H+ uptake increases GTP-induced connection of inositol 1,4,5-trisphosphate- and caffeine-sensitive calcium pools in pancreatic microsomal vesicles.

Evidence suggests that GTP but not GTP gamma S activates Ca2+ movement between myo-inositol 1,4,5-trisphosphate (IP3)-sensitive and -insensitive Ca2+ pools (1). Measuring 45Ca2+ uptake into pancreatic microsomal vesicles we have determined the sizes of three different Ca2+ pools which release Ca2+ in response 1) to IP3, 2) to caffeine, and 3) to both IP3 and caffeine ("common" Ca2+ pool). In the presence of GTP the size of the IP3-sensitive Ca2+ pool is decreased whereas the "common" Ca2+ pool is increased as compared to control Ca2+ pool sizes in the presence of GTP gamma S. This effect of GTP is inhibited by bafilomycin B1, a specific inhibitor of vacuolar type H+ ATPases (2). We conclude that GTP induced connection between IP3- and caffeine-sensitive Ca2+ pools is triggered by intravesicular acidification and involves function of small GTP-binding proteins, known to mediate interorganelle transfer.     

GTP-sensitivity of the energy-dependent Ca2+ storage pool in permeabilized pancreatic acinar cells

Isolated rabbit pancreatic acinar cells, permeabilized by saponin treatment and incubated in the presence of 0.1 microM free Ca2+, accumulated 3.3 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Part of this energy-dependent pool could be released by GTP in a polyethylene glycol-dependent manner. The kinetics of GTP-induced release of Ca2+ showed a biphasic pattern with an initial rapid phase followed by a sustained slower phase. In contrast, IP3-induced release of Ca2+ was completed within 30 s following addition of IP3. No reuptake of Ca2+ was observed following GTP- or IP3-induced release of Ca2+. The GTP effect was independent of IP3 and not inhibited by Ca2+, indicating that the IP3-operated Ca2+ channel is not involved in GTP-induced release of Ca2+. The size of the IP3-releasable pool was not affected by GTP, indicating that GTP, when added to permeabilized acinar cells, does not promote the coupling between IP3-insensitive and IP3-sensitive Ca2+ accumulating organelles. Thus, in permeabilized acinar cells, GTP and IP3 act on different Ca2+ sequestering pools. Interestingly, however, comparison of the size of the GTP-releasable pool with that of the IP3-releasable pool for the cell preparations used in the present study, revealed an inversed relationship, indicating that at the time of permeabilization the GTP-releasable pool can be coupled to a greater or lesser extent to the IP3-releasable pool. This suggests that, in the intact cell, a GTP-dependent mechanism may exist that controls the size of the IP3-releasable pool by coupling IP3-insensitive to IP3-sensitive organelles. Moreover, this suggests that the extent of coupling is preserved during permeabilization.     

Inositol 1,4,5-trisphosphate and intracellular Ca2+ homeostasis in clonal pituitary cells (GH3). Translocation of Ca2+ into mitochondria from a functionally discrete portion of the nonmitochondrial store

Ca2+-specific minielectrodes were used to monitor changes in the ambient free Ca2+ concentration [( Ca2+]a) maintained by the intracellular organelles of permeabilized GH3 cells. Mitochondria maintained a [Ca2+]a steady state of around 500 nM and displayed a very high capacity for Ca2+ uptake. A nonmitochondrial pool, tentatively identified as the endoplasmic reticulum (ER), displayed higher affinity for Ca2+ by maintaining a steady state of approximately 170 nM. The capacity of this pool was around 10 nmol/mg cell protein. Inositol 1,4,5-trisphosphate (InsP3) released Ca2+ specifically from the ER, with an EC50 of approximately 2 microM, and gave maximal release of around 4 nmol Ca2+/mg of cell protein. Repeated InsR3 additions under conditions allowing for functional mitochondrial transport resulted in successively attenuated peaks, leading eventually to the depletion of the InsP3 sensitive portion of the ER. However, Ca2+ could still be released from the total ER pool with the ATPase inhibitor, vanadate. This InsP3-insensitive store did not reaccumulate InsP3 releasable Ca2+ nor could it directly refill the sensitive pool. However, the attenuation of the InsP3 responses could be overcome by repleting the sensitive pool with exogenous Ca2+ or by inhibiting Ca2+ uptake into the mitochondria. The results suggest: 1) the ER is the major intracellular organelle buffering Ca2+ in nonstimulated GH3 cells; 2) InsP3 releases Ca2+ from only a portion of the ER; 3) the InsP3-sensitive and -insensitive ER pools are functionally distinct; 4) InsP3 addition results in a transfer of Ca2+ from the ER to the mitochondria.     

