Inositol trisphosphate-induced Ca2+ release from rat parotid subcellular fractions

Inositol trisphosphate (IP3) has been shown to induce a release of sequestered calcium in a large (7,000-48,000 g) microsomal fraction isolated from rat parotid gland. This effect was also observed after the calcium uptake had been stopped by EGTA and thus was not the result of an inhibition of the active calcium transport. IP3-induced Ca2+ release was also demonstrated in a more purified microsomal fraction (16,000-48,000 g) apparently free of mitochondria, and in a fraction enriched in rough endoplasmic reticulum markers. These results support the hypothesis that IP3 may be a second messenger for intracellular calcium mobilization during stimulation of the parotid gland by calcium mobilising agonists.     

Ca2+- and inositol-1,4,5-triphosphate induced release of Ca2+ in the microsomal fraction of the rat brain

Using the fluorescent probes, Quin 2 and chlortetracycline, a comparative study of the Ca2+ and inositol-1.4.5-triphosphate (IP3)-induced Ca2+ release from rabbit skeletal muscle sarcoplasmic reticulum (SR) terminal cisterns and rat brain microsomal vesicles was carried out. It was shown that Ca2+ release from rat brain microsomal vesicles is induced both by IP3 and Ca2+, whereas that in SR terminal cisterns is induced only by Ca2+. Data from chlorotetracycline fluorescence analysis revealed that CaCl2 (50 microM) causes the release of 15-20% and 40-50% of the total Ca2+ pool accumulated in rat brain microsomal vesicles and rabbit SR terminal cisterns, respectively. Using Quin 2, it was found that IP3 used at the optimal concentration (1.5 mM) caused the release of 0.4-0.6 nmol of Ca2+ per mg microsomal protein, which makes up to 10-15% of the total Ca2+ pool. IP3 does not induce Ca2+ release in SR. Preliminary release of Ca2+ from brain microsomes induced by IP3 diminishes the liberation of this cation induced by Ca2+. It is suggested that brain microsomes contain a Ca2+ pool which is exhausted under the action of the both effectors, Ca2+ and IP3.     

Mobilization of calcium by inositol trisphosphates from permeabilized rat parotid acinar cells. Evidence for translocation of calcium from uptake to release sites within the inositol 1,4,5-trisphosphate- and thapsigargin-sensitive calcium pool

D-myo-Inositol (1,4,5)-trisphosphate ((1,4,5)IP3)-induced Ca2+ release and subsequent Ca2+ reuptake were investigated in saponin-permeabilized rat parotid acinar cells. Following the rapid release of Ca2+ by (1,4,5)IP3, Ca2+ was resequestered. The sequential addition of submaximal concentrations of (1,4,5)IP3 resulted in sequential Ca2+ release. However, when the cells were challenged with the poorly metabolized (1,4,5)IP3 analogues, (1,4,5)IPS3 or (2,4,5)IP3, or under conditions where the metabolism of authentic (1,4,5)IP3 was reduced, Ca2+ reuptake again occurred, but sequestered Ca2+ was not released by subsequent additions of (1,4,5)IP3. The sequestered Ca2+ was, however, released by thapsigargin, an agent which inhibits active Ca2+ uptake into the (1,4,5)IP3-sensitive pool. Furthermore, the rate of thapsigargin-induced release was significantly increased in the continued presence of an (1,4,5)IP3 stimulus. Thus, Ca2+ reuptake apparently occurred into the (1,4,5)IP3- and thapsigargin-sensitive Ca2+ store and (1,4,5)IP3 continued to influence the permeability of this pool to Ca2+ during Ca2+ reuptake. In contrast to the findings in permeabilized cells, Ca2+ reuptake did not occur in the sustained presence of (1,4,5)IP3 in intact parotid cells. We conclude that cell permeabilization reveals a kinetic, and presumably structural, separation of Ca2+ uptake and release sites within the (1,4,5)IP3-regulated intracellular organelle.     

Postnatal expression of the inositol 1,4,5-trisphosphate receptor in canine cerebellum.

1. Inositol 1,4,5-trisphosphate (IP3), an intracellular second messenger, has been shown to be the link between activation of several plasma membrane receptors and Ca2+ release from intracellular, membrane-bound compartments. In this study, the postnatal expression of the canine cerebellum IP3 receptor was investigated by biochemical, ligand binding and immunocytochemical methods. 2. Specific receptor sites for IP3 and the extent of IP3-induced Ca2+ release were quantitated in microsomal fractions isolated from cerebella of developing (0-28 day-old) and adult dogs. The IP3 receptor was detected in newborn animals and adult levels were attained within 3-4 weeks. 3. The time-course of IP3 receptor ontogeny paralleled both growth of Purkinje neurons, as indicated by immunofluorescence of cerebellum cortex cryosections with anti-IP3 receptor antibodies, and synaptogenesis, as judged by Western blotting of the microsomal fractions with anti-synaptophysin antibodies.     

Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.     

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.     

