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.     

Secretory responses of rat peritoneal mast cells to high intracellular calcium

The patch-clamp technique was used to investigate the secretory responses of rat peritoneal mast cells at various intracellular calcium concentrations ([Ca2+]i). When Calcium was introduced into the cell with pipette-loaded dibromo-BAPTA, elevation of [Ca2+]i into the range 1-10 microM induced membrane capacitance increases indicative of exocytosis in a concentration-dependent manner. At higher concentrations a decrease of the response was observed. Cells that were exposed to micromolar [Ca2+]i underwent morphological alterations resulting in swelling, which is indicative of cytoskeletal alterations. The presence of dibromo-BAPTA (4 mM) strongly inhibited secretion induced by GTP-gamma-S, thus hampering the contribution of G-protein-mediated stimulation. Application of the Ca2+ ionophore ionomycin resulted in transient increases in [Ca2+]i which were parallelled by Ca2+-dependent secretion. Effective buffering of the cytosolic calcium level below 1 microM abolished the secretory response. Our results show that an increase in [Ca2+]i can trigger secretion, but only if it is high and sustained. During physiological stimulation, however, secretion proceeds at [Ca2+]i below 1 microM. It is, therefore, concluded that mast cell degranulation under physiological conditions is not simply a result of an increase in [Ca2+]i, but that other second messenger systems in conjunction with calcium act synergistically in order to ensure fast and efficient secretion.     

Extracellular magnesium decreases the secretory response of rat peritoneal mast cells to compound 48/80 in vitro.

Exposure of rat peritoneal mast cells to magnesium in the absence of extracellular calcium resulted in a time- and dose-dependent decrease in the secretory response induced by compound 48/80. The decrease was prevented by a low extracellular concentration of calcium. Furthermore, the decreased secretory responsiveness was dose-dependently restored by the addition of calcium to the cells simultaneously with compound 48/80. Preincubation with magnesium also inhibited antigen-induced histamine secretion in a dose-dependent manner. This was reversed by the simultaneous addition of calcium and the secretory stimulus. A dose-dependent decrease in antigen induced histamine secretion that was reversed by calcium was also observed. Exposure of the mast cells to magnesium for 15 min resulted in a parallel decrease in histamine secretion and in the cellular content of 45Ca2+. These observations suggest that magnesium may decrease the secretory response by displacing the cellular calcium which is utilized in stimulus-secretion coupling.     

Loss of proteins from digitonin-permeabilized adrenal chromaffin cells essential for exocytosis

Cultured chromaffin cells can be permeabilized with digitonin; the cell interior is then accessible to the cytoplasm, and addition of calcium provokes release of catecholamines. Increasing the incubation time between the permeabilization step and calcium-induced stimulation resulted in a progressive inhibition of secretion reaching 60% after 20 min. Cytosoluble proteins which leak from detergent-permeabilized cells were collected, dialyzed, and concentrated. When these proteins were added back to permeabilized cells which were unable to secrete, catecholamine release was fully restored, suggesting that certain proteins necessary for exocytosis had been dialyzed from these cells. One of the released proteins was characterized as calmodulin. However, addition of calmodulin alone was ineffective in maintaining or restoring secretory activity in digitonin-permeabilized cells, excluding calmodulin as the sole factor responsible for the loss of release. Protein kinase C was also identified as one of the leaked proteins. This enzyme is known to be retained in cells in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). However, under TPA-dependent conditions, there was also a loss of secretory activity. The present paper shows that among the proteins leaked from digitonin-permeabilized cells, there are specific proteins crucial to the exocytotic mechanism.     

Peripheral actin filaments control calcium-mediated catecholamine release from streptolysin-O-permeabilized chromaffin cells

