Regulatory Role of the GTP-Binding Protein, Go, in the Mechanism of Exocytosis in Adrenal Chromaffin Cells

To elucidate the possible involvement of GTP-binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and antibodies against the G proteins (Gi and G(o)) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine-evoked catecholamine release. High K(+)-, histamine-, or gamma-aminobutyric acid-evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10(-4) M acetylcholine was decreased significantly in PTX-treated cells. In digitonin-permeabilized cells, PTX pretreatment resulted in a decrease of the half-maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP-gamma-S (a nonhydrolyzable GTP analogue) inhibited Ca(2+)-dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the alpha subunit of the PTX-sensitive G protein, G(o). Treatment with similar antibodies against the alpha subunit of Gi had no effect. These findings suggest that G(o) directly controls the Ca(2+)-triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.     

ATP-dependent and ATP-independent pathways of exocytosis revealed by interchanging glutamate and chloride as the major anion in permeabilized mast cells.

Most investigations of the mechanism of regulated exocytosis have involved the use of secretory cells permeabilized in glutamate-based electrolyte solutions. In our previous work we have used NaCl-based electrolyte solutions. For secretion to occur from rat mast cells under these latter conditions, a dual effector system comprising Ca2+ and a guanine nucleotide are required; together they are sufficient. Here we compare the secretion from mast cells permeabilized in solutions of different electrolytes. Replacement of Na+ by K+ had little effect. Replacement of Cl- by Br-, SO4-, gluconate, isethionate, acetate, tartrate, succinate, etc. affected the maximal extent of secretion elicited by the dual effectors Ca2+ and guanosine-5'-O-(3-thiotriphosphate) (Ca2(+)-plus-GTP-gamma-S) but had little influence on the effective affinity for Ca2+. The dicarboxylic amino acids (L- and D-glutamate, and L-aspartate) permitted exocytosis to be elicited by Ca2+ or GTP-gamma-S alone. Secretion stimulated by GTP-gamma-S is strongly inhibited by Cl- (50% inhibition by 20 mM Cl-), whereas the extent of Ca2(+)-induced secretion is proportional to the concentration of glutamate in mixed electrolyte buffers. Unlike dual-effector stimulation, secretion due to the single effectors requires adenosine triphosphate (ATP) and is prevented by inhibitors of protein kinase C. These results point to the existence of two parallel pathways for control of exocytosis in permeabilized cells, one ATP dependent, the other ATP independent.     

Evidence for protein dephosphorylation as a permissive step in GTP-gamma-S-induced exocytosis from permeabilized mast cells.

Mast cells permeabilized by streptolysin O secrete histamine and lysosomal enzymes in response to provision of a dual effector system comprising Ca2+ and a guanine nucleotide (e.g., GTP-gamma-S2) at concentrations in the micromolar range. These are both necessary and together they are sufficient. There is no requirement for adenosine triphosphate (ATP) and hence no obligatory phosphorylation reaction in the terminal stages of the exocytotic pathway. When exocytosis is induced by Ca2(+)-plus-GTP-gamma-S (i.e., no ATP) added at times after permeabilization (the permeabilization interval), cellular responsiveness declines so that there is no response to provision of the two effectors (both at 10(-5)M) if they are initially withheld and then added after 5 min. Here we show that this decline in responsiveness is characterized by a time-dependent reduction in the effective affinity for Ca2+. Affinity for Ca2+ and hence secretory competence can then be restored if ATP is added alongside the stimulus. Unlike cells stimulated to secrete at the time of permeabilization, exocytosis from cells that have undergone the cycle of permeabilization-induced refractoriness followed by ATP-induced restoration can be triggered by Ca2+ alone: after such conditioning there is no requirement for guanine nucleotide. In contrast, dependence on guanine nucleotide remains mandatory in cells that have been pretreated (i.e., before permeabilization) with okadaic acid (understood to be an inhibitor of protein phosphatases 1 and 2A) or phorbol myristate acetate (an activator of protein kinase C). These results indicate that obligatory dependence on guanine nucleotide is retained when the cells are treated under conditions conducive to maintained phosphorylation. It is concluded that the exocytotic mechanism of permeabilized mast cells is enabled by a dephosphorylation reaction and that the effector of the guanosine triphosphate (GTP)-binding protein (G epsilon) that mediates exocytosis is likely to be a protein phosphate.     

