermatogenesis.
Animals↗
Amylase release from streptolysin O-permeabilized pancreatic acinar cells. Effects of Ca2+, guanosine 5'-[gamma-thio]triphosphate, cyclic AMP, tetanus toxin and botulinum A toxin.
The molecular requirements for amylase release and the intracellular effects of botulinum A toxin and tetanus toxin on amylase release were investigated using rat pancreatic acinar cells permeabilized with streptolysin O. Micromolar concentrations of free Ca2+ evoked amylase release from these cells. Maximal release was observed in the presence of 30 microM free Ca2+. Ca(2+)-stimulated, but not basal, amylase release was enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) (3-4 fold) or cyclic AMP (1.5-2 fold). Neither the two-chain forms of botulinum A toxin and tetanus toxin, under reducing conditions, nor the light chains of tetanus toxin, inhibited amylase release triggered by Ca2+, or combinations of Ca2+ + GTP[S] or Ca2+ + cAMP. The lack of inhibition was not due to inactivation of botulinum A toxin or tetanus toxin by pancreatic acinar cell proteolytic enzymes, as toxins previously incubated with permeabilized pancreatic acinar cells inhibited Ca(2+)-stimulated [3H]noradrenaline release from streptolysin O-permeabilized adrenal chromaffin cells. These data imply that clostridial neurotoxins inhibit a Ca(2+)-dependent mechanism which promotes exocytosis in neural and endocrine cells, but not in exocrine cells.
Amylases↗
Cultured microvascular endothelial cells derived from the bovine corpus luteum possess NCAM-140.
Previously, five phenotypically different, stable types of microvascular endothelial cells (MVE) were isolated from the bovine corpus and cultured successfully. We found that three out of these five types of MVE express the neural cell adhesion molecule (NCAM). As shown by immunocytochemistry, weak NCAM immunoreactivity occurred mainly in the perinuclear area of cell type 1. Monolayers of types 2 and 5 revealed heavy NCAM immunoreactivity, which was localized predominantly at the lateral cell surface outlining the contact zones of adjacent cells. In contrast, cell types 3 and 4 were not NCAM immunoreactive. Western blot analyses substantiated these results: While cell type 1 showed a weak immunoreactive band, cell types 2 and 5 displayed strong NCAM-immunoreactive bands of a molecular weight of approximately 140 kDa (NCAM-140), which was absent in cell types 3 and 4. These results reveal for the first time that NCAM can be expressed by cultured MVE and may serve in mediating endothelial cell contacts. Since luteal cells also express NCAM-140, this adhesion molecule could in addition be involved in the interactions of luteal cells with MVE.
Animals↗
The light chain of tetanus toxin inhibits calcium-dependent vasopressin release from permeabilized nerve endings.
The effects of tetanus toxin and its light and heavy chain subunits on vasopressin release were investigated in digitonin-permeabilized neurosecretory nerve terminals isolated from the neural lobe of the rat pituitary gland. Exocytosis was induced by challenging the permeabilized nerve endings with micromolar calcium concentrations. Tetanus toxin inhibited vasopressin release only in the presence of the reducing agent dithiothreitol. This effect was irreversible. The purified light chain of tetanus toxin strongly inhibited exocytosis in a dose-dependent manner with half-maximal effect at c. 10 nM. The action of the light chain was observed after only 2.5 min of preincubation. Separated heavy chain subunit had no effect on hormone secretion. Inhibition of vasopressin release could be prevented by preincubating the light chain of tetanus toxin with an immune serum against tetanus toxin. The data clearly demonstrate that in mammalian neurosecretory nerve endings tetanus toxin acts at a step downstream from the activation by Ca2+ of the exocytotic machinery and that the functional domain of this toxin is confined to its light chain.
Animals↗
Leydig cells express neural cell adhesion molecules in vivo and in vitro.
The neural cell adhesion molecule (NCAM) polypeptides are expressed by numerous tissues during embryonic development, where they are involved in cell-cell interactions. In the adult, NCAM expression is confined to a few cell types, including neurons and peptide-hormone-producing cells. Here we demonstrate that the Leydig cells of the adult rat, mouse, and hamster testes express NCAM as well. Western blotting showed that an NCAM of approximately 120 kDa was present in the adult testes of all three species investigated. This form was also found in freshly isolated mouse Leydig cells and in Leydig cells after 2 days in culture. After 4 days in culture, mouse Leydig cells expressed additional NCAM isoforms of approximately 140 and 180 kDa, indicating changes in alternative splicing of NCAM primary transcripts. Also, NCAM mRNA of all isoforms, as detected by S1-nuclease protection assays, increased with time in culture. The expression of the cell adhesion molecule NCAM by adult Leydig cells may explain the aggregation of Leydig cells in clusters in rodent testes, which could be a prerequisite for functional coordination of groups of Leydig cells. Furthermore, the presence of this neural and endocrine marker may indicate a closer relationship between Leydig cells and neural and peptide-hormone-producing cells than is considered to exist at the present time.
Animals↗
Chromogranin A in neurons of the rat cerebellum and spinal cord: quantification and sites of expression.
Chromogranin A (CGA) is an abundant protein of dense-cored secretory vesicles in endocrine and neuronal cells. The present study, for the first time, compares CGA of neurons of the central nervous system with the CGA of adrenal origin. By S1 nucleus protection assay, we found that the 3' part of the CGA mRNA between exons 5-8 of the cerebellum and the spinal cord of the rat is homologous to that of the adrenal. In situ hybridization histochemistry revealed that CGA mRNA in the cerebellar cortex is present in cell bodies of Purkinje cells and in neurons of the deep cerebellar nuclei. The perikarya of these cells also exhibit CGA-like immunoreactivity. CGA mRNA and CGA-like immunoreactivity are also present in the motoneurons of the ventral, lateral, and dorsal horns of the rat spinal cord. The amounts of CGA, as determined by radioimmunoassay in cerebellum and spinal cord, were about one tenth of the amounts detected in the adrenal, adenohypophysis, or the olfactory bulb. The sites of CGA expression suggest that CGA may be involved in signal transduction in the motor system.
