Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 microM IP3 was half maximally inhibited at 0.5 microM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 microM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTP gamma S could not replace GTP. Desensitization still occurred when GTP gamma S was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release.     

Cyclic ADP-ribose and inositol 1,4,5-trisphosphate mobilizes Ca from distinct intracellular pools in permeabilized lacrimal acinar cells

In permeabilized lacrimal acinar cells, cyclic ADP-ribose (cADP-ribose) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) release Ca²⁺ in a dose dependent manner from distinct thapsigargin-sensitive Ca²⁺ pools. Ryanodine specifically blocks the Ca²⁺ response to cADP-ribose, whereas heparin strongly reduces the response to Ins(1,4,5)P₃ application. GTP causes a rapid Ca²⁺ release by a ryanodine- and heparin-insensitive mechanism and potentiates Ins(1,4,5)P₃ but not cADP-ribose evoked Ca²⁺ release. It is estimated that cADP-ribose can release 16 μmol Ca²⁺/I cells, whereas Ins(1,4,5)P₃ can mobilize 55 μmol Ca²⁺/I cells. The results suggest that cADP-ribose and Ins(1,4,5)P₃ release Ca²⁺ from distinct internal stores and that a third Ca²⁺ pool exists which can selectively interact with the Ins(1,4,5)P₃-sensitive Ca²⁺ store by a GTP-mediated process.     

Ca2(+)-sensitivity of inositol 1,4,5-trisphosphate-mediated Ca2+ release in permeabilized pancreatic acinar cells.

Hormonal and phorbol ester pretreatment of pancreatic acinar cells markedly decreases the Ins(1,4,5)P3-induced release of actively stored Ca2+ [Willems, Van Den Broek, Van Os & De Pont (1989) J. Biol. Chem. 264, 9762-9767]. Inhibition occurred at an ambient free Ca2+ concentration of 0.1 microM, suggesting a receptor-mediated increase in Ca2(+)-sensitivity of the Ins(1,4,5)P3-operated Ca2+ channel. To test this hypothesis, the Ca2(+)-dependence of Ins(1,4,5)P3-induced Ca2+ release was investigated. In the presence of 0.2 microM free Ca2+, permeabilized cells accumulated 0.9 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Uptake into this pool increased 2.2- and 3.3-fold with 1.0 and 2.0 microM free Ca2+ respectively. At 0.2, 1.0 and 2.0 microM free Ca2+, Ins(1,4,5)P3 maximally released 0.53 (56%), 0.90 (44%) and 0.62 (20%) nmol of Ca2+/mg of acinar protein respectively. Corresponding half-maximal stimulatory Ins(1,4,5)P3 concentrations were calculated to be 0.5, 0.6 and 1.4 microM, suggesting that the affinity of Ins(1,4,5)P3 for its receptor decreases beyond 1.0 microM free Ca2+. The possibility that an inhibitory effect of sub-micromolar Ca2+ is being masked by the concomitant increase in size of the releasable store is excluded, since Ca2+ release from cells loaded in the presence of 0.1 or 0.2 microM free Ca2+ and stimulated at higher ambient free Ca2+ was not inhibited below 1.0 microM free Ca2+. At 2.0 and 10.0 microM free Ca2+, Ca2+, Ca2+ release was inhibited by approx. 30% and 75% respectively. The results presented show that hormonal pretreatment does not lead to an increase in Ca2(+)-sensitivity of the release mechanism. Such an increase in Ca2(+)-sensitivity to sub-micromolar Ca2+ is required to explain sub-micromolar oscillatory changes in cytosolic free Ca2+ by a Ca2(+)-dependent negative-feedback mechanism.     