Adrenal medullary chromaffin cells were permeabilized by treatment with a streptococcal cytotoxin streptolysin O (SLO) which generates pores of macromolecular dimensions in the plasma membrane. SLO did not provoke spontaneous release of catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However, the addition of micromolar free calcium concentration induced the corelease of noradrenaline and chromogranin A, indicating that secretory products are liberated by exocytosis. Calcium-dependent exocytosis from SLO-permeabilized cells required Mg-ATP and could not occur in the presence of other nucleotides. The pores generated by the toxin were large enough to introduce proteins, e.g., immunoglobulins, but also caused efflux of the cytosolic marker lactate dehydrogenase. Despite this, the cells remained responsive to calcium for up to 30 min after permeabilization, indicating that they retained their secretory machinery. In the search for a functional role of cytoskeletal proteins in the secretory process, we used SLO-permeabilized cells to examine the localization of filamentous actin, using rhodamine-phalloidin, and that of the actin-severing protein, gelsolin, using specific antibodies. It was found that both F-actin and gelsolin were exclusively localized in the subplasmalemmal region of the cell. We examined the relationship between actin disassembly, the elevation of intracellular calcium and secretion in SLO-treated cells. F-Actin destabilizing agents such as cytochalasin D or DNase I were found to potentiate calcium-stimulated release. The maximal effect was observed at low calcium concentrations (1-4 microM) and at the later stages of the secretory response (after 10 min stimulation). In addition, using rhodamine-phalloidin, we observed that calcium provoked simultaneously both cortical actin disassembly and catecholamine release in SLO-permeabilized cells. These results demonstrate that a close relationship exists between the secretory response and actin disassembly and provide further evidence that intracellular calcium controls the subplasmalemmal cytoskeletal actin organization and thereby the access of secretory granules to exocytotic sites.     

Pancreatic acinar cell: New insights into the control of secretion

Pancreatic acinar cells secrete fluid and digestive enzymes. Both types of secretion are activated by a rise in intracellular calcium but how the stimulus-secretion cascade actually regulates secretory output is not well understood. It has long been known that the calcium response of acinar cells to physiological stimulation is complex. Dependent on the type and concentration of agonist, it consists of either local or global calcium increases as well as spreading waves of calcium across the cell. In the past it has been speculated that these different calcium signals drive different secretory responses. Now, recent employment of two-photon microscopy has enabled the simultaneous recording of both enzyme secretion and calcium signals and is beginning to resolve this issue. The data shows that local calcium responses exclusively drive fluid secretion. Where-as, global calcium responses drive both fluid and enzyme secretion. This differential control of secretory output is likely central to controlling the physiological responses of pancreatic acinar cells.     

Protein phosphorylation and the dependence on Ca2+ and GTP-gamma-S for exocytosis from permeabilised mast cells

Exocytosis in permeabilised mast cells requires only that the concentrations of Ca2+ and GTP-gamma-S (the essential effectors) are elevated into the micromolar range of concentrations. These act through an unidentified Ca2(+)-binding protein and an uncharacterized G-protein (GE). There is no requirement for ATP in the final stages of the secretory pathway. However, mast cells permeabilised in the absence of ATP rapidly become refractory to stimulation due to a reduction in the affinity for the essential effectors. Here, we show that responsiveness may be restored by the addition of ATP. The characteristics of such ATP-dependent secretion have been examined. Preincubation (prior to permeabilization) of the cells with phorbol ester enhances affinity to Ca2+, and introduction of neomycin reduces Ca2+ affinity. AMG.C16, an ether-linked analogue of diglyceride, inhibits secretion in a manner which can be partially reversed by elevating the concentration of ATP. These observations indicate that while protein phosphorylation does not comprise a step in the triggering of exocytosis, a primed condition most likely involving a state of protein phosphorylation, and maintained by reactions catalysed by protein kinase C, is essential.     

Degranulation of individual mast cells in response to Ca2+ and guanine nucleotides: an all-or-none event

Widespread experience indicates that application of suboptimal concentrations of stimulating ligands (secretagogues) to secretory cells elicits submaximal extents of secretion. Similarly, for permeabilized secretory cells, the extent of secretion is related to the concentration of applied intracellular effectors. We investigated the relationship between the extent of secretion from mast cells (assessed as the release of hexosaminidase) and the degranulation (exocytosis) responses of individual cells. For permeabilized mast cells stimulated by the effector combination Ca2+ plus GTP-gamma-S and for intact cells stimulated by the Ca2+ ionophore ionomycin, we found that exocytosis has the characteristics of an all-or-none process at the level of the individual cells. With a suboptimal stimulus, the population comprised only totally degranulated cells and fully replete cells. In contrast, a suboptimal concentration of compound 48/80 applied to intact cells induced a partial degree of degranulation. This was determined by observing the morphological changes accompanying degranulation by light and electron microscopy and also as a reduction in the intensity of light scattered at 90 degrees, indicative of a change in the cell-refractive index. These results may be explained by the existence of a threshold sensitivity to the combined effectors that is set at the level of individual cells and not at the granule level. We used flow cytometry to establish the relationship between the extent of degranulation in individual rat peritoneal mast cells and the extent of secretion in the population (measured as the percentage release of total hexosaminidase). For comparison, secretion was also elicited by applying the Ca2+ ionophore ionomycin or compound 48/80 to intact cells. For permeabilized cells and also for intact cells stimulated with the ionophore, levels of stimulation that generate partial secretion gave rise to bimodal frequency distributions of 90 degrees light scatter. In contrast, a partial stimulus to secretion by compound 48/80 resulted in a single population of partially degranulated cells, the degree of degranulation varying across the cell population. The difference between the all-or-none responses of the permeabilized or ionophore-treated cells and the graded responses of cells activated by compound 48/80 is likely to stem from differences in the effective calcium stimulus. Whereas cell stimulated with receptor-directed agonists can undergo transient and localized Ca2+ changes, a homogeneous and persistent stimulus is sensed at every potential exocytotic site in the permeabilized cells.     

