Phosphomimetic mutation of Ser-187 of SNAP-25 increases both syntaxin binding and highly Ca2+-sensitive exocytosis

The phosphorylation targets that mediate the enhancement of exocytosis by PKC are unknown. PKC phosporylates the SNARE protein SNAP-25 at Ser-187. We expressed mutants of SNAP-25 using the Semliki Forest Virus system in bovine adrenal chromaffin cells and then directly measured the Ca2+ dependence of exocytosis using photorelease of caged Ca2+ together with patch-clamp capacitance measurements. A flash of UV light used to elevate [Ca2+](i) to several microM and release the highly Ca2+-sensitive pool (HCSP) of vesicles was followed by a train of depolarizing pulses to elicit exocytosis from the less Ca2+-sensitive readily releasable pool (RRP) of vesicles. Carbon fiber amperometry confirmed that the amount and kinetics of catecholamine release from individual granules were similar for the two phases of exocytosis. Mimicking PKC phosphorylation with expression of the S187E SNAP-25 mutant resulted in an approximately threefold increase in the HCSP, whereas the response to depolarization increased only 1.5-fold. The phosphomimetic S187D mutation resulted in an approximately 1.5-fold increase in the HCSP but a 30% smaller response to depolarization. In vitro binding assays with recombinant SNARE proteins were performed to examine shifts in protein-protein binding that may promote the highly Ca2+-sensitive state. The S187E mutant exhibited increased binding to syntaxin but decreased Ca2+-independent binding to synaptotagmin I. Mimicking phosphorylation of the putative PKA phosphorylation site of SNAP-25 with the T138E mutation decreased binding to both syntaxin and synaptotagmin I in vitro. Expressing the T138E/ S187E double mutant in chromaffin cells demonstrated that enhancing the size of the HCSP correlates with an increase in SNAP-25 binding to syntaxin in vitro, but not with Ca2+-independent binding of SNAP-25 to synaptotagmin I. Our results support the hypothesis that exocytosis triggered by lower Ca2+ concentrations (from the HCSP) occurs by different molecular mechanisms than exocytosis triggered by higher Ca2+ levels.     

A highly Ca2+-sensitive pool of granules is regulated by glucose and protein kinases in insulin-secreting INS-1 cells

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca(2+) to directly measure the Ca(2+) sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca(2+) sensitivity of release in that a small proportion of granules makes up a highly Ca(2+)-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca(2+). A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca(2+) are not located close to Ca(2+) channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca(2+)-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca(2+), suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca(2+) sensor that is poised to respond to modest, global elevations of [Ca(2+)](i).     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells.

Oscillations of free intracellular Ca2+ concentration ([Ca2+]i) are known to occur in many cell types during physiological cell signaling. To identify the basis for the oscillations, we measured both [Ca2+]i and extracellular Ca2+ concentration ([Ca2+]o) to follow the fate of Ca2+ during stimulation of [Ca2+]i oscillations in pancreatic acinar cells. [Ca2+]i oscillations were initiated by either t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Nle-Asp-2-phenylethyl ester (CCK-J), which mobilized Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-insensitive pool, or low concentration of cholecystokinin octapeptide (CCK-OP), which mobilized Ca2+ from the IP3-sensitive internal pool. Little Ca2+ efflux occurred during the oscillations triggered by CCK-J or CCK-OP in spite of a large average increase in [Ca2+]i. When internal store Ca2+ pumps were inhibited with thapsigargin (Tg) during [Ca2+]i oscillations, a rapid Ca2+ efflux occurred similar to that measured in intensely stimulated, nonoscillatory cells. Tg also stimulated 45Ca efflux from internal pools of cells stimulated with CCK-J or a low concentration of CCK-OP. Hence, a large fraction of the Ca2+ released during each spike is reincorporated by the internal store Ca2+ pumps. Surprisingly, when the increase in [Ca2+]i during stimulation of oscillations was prevented by loading the cells with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, a persistent activation of Ca2+ release and Ca2+ efflux occurred. This was reflected as a persistent increase in [Ca2+]o in cells suspended at low [Ca2+]o or persistent efflux of 45Ca from internal stores of cells maintained at high [Ca2+]o. Since agonist-stimulated Ca2+ release evidently remains activated when [Ca2+]i is highly buffered, the primary mechanism determining Ca2+ oscillations must include an inhibition of Ca2+ release by [Ca2+]i. Loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no apparent effect on the levels or kinetics of IP3 formation in agonist-stimulated cells. This suggests that [Ca2+]i regulated the oscillation by inhibition of Ca2+ release independent of its possible effects on cellular levels of IP3.     

