Role of Thapsigargin-Sensitive Intracellular Ca2+ Pools in Secretion Induced by Muscarinic Agonists in Porcine Adrenal Chromaffin Cells

The role of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-sensitive Ca2+ pools in secretion, induced by muscarinic agonists in porcine adrenal chromaffin cells, was studied. Activation of muscarinic receptors, as in other species, was found to increase inositol phosphate production including that of Ins(1,4,5)P3. Treatment of cells with thapsigargin, which is known to deplete Ins(1,4,5)P3-sensitive Ca2+ pools, eliminated the initial transient component of increases in the cytosolic free Ca2+ concentration ([Ca2+]in) induced by the muscarinic agonist, methacholine, in both the presence and the absence of extracellular Ca2+. Thapsigargin treatment also decreased methacholine-induced secretion by about 30% in the presence of extracellular Ca2+ and essentially eliminated secretion that occurred independently of extracellular Ca2+ (which was about 30% of the secretory response that occurred in the presence of extracellular Ca2+). Thapsigargin itself had no effect on inositol phosphate production. These results indicate that about 30% of muscarinic agonist-induced secretion is mediated by the release of Ca2+ from Ins(1,4,5)P3- and thapsigargin-sensitive intracellular Ca2+ pools. These results also suggest that Ca2+ influx activated by muscarinic agonists is not due to depletion of intracellular Ca2+ pools, as prior depletion of these pools had no effect on the portion of the methacholine-induced secretory response and [Ca2+]in signal that was dependent on extracellular Ca2+.     

Agonist-induced endocytosis and signal generation in adrenal glomerulosa cells. A potential mechanism for receptor-operated calcium entry

The relationships between receptor-mediated endocytosis and the generation of intracellular signals were analyzed in angiotensin II (AII)-stimulated adrenal glomerulosa cells. In cells equilibrated with 125I-AII analogs at 4 degrees C, specifically bound agonist but not antagonist AII derivatives were rapidly internalized at 37 degrees C. AII-induced internalization was not influenced by the presence or absence of extracellular Ca2+ but was inhibited by treatment with phenylarsine oxide (PAO) or by arresting coated pit formation with hypotonic shock and potassium depletion. Inhibition of internalization by PAO was prevented by the bifunctional sulfhydryl reagent dithiothreitol but only partially reversed by mercaptoethanol, and readdition of K+ restored internalization in K(+)-depleted cells. Treatment with PAO did not impair the initial AII-induced elevations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and cytoplasmic calcium [( Ca2+]i) but reduced the sustained phase of the Ins(1,4,5)P3 response by 85% and abolished the second phase of the cytoplasmic Ca2+ response; these responses were restored by concomitant treatment with dithiothreitol. Inhibition of AII-receptor internalization by K+ depletion also caused selective loss of the sustained phase of the AII-induced Ca2+ response. Thus, blockade of AII-receptor internalization has similar effects as extracellular Ca2+ deficiency, which abolishes the sustained but not the early AII-induced increases in Ins(1,4,5)P3 production and [Ca2+]i. The close correlations between AII-induced internalization and the generation of Ins(1,4,5)P3 and [Ca2+]i responses suggest that endocytosis of the agonist-receptor complex is necessary to maintain the production of these intracellular signals. It is also possible that receptor-operated vesicular uptake of extracellular Ca2+ makes a significant contribution to the sustained [Ca2+]i responses of certain agonist-stimulated target cells.     

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.     

Intercellular communication between follicular angiotensin receptors and Xenopus laevis oocytes: medication by an inositol 1,4,5- trisphosphate-dependent mechanism

In Xenopus laevis oocytes, activation of angiotensin II (AII) receptors on the surrounding follicular cells sends a signal through gap junctions to elevate cytoplasmic calcium concentration ([Ca2+]i) within the oocyte. The two major candidates for signal transfer through gap junctions into the oocyte during AII receptor stimulation are Ins(1,4,5)P3 and Ca2+. In [3H]inositol-injected follicular oocytes, AII stimulated two- to fourfold increases in phosphoinositide hydrolysis and production of inositol phosphates. Injection of the glycosaminoglycan, heparin, which selectively blocks Ins(1,4,5)P3 receptors, prevented both AII-stimulated and Ins(1,4,5)P3-induced Ca2+ mobilization in Xenopus follicular oocytes but did not affect mobilization of Ca2+ by ionomycin or GTP. These results indicate that the AII-regulated process of gap junction communication between follicular cells and the oocyte operates through an Ins(1,4,5)P3-dependent mechanism rather than through transfer of Ca2+ into the ooplasm and subsequent Ca(2+)-induced Ca2+ release.     