Characterization of calcium-triggered secretion in permeabilized rat basophilic leukemia cells. Possible role of vectorially acting G proteins.

Strong, albeit indirect, evidence suggests that a GTP-binding (G) protein(s) can act directly on the secretory machinery by a post-second messenger mechanism. The type and function of this putative Ge (exocytosis) protein were investigated in streptolysin-O-permeabilized rat basophilic leukemia (RBL) cells. The exocytotic response to calcium was first characterized both morphologically and biochemically using the release of preloaded [3H]serotonin as an index of exocytosis. Calcium-induced secretion (EC50 about 3 microM) in RBL cells requires ATP (EC50 about 2.5 mM) and is modulated by pH, the optimal value being 7.2. Another requirement for calcium-induced secretion is an activated G protein, since inactivators of G proteins such as GDP beta S (EC50 about 800 microM) inhibit the secretagogue effect of 10 microM free calcium. Conversely, GTP gamma S (EC50 about 1 microM) and other nonhydrolyzable analogs of GTP, which keep G proteins in a permanently active conformation, potentiate the effect of calcium. GTP gamma S alone is without effect. The effect of GTP gamma S on exocytosis is apparently not mediated by known second messengers, suggesting that a Ge protein is involved. Electron microscopic images show that in resting cells, secretory granules are clustered in the perinuclear area, whereas they become scattered upon calcium stimulation. A paradoxical effect of GTP gamma S is observed when applied during permeabilization; under these conditions, in fact, the nucleotide inhibits the subsequent secretory response to calcium. The scattering of granules is also inhibited. This effect of GTP gamma S is counteracted by coadministration of GTP. These responses to guanine nucleotides are typical of vectorially acting G proteins involved in protein synthesis and in intracellular vesicle transport. Taken together, the data presented suggest that calcium-dependent release requires a vectorially acting G protein controlling the movement of secretory granules. This and alternative models are discussed.     

TRPM4 controls insulin secretion in pancreatic beta-cells

TRPM4 is a calcium-activated non-selective cation channel that is widely expressed and proposed to be involved in cell depolarization. In excitable cells, TRPM4 may regulate calcium influx by causing the depolarization that drives the activation of voltage-dependent calcium channels. We here report that insulin-secreting cells of the rat pancreatic beta-cell line INS-1 natively express TRPM4 proteins and generate large depolarizing membrane currents in response to increased intracellular calcium. These currents exhibit the characteristics of TRPM4 and can be suppressed by expressing a dominant negative TRPM4 construct, resulting in significantly decreased insulin secretion in response to a glucose stimulus. Reduced insulin secretion was also observed with arginine vasopressin stimulation, a Gq-coupled receptor agonist in beta-cells. Moreover, the recruitment of TRPM4 currents was biphasic in both INS-1 cells as well as HEK-293 cells overexpressing TRPM4. The first phase is due to activation of TRPM4 channels localized within the plasma membrane followed by a slower secondary phase, which is caused by the recruitment of TRPM4-containing vesicles to the plasma membrane during exocytosis. The secondary phase can be observed during perfusion of cells with increasing [Ca(2+)](i), replicated with agonist stimulation, and coincides with an increase in cell capacitance, loss of FM1-43 dye, and vesicle fusion. Our data suggest that TRPM4 may play a key role in the control of membrane potential and electrical activity of electrically excitable secretory cells and the dynamic translocation of TRPM4 from a vesicular pool to the plasma membrane via Ca(2+)-dependent exocytosis may represent a key short- and midterm regulatory mechanism by which cells regulate electrical activity.     