Ca2+ entry in T cells is activated by emptying the inositol 1,4,5-triphosphate sensitive Ca2+ pool

Using alpha-linolenic acid (ALA), one of several polyunsaturated fatty acids (PUFAs) that have previously been shown to both mobilize intracellular Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool independently of IP3 production and inhibit Ca2+ influx, the relationship between Ca2+ mobilization from intracellular stores and Ca2+ influx in T cells (JURKAT) was studied. JURKAT cells were treated with 30 microM ALA to deplete the IP3-sensitive Ca2+ pool. When the intracellular free Ca2+ concentration [( Ca2+]i) returned to basal level, fatty acid free bovine serum albumin (BSA) was added to remove extracellular and membrane bound ALA. This resulted in a sustained increase in [Ca2+]i in the absence of inositol phosphates' formation. This sustained increase in [Ca2+]i was insensitive to protein kinase C activation but was inhibited by Ni2+ ions. The extent of Ca2+ influx was found to be correlated to the amount of Ca2+ initially discharged from the IP3-sensitive Ca2+ pool by sub-optimal concentrations of ALA. Ligation of the CD3 complex of the T cell antigen receptor with an anti-CD3 antibody (OKT3) during the sustained [Ca2+]i increased (induced by a sub-optimal concentration of ALA), produced a greater response. No increase in the sustained response was observed when the CD3 complex was activated in cells pretreated with an optimal concentration of ALA. In summary, Ca2+ entry in T cells is activated by emptying of the IP3-sensitive Ca2+ pool which can be dissociated from inositol phosphate production. The rate of Ca2+ influx appears to be closely correlated to the initial discharge of Ca2+ from the IP3-sensitive Ca2+ pool, suggesting that Ca2+ may first enter the depleted pool and then is released into the cytosol.     

Dissection of three Ca2+-dependent steps leading to secretion in chromaffin cells from mouse adrenal slices

In neurosecretory cells, intracellular Ca2+ ([Ca2+]i) not only acts as the trigger for secretion but also regulates earlier steps in the secretory pathway. Here, a novel approach was developed to control [Ca2+]i over a broad concentration range, which allowed the quantification of three distinct actions of [Ca2+]i on large dense-core vesicle (LDCV) fusion in chromaffin cells from mouse adrenal slices. Basal [Ca2+]i regulated the transfer of vesicles toward a slowly releasable state, whereas further maturation to the readily releasable state was Ca2+ independent. [Ca2+]i levels above 3 microM triggered exocytosis of all readily and slowly releasable vesicles in two parallel, kinetically distinct fusion reactions. In a molecular context, these results suggest that Ca2+ acts both before and after trans-SNARE complex formation to regulate fusion competence and fusion kinetics of LDCVs.     

Plasticity and adaptation of Ca2+ signaling and Ca2+-dependent exocytosis in SERCA2(+/-) mice

Darier's disease (DD) is a high penetrance, autosomal dominant mutation in the ATP2A2 gene, which encodes the SERCA2 Ca2+ pump. Here we have used a mouse model of DD, a SERCA2(+/-) mouse, to define the adaptation of Ca2+ signaling and Ca2+-dependent exocytosis to a deletion of one copy of the SERCA2 gene. The [Ca2+]i transient evoked by maximal agonist stimulation was shorter in cells from SERCA2(+/-) mice, due to an up-regulation of specific plasma membrane Ca2+ pump isoforms. The change in cellular Ca2+ handling caused approximately 50% reduction in [Ca2+]i oscillation frequency. Nonetheless, agonist-stimulated exocytosis was identical in cells from wild-type and SERCA2(+/-) mice. This was due to adaptation in the levels of the Ca2+ sensors for exocytosis synaptotagmins I and III in cells from SERCA2(+/-) mice. Accordingly, exocytosis was approximately 10-fold more sensitive to Ca2+ in cells from SERCA2(+/-) mice. These findings reveal a remarkable plasticity and adaptability of Ca2+ signaling and Ca2+-dependent cellular functions in vivo, and can explain the normal function of most physiological systems in DD patients.     

