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.     

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)     

Actions of inositol phosphates on Ca2+ pools in guinea-pig hepatocytes.

In permeabilized hepatocytes, inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate and inositol 4,5-bisphosphate induced rapid release of Ca2+ from an ATP-dependent, non-mitochondrial vesicular pool, probably endoplasmic reticulum. The order of potency was inositol 1,4,5-trisphosphate greater than inositol 2,4,5-trisphosphate greater than inositol 4,5-bisphosphate. The Ca2+-releasing action of inositol 1,4,5-trisphosphate is not inhibited by high [Ca2+], nor is it dependent on [ATP] in the range of 50 microM-1.5 mM. These results suggest a role for inositol 1,4,5-trisphosphate as a second messenger in hormone-induced Ca2+ mobilisation, and that a specific receptor is involved in the Ca2+-release mechanism.     

Ca(2+)-induced Ca2+ release in insulin-secreting cells.

The sulphydryl reagent thimerosal (50 microM) released Ca2+ from a non-mitochondrial intracellular Ca2+ pool in a dose-dependent manner in permeabilized insulin-secreting RINm5F cells. This release was reversed after addition of the reducing agent dithiothreitol. Ca2+ was released from an Ins(1,4,5)P3-insensitive pool, since release was observed even after depletion of the Ins(1,4,5)P3-sensitive pool by a supramaximal dose of Ins(2,4,5)P3 or thapsigargin. The Ins(1,4,5)P3-sensitive pool remained essentially unaltered by thimerosal. Thimerosal-induced Ca2+ release was potentiated by caffeine. These findings suggest the existence of Ca(2+)-induced Ca2+ release also in insulin-secreting cells.     

Identification of intracellular calcium pools. Selective modification by thapsigargin

Recent studies have identified inositol 1,4,5-tris-phosphate(InsP3)-sensitive and -insensitive Ca2+ pools and a GTP-dependent mechanism that transfers Ca2+ between them. Here, the Ca2+ pump-inhibitory sesquiterpene lactone, thapsigargin, is shown to distinguish these two Ca2+ pools and identify a third Ca2+ pumping pool unresponsive to InsP3 or GTP. Using saponin-permeabilized DDT1MF-2 smooth muscle cells, approximately 75% of total intracellular ATP-dependent Ca2+ accumulation is blocked by thapsigargin with an IC50 of 30 nM. In contrast, 1 mM vanadate or 5 microM A23187 block 100% of Ca2+ accumulation. The thapsigargin-responsive Ca2+ pool corresponds exactly to that released by 10 microM InsP3 in the presence of 10 microM GTP. Indeed, addition of InsP3 with GTP has no effect on Ca2+ accumulated in the presence of 3 microM thapsigargin whereas A23187 releases all the remaining Ca2+. Added after maximal Ca2+ uptake, thapsigargin induces only slow Ca2+ release consistent with blockade of pumping activity. Unlike InsP3, the action of thapsigargin is entirely heparin insensitive. The large increment in Ca2+ uptake caused by 12 mM oxalate is completely reversed by thapsigargin, indicating that thapsigargin functions on an oxalate-permeable pool. Moreover, the still larger uptake induced by GTP in the presence of oxalate is also completely reversed by either thapsigargin or InsP3. The results indicate that thasigargin blocks Ca2+ uptake into two discrete pools: the InsP3-sensitive, oxalate-permeable Ca2+ pool and the InsP3-insensitive, oxalate-impermeable Ca2+ pool that can be "recruited" into the InsP3-sensitive pool by GTP-dependent Ca2+ translocation (Ghosh, T. K., Mullaney, J.M., Tarazi, F.I., and Gill, D.L. (1989) Nature 340, 236-239). Additionally, a third Ca2+ pool is defined, unreleasable by InsP3 or GTP, and containing a thapsigargin-insensitive Ca2+ pump.     

Free cytoplasmic Ca2+ concentration oscillations in thapsigargin-treated parotid acinar cells are caffeine- and ryanodine-sensitive.