Enhancement of the inositol 1,4,5-trisphosphate-releasable Ca2+ pool by GTP in permeabilized hepatocytes

Permeabilized rat hepatocytes were used to study the effects of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and GTP on Ca2+ uptake and release by ATP-dependent intracellular Ca2+ storage pools. Under conditions where these Ca2+ pools were completely filled, maximal doses of Ins(1,4,5)P3 released only 25-30% of the sequestered Ca2+. The residual Ca2+ was freely releasable with the Ca2+ ionophore ionomycin. Addition of GTP in the absence of Ins(1,4,5)P3 did not cause Ca2+ release and had no effect on the steady-state level of Ca2+ accumulation by intracellular storage pools. However, after a 3-4-min treatment with GTP the size of the Ins(1,4,5)P3-releasable Ca2+ pool was increased by about 2-fold, with a proportional decrease in the residual Ca2+ available for release by ionomycin. In contrast to the situation with freshly permeabilized cells, permeabilized hepatocytes from which cytosolic components had been washed out exhibited direct Ca2+ release in response to GTP addition. The potentiation of Ins(1,4,5)P3-induced Ca2+ release by GTP in permeabilized hepatocytes was concentration-dependent with half-maximal effects at about 5 microM GTP. The dose response to Ins(1,4,5)P3 was not shifted by GTP; instead GTP increased the amount of Ca2+ released at all Ins(1,4,5)P3 concentrations. The effects of GTP were not mimicked by other nucleotides or nonhydrolyzable GTP analogues. In fact, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) inhibited the actions of GTP. However, this inhibition only occurred when GTP gamma S was added prior to GTP, suggesting that the GTP effect is not readily reversible once the cells have been permeabilized. Experiments using vanadate to inhibit the ATP-dependent Ca2+ uptake pump showed that Ins(1,4,5)P3 releases all of the Ca2+ within the Ins(1,4,5)P3-sensitive Ca2+ pool even in the absence of GTP. The increase of Ins(1,4,5)P3-induced Ca2+ release brought about by GTP was also unaffected by vanadate. It is concluded that GTP increases the proportion of the sequestered Ca2+ which is available for release by Ins(1,4,5)P3, either by unmasking latent Ins(1,4,5)P3-sensitive Ca2+ release sites or by allowing direct Ca2+ movement between Ins(1,4,5)P3-sensitive and Ins(1,4,5)P3-insensitive Ca2+ storage pools.     

Activation of calcium oscillations by thapsigargin in parotid acinar cells.

The tumor promoter thapsigargin releases Ca2+ from intracellular stores by specific inhibition of microsomal Ca-ATPase activity without inositol phosphate formation. Recent studies of the actions of thapsigargin support the concept that the level of Ca2+ within the inositol (1,4,5)-trisphosphate (IP3)-sensitive intracellular pool regulates the Ca2+ permeability of the plasma membrane. We examined the effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) in single rat parotid cells using digital fluorescence microscopy. In the absence of extracellular Ca2+ (Ca2+o), thapsigargin transiently increased [Ca2+]i. Following the thapsigargin-induced [Ca2+]i transient, carbachol in the continued absence of Ca2+o was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes Ca2+ from the IP3-sensitive store. In the converse experiment, carbachol prevented a rise of [Ca2+]i by thapsigargin, suggesting that the IP3- and thapsigargin-sensitive Ca2+ pools are the same. Depletion of Ca2+ from the IP3-sensitive pool by thapsigargin enhanced plasma membrane Ca2+ permeability. Thapsigargin triggered sustained Ca2+ oscillations in Ca2(+)-containing medium which are highly reminiscent of agonist-induced oscillations in these cells. Carbachol addition rapidly raised IP3 levels during oscillations triggered by thapsigargin but did not elevate [Ca2+]i, indicating that the IP3-sensitive pool remains continuously depleted during [Ca2+]i fluctuations. The results from this study rule out the involvement of the IP3-sensitive pool in the mechanisms involved in thapsigargin-induced (and by analogy, agonist-induced) oscillations in parotid cells.     