The type 2 inositol (1,4,5)-trisphosphate (InsP3) receptor determines the sensitivity of InsP3-induced Ca2+ release to ATP in pancreatic acinar cells

Calcium release through inositol (1,4,5)-trisphosphate receptors (InsP(3)R) is the primary signal driving digestive enzyme and fluid secretion from pancreatic acinar cells. The type 2 (InsP(3)R2) and type 3 (InsP(3)R3) InsP(3)R are the predominant isoforms expressed in acinar cells and are required for proper exocrine gland function. Both InsP(3)R2 and InsP(3)R3 are positively regulated by cytosolic ATP, but InsP(3)R2 is 10-fold more sensitive than InsP(3)R3 to this form of modulation. In this study, we examined the role of InsP(3)R2 in setting the sensitivity of InsP(3)-induced Ca(2+) release (IICR) to ATP in pancreatic acinar cells. IICR was measured in permeabilized acinar cells from wild-type (WT) and InsP(3)R2 knock-out (KO) mice. ATP augmented IICR from WT pancreatic cells with an EC(50) of 38 mum. However, the EC(50) was 10-fold higher in acinar cells isolated from InsP(3)R2-KO mice, indicating a role for InsP(3)R2 in setting the sensitivity of IICR to ATP. Consistent with this idea, heterologous expression of InsP(3)R2 in RinM5F cells, which natively express predominately InsP(3)R3, increased the sensitivity of IICR to ATP. Depletion of ATP attenuated agonist-induced Ca(2+) signaling in WT pancreatic acinar cells. This effect was more profound in acinar cells prepared from InsP(3)R2-KO mice. These data suggest that the sensitivity of IICR to ATP depletion is regulated by the particular complement of InsP(3)R expressed in an individual cell. The effects of metabolic stress on intracellular Ca(2+) signals can therefore be determined by the relative amount of InsP(3)R2 expressed in cells.     

Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release in permeabilized pancreatic acinar cells by hormonal and phorbol ester pretreatment

Isolated rabbit pancreatic acinar cells, permeabilized by saponin treatment and incubated in the presence of 0.1 microM free Ca2+, accumulated 0.9-1.5 nmol of Ca2+/mg acinar protein in an energy-dependent pool. Uptake into this pool was not altered by pretreatment of acinar cells with the Ca2+ mobilizing secretagogues carbamylcholine and cholecystokinin-octapeptide or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, indicating that the Ca2+ pump of the internal Ca2+ store was not affected by prolonged activation of the Ca2+ messenger system. Inositol 1,4,5-trisphosphate (1,4,5-IP3) caused a rapid decrease in Ca2+ content of the internal Ca2+ store. The response was maximal within 30 s following addition of 1,4,5-IP3 and no reuptake of Ca2+ was observed over the next 60 s. Up to 55% of the amount of Ca2+ stored in the energy-dependent pool was 1,4,5-IP3 releasable with an EC50 of 1.0 microM. Pretreatment of acinar cells with carbamylcholine or cholecystokinin-octapeptide significantly reduced the effectivity of 1,4,5-IP3 to release Ca2+ from the internal store. The dose-response curve for 1,4,5-IP3-induced release of actively stored Ca2+ from carbamylcholine-treated acinar cells showed both a rightward shift (EC50 value of 1.7 microM) and a decreased maximal response (maximal release value of 44%), which suggests that the affinity of 1,4,5-IP3 for its receptor as well as the number of 1,4,5-IP3 receptors or 1,4,5-IP3-operated Ca2+ channels was reduced upon prolonged activation of the Ca2+ messenger system. Moreover, analysis of the release values in a Hill plot suggested positive cooperativity for 1,4,5-IP3-induced Ca2+ release from the internal store (n values of 1.3 and 1.7 for saline- and carbamylcholine-treated permeabilized acinar cells, respectively). Pretreatment of acinar cells with 12-O-tetradecanoylphorbol 13-acetate partly mimicked the inhibitory effect of carbamylcholine on 1,4,5-IP3-induced release of actively stored Ca2+ in that the dose-response curve was shifted to the right but the maximal response was not affected, suggesting that the effects of carbamylcholine were at least in part mediated by protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

The inositol 1,4,5-trisphosphate (InsP3) receptor

Conclusion In this review, we have described the functional properties and regulation of the InsP₃R. Not all aspects of InsP₃R function and regulation were covered, the main focus was on the most recent and physiologically important data. Information about the structure, heterogeneity, functional properties, and regulation of the InsP₃R is useful for understanding the spatiotemporal aspects of Ca signaling. The combination of biochemical, biophysical and molecular biological techniques has revealed the intricacies of the InsP₃R over the past decade. However, questions about the functional differences between various isoforms and splice variants of the InsP₃R, the structural determinants responsible for regulation of InsP₃R by Ca and ATP, the functional effects of InsP₃R phosphorylation and many others remain to be elucidated. Future investigations can be expected to provide answers to these important questions.We thank S. Bezprozvannaya for expert technical assistance. This work was supported by National Institutes of Health grants HL 33026 and GM 39029, and a Grant-in-Aid from the Patrick and Catherine Weldon Donaghue Medical Research Foundation.     