The microsomal Ca-ATPase inhibitor thapsigargin induces in rat salivary acinar cells [Ca2+]i oscillations which, though similar to those activated by agonists, are independent of inositol phosphates or inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores (Foskett, J. K., Roifman, C., and Wong, D. (1991) J. Biol. Chem. 266, 2778-2782). To examine whether the oscillation mechanism resides in another, thapsigargin- and IP3-insensitive intracellular store, we examined the effects of caffeine and ryanodine, known modulators of Ca2+ release from sarcoplasmic reticulum in excitable cells. Oscillations were induced by caffeine (1-20 mM) in nonoscillating thapsigargin-treated acinar cells, which required the continued presence of caffeine, whereas caffeine was without effect or reduced oscillation amplitude in oscillating cells. Ryanodine (10-50 microM) inhibited oscillations in most of the cells. These results suggest that Ca2+ oscillations in parotid acinar cells are driven by periodic Ca2+ release from an IP3-insensitive Ca2+ store with properties similar to sarcoplasmic reticulum of excitable cells.     

Reassessment of the Ca2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurement of inositol 1,4,5-trisphosphate and Ca2+ in single cells

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca(2+) within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca(2+), increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca(2+) concentration. Under nominally Ca(2+)-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca(2+) concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca(2+) concentration absent. Depletion of intracellular Ca(2+) stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca(2+) and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca(2+) concentration. However, prolonged activation of phospholipase C requires extracellular Ca(2+), while the initial burst of activity is highly dependent on Ca(2+) mobilization from intracellular stores.     

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.     

Prolonged inactivation of nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release mediates a spatiotemporal Ca2+ memory

Although numerous extracellular stimuli are coupled to increases in intracellular Ca(2+), different stimuli are thought to achieve specificity by eliciting different spatiotemporal Ca(2+) increases. We investigated the effect of nicotinic acid adenine dinucleotide phosphate (NAADP) inactivation on spatiotemporal Ca(2+) signals in intact sea urchin eggs. The photorelease of NAADP but not inositol 1,4,5-trisphosphate or cyclic ADP-ribose resulted in self-inactivation. When NAADP was released first locally and subsequently globally, the spatial pattern of the first response shaped that of the second. Specifically, the local release of NAADP created a Ca(2+) gradient that was reversed during the subsequent global release of NAADP. Neither cyclic ADP-ribose nor inositol 1,4,5-trisphosphate showed a similar effect. In contrast to homogenates, NAADP inactivation was reversible in intact eggs with resensitization occurring in approximately 20 min. Because initial NAADP responses affect later responses, NAADP can serve as a mechanism for a Ca(2+) memory that has both spatial and temporal components. This NAADP-mediated Ca(2+) memory provides a novel mechanism for cells to control spatiotemporal Ca(2+) increases.     

Modulation of intracellular free Ca2+ concentration by IP3-sensitive and IP3-insensitive nonmitochondrial Ca2+ pools

