Release In Permeabilized Cells Research Articles

Loss of INPP5K attenuates IP3-induced Ca2+ responses in the glioblastoma cell line U-251 MG cells

INPP5K (inositol polyphosphate 5-phosphatase K) is an endoplasmic reticulum (ER)-resident enzyme that acts as a phosphoinositide (PI) 5-phosphatase, capable of dephosphorylating various PIs including PI 4,5-bisphosphate (PI(4,5)P2), a key phosphoinositide found in the plasma membrane. Given its ER localization and substrate specificity, INPP5K may play a role in ER-plasma membrane contact sites. Furthermore, PI(4,5)P2 serves as a substrate for phospholipase C, an enzyme activated downstream of extracellular agonists acting on Gq-coupled receptors or tyrosine-kinase receptors, leading to IP3 production and subsequent release of Ca2+ from the ER, the primary intracellular Ca2+ storage organelle. In this study, we investigated the impact of INPP5K on ER Ca2+ dynamics using a previously established INPP5K-knockdown U-251 MG glioblastoma cell model. We here describe that loss of INPP5K impairs agonist-induced, IP3 receptor (IP3R)-mediated Ca2+ mobilization in intact cells, while the ER Ca2+ content and store-operated Ca2+ influx remain unaffected. To further elucidate the underlying mechanisms, we examined Ca2+ release in permeabilized cells stimulated with exogenous IP3. Interestingly, the absence of INPP5K also disrupted IP3-induced Ca2+ release events. These results suggest that INPP5K may directly influence IP3R activity through mechanisms yet to be resolved. The findings from this study point towards role of INPP5K in modulating ER calcium dynamics, particularly in relation to IP3-mediated signaling pathways. However, further work is needed to establish the general nature of our findings and to unravel the exact molecular mechanisms underlying the interplay between INNP5K function and Ca2+ signaling.

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release from the endoplasmic reticulum by AMP-activated kinase modulators

The 5' AMP-activated protein kinase (AMPK) is a nutrient-sensitive kinase that plays a key role in the control of cellular energy metabolism. We have explored here the relationship between AMPK and Ca2+ signaling by looking at the effect of an AMPK activator (A769662) and an AMPK inhibitor (dorsomorphin) on histamine-induced Ca2+-release from the endoplasmic reticulum (ER) in HeLa cells. Our data show that incubation with A769662 (EC50 = 29 μM) inhibited histamine-induced Ca2+-release from the ER in intact cells, as well as inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release in permeabilized cells. On the contrary, dorsomorphin (EC50 = 0.4 μM) activated both histamine and IP3-induced Ca2+-release and reversed the effect of A769662. These results suggest a direct effect of AMPK regulation on IP3 receptor (IP3R) function. A phosphoproteomic study did not reveal changes in IP3R phosphorylation, but showed significant changes in phosphorylation of proteins placed upstream in the IP3R interactome and in several proteins related with Ca2+ metabolism, which could be candidates to mediate the effects observed. In conclusion, our data suggest that AMPK negatively regulates IP3R. This effect constitutes a novel and very important link between Ca2+ signaling and the AMPK pathway.

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca(2+) signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP(3)R), an intracellular Ca(2+) channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP(3)R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2(-/-) mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca(2+) release in intact cells and IP(3)-induced Ca(2+) release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca(2+) signal by a local Ca(2+)-induced Ca(2+)-release mechanism, which only occurred in the presence of the TRPP2-IP(3)R interaction, and not via altered IP(3)R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca(2+) microdomains necessary for initiating Ca(2+)-induced Ca(2+) release. The data strongly suggest that defects in this mechanism may account for the altered Ca(2+) signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.

Targeting Bcl-2-IP3 Receptor Interaction to Reverse Bcl-2's Inhibition of Apoptotic Calcium Signals

The antiapoptotic protein Bcl-2 inhibits Ca2+ release from the endoplasmic reticulum (ER). One proposed mechanism involves an interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel localized with Bcl-2 on the ER. Here we document Bcl-2-IP3R interaction within cells by FRET and identify a Bcl-2 interacting region in the regulatory and coupling domain of the IP3R. A peptide based on this IP3R sequence displaced Bcl-2 from the IP3R and reversed Bcl-2-mediated inhibition of IP3R channel activity in vitro, IP3-induced ER Ca2+ release in permeabilized cells, and cell-permeable IP3 ester-induced Ca2+ elevation in intact cells. This peptide also reversed Bcl-2's inhibition of T cell receptor-induced Ca2+ elevation and apoptosis. Thus, the interaction of Bcl-2 with IP3Rs contributes to the regulation of proapoptotic Ca2+ signals by Bcl-2, suggesting the Bcl-2-IP3R interaction as a potential therapeutic target in diseases associated with Bcl-2's inhibition of cell death.