Rundown of secretion after depletion of intracellular calcium stores in bovine adrenal chromaffin cells

In this study, the relationship between intracellular calcium stores and depolarization-evoked stimulation was examined in bovine chromaffin cells, using changes in membrane capacitance to monitor both exocytosis and endocytosis. Cells were voltage-clamped using the perforated whole-cell patch configuration to minimize alterations in intracellular constituents. Control cells exhibited reproducible secretory responses each time the cell was stimulated. However, the same stimulation protocol elicited progressively smaller secretory responses in cells where their intracellular calcium store was emptied by thapsigargin. Transient elevation of the intracellular calcium concentration with a brief histamine treatment enhanced subsequent secretory responses in control but not in thapsigargin-treated cells. A series of depolarizations to -20 mV, which allowed small amounts of Ca(2+) influx but which by itself did not trigger catecholamine secretion, enhanced subsequent exocytosis in both control and thapsigargin-treated cells. Caffeine-pretreated cells exhibited a rundown in the secretory response that was similar to that produced by thapsigargin. These results suggest that brief elevations of [Ca(2+)](i) could enhance subsequent secretory responses. In addition, the data suggest that intracellular calcium stores are vital for the maintenance of exocytosis during repetitive stimulation.     

Thapsigargin potentiates histamine-stimulated HCl secretion in gastric parietal cells but does not mimic cholinergic responses.

The role of calcium in control of HCl secretion by the gastric parietal cell was examined using a recently available intracellular calcium-releasing agent, thapsigargin, which has been shown, in some cell types, to induce sustained elevation of intracellular calcium ([Ca2+]i), an action that appears to be independent of inositol lipid breakdown and protein kinase C activation and to be mediated, at least partially, by selective inhibition of endoplasmic reticulum Ca2(+)-ATPase. Using the calcium-sensitive fluorescent probe, fura-2, in combination with digitized video image analysis of single cells as well as standard fluorimetric techniques, we found that thapsigargin induced sustained elevation of [Ca2+]i in single parietal cells and in parietal cells populations. Chelation of medium calcium led to a transient rise and fall in [Ca2+]i, indicating that the sustained elevation in [Ca2+]i in response to thapsigargin was due to both intracellular calcium release and influx. Although thapsigargin appeared to affect the same calcium pool(s) regulated by the cholinergic agonist, carbachol, and the pattern of thapsigargin-induced increases in [Ca2+]i were similar to the plateau phase of the cholinergic response, thapsigargin did not induce acid secretory responses of the same magnitude as those initiated by carbachol (28 vs 600% of basal). The protein kinase C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) potentiated the secretory response to thapsigargin but this combined response also did not attain the same magnitude as the maximal cholinergic response. In the presence but not the absence of medium calcium, thapsigargin potentiated acid secretory responses to histamine, which elevate both cyclic AMP (cAMP) and [Ca2+]i in parietal cells, as well as forskolin and cAMP analogues but had no effect on submaximal and an inhibitory effect on maximal cholinergic stimulation. Furthermore, thapsigargin did not fully mimic potentiating interactions between histamine and carbachol, either in magnitude or in the pattern of temporal response. Assuming that the action of thapsigargin is specific for intracellular calcium release mechanisms, these data suggest that 1) sustained influx of calcium is necessary but not sufficient for cholinergic activation of parietal cell HCl secretion and for potentiating interactions between cAMP-dependent agonists and carbachol; 2) mechanisms in addition to elevated [Ca2+]i and protein kinase C activation may be involved in cholinergic regulation; and 3) increases in [Ca2+]i in response to histamine are not directly involved in the mechanism of histamine-stimulated secretion.     

Activation of paracrine TGF-beta1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase.

As a source of transforming growth factor beta1 (TGF-beta1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF-beta1 have remained vague. We show here by means of RT-PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF-beta1 in their chymase 1-containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF-beta1, which is converted by chymase 1 into an active form recognized by the type II TGF-beta serine/threonine kinase receptor (TbetaRII). Thus, mast cells secrete active TGF-beta1 by a unique secretory mechanism in which latent TGF-beta1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF-beta1 specifically by chymase was verified using recombinant human latent TGF-beta1 and recombinant human chymase. In isolated TbetaRI- and TbetaRII-expressing peritoneal macrophages, the activated TGF-beta1 induces the expression of the plasminogen activator inhibitor 1 (PAI-1), whereas in the mast cells, the levels of TbetaRI, TbetaRII, and PAI-1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF-beta1 in peritoneal mast cells and of TbetaRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF-beta1 and by enhancing its response in target cells.     