Urokinase receptor (CD87) aggregation triggers phosphoinositide hydrolysis and intracellular calcium mobilization in mononuclear phagocytes

Leukocytes utilize urokinase receptors (uPAR; CD87) in adhesion, migration, and matrix proteolysis. uPAR aggregate at cell-substratum interfaces and at leading edges of migrating cells, so this study was undertaken to determine whether uPAR aggregation is capable of initiating activation signaling. Monocyte-like U937 cells were labeled with fluo-3-acetoxymethyl ester to quantitate intracellular Ca2+ concentrations ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by mAb cross-linking. uPAR aggregation induced highly reproducible increases in [Ca2+]i of 103.0 +/- 10.9 nM (p < 0.0001) and >3-fold increases in cellular d-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Similar increases in [Ca2+]i were also elicited by uPAR aggregation in human monocytes, but cross-linking a control IgG2a had no effect on [Ca2+]i. Selectively cross-linking uPA-occupied uPAR with an anti-uPA mAb produced smaller increases in [Ca2+]i, but fully saturating uPAR with exogenous uPA enhanced the [Ca2+]i response to equal the effect of aggregating uPAR directly. Increased [Ca2+]i was inhibited by thapsigargin, herbimycin A, and U73122, but only partially reduced by low extracellular [Ca2+], indicating that uPAR aggregation increases [Ca2+]i by activating phospholipase C through a tyrosine kinase-dependent mechanism, generating Ins(1,4,5)P3 and releasing Ca2+ from Ins(1,4, 5)P3-sensitive intracellular stores. Cross-linking the beta2 integrin CR3 could not duplicate the effect of uPAR cross-linking, and uPAR-triggered Ca2+ mobilization was not blocked by anti-CR3 mAbs. These results indicate that uPAR aggregation initiates phosphoinositide hydrolysis by mechanisms that are not strictly dependent on associated uPA or CR3.     

Modulation of inositol(1,4,5)trisphosphate-sensitive calcium store content during continuous receptor activation and its effects on calcium entry.

Changes in intracellular Ca2+ concentration ([Ca2+]i) following the activation of muscarinic receptors with carbachol were studied in cells from the exocrine avian nasal gland that had been maintained in culture for 40-48 h. In these cells, the carbachol-induced sustained increase in [Ca2+]i could be further increased by the subsequent addition of thapsigargin. This increase was due to an additional release of intracellular Ca2+ and a corresponding further enhancement of Ca2+ entry. However, thapsigargin-sensitive and Ins(1,4,5)P3-sensitive stores appeared to be coincident and the initial carbachol stimulus was sufficient to completely empty these stores. It was concluded that the subsequent effect of thapsigargin was due to a partial refilling of the Ins(1,4,5)P3-sensitive stores despite the continued presence of agonist, an effect that was not the result of any decline in levels of cellular Ins(1,4,5)P3 or changes in the generation of Ins(1,3,4,5)P4, which were sustained throughout. Possible explanations for this refilling response include compartmentalization of intracellular Ins(1,4,5)P3, or a desensitization of the Ins(1,4,5)P3 receptor/Ca(2+)-release channel. Alternatively, the data are also compatible with a recently proposed kinetic separation of Ca2+ uptake and release sites. An important implication of this particular interpretation of our findings would be an apparent dependence of Ca2+ entry specifically on the status of the Ca(2+)-uptake component of the agonist-sensitive store, rather than the Ca(2+)-release component.     