Modulation of Histamine-induced Ca2+ Release by Protein Kinase C: EFFECTS ON CYTOSOLIC AND MITOCHONDRIAL [Ca2+] PEAKS

In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.

Inhibition of type I and III IP3Rs by TGF-β is associated with impaired calcium release in mesangial cells

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) mediate cytosolic free calcium concentration ([Ca(2+)](c)) signals in response to a variety of agonists that stimulate mesangial cell contraction and proliferation. In the present study, we demonstrate that mesangial cells express both type I and III IP(3)Rs and that these receptors occupy different cellular locations. Chronic treatment with transforming growth factor-beta1 (TGF-beta1; 10 ng/ml, 24 h) leads to downregulation of both type I and III IP(3)Rs as measured by immunoblot and confocal analysis. TGF-beta1 treatment does not affect IP(3) levels, and downregulation of type I IP(3)R is not due to enhanced degradation of the protein, as the half-life of type I IP(3)R is unchanged in the presence or absence of TGF-beta1. Functional effects of TGF-beta1-induced downregulation of the IP(3)Rs were evaluated by measuring [Ca(2+)](c) changes in response to epidermal growth factor (EGF) in intact cells and sensitivity of [Ca(2+)](c) release to IP(3) in permeabilized cells. TGF-beta1 pretreatment led to a significant decrease of [Ca(2+)](c) release induced by EGF in intact cells and by submaximal IP(3) (400 nm) in permeabilized cells. Total IP(3)-sensitive [Ca(2+)](c) stores were not changed, as assessed by stimulation with maximal doses of IP(3) (10.5 microm) and thapsigargin-mediated calcium release in permeabilized cells. We conclude that prolonged exposure to TGF-beta1 leads to downregulation of both type I and III IP(3)Rs in mesangial cells and this is associated with impaired sensitivity to IP(3).

Inositol 1,4,5-Trisphosphate Receptor Down-regulation Is Activated Directly by Inositol 1,4,5-Trisphosphate Binding: STUDIES WITH BINDING-DEFECTIVE MUTANT RECEPTORS

Activation of certain phosphoinositidase C-linked cell surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate (InsP3) receptors and, thus, lead to InsP3 receptor down-regulation. To gain insight into this process, we examined whether or not InsP3 receptor degradation is a direct consequence of InsP3 binding by analyzing the down-regulation of exogenous wild-type and binding-defective mutant InsP3 receptors expressed in SH-SY5Y human neuroblastoma cells. Stimulation of these cells with carbachol showed that wild-type exogenous receptors could be down-regulated but that the binding-defective mutant exogenous receptors were not. Thus, InsP3 binding appears to mediate down-regulation. To validate this conclusion, a comprehensive analysis of the effects of the exogenous receptors was undertaken. This showed that exogenous receptors (i) are localized appropriately within the cell, (ii) enhance InsP3-induced Ca2+ release in permeabilized cells, presumably by increasing the number of InsP3-sensitive Ca2+ channels, (iii) have minimal effects on Ca2+ mobilization and InsP3 formation in intact cells, (iv) form heteromers with endogenous receptors, and (v) do not alter the down-regulation of endogenous receptors. In total, these data show that the introduction of exogenous receptors into SH-SY5Y cells does not compromise intracellular signaling or the down-regulatory process. We can thus conclude that InsP3 binding directly activates InsP3 receptor degradation. Because InsP3 binding induces a conformational change in the InsP3 receptor, these data suggest that this change provides the signal for accelerated proteolysis.