Relationship between secretagogue-induced Ca2+ release and inositol polyphosphate production in permeabilized pancreatic acinar cells

We have previously shown that inositol trisphosphate (IP3) releases Ca2+ from a nonmitochondrial pool of permeabilized rat pancreatic acinar cells (Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1984) Nature 306, 67-69). This pool was later identified as endoplasmic reticulum (Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F., and Schulz, I. (1984) J. Membr. Biol. 81, 241-253). As IP3 is produced by hydrolysis of phosphatidylinositol bisphosphate on activation of many "Ca2+-mobilizing receptors," our observation supported the proposal that IP3 functions as a second messenger to release Ca2+ from the endoplasmic reticulum. We have here used the same preparation of permeabilized acinar cells to study the relationship of secretagogue-induced Ca2+ release and IP3 production. We show that: 1) secretagogue-induced Ca2+ release in permeabilized cells is accompanied by a parallel production of inositol trisphosphate. 2) When the secretagogue-induced increase in intracellular free Ca2+ concentration was abolished by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering, secretagogue-induced IP3 production was unimpaired. 3) When secretagogue-induced IP3 production was reduced by inhibiting phospholipase C with neomycin, secretagogue-induced Ca2+ release was also abolished. 4) When the IP3 breakdown was reduced either by lowering the free Mg2+ concentration of the incubation medium or by adding 2.3-diphosphoglyceric acid, the rise in IP3 and the release of Ca2+ induced by secretagogues were both increased. These results further support the role of IP3 as a second messenger to induce Ca2+ mobilization.     

Ca signalling in exocrine acinar cells: the diffusional properties of cellular inositol 1,4,5-trisphosphate and its role in the release of Ca

The correlation between acetylcholine induced changes in the intracellular free, Ca²⁺ concentration ([Ca²⁺]_i), and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) content in isolated acini from the rat parotid and lacrimal glands was investigated. Applying digital image processing on Fura-2 loaded acini, we observed that Ca²⁺ increases either simultaneously throughout the acinar configurations or that occasionally, the rise near the lumen can precede the rise near the basal part by 50–100 ms. Measurements on cell suspensions revealed a correlation between changes in [Ca²⁺]_i and changes in the cellular Ins(1,4,5)P₃ content, and it is concluded that in the individual cells Ins(1,4,5)P₃ is released to the cytosol within the first second after stimulation. Applying a diffusion coefficient for cytoplasmic Ins(1,4,5)P₃ of 2.83 × 10⁻⁶ cm²/s (Allbritton et al., 1992, Science, 258, 1812–1815), we have calculated the concentration profile for this messenger in a sphere with a radius of 10 μm where Ins(1,4,5)P₃ is released in the center following a monoexponential function with a rate constant of 4 s⁻¹. Assuming that Ins(1,4,5)P₃ concentrations of 1 or 5% of the maximum value is able to release Ca²⁺, we calculated that Ca²⁺ waves can appear at a rate of 100 or 40 μm/s. The present data are consistent with Ins(1,4,5)P₃ being a cellular messenger, that by diffusion, initiates the Ca²⁺ release from the cellular pools within the first fraction of a second.     