Functional homogeneity of the non-mitochondrial Ca2+ pool in intact mouse lacrimal acinar cells.

In the absence of extracellular Ca2+, treatment of mouse lacrimal acinar cells with maximal concentrations of methacholine released Ca2+ from intracellular stores. No additional Ca2+ was mobilized by subsequent application of the intracellular Ca(2+)-ATPase inhibitor, thapsigargin, the stable inositol 1,4,5-trisphosphate ((1,4,5)IP3) analog, inositol 2,4,5-trisphosphate ((2,4,5)IP3) (by microinjection), or the Ca2+ ionophore, ionomycin. However, following prolonged activation of cells by methacholine in the presence of extracellular Ca2+, Ca2+ accumulated into a pool which was released by ionomycin but not by thapsigargin. This latter accumulation was blocked by prior microinjection of ruthenium red, indicating that it represents mitochondrial uptake. In saponin-permeabilized lacrimal cells, two Ca(2+)-sequestering pools were detected: (i) a ruthenium red-sensitive, thapsigargin-insensitive pool, presumed to be the mitochondria; and (ii) a ruthenium red-insensitive, thapsigargin-sensitive pool. Only the thapsigargin-sensitive pool accumulated Ca2+ at concentrations similar to those in unstimulated cells. The thapsigargin-sensitive Ca2+ pool was sensitive to (1,4,5)IP3; however, in contrast to findings in intact cells, only 44% of this pool was releasable by (1,4,5)IP3 or (2,4,5)IP3. These data indicate that, in intact lacrimal acinar cells, all exchangeable (ionomycin-sensitive) Ca2+ residues in a pool which responds homogeneously to agonists, (1,4,5)IP3, and thapsigargin. Prolonged elevation of [Ca2+]i results in Ca2+ accumulation into a second, ruthenium red-sensitive pool, presumably mitochondria. Finally, permeabilization of the cells fragments the non-mitochondrial pool, resulting in two pools, one sensitive and one insensitive to (1,4,5)IP3.     

Characterisation of Distinct Inositol 1,4,5-Trisphosphate-Sensitive and Caffeine-Sensitive Calcium Stores in Digitonin-Permeabilised Adrenal Chromaffin Cells

The effect of inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] and caffeine on Ca2+ release from digitonin-permeabilised bovine adrenal chromaffin cells was examined by using the Ca2+ indicator fura-2 to monitor [Ca2+]. Permeabilised cells accumulated Ca2+ in the presence of ATP and addition of either Ins(1,4,5)P3 or caffeine released 17% or 40-50%, respectively, of the accumulated Ca2+, indicated by sustained rises in [Ca2+] in the cell suspension. Prior addition of Ins(1,4,5)P3 had no effect on the magnitude of the response to a subsequent addition of caffeine. The response to Ins(1,4,5)P3 was prevented by prior addition of caffeine or CaCl2, indicating that the Ins(1,4,5)P3 response was blocked by elevated [Ca2+]. The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, indicating that the Ca2+ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca2+ accumulation into the Ins(1,4,5)P3- or caffeine-sensitive stores. Ca2+ release from the caffeine-sensitive store was selectively blocked by ryanodine. The Ins(1,4,5)P3-sensitive store was emptied by thapsigargin, which had no effect on caffeine responses. These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca2+ stores sensitive to either Ins(1,4,5)P3 or caffeine and support previous conclusions that these stores possess different Ca2(+)-ATPases.     

Control of cytosolic free calcium by intracellular organelles in bovine adrenal glomerulosa cells. Effects of sodium and inositol 1,4,5-trisphosphate