Intracellular Ca2+ pools play an important role in the adjustment of cytosolic free Ca2+ concentrations. This review summarizes the recent knowledge on receptor-mediated Ca2+ release and Ca2+ uptake mechanisms in Ca2+ stores of exocrine cells taking the exocrine pancreas and the parotid gland as an example. The intracellular mediator for agonist-induced Ca2+ release is inositol 1,4,5-trisphosphate (IP3) which acts by opening Ca2+ channels from the endoplasmic reticulum or a more specialized organelle called 'calciosome'. This Ca2+ release is the major event to increase cytosolic free Ca2+ concentrations of exocrine glands from a resting level of approximately 10(-7) mol/l to approximately 10(-6) mol/l. Subsequently also Ca2+ influx from the extracellular fluid into the cell is increased which involves the action of inositol 1,3,4,5-tetrakisphosphate (IP4). Intracellular nonmitochondrial Ca2+ reuptake occurs into IP3-sensitive (IsCaP) as well as into IP3-insensitive Ca2+ pools Ca2+ pools (IisCaP). While Ca2+ uptake into the IisCaP is mediated by a vanadate-sensitive Ca2+ pump, Ca2+ uptake into the IsCaP is mediated by a Ca2+/H+ exchanger at the expense of an H+ gradient which is established by a vacuolar type H+ pump present in the same Ca2+ pool. During stimulation both Ca2+ pools, IsCaP and IisCaP, are probably connected, the nature of which has not yet been clarified. It is suggested that GTP and/or IP4 control Ca2+ conveyance between intracellular Ca2+ pools by forming Ca2+-carrying junctions between membranes. Other models propose that Ca2+, which is released by IP3, induces Ca2+ release from another Ca2+ pool. Taking into account that H+ transport is present in IP3-sensitive Ca2+ pools the possibility of pH-regulated Ca2+ channels in the IisCaP, located in close neighbourhood to the IsCaP, is also considered.     

Multiple effects of caffeine on Ca2+ release and influx in human B lymphocytes

Caffeine has been used as a pharmacological tool to study the ryanodine receptor (RYR)-mediated Ca2+ release from caffeine-sensitive, inositol 1,4,5,-trisphosphate (IP3)-insensitive pools. In the present study, we demonstrate multiple effects of caffeine on Ca2+ homeostasis in human B lymphocytes. Although B cells express a functional RYR, which can be activated by 4-chloro-m-cresol following depletion of IP(3)-sensitive pools, caffeine does not activate RYR-mediated Ca2+ release. Instead, caffeine dose-dependently inhibited IP3 receptor (IP3R)-mediated Ca2+ release, RYR-mediated Ca2+ release and B cell receptor-initiated Ca2+ influx, while high concentrations of caffeine (> or = 25 mM) induced a Ca2+ influx. In contrast with its ability to suppress receptor-stimulated Ca2+ influx, caffeine had no significant effect on the store-operated Ca2+ (SOC) channel-dependent Ca2+ influx induced by thapsigargin. Thus, caffeine may act as an inhibitor on a single or multiple site(s) responsible for regulating the IP3R channel, RYR channel and presumably the receptor-mediated SOC channel. The present report may be the first demonstration of multiple effects of caffeine on Ca2+ mobilization in single cell type. Our results suggest the need for caution regarding use of caffeine simply as a RYR-activator to study Ca2+ homeostasis in eucaryotic cells.     

Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells. Evidence that an intracellular calcium pool and not an inositol phosphate regulates calcium fluxes at the plasma membrane

The depletion of an inositol 1, 4,5-trisphosphate-sensitive intracellular Ca2+ pool has been proposed to be the signal for Ca2+ entry in agonist-activated cells. Consistent with this idea, thapsigargin, which releases intracellular Ca2+ without inositol phosphate formation, has been reported to activate Ca2+ entry in certain cells. We now report the effects of thapsigargin on Ca2+ entry in parotid acinar cells. In fura-2-loaded parotid acinar cells, thapsigargin caused a sustained elevation of [Ca2+], but did not increase inositol phosphate formation. In the absence of extracellular Ca2+, the increase in [Ca2+], was transient, suggesting that thapsigargin activates both the release of Ca2+ from intracellular stores and the entry of Ca2+ from the extracellular space. In the absence of extracellular Ca2+, pretreatment with methacholine, an agonist believed to mobilize Ca2+ through the production of inositol 1,4,5-trisphosphate, inhibited but did not completely block the response to thapsigargin; likewise, pretreatment with thapsigargin inhibited the response to methacholine. In permeabilized cells, thapsigargin gradually released Ca2+, whereas inositol 1,4,5-trisphosphate caused a rapid and transient discharge of Ca2+. The simultaneous addition of thapsigargin with inositol 1,4,5-trisphosphate evoked a maximum Ca2+ release similar to that for inositol 1,4,5-trisphosphate alone, but the reuptake seen with inositol 1,4,5-trisphosphate alone was abolished. In intact cells, methacholine and thapsigargin together produced a greater initial release of Ca2+ than either alone, but they were not additive in the sustained phase of Ca2+ mobilization. These results demonstrate that the mechanisms for activation of Ca2+ entry by thapsigargin and methacholine are the same and are consistent with the idea that entry is initiated by the depletion of the intracellular inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. The results also indicate that, in contrast to previously proposed models, Ca2+ entry into agonist-activated cells occurs directly across the plasma membrane to the cytoplasm rather than through a cycle of uptake and release by the intracellular Ca2+ pool.     