Stochastic properties of Ca(2+) release of inositol 1,4,5-trisphosphate receptor clusters

Intracellular Ca(2+) release is controlled by inositol 1,4,5-trisphosphate (IP(3)) receptors or ryanodine receptors. These receptors are typically distributed in clusters with several or tens of channels. The random opening and closing of these channels introduces stochasticity into the elementary calcium release mechanism. Stochastic release events have been experimentally observed in a variety of cell types and have been termed sparks and puffs. We put forward a stochastic version of the Li-Rinzel model (the deactivation binding process is described by a Markovian scheme) and a computationally more efficient Langevin approach to model the stochastic Ca(2+) oscillation of single clusters. Statistical properties such as Ca(2+) puff amplitudes, lifetimes, and interpuff intervals are studied with both models and compared with experimental observations. For clusters with tens of channels, a simply decaying amplitude distribution is typically observed at low IP(3) concentration, while a single peak distribution appears at high IP(3) concentration.     

Ruthenium red selectively depletes inositol 1,4,5-trisphosphate-sensitive calcium stores in permeabilized rabbit pancreatic acinar cells

Rabbit pancreatic acinar cells, permeabilized by saponin treatment, rapidly accumulated 3.5 nmol of Ca²⁺/mg protein in an energy-dependent pool when incubated at an ambient free Ca²⁺ concentration of 100 nm. Maximal loading of the internal stores was reached at 10 min and remained unchanged thereafter. Complete inhibition of the Ca²⁺ pump with thapsigargin revealed that this plateau was the result of a steady-state between slow Ca²⁺ efflux and ATP-driven Ca²⁺ uptake. Sixty percent of the pool could be released by Ins(1,4,5)P₃, whereas GTP released another twenty percent. The striking finding of this study is that the energy-dependent store could also be released by ruthenium red. Uptake experiments in the presence of ruthenium red revealed that the dye, at concentrations below 100 m, selectively reduced the size of the Ins(1,4,5)P₃-releasable pool. Ruthenium red had no effect on the half-maximal stimulatory concentration of Ins(1,4,5)P₃. At concentrations beyond 100 m, the dye also affected the GTP-releasable pool. Comparison with thapsigargin revealed that ruthenium red released Ca²⁺ from stores loaded to steady-state at a rate markedly faster than can be explained by inhibition of the ATPase alone. From the data presented, we concluded that ruthenium red selectively releases Ca²⁺ from the Ins(1,4,5)P₃-sensitive store by activating a Ca²⁺ release channel, whereas Ca²⁺ release from the GTP-sensitive store is predominantly caused by inhibition of the Ca²⁺ pump. The postulated ruthenium red-sensitive Ca²⁺ release channel might be similar to the ryanodine-receptor in muscle.The research of Dr. P.H.G.M. Willems has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences.     

Inositol 1,3,4,5-tetrakisphosphate causes release of Ca2+ from permeabilized mouse lymphoma L1210 cells by its conversion into inositol 1,4,5-trisphosphate.

Addition of Ins(1,3,4,5)P4 at micromolar concentrations causes release of Ca2+ from electroporated L1210 cells, but not from digitonin-permeabilized cells. This was shown to be due to its conversion into Ins(1,4,5)P3, because only the electroporated cells convert Ins(1,3,4,5)P4 into Ins(1,4,5)P3. Thus electroporation appears to activate or expose an Ins(1,3,4,5)P4 3-phosphatase.     

Calmodulin inhibits inositol 1,4,5-trisphosphate-induced calcium release through the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1.

Our previous studies have demonstrated that calmodulin binds to IP3R type I (IP3R1) in a Ca2+ dependent manner, which suggests that calmodulin regulates the IP3R1 channel. In the present study, we investigated real-time kinetics of interactions between calmodulin and IP3R1 as well as effects of calmodulin on IP3-induced Ca2+ release by purified and reconstituted IP3R1. Kinetic analysis revealed that calmodulin binds to IP3R1 in a Ca2+ dependent manner and that both association and dissociation phase consist of two components with time constants of k(a) = 4.46 x 10(2) and > 10(4) M(-1) s(-1) k(d) = 1.44 x 10(-2) and 1.17 x 10(-1) s(-1). The apparent dissociation constant was calculated to be 27.3 microM. The IP3-induced Ca2+ release through the purified and reconstituted IP3R1 was inhibited by Ca2+/calmodulin, in a dose dependent manner. We interpret our findings to mean that calmodulin binds to IP3R1 in a Ca2+ dependent manner to exert inhibitory effect on IP3R channel activity. This event may be one of the mechanisms governing the negative feedback regulation of IP3-induced Ca2+ release by Ca2+.     