The regulation of cytosolic free Ca2+ concentration ([Ca2+]c) by intracellular organelles was studied in permeabilized bovine adrenal glomerulosa cells. Two compartments, with distinct characteristics, were able to pump Ca2+. A first pool, sensitive to ruthenium red and presumably mitochondrial, required respiratory chain substrates to maintain [Ca2+]c around 700 nM. Ca2+ efflux from this compartment was activated by Na+ (ED50 = 5 mM). Inositol 1,4,5-trisphosphate (IP3) had no effect on this pool. A second nonmitochondrial pool required ATP to lower [Ca2+]c to about 200 nM and released Ca2+ transiently upon addition of IP3. When the two systems were allowed to work simultaneously, the nonmitochondrial pool regulated [Ca2+]c and IP3 released Ca2+ in a concentration-dependent manner (EC50 = 0.6 microM). Under these conditions the mitochondria seemed Ca2+ depleted. Upon repeated stimulations with IP3, a marked attenuation of the response was observed. This phenomenon was due to Ca2+ sequestration by a nonmitochondrial IP3-insensitive pool. Neither dantrolene (200 microM) nor 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (10 microM) were able to abolish IP3-induced Ca2+ release, though both compounds efficiently inhibited aldosterone production in intact cells stimulated with angiotensin II (10 nM) or K+ (12 mM). These results suggest that in permeabilized adrenal glomerulosa cells: the nonmitochondrial pool is responsible for buffering [Ca2+]c and for releasing Ca2+ in response to IP3; at resting [Ca2+]c levels, the mitochondria appear Ca2+ depleted; when [Ca2+]c rises above their set point, the mitochondria accumulate Ca2+ as a function of [Na+]c; 4) the mitochondria are not involved in the desensitization mechanism of the response to IP3.     

The temporal integration of the aldosterone secretory response to angiotensin occurs via two intracellular pathways

Angiotensin II (AII) regulates the secretion of aldosterone from adrenal glomerulosa cells by a calcium-dependent mechanism which involves both the uptake of calcium from the extracellular pool, and the release of calcium from a dantrolene-sensitive intracellular pool. In the present study, it was shown that AII induces the rapid (10 s) hydrolysis of phosphatidylinositol 4-phosphate and -4,5-bisphosphate, leading to the sustained production of inositol bis- and trisphosphate (Ins-P3), and diacylglycerol rich in arachidonic acid. Saponin-permeabilized glomerulosa cells accumulate calcium into a nonmitochondrial pool by an ATP-dependent manner. Ins-P3 (0.5-5 microM) induces a release of Ca2+ from this pool. This release was blocked by dantrolene (10 microM). Adrenal glomerulosa cells were shown to contain the calcium-activated, phospholipid-dependent protein kinase (C-kinase). Perfusion of glomerulosa cells with combined 12-O-tetradecanoyl phorbol 13-acetate and A23187 induced an immediately developing, sustained, maximal secretory response similar to that induced by AII. These data are interpreted in terms of a model in which, after AII addition, there is a flow of information through two separate branches of the calcium messenger system, each with its unique temporal role: a calmodulin branch activated by the transient rise in the [Ca2+] in the cell cytosol, which is largely responsible for the initial transient cellular response; and a C-kinase branch activated by the increase in both cytosolic [Ca2+] and the diacylglycerol content of the plasma membrane, which is largely responsible for the sustained phase of the cellular response. The temporal integration of these two phases underlies the observed pattern of cellular response.     

Okadaic acid inhibits angiotensin II stimulation of Ins(1,4,5)P3 and calcium signalling in rat hepatocytes.

OKA2 and CL-A significantly inhibit the ability of angiotensin II, ATP and vasopressin to raise [Ca2+]i in rat hepatocytes, with a partial inhibition of the initial spike, and a complete inhibition of the following plateau. In contrast, the [Ca2+]i response to thapsigargin, which releases intracellular calcium stores through a mechanism independent of inositol phosphates, is much less affected. The ability of angiotensin II to stimulate Ins(1,4,5)P3 production is also reduced by OKA, with kinetics consistent with the inhibited [Ca2+]i response. Since OKA and CL-A are potent and selective inhibitors of phosphoprotein phosphatases, these results provide further evidence that agonist-stimulated Ins(1,4,5)P3 signalling can be inhibited by protein phosphorylation.     

Interaction between mitogens upon intracellular Ca2+ pools in murine fibroblasts.