Relationship between agonist- and thapsigargin-sensitive calcium pools in adrenal glomerulosa cells. Thapsigargin-induced Ca2+ mobilization and entry

The relationships between agonist-sensitive calcium pools and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in intact bovine adrenal glomerulosa cells and a subcellular adrenocortical membrane fraction. In Fura-2-loaded glomerulosa cells, angiotensin II (AII) stimulated a rapid increase in cytoplasmic Ca2+ concentration ([Ca2+]i) followed by a smaller plateau phase that was dependent on extra-cellular Ca2+. In such cells thapsigargin caused a sustained and dose-dependent increase in [Ca2+]i which was diminished in Ca(2+)-deficient medium. The contribution of an influx component to the thapsigargin-induced [Ca2+]i response was demonstrated by measurement of 45Ca influx rate in glomerulosa cells. Thapsigargin-induced Ca2+ entry was significantly less than that evoked by AII, and its kinetics were similar to those of the concomitant increase in [Ca2+]i. The rate of emptying of the agonist-responsive Ca2+ pool after thapsigargin treatment, as indicated by the progressive decrease in the size of the AII-induced Ca2+ transient, showed a rapid initial (t1/2 = 1.7 min) component that accounted for about 80% of the response and a slowly decreasing phase with t1/2 = 112 min. The latter thapsigargin-resistant component was abolished by the removal of extracellular Ca2+. Pretreatment with AII dose-dependently attenuated but did not abolish the subsequent Ca2+ response to thapsigargin and also increased the rate of the Ca2+ rise induced by thapsigargin. In bovine adrenocortical microsomes, thapsigargin inhibited the ATP-dependent filling of Ca2+ pools and caused a dose-dependent rise in extravesicular Ca2+ levels when added to previously loaded microsomes. The thapsigargin-releasable Ca2+ pool in adrenal microsomes was larger than the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool but only slightly greater than the GTP-releasable pool. Ins(1,4,5)P3-induced Ca2+ release was reduced markedly when ATP-dependent Ca2+ loading of the microsomes was prevented by prior addition of thapsigargin. However, the subsequent Ca2+ response to Ins(1,4,5)P3 was consistently better preserved after the addition of thapsigargin to microsomes preloaded with Ca2+. This difference suggests that although Ca2+ uptake by the Ins(1,4,5)P3-responsive pool is also sensitive to thapsigargin, once filled, this pool shows a slower passive leakage than other thapsigargin-sensitive pools. These findings indicate that thapsigargin increases [Ca2+]i by inhibiting Ca2+ uptake into multiple intracellular Ca2+ pools and by also promoting entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of m-3m3FBS on Ca2+ handling and viability in OC2 human oral cancer cells

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-60 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. M-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, 30 μM m-3M3FBS pretreatment inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. At concentrations between 5 and 100 μM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 μM) induced apoptosis in a Ca2+-independent manner. Collectively, in OC2 cells, m-3M3FBS induced [Ca2+]i rise by causing inositol 1,4,5-trisphosphate-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive store-operated Ca2+ channels. M-3M3FBS also induced Ca2+-independent cell death and apoptosis.     

Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.     

The caffeine-sensitive Ca2+ store in bovine adrenal chromaffin cells; an examination of its role in triggering secretion and Ca2+ homeostasis

The effect of caffeine on catecholamine secretion and intracellular free Ca2+ concentration [( Ca2+]i) in bovine adrenal chromaffin cells was examined using single fura-2-loaded cells and cell populations. In cell populations caffeine elicited a large (approximately 200 nM) transient rise in [Ca2+]i that was independent of external Ca2+. This rise in [Ca2+]i triggered little secretion. Single cell measurements of [Ca2+]i showed that most cells responded with a large (greater than 200 nM) rise in [Ca2+]i, whereas a minority failed to respond. The latter, whose caffeine-sensitive store was empty, buffered a Ca2+ load induced by a depolarizing stimulus more effectively than those whose store was full. The caffeine-sensitive store in bovine chromaffin cells may be involved in Ca2+ homeostasis rather than in triggering exocytosis.     

Regulation of zymogen granule exocytosis by Ca2+, cAMP, and PKC in pancreatic acinar cells

The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10 microM), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a PKC activator) alone did not elicit any [Ca2+]c rise or zymogen granule exocytosis, but co-stimulation with ACh led to exocytosis in that the total number of secreted granules increased markedly without a significant difference in [Ca2+]c rises. When we evoked exocytosis by [Ca2+]c ramps, pretreatment with forskolin or PMA elicited exocytosis at lower [Ca2+]c levels. These results indicate that PKC or cAMP alone could not directly elicit zymogen granule exocytosis, but that they increase the total releasable pool by rendering zymogen granules more sensitive to Ca2+.     