Discrimination between plasma membrane and intracellular target sites of sphingosylphosphorylcholine

On the background of the emerging concept of G protein-coupled sphingolipid receptors, Ca 2+ mobilization by sphingosylphosphorylcholine (SPPC) in intact cells and SPPC-induced Ca 2+ release in permeabilized cells, both occurring at similar, micromolar concentrations, were characterized and compared. In intact human embryonic kidney (HEK-293) cells, SPPC rapidly increased [Ca 2+] i by mobilization of Ca 2+ from thapsigargin-sensitive stores. In saponin-permeabilized HEK-293 cells, SPPC released stored Ca 2+, in a manner similar to but independent of inositol 1,4,5-trisphosphate. Only the action of SPPC on intact cells, but not that in permeabilized cells, was, at least in part, sensitive to pertussis toxin. In addition and most important, Ca 2+ release by SPPC in permeabilized cells was not stereoselective, whereas in intact cells only the naturally occurring d- erythro-SPPC, but not l- threo-SPPC, increased [Ca 2+] i. Stereoselectivity of SPPC-induced [Ca 2+] i increase was also demonstrated in bovine aortic endothelial cells. In conclusion, Ca 2+ mobilization by SPPC in intact cells is independent of the previously described SPPC-gated Ca 2+ channel on endoplasmic reticulum but probably mediated by a membrane sphingolipid receptor. Thus, SPPC can regulate Ca 2+ homeostasis by acting apparently at two cellular targets, which exhibit clearly distinct recognition patterns.

Differences in intracellular CA2+ stores of submandibular cells of adult and newborn rats.

The intracellular Ca2+ stores of submandibular acinar cells of adult and newborn rats were compared by measuring changes in cytosolic Ca2+ ([Ca2+]i) with the Ca2+-sensitive fluorescent probe, fura-2, in intact cells and 45Ca2+ release in permeabilized cells. In cells of adult rats, acetylcholine (ACh) and thapsigargin (TG) elicited, respectively, a 105% and 125% net increase in [Ca2+]i in Ca2+-free medium. In cells of newborn animals, the ACh-induced [Ca2+]i increase (218%) was larger; but the TG-induced increase was significantly smaller (77%), suggesting that the TG-sensitive Ca2+ pump of the store is less developed, abundant, or responsive in these cells. After ACh or TG, ionomycin elicited a further Ca2+ release. Ionomycin by itself completely discharged the ACh- and TG-sensitive store in cells of mature glands but not in cells of immature glands, suggesting a further difference in the distribution of the agonist and ionomycin sensitivities in the two types of cells. After exposure to ACh + ionomycin or TG + ionomycin, collapsing the intracellular pH gradient with monensin caused a third and large Ca2+ release (262% and 289%, respectively) in cells of adult rats, but a significantly smaller release (96%, P < 0.001, and 57%, P < 0.001, respectively) in cells of newborn rats, suggesting that a third, IP3-insensitive acidic store is significantly smaller in cells of immature glands. In permeabilized and 45Ca2+-loaded cells, inositol-1,4,5-trisphosphate (IP3, 5 microM) induced an identical 45Ca2+ release (34% and 33%, respectively) in both types of cells, but TG induced a significant smaller 45Ca2+ release in cells of immature glands (15%) than in cells of mature glands (33%). Monensin discharged <10% of loaded 45Ca2+ in both types of cells, indicating that the radiotracer was not loaded into the monensin-sensitive store. These results suggest that in cells of immature glands: (1) the TG-sensitive Ca2+ pump of the IP3-sensitive store is not well developed; (ii) the IP3-sensitive store is not completely discharged by ionomycin; and (iii) an acidic store likely to be associated with secretory granules is also underdeveloped and of a smaller size than in cells of mature glands.

Functional dissociation between glucocorticoid-induced decrease in arachidonic acid release and inhibition of adrenocorticotropic hormone secretion in AtT-20 corticotrophs.

The biochemical basis of the short-term inhibitory effects of glucocorticoids on corticotropin release from pituitary corticotrophs is still obscure. A well-characterized effect of glucocorticoids in several cell types is the inhibition of arachidonic acid (AA) generation by phospholipase A2 (PLA2). Arachidonic acid and its metabolites have been implicated in the secretory process from a number of pituitary cells, such as the corticotrophs. We have thus examined the role of AA in the anti-secretagogue effects of glucocorticoids in a corticotropin-secreting clonal corticotroph line (AtT-20 D16/16). Glucocorticoids decreased AA release induced by melittin, a bee venom protein related to extracellular PLA2. When a possible role of AA in corticotropin release was studied, the following results were obtained: (a) all corticotropin secretagogues tested, including corticotropin-releasing factor (CRF), did not alter AA generation; (b) calcium and guanine nucleotides, which stimulate corticotropin release in permeabilized cells, inhibited the release of AA under the same conditions; (c) administration of melittin or of exogenous AA had no effect on basal and CRF-stimulated corticotropin release; (d) administration of large amounts of exogenous AA was unable to restore the ability to secrete corticotropin under suppression by glucocorticoids. Altogether, the data suggest that whereas glucocorticoids can inhibit both AA generation and corticotropin release, these two effects appear to be causally unrelated.