Mammalian target of rapamycin (mTOR) phosphorylates inositol 1,4,5-trisphosphate receptor type 2 and increases its Ca release activity

There is substantial evidence that crosstalk between the proliferation and Ca²⁺-signaling pathways plays a critical role in the regulation of normal physiological functions as well as in the pathogenesis of a variety of abnormal processes. In non-excitable cells, intracellular Ca²⁺ is mobilized through inositol 1,4,5-trisphosphate sensitive Ca²⁺ channels (IP₃R) expressed on the endoplasmic reticulum. Here we report that mTOR, a point of convergence for signals from mitogenic growth factors, nutrients and cellular energy levels, phosphorylates the IP₃R-2, the predominant isoform of IP₃R in AR4-2J cells. Pretreatment with the mTOR inhibitor rapamycin, decreased carbachol-induced Ca²⁺ release in AR4-2J cells. Rapamycin also decreased IP₃-induced Ca²⁺ release in permeabilized AR4-2J cells. We also showed that IGF-1 potentiates carbachol-induced Ca²⁺ release in AR4-2J cells, an effect that was prevented by rapamycin. Rapamycin also decreased carbachol-induced Ca²⁺ release in HEK 293A cells in which IP₃R-1 and IP₃R-3 had been knocked down. These results suggest that mTOR potentiates the activity of IP₃R-2 by a phosphorylation mechanism. This conclusion supports the concept of crosstalk between Ca²⁺ signaling and proliferation pathways and thus provides another way by which intracellular Ca²⁺ signals are finely encoded.     

Agonist-mediated Ca2+ release in permeabilized UMR-106-01 cells. Transport properties and generation of inositol 1,4,5-trisphosphate

Permeabilized and intact UMR-106-01 cells attached to culture plates or coverslips were used to evaluate compartmentalized generation and the effective concentration of inositol 1,4,5-trisphosphate (In-1,4,5-P3) during agonist-mediated Ca2+ release. In permeabilized cells, Ca2+ release had the following characteristics. In-1,4,5-P3 released approximately 65% of the Ca2+ incorporated into intracellular stores. Prostaglandin F2 alpha (PGF2 alpha), endothelin, or GTP(gamma S) alone released a small amount or no Ca2+. However, the agonists together with GTP(gamma S) were as effective as In-1,4,5-P3 in releasing Ca2+. Both agonist- and In-1,4,5-P3-mediated Ca2+ release required the presence of permeable ion. Agonists, like In-1,4,5-P3, stimulated 45Ca uptake from low Ca2+ medium devoid of permeable ions into Ca2(+)-loaded intracellular stores. The permeabilized cell system was then used to evaluate compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation. Mass measurement shows that in intact resting cells In-1,4,5-P3 concentration was 1.4 microM and was reduced to 0.05 microM following permeabilization. Stimulation with agonists increases In-1,4,5-P3 concentration from 0.05 to 0.34 microM. Ca2+ release by this concentration of In-1,4,5-P3 evenly distributed in the cytosol can account for only part of the agonist-mediated Ca2+ release. However, the effects of saturating In-1,4,5-P3 concentration and agonists were blocked by the specific inhibitor heparin. Measurement of heparin dependency of In-1,4,5-P3-mediated Ca2+ release was used to calculate an affinity for In-1,4,5-P3 of 0.39 microM. Similar measurements with agonists show that In-1,4,5-P3 concentration at the site of Ca2+ release during agonist stimulation is 11.2 microM. Hence, the total increase in In-1,4,5-P3 is reflected in considerably higher localized concentrations. This is interpreted to suggest compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation.     

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.     