Changes in the state of actin during the exocytotic reaction of permeabilized rat mast cells

The major part of mast cell actin is Triton-soluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, through filamentous actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be approximately 2.5 micrograms/10(6) cells (cytosolic concentration approximately 110 microM. After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, approximately 60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP-gamma-S together with Ca2+. In the presence of cytochalasin E the requirement for Ca2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by Ca2(+)-dependent actin regulatory proteins; their presence is indicated by a Ca2(+)-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca2+ and cause variable extents of Ca2(+)-independent secretion. Exposing the permeabilized cells to increasing concentrations of Ca2+ caused a progressive decrease in F-actin levels as measured by flow cytometry of RP-stained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP-gamma-S was not required for the Ca2(+)-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur.     

Mechanisms of granule enzyme secretion from permeabilized guinea pig eosinophils. Dependence on Ca2+ and guanine nucleotides

The mechanisms of granule protein secretion have been studied in streptolysin-O-permeabilized guinea pig eosinophils. Secretion of the granule-associated enzyme N-acetyl-beta-D-glucosaminidase was dependent on both Ca2+ and a nonhydrolyzable GTP analogue, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting roles for both calcium and GTP binding proteins. Secretion was maximal by 7 min, and varied between 35 and 60% of the total enzyme activity. Other GTP analogues also elicited secretion, with rank order GTP-gamma-S greater than guanylyl-imidophosphate greater than guanylyl (beta-gamma-methylene-diphosphate). Unrelated nucleotide triphosphates showed little or no effect confirming the specificity of the G protein. Transmission electronmicroscopy confirmed that permeabilization alone did not result in loss of granules and that exocytosis was dependent on the addition of the effectors, Ca2+ and GTP-gamma-S. ATP enhanced the magnitude of the secretory response and also enhanced the effective affinities for both Ca2+ and GTP-gamma-S. In the presence of 10(-5) M GTP-gamma-S the ED50 (Ca2+) was pCa 5.57 +/- 0.04 (2.69 microM) in the absence of ATP and declined to pCa 6.16 +/- 0.03 (0.69 microM) in the presence of ATP (p less than 0.0001). Furthermore, ATP served to restore responsiveness in cells that had been rendered refractory by delaying stimulation after permeabilization. Pretreatment with PMA (an activator of PKC) inhibited the induction of a refractory state, whereas inhibition of PKC partially countered the ability of ATP to restore responsiveness, both observations pointing to a requirement for a specific component of the secretory mechanism to be in a phosphorylated state in order to condone the secretion process. These observations show that secretory mechanisms in eosinophils are similar to those in other myeloid cells, in particular neutrophils and mast cells, although the time course of secretion is more protracted.     

Loss and Ca2+-Dependent Retention of Scinderin in Digitonin-Permeabilized Chromaffin Cells: Correlation with Ca2+-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.     

Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilized rat mast cells

Rat mast cells, pretreated with metabolic inhibitors and permeabilized by streptolysin-O, secrete histamine when provided with Ca2+ (buffered in the micromolar range) and nucleoside triphosphates. We have surveyed the ability of various exogenous nucleotides to support or inhibit secretion. The preferred rank order in support of secretion is ITP greater than XTP greater than GTP much greater than ATP. Pyrimidine nucleotides (UTP and CTP) are without effect. Nucleoside diphosphates included alongside Ca2+ plus ITP inhibit secretion in the order 2'-deoxyGDP greater than GDP greater than o-GDP greater than ADP approximately equal to 2'deoxyADP approximately equal to IDP. Secretion from the metabolically inhibited and permeabilized cells can also be induced by stable analogues of GTP (GTP-gamma-S greater than GppNHp greater than GppCH2p) which synergize with Ca2+ to trigger secretion in the absence of phosphorylating nucleotides. ATP enhances the effective affinity for Ca2+ and GTP analogues in the exocytotic process but does not alter the maximum extent of secretion. The results suggest that the presence of Ca2+ combined with activation of events controlled by a GTP regulatory protein provide a sufficient stimulus to exocytotic secretion from mast cells.     

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.     

A role for soluble NSF attachment proteins (SNAPs) in regulated exocytosis in adrenal chromaffin cells.