Characterization of the effects of Mg2+ on Ca2+- and Sr2+-activated tension generation of skinned skeletal muscle fibers

Changes in [Mg2+] in a millimolar range have a significant inverse effect on the Ca2+- (or Sr2+)activated tension generation of skeletal muscle fibers. Single frog (Rana pipiens) semitendinosus muscle fibers were "skinned" (sarcolemma removed) and contracted isometrically in bathing solutions of varying [Ca2+] or [Sr2+] and [Mg2+] but a constant pH, [MgATP2-], [K+], [CP2-], [CPK], and ionic strength. Ca2+- (or Sr2+- )activated steady-state tensions were recorded for three [Mg2+]'s: 5 X 10(-5)M, 1 X 10(-3) M, and 2 X 10(-3) M; and these tensions were expressed as the percentages of maximum tension generation of the fibers for the same [Mg2+]. Maximum tension was not affected by [Mg2+] within Ca2+-activating or Sr2+-activating sets of solutions; however, the submaximum Ca2+-(or Sr2+)activated tension is strongly affected in an inverse fashion by increasing [Mg2+]. Mg2+ behaves as a competitive inhibitor of Ca2+ and also affects the degree of cooperativity in the system. At [Mg2+] = 5 X 10(-5)M the shape of tension versus [Ca2+] (or [Sr2+]) curve showed evidence of cooperativity of Ca2+ (or Sr2+) binding or activation of the contractile system. As [Mg2+] increased, the apparent affinity for Ca2+ or Sr2+ and cooperativity of the contractile system declined. The effect on cooperativity suggests that as [Mg2+] decreases a threshold for Ca2+ activation appears.     

2-O-methyl PAF as a Ca2+ mobilizer in Madin Darby canine kidney cells

In Madin-Darby canine kidney (MDCK) cells, the effect of 2-O-methyl PAF, an inactive analogue of platelet activating factor (PAF), on intracellular Ca2+ concentration ([Ca2+]i) was measured by using the Ca2+-sensitive fluorescent dye fura-2. 2-O-methyl PAF (> or = 15 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. 2-O-methyl PAF-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. 2-O-methyl PAF-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. The 2-O-methyl PAF-induced Ca2+ influx was blocked by nifedipine, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which 2-O-methyl PAF failed to increase [Ca2+]i; also, pretreatment with 2-O-methyl PAF depleted thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not 2-O-methyl PAF)-induced [Ca2+]i rise. These findings suggest that 2-O-methyl PAF evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release.     

Ca2+ triggers massive exocytosis in Chinese hamster ovary cells.

We have tracked the cell surface area of CHO cells by measuring the membrane capacitance, Cm. An increase in cytosolic [Ca2+], [Ca2+]i, increased the cell surface area by 20-30%. At micromolar [Ca2+]i the increase occurred in minutes, while at 20 microM or higher [Ca2+]i it occurred in seconds and was transient. GTPgammaS caused a 3% increase even at 0.1 microM [Ca2+]i. We conclude that CHO cells, previously thought capable only of constitutive exocytosis, can perform Ca2+-triggered exocytosis that is both massive and rapid. Ca2+-triggered exocytosis was also observed in 3T3 fibroblasts. Our findings add evidence to the view that Ca induces exocytosis in cells other than known secretory cells.     

Overexpression of calreticulin increases the Ca2+ capacity of rapidly exchanging Ca2+ stores and reveals aspects of their lumenal microenvironment and function

A molecularly tagged form of calreticulin (CR), a low affinity-high capacity Ca2+ binding protein that resides in the ER lumen, was transiently transfected into HeLa cells to specifically modify the Ca2+ buffering capacity of the intracellular Ca2+ stores. Fluorescence and confocal microscope immunocytochemistry revealed the tagged protein to be expressed by over 40% of the cells and to overlap in its distribution the endogenous CR yielding a delicate cytoplasmic network, i.e., the typical pattern of ER. In contrast, no signal was observed associated with the plasmalemma (marked by ConA) and within the nucleus. One- and two-dimensional Western blots revealed the transfected to exceed the endogenous CR of approximately 3.5-fold and to maintain its Ca2+ binding ability, whereas the expression of other ER proteins was unchanged. Ca2+ homeostasis in the transfected cells was investigated by three parallel approaches: (a) 45Ca equilibrium loading of cell populations; (b) [Ca2+]c measurement with fura-2 followed by quantitative immunocytochemistry of single cells and iii) [Ca2+]c measurement of cell population upon cotransfection with the Ca(2+)-sensitive photoprotein, aequorin. The three approaches revealed different aspects of Ca2+ homeostasis, yielding results which were largely complementary. In particular, the following conclusions were established: (a) both endogenous and transfected CR participate in Ca2+ buffering within the IP3-sensitive, rapidly exchanging, Ca2+ stores; the other pools of the cells were in contrast unaffected by CR transfection; (b) the Ca2+ capacity of the stores is not the main limiting factor of individual IP3-mediated Ca2+ release responses triggered by receptor agonists; (c) in control cells, the contribution of CR to Ca2+ buffering within the IP3-sensitive stores accounts for approximately 45% of the total, the rest being probably contributed by the other lumenal (and also membrane) Ca2+ binding proteins; (d) the free [Ca2+] within the lumen of the IP3-sensitive stores, revealed by the degree of Ca2+ binding to the transfected CR protein, amounts to values in (or approaching) the millimolar range; and (e) Ca2+ influx across the plasmalemma activated by depletion of the stores is directly dependent on the lumenal [Ca2+].     