A comparison of the effect of platelet-derived growth factor (PDGF) and bombesin on intracellular Ca2+ stores was carried out in Swiss 3T3 cells loaded with Fura-2. It was found that the tumor promoter thapsigargin (Tg) almost completely inhibited both the PDGF- and the bombesin-induced intracellular Ca2+ concentration ([Ca2+]i) rise, indicating that the two mitogens mobilize Ca2+ from intracellular pool(s) sensitive to the tumor promoter. It was also found that pre-treatment with PDGF almost totally and persistently (up to at least 30 min) inhibited the bombesin-, Tg- and ionomycin-induced rise in [Ca2+]i, whereas pre-treatment with bombesin had only a partial inhibitory effect on the PDGF, Tg and ionomycin [Ca2+]i response, both in the absence and in the presence of external Ca2+. On the other hand, vasopressin and bradykinin, which also stimulate hydrolysis of phosphoinositides in these cells, did not affect the [Ca2+]i response induced by the same agents. These results indicate that, despite the poor production of inositol 1,4,5-trisphosphate (InsP3), PDGF was capable of totally discharging and maintaining discharged the InsP3-sensitive stores of intracellular Ca2+, regardless of whether extracellular Ca2+ was present in the medium. Bombesin only partially caused this effect. On the contrary, bradykinin and vasopressin, after releasing intracellular Ca2+ allowed an almost total refilling of the pools. It is interesting to note that, at variance with PDGF and bombesin, neither bradykinin nor vasopressin are able to induce a mitogenic response in Swiss 3T3 cells.     

Quantitative analysis of the cytosolic-free-Ca2+-dependency of aldosterone production in bovine adrenal glomerulosa cells. Different requirements for angiotensin II and K+.

Angiotensin II (AII) and K+ raise the cytosolic free Ca2+ concentration [( Ca2+]i) and stimulate aldosterone production in isolated bovine adrenal glomerulosa cells. The mechanisms leading to an elevation of [Ca2+]i were analysed with the fluorescent Ca2+ probe quin 2. (1) Whereas [Ca2+]i rose transiently and returned to basal values within 5 min in response to AII, the effect of K+ was sustained for at least 15 min. (2) AII released Ca2+ from intracellular stores, whereas the [Ca2+]i response to K+ depended solely on extracellular [Ca2+]. (3) When added after K+ stimulation, AII provoked a dramatic decrease in [Ca2+]i to below the resting value. The role of [Ca2+]i in stimulating steroidogenesis was determined by manipulating the concentration of this cation. (4) In a cell superfusion system, the aldosterone response to AII is biphasic. Suppressing the transient [Ca2+]i elevation triggered by AII resulted in the disappearance of the initial secretory peak, but the final production rate was similar to that of control cells. (5) Normal basal [Ca2+]i levels were, however, necessary to maintain continuous AII-induced steroidogenesis. (6) When added after AII, the antagonist analogue [Sar1,Ala8]AII suppressed steroidogenesis without affecting [Ca2+]i levels. (7) In contrast, continuously elevated [Ca2+]i values were required for the initiation and the maintenance of K+-stimulated aldosterone production. These results demonstrate important differences in the mechanisms through which AII and K+ activate the Ca2+ messenger system. Moreover, functional correlations have shown that K+, but not AII, depends solely on a sustained [Ca2+]i response for its steroidogenic effect. However, the AII-induced effect is also a Ca2+-requiring process: the initial [Ca2+]i transient accelerates the onset of steroidogenesis, which is subsequently extremely sensitive to [Ca2+]i decreases below normal basal levels.     