Digitonin-permeabilized chromaffin cells secrete catecholamines by exocytosis in response to micromolar Ca2+ concentrations, but lose the ability to secrete in response to Ca2+ as the cells lose soluble proteins through the plasma membrane pores. Such secretory run-down can be retarded by cytosolic fractions, thus providing an assay for proteins potentially involved in the exocytotic process. We have used this assay to investigate the role of N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs) in regulated exocytosis. Recombinant alpha- and gamma-SNAP stimulated Ca(2+)-dependent exocytosis, although recombinant NSF was ineffective, despite the fact that NSF and alpha-SNAP leak from the permeabilized cells with similar time courses. However, around one third of cellular NSF was found to be present in a non-cytosolic form and so it is possible that this is sufficient for exocytosis and that exogenous SNAPs stimulate the exocytotic mechanism by acting on the leakage-insensitive NSF. The stimulatory effect of alpha-SNAP displayed a biphasic dose-response curve and was maximal at 20 micrograms/ml. The effect of alpha-SNAP was Ca(2+)- and MgATP-dependent and was inhibited by N-ethylmaleimide and botulinum A neurotoxin, indicating a bona fide action on the exocytotic mechanism. Furthermore, Ca2+ concentrations which trigger catecholamine secretion acted to prevent the leakage of NSF and alpha-SNAP from permeabilized cells. These findings provide functional evidence for a role of SNAPs in regulated exocytosis in chromaffin cells.     

Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol

The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.     

Mechanisms of luteinizing-hormone exocytosis in Staphylococcus aureus-alpha-toxin-permeabilized sheep gonadotropes.

We have used primary gonadotropes permeabilized with the pore-forming protein Staphylococcus aureus alpha-toxin to investigate luteinizing hormone (lutropin, LH) exocytosis. The diameter of the alpha-toxin pores (2-3 nm) allows the exchange of small molecules, whereas larger cytosolic proteins are retained. Because of the slow exchange of small molecules through the pores, we have developed a protocol which combines prolonged pre-equilibration of the permeabilized cells at 0 degrees C before stimulation with strong Ca2+ buffering. Under these conditions, increasing the free Ca2+ concentration from 0.1 microM to 10 microM [EC50 (concentration effecting half-maximal response) 2-3 microM] resulted in a 15-20-fold increase in LH exocytosis. LH exocytosis was maximal in the first 3 min and completed by 12 min. When permeabilized cells were equilibrated for prolonged periods in the absence of MgATP, Ca2(+)-stimulated LH secretion gradually declined (greater than 90% decrease by 60 min). Addition of MgATP (5 mM) rapidly restored full Ca2(+)-stimulated LH secretion. MgATP supported Ca2(+)-stimulated LH secretion at a half-maximal concentration of 1.5 mM. UTP and adenosine 5'-[gamma-thio]triphosphate were 40 and 31% as effective as MgATP, whereas other nucleotide triphosphates were ineffective. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 50 nM) stimulated LH exocytosis at free Ca2+ concentrations as low as 1 nM and was additive with Ca2+ at higher free Ca2+ concentrations. PMA-stimulated exocytosis required MgATP at concentrations similar to those required for Ca2(+)-stimulated LH exocytosis. These results demonstrate that LH exocytosis can be triggered both by micromolar Ca2+ concentrations or, in the virtual absence of Ca2+, by PKC activation. Both mechanisms of stimulated exocytosis have an absolute requirement for millimolar ATP. Because they retain cytosolic proteins, alpha-toxin-permeabilized cells may have advantages over alternative permeabilization methods provided that conditions are used that compensate for slow diffusion through alpha-toxin pores.     

Norepinephrine and GTP-gamma-S increase myofilament Ca2+ sensitivity in alpha-toxin permeabilized arterial smooth muscle

A new method for preparing permeabilized smooth muscle fibers from rabbit mesenteric artery has been developed using alpha-toxin, a transmembrane pore-making exo-protein produced by Staphylococcus aureus. After alpha-toxin treatment the fibers developed tension as a function of Ca2+ concentration (EC50 = 890 nM). But they could not contract without added ATP, indicating ATP is permeable. When the sarcoplasmic reticulum was loaded with 5 X 10(-7) M Ca2+ solution, NE induced a transient contraction in 2 mM EGTA 0 M Ca2+ solution and a transient and maintained contraction in 5 X 10(-7) M Ca2+ solution. GTP-gamma-S, a non-hydrolyzable analogue of GTP, substituted for NE in producing these contractile effects. The analysis of the relationship between Ca2+ and maintained tension revealed that NE and GTP-gamma-S cause increases in Ca2+ sensitivity of myofilament shifting the EC50 to 280 nM and 160 nM, respectively. We conclude that NE or GTP-gamma-S causes an increase in myofilament Ca2+ sensitivity and that G protein may be involved in receptor signal transduction system. alpha-Toxin is a useful tool to permeabilize the smooth muscle tissue to ions and small molecules without any damage of receptor and signal transduction system.     