Mercury-induced Ca2+ increase and cytotoxicity in renal tubular cells

The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 microM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+ -induced [Ca2+]i increase by 27%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+ -free medium, the Hg2+ -induced [Ca2+]i increase was nearly abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+ -induced Ca2+ release was not altered by inhibition of phospholipase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 microM Hg2+ did not alter cell proliferation rate and mitochondrial activity, but 10 microM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.     

Ca2+ release from subplasmalemmal stores as a primary event during exocytosis in Paramecium cells

A correlated electrophysiological and light microscopic evaluation of trichocyst exocytosis was carried out the Paramecium cells which possess extensive cortical Ca stores with footlike links to the plasmalemma. We used not only intra- but also extracellular recordings to account for polar arrangement of ion channels (while trichocysts can be released from all over the cell surface). With three widely different secretagogues, aminoethyldextran (AED), veratridine and caffeine, similar anterior Nain and posterior Kout currents (both known to be Ca(2+)-dependent) were observed. Direct de- or hyperpolarization induced by current injection failed to trigger exocytosis. For both, exocytotic membrane fusion and secretagogue-induced membrane currents, sensitivity to or availability of Ca2+ appears to be different. Current responses to AED were blocked by W7 or trifluoperazine, while exocytosis remained unaffected. Reducing [Ca2+]o to < or = 0.16 microM (i.e., resting [Ca2+]i) suppressed electrical membrane responses triggered with AED, while we had previously documented normal exocytotic membrane fusion. From this we conclude that the primary effect of AED (as of caffeine) is the mobilization of Ca2+ from the subplasmalemmal pools which not only activates exocytosis (abolished by iontophoretic EGTA injection) but secondarily also spatially segregated plasmalemmal Ca(2+)-dependent ion channels (indicative of subplasmalemmal [Ca2+]i increase, but irrelevant for Ca2+ mobilization). The 45Ca2+ influx previously observed during AED triggering may serve to refill depleted stores. Apart from the insensitivity of our system to depolarization, the mode of direct Ca2+ mobilization from stores by mechanical coupling to the cell membrane (without previous Ca(2+)-influx from outside) closely resembles the model currently discussed for skeletal muscle triads.     

Ca2(+)-mobilizing hormones stimulate Ca2+ efflux from hepatocytes

Treatment of hepatocytes with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a novel mobilizer of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool, produces a sustained elevation of [Ca2+]i (Kass, G. E. N., Duddy, S. K., and Orrenius, S. (1989) J. Biol. Chem. 264, 15192-15198). Exposure of hepatocytes to the Ca2(+)-mobilizing hormones, vasopressin, angiotensin II, or ATP following [Ca2+]i elevation by tBuBHQ produced a rapid return of [Ca2+]i to basal or near basal levels. Release of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool by tBuBHQ following pretreatment with vasopressin or angiotensin II resulted in a [Ca2+]i transient and not the sustained [Ca2+]i elevation observed in the absence of the Ca2(+)-mobilizing hormones. The G-protein activator, NaF plus AlCl3, mimicked both effects of the Ca2(+)-mobilizing hormones on [Ca2+]i. The mechanism for Ca2+ removal from the cytosol by Ca2(+)-mobilizing hormones did not involve cyclic nucleotides nor did it require protein kinase C activation or cyclo- and lipoxygenase-dependent metabolites of arachidonic acid. Furthermore, the hormone-mediated decrease in [Ca2+]i did not involve the pertussis toxin-sensitive Gi-protein. Removal of the tBuBHQ-mobilized Ca2+ from the cytosol of hepatocytes by Ca2(+)-mobilizing hormones was mediated by stimulation of a Ca2+ efflux pathway. Thus, in addition to initiating [Ca2+]i transients by releasing Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ store and stimulating Ca2+ influx, Ca2(+)-mobilizing hormones also regulate the termination of the [Ca2+]i transient by stimulating a Ca2+ efflux pathway.