Factors affecting habituation of PC12 cells to ATP.

Extracellular ATP triggers catecholamine secretion from PC12 cells by activating ionotropic purine receptors. Repeated stimulation by ATP leads to habituation of the secretory response. In this paper, we use amperometric detection to monitor the habituation of PC12 cells to multiple stimulations of ATP or its agonist. Cells habituate to 30 microm ATP slower than they do to 300 or 600 microm ATP. Modifying external Mg2+ affects the response of cells to 30 microm ATP, but does not affect habituation, suggesting that habituation does not necessarily correspond to either stimulus intensity or cellular response. Mg2+ affects the initial response of PC12 cells to 2MeSATP in a manner similar to ATP. Increasing external [Mg2+] to 3.0 mm, however, eliminates habituation to 2MeSATP. This habituation can be partially restored by costimulation with 100 microm UTP. Background application of UTP increases habituation to both ATP and 2MeSATP. This suggests that ATP-sensitive metabotropic (P2Y) receptors play a role in the habituation process. Finally, although Ca2+ influx through voltage-operated calcium channels does not appear to contribute to secretion during ATP stimulation, blocking these channels with nicardipine increases habituation. This suggests a role for voltage-operated calcium channels in the habituation process.     

The small GTP-binding proteins, Rac and Rho, regulate cytoskeletal organization and exocytosis in mast cells by parallel pathways.

In mast cells, activation of GTP-binding proteins induces centripetal reorganization of actin filaments. This effect is due to disassembly, relocalization, and polymerization of F-actin and is dependent on two small GTPases, Rac and Rho. Activities of Rac and Rho are also essential for the secretory function of mast cells. In response to GTP-gamma-S and/or calcium, only a proportion of permeabilized mast cells is capable of secretory response. Here, we have compared actin organization of secreting and nonsecreting cell populations. We show that the cytoskeletal and secretory responses are strongly correlated, indicating a common upstream regulator of the two functions. The secreting cell population preferentially displays both relocalization and polymerization of actin. However, when actin relocalization or polymerization is inhibited by phalloidin or cytochalasin, respectively, secretion is unaffected. Moreover, the ability of the constitutively active mutants of Rac and Rho to enhance secretion is also unaffected in the presence of cytochalasin. Therefore, Rac and Rho control these two functions by divergent, parallel signaling pathways. Cortical actin disassembly occurs in both secreting and nonsecreting populations and does not, by itself, induce exocytosis. A model for the control of exocytosis is proposed that includes at least four GTP-binding proteins and suggests the presence of both shared and divergent signaling pathways from Rac and Rho.     

Serglycin is the major secreted proteoglycan in macrophages and has a role in the regulation of macrophage tumor necrosis factor-alpha secretion in response to lipopolysaccharide

It has recently been shown that serglycin is essential for maturation of mast cell secretory granules. However, serglycin is expressed also by other cell types, and in this study we addressed the role of serglycin in macrophages. Adherent cells were prepared from murine peritoneal cell populations and from spleens, and analyzed for proteoglycan synthesis by biosynthetic labeling with [35S]sulfate. Conditioned media from serglycin-/- peritoneal macrophages and adherent spleen cells displayed a 65-80% reduction of 35S-labeled proteoglycans, compared with corresponding material from serglycin+/+ cells, indicating that serglycin is the dominant secretory proteoglycan in macrophages of these origins. In contrast, the levels of intracellular proteoglycans were similar in serglycin+/+ and serglycin-/- cells, suggesting that serglycin is not stored intracellularly to a major extent in macrophages. This is in contrast to mast cells, in which serglycin is predominantly stored intracellularly. Transmission electron microscopy revealed that the absence of serglycin did not cause any major morphological effects on peritoneal macrophages, in contrast to dramatic defects in intracellular storage vesicles in peritoneal mast cells. Several secretory products were not found to be affected by the lack of serglycin. However, the secretion of tumor necrosis factor-alpha in response to lipopolysaccharide stimulation was markedly higher in serglycin-/- cultures than in those of serglycin+/+. The present report thus demonstrates that serglycin is the major proteoglycan secreted by peritoneal macrophages and suggests that the macrophage serglycin may have a role in regulating secretion of tumor necrosis factor-alpha.     