Differential up-regulation of specific and azurophilic granule membrane markers in electropermeabilized neutrophils.

We have developed an alternative method to study the degranulation in electropermeabilized human neutrophils by measuring the up-regulation of the specific membrane markers CD63 (residing in the azurophilic granules of resting neutrophils) and CD67 (present in specific granules). The expression of these marker proteins was measured by the binding of specific antibodies to paraformaldehyde-fixed cells and subsequent flow cytometry. We first investigated whether the changes in CD63 and CD67 expression after stimulation of intact cells were comparable with earlier measurements of neutrophil degranulation, in which the release of soluble marker proteins was measured. These experiments indicated that this new method compares favourably with earlier studies, both with respect to kinetics and stimulus dependency. Subsequently, we applied this method (which does not include centrifugation of the cells) to study degranulation in electropermeabilized neutrophils. In permeabilized neutrophils, a clear up-regulation of the specific granule marker CD67 was observed upon incubation with a free Ca2+ concentration of 1 microM, a value of the cytosolic free Ca2+ concentration occurring in formylmethionyl-leucyl-phenylalanine (FMLP)-activated neutrophils. The azurophilic granule marker CD63 required GTP-gamma-S besides 1 microM Ca2+ for a significant up-regulation. Hence, our study indicates a different requirement for intracellular signals of the two main types of granules in human neutrophils.     

Stoichiometry of contraction and Ca2+ mobilization by inositol 1,4,5-trisphosphate in isolated gastric smooth muscle cells

Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach and permeabilized by brief exposure to saponin. Both permeabilized and intact muscle cells contracted in response to cholecystokinin octapeptide (CCK-8) and acetylcholine, but only permeabilized muscle cells contracted in response to inositol 1,4,5-trisphosphate (InsP3). The contractile response to InsP3 was prompt (peak less than 5 s), concentration-dependent (EC50-0.3 microM), and insensitive to antimycin or oligomycin. Contraction induced by either InsP3 or CCK-8 was accompanied by a concentration-dependent increase in free Ca2+ that was directly correlated with the magnitude of contraction. Both InsP3 and CCK-8 caused rapid net efflux of Ca2+ from cells preloaded with 45Ca2+. Contraction, increase in free Ca2+ concentration, and net 45Ca2+ efflux elicited by a combination of maximal concentrations of InsP3 and CCK-8 were not significantly different from those elicited by maximal concentrations of either agent alone. Repeated stimulation of single muscle cells with either InsP3 or CCK-8 in Ca2+-free medium caused eventual loss of the contractile response to all agents. The response to all agents was restored upon re-exposure of the cell to a cytosol-like concentration of Ca2+, implying equal access of InsP3 and receptor-linked agonists to the same intracellular Ca2+ store. The results demonstrate that InsP3 mimics the effects of receptor-linked agonists on contraction and mobilization of intracellular Ca2+ in permeabilized smooth muscle cells that retain the functional properties of intact smooth muscle cells and support a role for InsP3 as membrane-derived messenger responsible for mobilization of intracellular Ca2+ in smooth muscle cells.     

Functional shift from muscarinic to nicotinic cholinergic receptors involved in inositol trisphosphate and cyclic GMP accumulation during the primary culture of adrenal chromaffin cells.

The incorporation of 32P from [gamma-32P]ATP into intracellular proteins was studied in electrically permeabilized rat islets of Langerhans. Ca2+ (10 microM), cyclic AMP (100 microM) and a protein kinase C-activating phorbol ester, phorbol 13-myristate 12-acetate (PMA; 100 nM) produced marked changes in the phosphorylation state of a number of proteins in permeabilized islets after incubation for 1 min at 37 degrees C. Ca2+ modified the effects of cyclic AMP and PMA on protein phosphorylation. Noradrenaline (10 microM) had no detectable effects on Ca2+-dependent protein phosphorylation, but significantly inhibited Ca2+-induced insulin secretion from electrically permeabilized islets. These results suggest that electrically permeabilized islets offer a useful model in which to study rapid events in protein phosphorylation as a mechanism of stimulus-secretion coupling. If the rapid Ca2+-induced effects on protein phosphorylation are involved in the control of insulin secretion, the results of this study also imply that part of the catecholamine inhibition of insulin secretion occurs at a stage in the secretory pathway beyond the activation of the regulated protein kinases.     