Intracellular calcium uptake activated by GTP. Evidence for a possible guanine nucleotide-induced transmembrane conveyance of intracellular calcium

The GTP-activated Ca2+ release process we recently described (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464) was revealed in the preceding report to operate via a mechanism likely to be induced by close membrane association but which appears not to involve membrane fusion (Chueh, S. H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864). To determine more about the GTP-activated Ca2+ translocation process, effects of GTP on cells loaded with Ca-oxalate were investigated. Using permeabilized cells of both the N1E-115 neuroblastoma and DDT1MF-2 smooth muscle cell lines, 10 microM GTP activates a profound uptake of Ca2+ in the presence of oxalate, as opposed to release observed without oxalate. GTP stimulation of Ca2+ uptake was observed at oxalate concentrations (2 mM) only slightly augmenting Ca2+ uptake without GTP; with 8 mM oxalate (which alone induces linear Ca2+ accumulation) GTP still increases the rate of uptake. GTP-activated uptake in the presence of oxalate is completely reversed by 1 mM vanadate. 3% polyethylene glycol enhances the effect of GTP although GTP-activated uptake is still observed without polyethylene glycol. The Km for GTP for activation of Ca2+ uptake is 0.9 microM. Uptake is not activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp); however, GTP gamma S (but not GppNHp) completely blocks the action of GTP. GDP gives a delayed uptake response which is blocked by ADP, indicating its action arises from conversion to GTP. In the presence of ADP, GDP blocks the action of GTP; guanosine 5'-O-(2-thio)diphosphate, which does not activate uptake, also blocks the action of GTP. These data reveal almost exact correlation between parameters affecting GTP-activated uptake and release, strongly suggesting the same process mediates both events. To explain the opposite effects of GTP in the absence and presence of oxalate, it is proposed that GTP activates a transmembrane conveyance of Ca2+ between oxalate-permeable and -impermeable compartments.     

Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth.

The intracellular Ca2+ pump inhibitor, thapsigargin, added to DDT1MF-2 smooth muscle cells in culture, irreversibly inhibited accumulation of Ca2+ within cells, permanently emptied the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool, and simultaneously induced profound alteration of cell growth. After only a brief (30-min) treatment of cultured cells with 3 microM thapsigargin followed by extensive washing, the total releasable InsP3-sensitive Ca2+ pool remained entirely empty, even after 7 days of culture without thapsigargin. After thapsigargin treatment, cells retained viability, usual morphology, and normal mitochondrial function. Despite the otherwise normal appearance and function of thapsigargin-treated cells, cell division was completely blocked by thapsigargin. DNA synthesis was completely inhibited when thapsigargin was added immediately after passaging, but was suppressed only slowly (4-6 h) when added to rapidly synthesizing cells (24 h after passaging). Protein synthesis was reduced by approximately 70% in thapsigargin-treated cells. The sensitivity of thapsigargin-mediated inhibition of cell division, DNA synthesis, protein synthesis, and Ca(2+)-pumping activity were all similar with the EC50 values for thapsigargin in each case being close to 10 nM. Upon application to DDT1MF-2 cells, thapsigargin transiently increased resting cytosolic Ca2+ (0.15 microM) to a peak of 0.3 microM within 50 s; thereafter, free Ca2+ declined to 0.2 microM by 150 s and continued to slowly decline toward resting levels. Cells treated with thapsigargin for 1-72 h in culture displayed normal resting cytosolic Ca2+ levels. However, application of thapsigargin or epinephrine to such cells resulted in no change in the intracellular Ca2+, indicating that the internal Ca2+ pool remained completely empty. These results suggest that emptying of Ca2+ from intracellular thapsigargin-sensitive Ca(2+)-pumping pools induces profound alteration of cell proliferation.     

Possible physiological role of guanosine triphosphate and inositol 1,4,5-trisphosphate in Ca2+ release in macrophages stimulated with chemotactic peptide.