Regulation of cADP-ribose-induced Ca2+ Release by Mg2+ and Inorganic Phosphate

cADP-ribose (cADPr) has recently been shown to release Ca2+ from an intracellular store of permeabilized T lymphocyte cell lines (Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Using permeabilized Jurkat and HPB. ALL T lymphocytes, the effects of varying concentrations of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release were investigated. cADPr-induced Ca2+ release was dependent on the concentration of inorganic phosphate, showing very low Ca2+ release activity between 0.5 and 2 mM inorganic phosphate. At 4 to 5 mM inorganic phosphate, the cADPr-induced Ca2+ release was much more pronounced, reaching maximal values at 10 mM inorganic phosphate. The underlying mechanism for this stimulatory effect was an increased loading of the cADPr-sensitive Ca2+ store, which was demonstrated by enhanced resequestration of Ca2+ selectively into the cADPr-sensitive Ca2+ store. The free Mg2+ concentration also influenced cADPr-induced Ca2+ release in permeabilized cells: at 0 and 8.58 mM the release was nearly completely abolished, whereas at 1.06 mM maximal Ca2+ release by cADPr was observed. High performance liquid chromatographic analysis of exogenously added cADPr revealed that the catabolism of cADPr at varying Mg2+ and Pi concentrations had only minor relevance for the modulatory effects observed. To correlate the effects of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release observed in the permeabilized cell preparations, measurements of these ions in intact Jurkat T lymphocytes were carried out. Intact Jurkat T cells stimulated via the T cell receptor middle dotCD3 complex did not respond with significant elevation of the free intracellular Mg2+ concentration. In contrast, stimulation via the T cell receptor middle dotCD3 complex resulted in an increase in the intracellular inorganic phosphate concentration. These data indicate a role for the intracellular inorganic phosphate concentration in the regulation of cADPr-mediated Ca2+ release in T lymphocytes.

Gonadotropin-releasing hormone-induced sensitization of calcium-dependent exocytosis in pituitary gonadotrophs.

Agonist-induced increases in cytoplasmic calcium concentration ([Ca2+]i) play a pivotal role in regulated exocytosis by promoting the fusion of secretory vesicles with the plasma membrane. In permeabilized and ATP-primed pituitary cells, increases in ambient [Ca2+]i stimulated the release of LH from gonadotrophs with an EC50 of 2-3 microM. In contrast, the responses of intact gonadotrophs to agonist stimulation by GnRH were characterized by transient [Ca2+]i elevations of up to 1.5 microM, followed by a plateau of 300-400 nM. The sensitivity of the exocytotic response of permeabilized cells to [Ca2+]i was significantly increased by GnRH, which reduced the EC50 for [Ca2+]i to the submicromolar concentration range. The stimulatory action of GnRH on LH release in permeabilized cells was not a consequence of intracellular Ca2+ release, but was associated with increased cytidine diphosphate diacylglycerol production. Activation of protein kinase C by phorbol esters caused a similar increase in the Ca2+ sensitivity of LH release from permeabilized cells, and this effect was not additive to that of GnRH. Furthermore, the GnRH-induced increase in the sensitivity of the exocytotic response to Ca2+ was attenuated by inhibitors of protein kinase C. These findings indicate that although elevated [Ca2+]i per se can promote LH release from permeabilized gonadotrophs, concomitant activation of protein kinase C is necessary to support exocytosis at the physiological [Ca2+]i levels that prevail in GnRH-stimulated intact cells. Such sensitization of the Ca(2+)-dependent secretory mechanism by protein kinase C may be an important step in the agonist-induced release of LH from pituitary gonadotrophs.