The inositol 1,4,5-trisphosphate receptor (Itpr) gene family in Xenopus: identification of type 2 and type 3 inositol 1,4,5-trisphosphate receptor subtypes

Studies in the Xenopus model system have provided considerable insight into the developmental role of intracellular Ca2+ signals produced by activation of IP3Rs (inositol 1,4,5-trisphosphate receptors). However, unlike mammalian systems where three IP3R subtypes have been well characterized, our molecular understanding of the IP3Rs that underpin Ca2+ signalling during Xenopus embryogenesis relate solely to the original characterization of the 'Xenopus IP3R' cloned and purified from Xenopus laevis oocytes several years ago. In the present study, we have identified Xenopus type 2 and type 3 IP3Rs and report the full-length sequence, genomic architecture and developmental expression profile of these additional IP3R subtypes. In the light of the emerging genomic resources and opportunities for genetic manipulation in the diploid frog Xenopus tropicalis, these data will facilitate manipulations to resolve the contribution of IP3R diversity in Ca2+ signalling events observed during vertebrate development.     

Receptor-mediated release of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate in rat basophilic leukemia RBL-2H3 cells permeabilized with streptolysin O

Antigen-mediated exocytosis in intact rat basophilic leukemia (RBL-2H3) cells is associated with substantial hydrolysis of membrane inositol phospholipids and an elevation in concentration of cytosol Ca2+ ([ Ca2+i]). Paradoxically, these two responses are largely dependent on external Ca2+. We report here that cells labeled with myo-[3H]inositol and permeabilized with streptolysin O do release [3H]inositol 1,4,5-trisphosphate upon stimulation with antigen or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at low (less than 100 nM) concentrations of free Ca2+. The response, however, is amplified by increasing free Ca2+ to 1 microM. The subsequent conversion of the trisphosphate to inositol 1,3,4,5-tetrakisphosphate is enhanced also by the increase in free Ca2+. Although [3H]inositol 1,4,5-trisphosphate accumulates in greater amounts than is the case in intact cells, [3H]inositol 1,4-bisphosphate is still the major product in permeabilized cells even when the further metabolism of [3H]inositol 1,4,5-trisphosphate is suppressed (by 77%) by the addition of excess (1000 microM) unlabeled inositol 1,4,5-trisphosphate and the phosphatase inhibitor 2,3-bisphosphoglycerate. It would appear that either the activity of the membrane 5-phosphomonoesterase allows virtually instantaneous dephosphorylation of the inositol 1,4,5-trisphosphate under all conditions tested or both phosphatidylinositol 4-monophosphate and the 4,5-bisphosphate are substrates for the activated phospholipase C. The latter alternative is supported by the finding that permeabilized cells, which respond much more vigorously to high (supraoptimal) concentrations of antigen than do intact RBL-2H3 cells, produce substantial amounts of [3H]inositol 1,4-bisphosphate before any detectable increase in levels of [3H]inositol 1,4,5-trisphosphate.     

Cell side-specific sensitivities of intracellular Ca2+ stores for inositol 1,4,5-trisphosphate, cyclic ADP-ribose, and nicotinic acid adenine dinucleotide phosphate in permeabilized pancreatic acinar cells from mouse.

In pancreatic acinar cells hormonal stimulation leads to a cytosolic Ca(2+) wave that starts in the apical cell pole and subsequently propagates toward the basal cell side. We used permeabilized pancreatic acinar cells from mouse and the mag-fura-2 technique, which allows direct monitoring of changes in [Ca(2+)] of intracellular stores. We show here that Ca(2+) can be released from stores in all cellular regions by inositol 1,4,5-trisphosphate. Stores at the apical cell pole showed a higher affinity to inositol 1,4,5-trisphosphate (EC(50) = 89 nm) than those at the basolateral side (EC(50) = 256 nm). In contrast, cADP-ribose, a modifier of Ca(2+)-induced Ca(2+) release, and nicotinic acid adenine dinucleotide phosphate (NAADP) were able to release Ca(2+) exclusively from intracellular stores located at the basolateral cell side. Our data agree with observations that upon stimulation Ca(2+) is released initially at the apical cell side and that this is caused by high affinity inositol 1,4,5-trisphosphate receptors. Moreover, our findings allow the conclusion that in Ca(2+) wave propagation from the apical to the basolateral cell side observed in pancreatic acinar cells Ca(2+)-induced Ca(2+) release, modulated by cADP-ribose and/or NAADP, might be involved.     