Preservation of the secretory response of peritoneal mast cells in the absence of extracellular calcium

The transfer of rat peritoneal mast cells from balanced salt solution to calcium-free buffer led to a time-dependent decrease in their response to compound 48/80 and to ionophore A23187. The concomittant absence of potassium from the calcium-free buffer enabled the mast cells to retain their secretory response. The increase in potassium level, with a parallel decrease in sodium to maintain osmolarity, led to a slight potentiation of the response to 48/80 and to a large but transient potentiation of the response to A23187. Mast cells can be considered nonexcitable. The apparent dependency upon extracellular calcium of mast cell secretory responses might be related to the presumed tight equilibrium between endoplasmic reticulum calcium stores and extracellular calcium. The control of this equilibrium by transmembrane gradients of monovalent ions is proposed.     

Inhibition of antigen and calcium ionophore induced secretion from RBL-2H3 cells by phosphatase inhibitors

The role of serine/threonine protein phosphatases PP1 and PP2A in mast cell secretion was investigated using the phosphatase inhibitors okadaic acid and calyculin A. Calyculin A (5-25 nm) inhibited antigen-induced secretion from a rat mucosal mast cell line (RBL-2H3) when added in conjunction with the activator. Okadaic acid (250-1000 nm) inhibited secretion only when added before activation and did so in a time- and concentration-dependent manner. Both inhibitors caused the cells to become rounder, but only calyculin A induced membrane blebbing and a loss of adherence. Okadaic acid also inhibited secretion induced by the calcium ionophore A23187, in the presence or absence of PMA, indicating that the phosphatase inhibitors act on a component of the secretory pathway downstream of calcium mobilization. Okadaic acid increased the phosphorylation of a number of proteins, as did an analogue methyl okadaate, which also inhibited secretion, but less effectively. Okadaic acid induced the phosphorylation of triton-insoluble proteins of 55, 18 and 16 kDa. The 55 kDa protein was identified as vimentin and okadaic acid induced its partial translocation to the triton-soluble fraction. Our data indicate that full secretory function in mucosal mast cells requires phosphatase activity.     

Role of calcium fluxes in the sustained phase of angiotensin II-mediated aldosterone secretion from adrenal glomerulosa cells

When angiotensin II stimulates aldosterone secretion, it causes a rapid but transient mobilization of calcium from an intracellular pool and a sustained increase in the influx of calcium in adrenal glomerulosa cells. The present studies were undertaken to determine the respective roles of the two angiotensin II-induced changes in cellular calcium metabolism in modulating events during the sustained phase of cellular response which is thought to be mediated by the C-kinase branch of the calcium messenger system. The sustained response to angiotensin II is only 50% of maximal in cells pretreated with dantrolene in a concentration sufficient to inhibit the angiotensin II-induced mobilization of intracellular calcium. Also, if A23187 is added to cells simultaneously with 1-oleoyl-2-acetylglycerol (OAG), the aldosterone secretory response is similar to that seen after angiotensin II. However, if A23187 is added first and the transient aldosterone secretory response allowed to decay, and OAG then added, the sustained aldosterone secretory response is only 45-50% of maximal. Addition of the calcium channel agonist, BAY K 8644, with OAG leads to an aldosterone secretory response which is only 50% of maximal, but if upon addition of OAG and BAY K 8644 the cells are also exposed for 5 min to media containing 8 mM K+, then the sustained secretory response is maximal. These data imply that the initial transient rise in the [Ca2+] of the cell cytosol plays a role in determining the extent to which C-kinase is shifted from its calcium-insensitive to its calcium-sensitive form. The second group of experiments examined the relationship between the sustained angiotensin II-induced increase in plasma membrane calcium influx and the sustained aldosterone secretory response. The results show that in the presence of 1 microM nitrendipine or 2 mM extracellular K+, angiotensin II causes no increase in calcium influx and only a transient rather than a sustained increase in the rate of aldosterone secretion indicating that the sustained phase of the response is dependent upon a continued high rate of Ca2+ influx which regulates the rate of turnover of the activated C-kinase.