Animals↗
Oxytocin at physiological concentrations evokes adrenocorticotropin (ACTH) release from corticotrophs by increasing intracellular free calcium mobilized mainly from intracellular stores. Oxytocin displays synergistic or additive effects on ACTH-releasing factor or arginine vasopressin-induced ACTH secretion, respectively.
The potency of oxytocin (OT) in evoking ACTH secretion by isolated, superfused rat adenohypophyseal corticotrophs and its enhancement by CRF and arginine vasopressin (AVP) were analyzed. Each secretagogue effectively released ACTH from adenohypophyseal cells when added separately in pulsatile fashion in physiological concentrations based on hypophyseal portal blood (OT, 10 nM; AVP, 0.5 nM; CRF, 0.1 nM). OT released ACTH at concentrations as low as 1 nM. Moreover, a dose-response relationship up to 10 microM was revealed. Combinations of a constant amount of CRF (0.1 nM) with increasing concentrations of OT exerted a synergistic effect on ACTH release. In contrast, OT given in various concentrations in combination with AVP (0.5 nM) produced an additive effect on ACTH release. To study the mechanism of action of OT on ACTH secretion, cytosolic free calcium levels in single pituitary cells exposed to OT or AVP were measured using the calcium-sensitive fluorescent indicator Fura-2. Corticotrophs among mixed adenohypophyseal cell types in the primary cultures were identified by immunocytochemistry. More than 500 cells were individually stimulated with OT or AVP. Basal cytosolic free calcium levels ranged between 80-130 nM free calcium. The addition of 100 nM OT or 1 microM AVP increased the cytosolic free calcium concentration within 3 sec to values ranging from 500-800 nM. An increase in intracellular calcium ranging from 200-500 nM due to OT could still be observed after extracellular calcium depletion. Taken together, our data demonstrate that physiological concentrations of OT stimulate ACTH secretion, independent of the other ACTH secretagogues, by mobilizing calcium mainly from intracellular stores.
Adrenocorticotropic Hormone↗
Carbachol increases intracellular free calcium concentrations in human granulosa-lutein cells.
We investigated whether the stimulation of human granulosa-lutein cells with muscarinic and nicotinic receptor agonists can cause increases in intracellular free calcium (Ca2+), using Fura-2 microfluorimetry. The addition of carbachol (a non-selective muscarinic and nicotinic receptor agonist) to cultured human granulosa-lutein cells increased intracellular free Ca2+ levels. Concentrations as low as 10 nmol/l were effective. In contrast, nicotine did not evoke elevations of intracellular free Ca2+. Basal Ca2+ levels ranged around 70-140 nmol/l and maximal, carbachol-induced peaks reached 1.1 mumol/l. The carbachol-induced Ca2+ signal was abolished after preincubation of the cells with the muscarinic receptor antagonists quinuclidinyl benzilate or atropine, but it was not affected by removal of extracellular Ca2+. Further evidence for the involvement of intracellular Ca2+ stores is provided by experiments in the absence of extracellular Ca2+. While thapsigargin (a blocker of ATP-driven Ca2+ uptake by intracellular stores) and ionomycin (an ionophore by which Ca2+ is released from intracellular stores) evoked small Ca2+ transients, cells pretreated with these agents did not respond to carbachol any more. These data suggest the presence of a functional muscarinic receptor on human granulosa-lutein cells and imply the involvement of intracellular Ca2+ stores during the cellular response. These results also suggest the participation of the nervous system, acting through muscarinic receptors, in the control of the function of human granulosa-lutein cells.
Atropine↗
Decavanadate displaces inositol 1,4,5-trisphosphate (IP3) from its receptor and inhibits IP3 induced Ca2+ release in permeabilized pancreatic acinar cells.
Inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release in digitonin permeabilized rat pancreatic acinar cells is specifically inhibited by decavanadate. The Ca2+ release induced with 0.18 microM IP3 is half maximally inhibited with approximately 5 microM decavanadate. Complete inhibition is achieved with around 20 microM decavanadate. Removal of decavanadate from the permeabilized cells fully restores sensitivity towards IP3, indicating the reversibility of the inhibition. Oligovanadate, which inhibits ATP dependent Ca2+ uptake into intracellular stores, does not influence IP3 induced Ca2+ release. In order to reveal the mechanism underlying the effects of the different vanadate species, binding of IP3 to the same cellular preparations was investigated. We found that binding of IP3 to a high affinity receptor site (Kd approx. 1.2 nM) could be abolished by decavanadate but not by oligovanadate. With 0.5 microM decavanadate, IP3 binding was half maximally inhibited. A similar potency of decavanadate was also found with adrenal cortex microsomes which bind IP3 with the same affinity (Kd approx. 1.4 nM) as permeabilized pancreatic acinar cells. Labelled IP3 was displaced from these subcellular membranes with similar kinetics by unlabelled IP3 and decavanadate. The data suggest that the inhibitory action of decavanadate on IP3 induced Ca2+ release is a consequence of its effect on binding of IP3 to its receptor.
Adrenal Cortex↗
Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells.
The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 microM IP3 was half maximally inhibited at 0.5 microM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 microM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTP gamma S could not replace GTP. Desensitization still occurred when GTP gamma S was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release.
Animals↗