The release of Ca2+ induced by inositol 1,4,5-trisphosphate (InsP3) in the presence of GTP was examined by using saponin-permeabilized macrophages. The origin and the amount of mobilized Ca2+ in intact macrophages stimulated with chemotactic peptide were also examined to assess the physiological significance of GTP and InsP3 on Ca2+-releasing activities. The total amount of Ca2+ released by 20 microM-A23187 from the unstimulated intact macrophages was 1.4 nmol/4 x 10(6) cells, and the mitochondrial uncoupler did not cause an efflux of Ca2+ from the cells. The Ca2+ accumulation by the non-mitochondrial pool(s) was inhibited by the presence of GTP, and the total amount of releasable Ca2+ (1.4 nmol/4 x 10(6) cells) was comparable with that accumulated by the non-mitochondrial pool(s) in the presence of GTP at a free Ca2+ concentration of 0.14 microM. The mobilized and subsequently effluxed Ca2+ in cells stimulated with chemotactic peptide was estimated to be 0.3 nmol/4 x 10(6) cells. Much the same amounts were released by about the half-maximal dose of InsP3 from the non-mitochondrial pool(s) of saponin-treated macrophages that had accumulated Ca2+ at a free concentration of 0.14 microM in the presence of GTP. These results suggest that the Ca2+-releasing activity induced by GTP may play a role in the long-term regulation of Ca2+ content in the non-mitochondrial pool(s) of macrophages, and that released by InsP3 can explain, quantitatively, the chemotactic-peptide-induced mobilization of Ca2+.     

Inositol trisphosphate and thapsigargin discriminate endoplasmic reticulum stores of calcium in rat brain

ATP dependent Ca2+ accumulation into oxalate-loaded rat brain microsomes is potently inhibited by thapsigargin with an IC50 of 2 nM and maximal inhibition at 10 nM. Approximately 15% of the total A23187-releasable microsomal calcium store is insensitive to thapsigargin concentrations up to 100 microM. Inositol-1,4,5-trisphosphate (IP3) maximally inhibits 40% of the net Ca2+ accumulation by whole brain microsomes. Its effects are non-additive with thapsigargin suggesting that the IP3-sensitive Ca2+ pool is a subset of the thapsigargin sensitive Ca2+ pool. Marked regional differences occur in Ca2+ transport rates and sensitivity to both thapsigargin and IP3.     

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.     

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.     

Analysis of Ca2+ fluxes and Ca2+ pools in pancreatic acini

45Ca2+ movements have been analysed in dispersed acini prepared from rat pancreas in a quasi-steady state for 45Ca2+. Carbamyl choline (carbachol; Cch) caused a quick 45Ca2+ release that was followed by a slower 45Ca2+ 'reuptake'. Subsequent addition of atropine resulted in a further transient increase in cellular 45Ca2+. The data suggest the presence of a Cch-sensitive 'trigger' pool, which could be refilled by the antagonist, and one or more intracellular 'storage' pools. Intracellular Ca2+ sequestration was studied in isolated acini pretreated with saponin to disrupt their plasma membranes. In the presence of 45Ca2+ (1 microM), addition of ATP at 5 mM caused a rapid increase in 45Ca2+ uptake exceeding the control by fivefold. Maximal ATP-promoted Ca2+ uptake was obtained at 10 microM Ca2+ (half-maximal at 0.32 microM Ca2+). In the presence of mitochondrial inhibitors it was 0.1 microM (half-maximal at 0.014 microM). 45Ca2+ release could still be induced by Cch but the subsequent reuptake was missing. The latter was restored by ATP and atropine caused further 45Ca2+ uptake. Electron microscopy showed electron-dense precipitates in the rough endoplasmic reticulum of saponin-treated cells in the presence of Ca2+, oxalate and ATP which were absent in intact cells or cells pretreated with A23187. The data suggest the presence of a plasma membrane-bound Cch-sensitive 'trigger' Ca2+ pool and ATP-dependent Ca2+ storage systems in mitochondria and rough endoplasmic reticulum of pancreatic acini. It is assumed that Ca2+ is taken up into these pools after secretagogue-induced Ca2+ release.U     

Calcium pools in Ehrlich carcinoma cells. A major, high affinity Ca pool is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin

To investigate the presence and the size of different non-mitochondria) Ca²⁺ pools of Ehrlich ascites tumor cells (EATCs) , digitonin-permeabilized cells were allowed to accumulate Ca²⁺ in the presence of mitochondrial inhibitors and treated with the reticular Ca²⁺-ATPase inhibitor thapsigargin, IP₃ and the Ca²⁺ ionophore A23187. Emptying of thapsigargin-sensitive Ca²⁺ stores prevented any Ca²⁺ release by IP₃, and, after IP₃ addition, little or no Ca²⁺ was released by thapsigargin. In both instances, a further Ca²⁺ release was accomplished by A23187. The IP₃-thapsigargin-sensitive pool and the residual A23187-sensitive one corresponded to approximately 60 and 37% of non-mitochondria) stored Ca²⁺, respectively. In intact EATCs, IP₃-dependent agonists and thapsigargin discharged Ca ²⁺ pools almost completely overlapping, and A32187 released a minor residual Ca²⁺ pool. The IP₃-insensitive pool appeared to have a relatively low affinity for Ca²⁺ (below 600 nM). The high affinity, IP₃-sensitive Ca²⁺ pool was discharged in a ‘quantal’ manner following step additions of sub maximal [IP₃], and the IP₃-induced fractional Ca²⁺ release was more marked at higher concentrations of stored (luminal) Ca²⁺, The IP₃-sensitive Ca²⁺ pool appeared to be devoid of the Ca²⁺-activated Ca²⁺ release channel since caffeine did not released any Ca²⁺ in intact and permeabilized EATCs, and Western blot analyses of EATC microsomal membranes failed to detect any known ryanodine receptor isoform.     

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.     

Calcium mobilization in permeabilized fibroblasts: effects of inositol trisphosphate, orthovanadate, mitogens, phorbol ester, and guanosine triphosphate

Utilizing a digitonin-permeabilized cell system, we have studied the release of calcium from a non-mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (+/- 6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3-sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP-dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down-regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.     

Inositol polyphosphates regulate Ca2+ efflux in a cardiac membrane subtype distinct from junctional sarcoplasmic reticulum

The membrane location and mechanism of inositol 1,3,4,5-tetrakisphosphate (InsP4)-regulated Ca2+ uptake in cardiac membrane vesicles was investigated. In canine and rat membranes separated by sucrose density gradient centrifugation, InsP4-regulated Ca2+ uptake was slightly more enriched in low density than in higher density membranes. Membranes supporting InsP4-regulated Ca2+ uptake were correspondingly enriched in type 1 InsP3 receptors. Junctional sarcoplasmic reticulum (J-SR), enriched in sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and ryanodine receptors, separated predominantly with higher density membranes. In membranes supporting InsP4-regulated Ca2+ uptake, Ca2+ uptake was facilitated by a high Ca2+ affinity carrier that was insensitive to thapsigargin. Ca2+ uptake in J-SR was mediated by thapsigargin-sensitive SERCA2a. Net Ca accumulation was enhanced by oxalate in both SR subtypes. Although Ca2+-carrier-mediated Ca2+ uptake was ATP independent, ATP indirectly regulated net Ca2+ accumulation by modifying Ca2+ efflux via a Ca2+ channel with properties of type 1 InsP3 receptors. In the presence of < or = 0.1 mM ATP, InsP4 enhanced Ca2+ accumulation whereas InsP4 inhibited Ca2+ uptake at higher ATP concentrations. In the presence of 0.15 mM ATP, InsP4 stimulated Ca2+ efflux from vesicles preloaded with Ca. Several other InsP4 isomers and 1,3,4-InsP3 also stimulated Ca2+ efflux but with slightly less potency than 1,3,4,5-InsP4. Ruthenium red enhanced net Ca accumulation by the Ca2+ carrier and reduced the potency of ATP, InsP4, and InsP3 to stimulate Ca2+ efflux in vesicles. In summary, this investigation shows that a Ca2+ carrier facilitates Ca loading in a sarcoplasmic reticulum subtype distinct from J-SR. InsP4 and InsP3 are proposed to regulate Ca2+ efflux in low density SR by acting on an ATP-modulated Ca2+ channel with properties of type 1 InsP3 receptors.     

Characterization of the inositol 1,4,5-trisphosphate-induced Ca2+ release in pancreatic beta-cells.