Comparison of and chromogranin effect on inositol 1,4,5-trisphosphate sensitivity of cytoplasmic and nucleoplasmic inositol 1,4,5-trisphosphate receptor/Ca2+ channels

The nucleus also contains the inositol 1,4,5-trisphosphate receptor (IP3R)/Ca2+ channels in the nucleoplasm proper independent of the nuclear envelope or the cytoplasm. The nuclear IP3R/Ca2+ channels were shown to be present in small IP3-dependent nucleoplasmic Ca2+ store vesicles, yet no information is available regarding the IP3 sensitivity of nuclear IP3R/Ca2+ channels. Here, we show that nuclear IP3R/Ca2+ channels are 3-4-fold more sensitive to IP3 than cytoplasmic ones in both neuroendocrine PC12 cells and nonneuroendocrine NIH3T3 cells. Given the presence of phosphoinositides and phospholipase C and the importance of IP3-mediated Ca2+ signaling in the nucleus, the high IP3 sensitivity of nuclear IP3R/Ca2+ channels seemed to reflect the physiological needs of the nucleus to finely control the IP3-dependent Ca2+ concentrations. It was further shown that the IP3R/Ca2+ channels of secretory cells are 7-8-fold more sensitive to IP3 than those of nonsecretory cells. This difference appeared to result from the presence of secretory cell marker protein chromogranins (thus secretory granules) in secretory cells; expression of chromogranins in NIH3T3 cells increased the IP3 sensitivity of both nuclear and cytoplasmic IP3R/Ca2+ channels by approximately 4-6-fold. In contrast, suppression of chromogranin A expression in PC12 cells changed the EC50 of IP3 sensitivity for cytoplasmic IP3R/Ca2+ channels from 17 to 47 nM, whereas suppression of chromogranin B expression changed the EC50 of cytoplasmic IP3R/Ca2+ channels from 17 to 102 nM and the nuclear ones from 4.3 to 35 nM. Given that secretion is the major function of secretory cells and is under a tight control of intracellular Ca2+ concentrations, the high IP3 sensitivity appears to reflect the physiological roles of secretory cells.     

Calcium release of heat-shocked porcine oocytes induced by thimerosal or inositol 1,4,5-trisphosphate (IP3)

The objective of this study was to determine the effect of heat shock (HS) on the Ca(2+) release and the subsequent development in matured porcine oocytes. Oocytes were matured in vitro and randomly allocated to different heat treatments at 41.5 degrees C for 1 (HS1h), 2 (HS2h) or 4h (HS4h). Control groups of oocytes were cultured for 0 or 4h without HS (39 degrees C, C0h, C4h). In Experiment 1 (eight replicates), matured oocytes were activated by thimerosal (200 microM, 10 min) following HS. Among all heated groups, maximal intracellular calcium concentration ([Ca(2+)](i)) was the highest in the HS2h. The lowest [Ca(2+)](i) peak among HS groups was observed in the HS4h, but it was higher than that in the non-heated C4h group (P<0.05). In Experiment 2 (12 replicates), each matured oocyte was injected with IP(3) (0.5mM) and the Ca(2+) transient was recorded. The peak [Ca(2+)](i) in the C4h group was still the lowest among all groups (P<0.05). Total Ca(2+) release in HS2h appeared the highest among all treatments, and it was significantly higher than that in HS1h and C4h groups (P<0.05). In order to clarify the effect of incubation time in vitro (Experiment 3), matured oocytes were cultured at 39 degrees C for 0, 2 and 4h prior to treatment with thimerosal or injected with IP(3) (three replicates). The Ca(2+) release of matured oocytes declined with the prolonged culture (P<0.05). Finally, the development of HS-oocytes was evaluated after parthenogenetic activation (Experiment 4, three replicates), and the proportion of embryos developing to the blastocysts were lower (P<0.05) in the HS groups (31+/-7% to 33+/-1%) than in the control groups (52+/-11% to 56+/-9%). We conclude that HS alters the Ca(2+)-releasing ability of matured pig oocytes, and that heat-shocked oocytes with greater Ca(2+) release incur a low developmental competence after parthenogenetic activation.