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.     

Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca(2+)-induced Ca2+ release and Ca2+ oscillations in hamster eggs.

The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.     

Properties of different Ca2+ pools in permeabilized rat thymocytes

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.     

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors identify a novel calcium pool in the central nervous system

Ca2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca2+ ATPase isoforms, which are all selectively inhibited by nanomolar concentrations of thapsigargin. Using ATP/Mg2+-dependent 45Ca2+ transport in rat brain microsomes, tissue sections, and permeabilized cells, as well as Ca2+ imaging in living cells we distinguish two ER Ca2+ pools in the rat CNS. Nanomolar levels of thapsigargin blocked one component of brain microsomal 45Ca2+ transport, which we designate as the thapsigargin-sensitive pool (TG-S). The remaining component was only inhibited by micromolar thapsigargin, and thus designated as thapsigargin resistant (TG-R). Ca2+ ATPase and [32P]phosphoenzyme assays also distinguished activities with differential sensitivities to thapsigargin. The TG-R Ca2+ uptake displayed unique anion permeabilities, was inhibited by vanadate, but was unaffected by sulfhydryl reduction. Ca2+ sequestered into the TG-R pool could not be released by inositol-1,4,5-trisphosphate, caffeine, or cyclic ADP-ribose. The TG-R Ca2+ pool had a unique anatomical distribution in the brain, with selective enrichment in brainstem and spinal cord structures. Cell lines that expressed high levels of the TG-R pool required micromolar concentrations of thapsigargin to effectively raise cytoplasmic Ca2+ levels. TG-R Ca2+ accumulation represents a distinct Ca2+ buffering pool in specific CNS regions with unique pharmacological sensitivities and anatomical distributions.     

Characterization of Ca2+ release from heterogeneous Ca2+ stores in sarcoplasmic reticulum isolated from arterial and gastric smooth muscle

Ca2+ transport was investigated in vesicles of sarcoplasmic reticulum subfractionated from bovine main pulmonary artery and porcine gastric antrum using digitonin binding and zonal density gradient centrifugation. Gradient fractions recovered at 15-33% sucrose were studied as the sarcoplasmic reticulum component using Fluo-3 fluorescence or 45Ca2+ Millipore filtration. Thapsigargin blocked active Ca2+ uptake and induced a slow Ca2+ release from actively loaded vesicles. Unidirectional 45Ca2+ efflux from passively loaded vesicles showed multicompartmental kinetics. The time course of an initial fast component could not be quantitatively measured with the sampling method. The slow release had a half-time of several minutes. Both components were inhibited by 20 microM ruthenium red and 10 mM Mg2+. Caffeine, inositol 1,4,5-trisphosphate, ATP, and diltiazem accelerated the slow component. A Ca2+ release component activated by ryanodine or cyclic adenosine diphosphate ribose was resolved with Fluo-3. Comparison of tissue responses showed that the fast Ca2+ release was significantly smaller and more sensitive to inhibition by Mg2+ and ruthenium red in arterial vesicles. They released more Ca2+ in response to inositol 1,4,5-trisphosphate and were more sensitive to activation by cyclic adenosine diphosphate ribose. Ryanodine and caffeine, in contrast, were more effective in gastric antrum. In each tissue, the fraction of the Ca2+ store released by sequential application of caffeine and inositol 1,4,5-trisphosphate depended on the order applied and was additive. The results indicate that sarcoplasmic reticulum purified from arterial and gastric smooth muscle represents vesicle subpopulations that retain functional Ca2+ channels that reflect tissue-specific pharmacological modulation. The relationship of these differences to physiological responses has not been determined.     