Pancreatic beta-cells isolated from obese-hyperglycaemic mice released intracellular Ca2+ in response to carbamoylcholine, an effect dependent on the presence of glucose. The effective Ca2+ concentration reached was sufficient to evoke a transient release of insulin. When the cells were deficient in Ca2+, the Ca2+ pool sensitive to carbamoylcholine stimulation was equivalent to that released by ionomycin. Unlike intact cells, cells permeabilized by high-voltage discharges failed to generate either inositol 1,4,5-triphosphate (InsP3) or to release Ca2+ after exposure to carbamoylcholine. However, the permeabilized cells released insulin sigmoidally in response to increasing concentrations of Ca2+. Also in the absence of functional mitochondria these cells exhibited a large ATP-dependent buffering of Ca2+, enabling the maintenance of an ambient Ca2+ concentration corresponding to about 150 nM even after several additional pulses of Ca2+. InsP3, maximally effective at 6 microM, promoted a rapid and pronounced release of Ca2+. The InsP3-sensitive Ca2+ pool was rapidly filled and lost its Ca2+ late after ATP depletion. The transient nature of the Ca2+ signal was not overcome by repetitive additions of InsP3. It was possible to restore the response to InsP3 after a delay of approx. 20 min, an effect which had less latency after the addition of Ca2+. These latter findings argue against degradation and/or desensitization as factors responsible for the transiency in InsP3 response. It is suggested that Ca2+ released by InsP3 is taken up by a part of the endoplasmic reticulum (ER) not sensitive to InsP3. On metabolism of InsP3, Ca2+ recycles to the InsP3-sensitive pool, implying that this pool indeed has a very high affinity for the ion. The presence of functional mitochondria did not interfere with the recycling process. The ER in pancreatic beta-cells is of major importance in buffering Ca2+, but InsP3 only modulates Ca2+ transport for a restricted period of time following immediately upon its formation. Thereafter the non-sensitive part of the ER takes over the continuous regulation of Ca2+ cycling.     

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)     

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.     

Granulocyte-macrophage colony-stimulating factor can stimulate macrophage proliferation via persistent activation of Na+/H+ antiport. Evidence for two distinct roles for Na+/H+ antiport activation.

Thapsigargin stimulates an increase of cytosolic free Ca2+ concentration [( Ca2+]c) in, and 45Ca2+ efflux from, a clone of GH4C1 pituitary cells. This increase in [Ca2+]c was followed by a lower sustained elevation of [Ca2+]c, which required the presence of extracellular Ca2+, and was not inhibited by a Ca2(+)-channel blocker, nimodipine. Thapsigargin had no effect on inositol phosphate generation. We used thyrotropin-releasing hormone (TRH) to mobilize Ca2+ from an InsP3-sensitive store. Pretreatment with thapsigargin blocked the ability of TRH to cause a transient increase in both [Ca2+]c and 45Ca2+ efflux. The block of TRH-induced Ca2+ mobilization was not caused by a block at the receptor level, because TRH stimulation of InsP3 was not affected by thapsigargin. Rundown of the TRH-releasable store by Ca2(+)-induced Ca2+ release does not appear to account for the action of thapsigargin on the TRH-induced spike in [Ca2+]c, because BAY K 8644, which causes a sustained rise in [Ca2+]c, did not block Ca2+ release caused by TRH. In addition, caffeine, which releases Ca2+ from intracellular stores in other cell types, caused an increase in [Ca2+]c in GH4C1 cells, but had no effect on a subsequent spike in [Ca2+]c induced by TRH or thapsigargin. TRH caused a substantial decrease in the amount of intracellular Ca2+ released by thapsigargin. We conclude that in GH4C1 cells thapsigargin actively discharges an InsP3-releasable pool of Ca2+ and that this mechanism alone causes the block of the TRH-induced increase in [Ca2+]c.     

Evidence for multiple intracellular calcium pools in GH4C1 cells: investigations using thapsigargin.

The actions of thapsigargin (Tg), a plant sesquiterpene lactone, on Ca2+ homeostasis were investigated in digitonin-permeabilized GH4C1 rat pituitary cells. Tg (1 microM) caused a rapid and sustained increase in ambient Ca2+ concentration [( Ca2+]) and inhibited the rise in [Ca2+] induced by subsequent addition of TRH (100 nM), inositol 1,4,5-trisphosphate (IP3, 10 microM), or the nonhydrolyzable GTP analogue guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S, 10 microM). However, neither IP3 nor GTP gamma S pretreatment, which themselves release sequestered Ca2+, prevented the Ca2+ accumulation induced by Tg. Pretreatment with heparin (100 micrograms/ml, 10 min), an IP3 receptor antagonist, did not affect Ca2+ accumulation induced by Tg, although it abolished the rise in [Ca2+] induced by IP3. The ability of Tg to increase [Ca2+] was dependent on added ATP. We conclude that, in GH4C1 cells, Tg acts, in part, on TRH-, IP3- and GTP gamma S-sensitive Ca2+ pools; however, Tg also acts on an ATP-dependent pool of intracellular Ca2+ which is not sensitive to TRH, IP3 or GTP gamma S, indicating a complexity of intracellular Ca2+ pools not previously appreciated in these cells.     

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.