Gonadotropin-releasing hormone-induced sensitization of calcium- dependent exocytosis in pituitary gonadotrophs

Agonist-induced increases in cytoplasmic calcium concentration ([Ca2+]i) play a pivotal role in regulated exocytosis by promoting the fusion of secretory vesicles with the plasma membrane. In permeabilized and ATP-primed pituitary cells, increases in ambient [Ca2+]i stimulated the release of LH from gonadotrophs with an EC50 of 2-3 microM. In contrast, the responses of intact gonadotrophs to agonist stimulation by GnRH were characterized by transient [Ca2+]i elevations of up to 1.5 microM, followed by a plateau of 300-400 nM. The sensitivity of the exocytotic response of permeabilized cells to [Ca2+]i was significantly increased by GnRH, which reduced the EC50 for [Ca2+]i to the submicromolar concentration range. The stimulatory action of GnRH on LH release in permeabilized cells was not a consequence of intracellular Ca2+ release, but was associated with increased cytidine diphosphate diacylglycerol production. Activation of protein kinase C by phorbol esters caused a similar increase in the Ca2+ sensitivity of LH release from permeabilized cells, and this effect was not additive to that of GnRH. Furthermore, the GnRH-induced increase in the sensitivity of the exocytotic response to Ca2+ was attenuated by inhibitors of protein kinase C. These findings indicate that although elevated [Ca2+]i per se can promote LH release from permeabilized gonadotrophs, concomitant activation of protein kinase C is necessary to support exocytosis at the physiological [Ca2+]i levels that prevail in GnRH-stimulated intact cells. Such sensitization of the Ca(2+)-dependent secretory mechanism by protein kinase C may be an important step in the agonist-induced release of LH from pituitary gonadotrophs.

Calcium ion-dependent signalling and mitochondrial dysfunction: mitochondrial calcium uptake during hormonal stimulation in intact liver cells and its implication for the mitochondrial permeability transition

Hormones that elevate cytosolic Ca 2+ concentrations ([Ca 2+] cyt) often use Ca 2+ as a messenger to activate intramitochondrial metabolic processes. However, the mitochondrial Ca 2+ level also regulates the activation of the mitochondrial permeability transition (MPT), a process that involves the assembly of a high conductance proteinaceous pore across the inner and outer membrane. Studies on intact liver cells indicate that the MPT is a critical step in the cell killing induced by anoxia or respiratory inhibitors. In this study, we used freshly isolated hepatocytes to investigate to what extent the elevation of [Ca 2+] cyt by vasopressin or other agonists causes Ca 2+ accumulation in the mitochondria and how this treatment affects the mitochondrial susceptibility to undergo the MPT. Hepatocytes were incubated with vasopressin, glucagon, or with thapsigargin (an inhibitor of the endoplasmic reticulum Ca 2+ pump) prior to permeabilization with digitonin. Mitochondrial Ca 2+ accumulation was determined by following the ionomycin-induced Ca 2+ release in permeabilized cells and mitochondrial swelling was studied by following cyclosporin A-sensitive light scattering changes induced by phenylarsenoxide and rotenone. The results indicate that agents that elevate [Ca 2+] cyt cause a significant Ca 2+ accumulation in the mitochondria. Excessive Ca 2+ accumulation (> 10-fold increase over basal levels) was obtained with the combination of vasopressin and glucagon or with incubations containing thapsigargin. These conditions were also associated with a marked increase in rotenone-induced mitochondrial swelling. However, the more modest increase in mitochondrial Ca 2+ content after treating cells with vasopressin alone did not enhance the swelling response; instead, vasopressin suppressed mitochondrial swelling compared to control incubations. Vasopressin also partly suppressed the swelling associated with thapsigargin treatment, although it did not significantly affect the Ca 2+ accumulation under these conditions. This effect of vasopressin was mimicked by phorbol ester, suggesting a role for protein kinase C. The data indicate that mitochondrial Ca 2+ accumulation following elevation of [Ca 2+] cyt enhances the susceptibility for activation of the MPT, a response that may increase cell injury during anoxia or in response to other challenges. However, hormones also activate protective responses in the cell that suppress the MPT.