Characteristics of bile acid-mediated Ca2+ release from permeabilized liver cells and liver microsomes

Saponin-treated liver cells and a microsomal fraction were used to characterize the mechanism of the Ca2+ release induced by different bile acids. The saponin-treated cells accumulated 0.8-1 nmol/mg of protein of the medium Ca2+ in a nonmitochondrial, high affinity, and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool. Three of five bile acids tested, lithocholate and the conjugates taurolithocholate and taurolithocholate sulfate, released 85% of the Ca2+ pool within 45-60 s and with ED50 from 16 to 28 microM. Ins(1,4,5)P3 released 80% from the same Ca2+ pool with an ED50 of 0.3 microM. The Ca2+-Mg2+-ATPase inhibitor vanadate (1 mM) had no effect on the Ca2+ released by the bile acids and Ins(1,4,5)P3. The Ins(1,4,5)P3-binding antibiotic neomycin (1 mM) and the receptor competitor heparin (16 micrograms/ml) abolished the releasing effect of Ins(1,4,5)P3 but had no effect on the bile acid-mediated Ca2+ release. The 45Ca2+ accumulated by the microsomal fraction (8 nmol of 45Ca2+/mg of protein) was released by the bile acids within 45-90 s and with an ED50 of 17 microM. In contrast, the bile acids had no effect on the Ca2+ permeability of other natural and artificial membranes. The resting 45Ca2+ influx of intact cells (0.45 nmol/mg of protein/min), the 45Ca2+ accumulated by mitochondria (2-13 nmol of 45Ca2+/mg of protein), and the 45Ca2+ trapped in sonicated phosphatidylcholine vesicles (5 mM 45Ca2+) were not altered by the different bile acids. These results suggest that the Ca2+ release initiated by lithocholate and its conjugates results from a direct action on the Ca2+ permeability of the Ins(1,4,5)P3-sensitive pool. It is not mediated by Ins(1,4,5)P3 or via activation of the Ins(1,4,5)P3 receptor, and it is specific for the membrane of the internal pool.     

Spontaneous [Ca2+]i fluctuations in rat chromaffin cells do not require inositol 1,4,5-trisphosphate elevations but are generated by a caffeine- and ryanodine-sensitive intracellular Ca2+ store

A considerable fraction (65%) of single rat chromaffin cells loaded with the fluorescent [Ca2+]i indicator fura-2 exhibited spontaneous rhythmic fluctuations with an average period of approximately 100 s. Parallel patch clamp experiments as well as fura-2 experiments carried out in Ca2(+)-free and other modified media in the presence of Ca2+ and Na+ channel blockers indicated an origin from intracellular stores. Appropriate concentrations of agonists (bradykinin and histamine) for receptors (B2 and H1) that trigger generation of inositol 1,4,5-trisphosphate induced increased fluctuation frequency, recruitment of silent cells, and large [Ca2+]i changes at high doses. These effects were blocked by cell pretreatment with neomycin, a drug that inhibits inositol 1,4,5-trisphosphate generation. In contrast, spontaneous fluctuations and the effects of another drug, caffeine, which also induced increased frequency and recruitment, were unaffected by neomycin. Ryanodine caused first a prolongation and then (approximately 10 min) a block of both spontaneous fluctuations and caffeine effects, where the single transients after bradykinin and histamine were maintained. Caffeine and ryanodine are known to affect selectively the process of calcium-induced Ca2+ release; this is the first demonstration of [Ca2+]i fluctuation activity arising from Ca2(+)-induced Ca2+ release in nonmuscle cells with no strict requirement for inositol 1,4,5-trisphosphate involvement.