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC12) permeabilized with staphylococcal alpha-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC12 cells. Permeabilization with alpha-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC12 cells.     

Modulation of smooth muscle contractility by CHASM, a novel member of the smoothelin family of proteins

Cyclic nucleotides acting through their associated protein kinases, the cGMP- and cAMP-dependent protein kinases, can relax smooth muscles without a change in free intracellular calcium concentration ([Ca2+]i), a phenomenon referred to as Ca2+ desensitization. The molecular mechanisms by which these kinases bring about Ca2+ desensitization are unknown and an understanding of this phenomenon may lead to better therapies for treating diseases involving defects in the contractile response of smooth muscles such as hypertension, bronchospasm, sexual dysfunction, gastrointestinal disorders and glaucoma. Utilizing a combination of real-time proteomics and smooth muscle physiology, we characterized a distinct subset of protein targets for cGMP-dependent protein kinase in smooth muscle. Among those phosphoproteins identified was calponin homology-associated smooth muscle (CHASM), a novel protein that contains a calponin homology domain and shares sequence similarity with the smoothelin family of smooth muscle specific proteins. Recombinant CHASM was found to evoke relaxation in a concentration dependent manner when added to permeabilized smooth muscle. A co-sedimentation assay with actin demonstrated that CHASM does not possess actin binding activity. Our findings indicate that CHASM is a novel member of the smoothelin protein family that elicits Ca2+ desensitization in smooth muscle.     

Thiophosphorylation causes Ca2+-independent norepinephrine secretion from permeabilized PC12 cells

Adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was used to examine the role of phosphorylation in the regulation of norepinephrine secretion by digitonin-permeabilized PC12 cells. While most kinases will use ATP gamma S to thiophosphorylate proteins, thiophosphorylated proteins are relatively resistant to dethiophosphorylation by protein phosphatases. Norepinephrine secretion by permeabilized PC12 cells was ATP- and Ca2+-dependent but resistant to calmodulin antagonists. Half-maximum secretion was obtained in 0.75 microM Ca2+. Permeabilized PC12 cells were incubated with ATP gamma S in the absence of Ca2+, the ATP gamma S was removed, and norepinephrine secretion was determined. Preincubation with ATP gamma S increased the amount of norepinephrine secreted in the absence of Ca2+, but it had no effect on the amount released in the presence of Ca2+. After a 15-min preincubation in 1 mM ATP gamma S, there was almost as much secretion in the absence of Ca2+ as in its presence. Inclusion of ATP in the preincubation inhibited the effect of ATP gamma S. Ca2+ stimulated the rate of modification by ATP gamma S as brief preincubations with ATP gamma S in the presence of Ca2+ resulted in higher levels of Ca2+-independent secretion than did preincubations with ATP gamma S in the absence of Ca2+. Similarly, brief preincubations of permeabilized cells with ATP in the presence of Ca2+ resulted in elevated levels of Ca2+-independent secretion. Secretion of norepinephrine from ATP gamma S-treated cells was ATP-dependent. These results suggest that norepinephrine secretion by PC12 cells is regulated by a Ca2+-dependent phosphorylation. Once this phosphorylation has occurred, secretion is still ATP-dependent, but it no longer requires Ca2+.     

Effects of Ca2+ on phosphoinositide breakdown in exocrine pancreas.

Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.     

Rho guanine nucleotide dissociation inhibitor protein (RhoGDI) inhibits exocytosis in mast cells.

Introducing non-hydrolysable analogues of GTP into the cytosolic compartment of mast cells results in exocytotic secretion through the activation of GTP binding proteins. The identity and mechanism of action of these proteins are not established. We have investigated the effects of Rho GDP dissociation inhibitor (RhoGDI) on exocytosis induced by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) in rat mast cells, introducing the protein into cells by means of a patch pipette and recording the progress of exocytosis by monitoring cell capacitance. To allow time for the protein to enter the cells and find its correct location, stimulation was provided 5-10 min after patch rupture by photolysing caged GTP-gamma-S included in the pipette solution. When bovine RhoGDI was introduced into mast cells, exocytosis was inhibited at concentrations of 200-400 nM for native protein and 800 nM to 8 microM for the recombinant form. Protein denatured by heat or N-ethylmaleimide treatment did not inhibit. In permeabilized cells, recombinant RhoGDI increased the rate at which cells lose their ability to respond to GTP-gamma-S. These data demonstrate that one or more small GTP binding proteins of the Rho family has a central role in the exocytotic mechanism in mast cells.