A pertussis-toxin-sensitive protein controls exocytosis in chromaffin cells at a step distal to the generation of second messengers

The role of GTP-binding proteins (G-proteins) in the secretory process in chromaffin cells was investigated by studying the effects of pertussis toxin (PTX) on catecholamine release and generation of various second messengers. PTX was found to stimulate the catecholamine secretion induced by nicotine, 59 mM-K+ or veratridine. PTX also potentiated Ca2(+)-evoked catecholamine release from permeabilized chromaffin cells, suggesting that PTX substrate(s) regulate the exocytotic machinery at a step distal to the rise in intracellular Ca2+. We have investigated the possible intracellular pathways involved in the stimulation of secretion by PTX. PTX did not modify the translocation of protein kinase C (PKC) to membranes in intact or permeabilized cells; in addition, neither inhibitors nor activators of PKC had any effect on catecholamine release induced by PTX. Thus it seems unlikely that the effect of PTX on secretion is mediated by activation of PKC. The effect of PTX is also cyclic AMP-independent, as PTX did not change cytoplasmic cyclic AMP levels. The relationship between PTX treatment and arachidonic acid release was also examined. We found that an increase in cytoplasmic arachidonic acid concentration enhanced Ca2(+)-evoked catecholamine release in permeabilized cells, but arachidonic acid did not mimic the effect of PTX on the Ca2(+)-dose-response curve for secretion. Furthermore, PTX did not significantly modify the release of arachidonic acid measured in resting or stimulated chromaffin cells, suggesting that the stimulatory effect of PTX on secretion is not mediated by an activation of phospholipase A2. Taken together, these results suggest that PTX may modulate the intracellular machinery of secretion at a step distal to the generation of second messengers. In alpha-toxin-permeabilized cells, full retention of the PTX-induced activation of secretion was observed even 30 min after permeabilization. In contrast, when chromaffin cells were permeabilized with streptolysin-O (SLO), there was a marked progressive loss of the PTX effect. We found that SLO caused the rapid leakage of three G-protein alpha-subunits which are specifically ADP-ribosylated by PTX. We propose that a PTX-sensitive G-protein may play an inhibitory role in the final stages of the Ca2(+)-evoked secretory process in chromaffin cells.

Guanine nucleotides inhibit agonist-stimulated arachidonic acid release in both intact and saponin-permeabilized human platelets

The effects of guanine nucleotides on arachidonic acid (AA) release were studied in intact and saponin-permeabilized human platelets. While GTP[S] itself caused a stimulation of AA release in permeabilized cells, GTP[S], GDP[S], GTP, ATP and other nucleotides inhibited AA release in response to thrombin and other agonists in intact, as well as permeabilized platelets. Inhibition of agonist-stimulated AA release by nucleotides was partially attenuated by addition of ADP, and was abolished by prior stimulation of platelets to discharge the ADP-containing dense granules. These results suggest: (i) that released ADP plays an important contributory role in agonist-stimulated platelet AA release, and (ii) that guanine nucleotides can modulate platelet activation through an extracellular action which is distinct from their effects on G-proteins.

Relationship between secretagogue-induced Ca2+ release and inositol polyphosphate production in permeabilized pancreatic acinar cells.

We have previously shown that inositol trisphosphate (IP3) releases Ca2+ from a nonmitochondrial pool of permeabilized rat pancreatic acinar cells (Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1984) Nature 306, 67-69). This pool was later identified as endoplasmic reticulum (Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F., and Schulz, I. (1984) J. Membr. Biol. 81, 241-253). As IP3 is produced by hydrolysis of phosphatidylinositol bisphosphate on activation of many "Ca2+-mobilizing receptors," our observation supported the proposal that IP3 functions as a second messenger to release Ca2+ from the endoplasmic reticulum. We have here used the same preparation of permeabilized acinar cells to study the relationship of secretagogue-induced Ca2+ release and IP3 production. We show that: 1) secretagogue-induced Ca2+ release in permeabilized cells is accompanied by a parallel production of inositol trisphosphate. 2) When the secretagogue-induced increase in intracellular free Ca2+ concentration was abolished by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering, secretagogue-induced IP3 production was unimpaired. 3) When secretagogue-induced IP3 production was reduced by inhibiting phospholipase C with neomycin, secretagogue-induced Ca2+ release was also abolished. 4) When the IP3 breakdown was reduced either by lowering the free Mg2+ concentration of the incubation medium or by adding 2.3-diphosphoglyceric acid, the rise in IP3 and the release of Ca2+ induced by secretagogues were both increased. These results further support the role of IP3 as a second messenger to induce Ca2+ mobilization.