Cytosolic multiple inositol polyphosphate phosphatase in the regulation of cytoplasmic free Ca2+ concentration

Multiple inositol polyphosphate phosphatase (MIPP) is an enzyme that, in vitro, has the interesting property of degrading higher inositol polyphosphates to the Ca2+ second messenger, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), independently of inositol lipid breakdown. We hypothesized that a truncated cytosolic form of the largely endoplasmic reticulum-confined MIPP (cyt-MIPP) could represent an important new tool in the investigation of Ins(1,4,5)P3-dependent intracellular Ca2+ homeostasis. To optimize our ability to judge the impact of cyt-MIPP on intracellular Ca2+ concentration ([Ca2+]i) we chose a poorly responsive beta-cell line (HIT M2.2.2) with an abnormally low [Ca2+]i. Our results show for the first time in an intact mammalian cell that cyt-MIPP expression leads to a significant enhancement of Ins(1,4,5)P3 concentration. This is achieved without a significant interference from other cyt-MIPP-derived inositol phosphates. Furthermore, the low basal [Ca2+]i of these cells was raised to normal levels (35 to 115 nm) when they expressed cyt-MIPP. Noteworthy is that the normal feeble glucose-induced Ca2+ response of HIT M2.2.2 cells was enhanced dramatically by mechanisms related to this increase in basal [Ca2+]i. These data support the use of cyt-MIPP as an important tool in investigating Ins(1,4,5)P3-dependent Ca2+ homeostasis and suggest a close link between Ins(1,4,5)P3 concentration and basal [Ca2+]i, the latter being an important modulator of Ca2+ signaling in the pancreatic beta-cell.     

Ca2+ homeostasis in permeabilized human neutrophils. Characterization of Ca2+-sequestering pools and the action of inositol 1,4,5-triphosphate

The regulation of Ca2+ transport by intracellular compartments was studied in digitonin-permeabilized human neutrophils, using a Ca2+-selective electrode. When incubated in a medium containing ATP and respiratory substrates, the cells lowered within 6 min the ambient [Ca2+] to a steady state of around 0.2 microM. A vesicular ATP-dependent and vanadate-sensitive non-mitochondrial pool maintained this low [Ca2+] level. In the absence of ATP, a higher Ca2+ steady state of 0.6 microM was seen, exhibiting the characteristics of a mitochondrial Ca2+ "set point." Both pools were shown to act in concert to restore the previous ambient [Ca2+] following its elevation. Thus, the mitochondria participate with the other pool(s) in decreasing [Ca2+] to the submicromolar range whereas only the nonmitochondrial pool(s) lowers [Ca2+] to the basal level. The action of inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a few cell types was studied. IP3 released (detectable within 2 s) Ca2+ accumulated in the ATP-dependent pool(s) but had no effect on the mitochondria. The response was transient and resulted in desensitization toward subsequent IP3 additions. Under experimental conditions in which the ATP-dependent Ca2+ influx was blocked, the addition of IP3 resulted in a very large Ca2+ release from nonmitochondrial pool. The results strongly suggest that IP3 is a second messenger mediating intracellular Ca2+ mobilization in human neutrophils. Furthermore, the nonmitochondrial pool appears to have independent influx and efflux pathways for Ca2+ transport, a Ca2+ ATPase (the influx component) and an IP3-sensitive efflux component activated during Ca2+ mobilization.     

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.     

An ATP-dependent and inositol trisphosphate-sensitive Ca2+ pool linked with microfilaments of the parietal cell

In digitonin-permeabilized parietal cells, myo-inositol 1,4,5-trisphosphate (Ins P3) or Ca2+ ionophore (A23187) increased the cytosolic Ca2+ concentration due to the intracellular Ca2+ release. Addition of ATP decreased the cytosolic Ca2+ concentration due to the rapid Ca2+ re-uptake into the same or similar pool which releases Ca2+ from a non-mitochondrial location (measured by quin2/AM and 45Ca2+). Cytochalasin B failed to increase the cytosolic Ca2+ concentration in response to Ins P3 or A23187 and even failed to decrease the cytosolic Ca2+ concentration in response to ATP. This implies that the ATP-dependent and Ins P3-sensitive Ca2+ pool is linked with the microfilaments of the parietal cell. In intact parietal cells, A23187 increased the amino[14C]pyrine accumulation (an index of acid secretion), that was independent of medium Ca2+. This increase of acid secretion was inhibited by the pretreatment with cytochalasin B. This suggests that medium Ca2+-independent acid secretion (by A23187) is regulated by the microfilaments. Therefore, there is a close relationship between the intracellular Ca2+ metabolism, microfilaments and acid secretion.