Distribution of endoplasmic reticulum and calciosome markers in membrane fractions isolated from different regions of the canine brain

Four regions of the canine brain (frontal lobe, parieto-occipital lobe, brainstem, and cerebellum) were each fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). Markers of endoplasmic reticulum (glucose-6-phosphate phosphatase and rotenone-insensitive NADPH cytochrome c reductase) and markers of the 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store ([3H]IP3 binding and IP3-induced Ca2+ release) were measured. No correlation was found between the two classes of markers, which suggests that the IP3 receptor does not belong to the endoplasmic reticulum in canine brain. Cerebellum P2 and P3 fractions displayed levels of [3H]IP3 binding 10- to 30-fold higher, and rates of IP3-induced Ca2+ release greater than 15-fold faster than the homologous cerebrum and brainstem fractions. Actively accumulated Ca2+ was only partially released by IP3, both before and after saponin disruption of the plasma membrane compartment. The proportion of the IP3-sensitive Ca2+ store relative to that of the total (IP3-sensitive and IP3-insensitive) Ca2+ store was variable; i.e., it was larger in cerebellum P2 (approximately 90%) than in cerebrum fractions (less than 30%). Cerebellum fractions constitute the best source from which an IP3-sensitive Ca2+ storing organelle can be purified.     

Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci

Ca2+ dependence of the inositol 1,4,5-trisphosphate (IP3)-induced Ca release was studied in saponin-skinned smooth muscle fiber bundles of the guinea pig taenia caeci at 20-22 degrees C. Ca release from the skinned fiber bundles was monitored by microfluorometry of fura-2. Fiber bundles were first treated with 30 microM ryanodine for 120 s in the presence of 45 mM caffeine to lock open the Ca-induced Ca release channels which are present in approximately 40% of the Ca store of the smooth muscle cells of the taenia. The Ca store with the Ca-induced Ca release mechanism was functionally removed by this treatment, but the rest of the store, which was devoid of the ryanodine-sensitive Ca release mechanism, remained intact. The Ca2+ dependence of the IP3-induced Ca release mechanism was, therefore, studied independently of the Ca-induced Ca release. The rate of IP3-induced Ca release was enhanced by Ca2+ between 0 and 300 nM, but further increase in the Ca2+ concentration also exerted an inhibitory effect. Thus, the rate of IP3-induced Ca release was about the same in the absence of Ca2+ and at 3 microM Ca2+, and was about six times faster at 300 nM Ca2+. Hydrolysis of IP3 within the skinned fiber bundles was not responsible for these effects, because essentially the same effects were observed with or without Mg2+, an absolute requirement of the IP3 phosphatase activity. Ca2+, therefore, is likely to affect the gating mechanism and/or affinity for the ligand of the IP3-induced Ca release mechanism. The biphasic effect of Ca2+ on the IP3-induced Ca release is expected to form a positive feedback loop in the IP3-induced Ca mobilization below 300 nM Ca2+, and a negative feedback loop above 300 nM Ca2+.     

GTP- and inositol 1,4,5-trisphosphate-activated intracellular calcium movements in neuronal and smooth muscle cell lines

Recent evidence has revealed that a highly sensitive and specific guanine nucleotide regulatory process controls intracellular Ca2+ release within N1E-115 neuroblastoma cells (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The present report documents GTP-induced Ca2+ release within quite distinct cell types, including the DDT1MF-2 smooth muscle cell line. GTP-induced Ca2+ release has similar GTP sensitivity and specificity among cells and rapidly mobilizes up to 70% of Ca2+ specifically accumulated within a nonmitochondrial Ca2+-pumping organelle within permeabilized DDT2MF-2 cells. Maximal GTP-induced release of Ca2+ is observed to be greater than inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (the latter being approximately 30% of total releasable Ca2+). After maximal IP3-induced release, further IP3 addition is ineffective, whereas subsequent addition of GTP further releases Ca2+ to equal exactly the extent of Ca2+ release observed by addition of GTP in the absence of IP3. This suggests that IP3 releases Ca2+ from the same pool as GTP, whereas GTP also releases from an additional pool. The effects of GTP appear to be reversible since simple washing of GTP-treated cells restores their previous Ca2+ uptake properties. Electron microscopic analysis of GTP-treated membrane vesicles reveals their morphology to be unchanged, whereas treatment of vesicles with 3% polyethylene glycol, known to enhance GTP-mediated Ca2+ release, clearly induces close coalescence of membranes. In the presence of 4 mM oxalate, GTP induces a rapid and profound uptake, as opposed to release, of Ca2+. The findings suggest that GTP-activated Ca2+ movement is a widespread phenomenon among cells, which can function on the same Ca2+ pool mobilized by IP3, and although activating Ca2+ movement by a mechanism distinct from IP3, does so via a process that does not appear to involve fusion between membranes.     

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.     

Phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum terminal cisternal membranes. Ca2+ flux and single channel studies.

We report here that the inositol 1,4,5-trisphosphate (IP3) precursor, L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent molecule (1 microM) which activates the ryanodine-sensitive Ca2+ release channel from rabbit skeletal muscle terminal cisternae incorporated into a phospholipid bilayer. It also stimulates Ca2+ release from these membrane vesicles. Therefore, it may play a modulating role in excitation-contraction coupling. In the bilayer, PIP2 added on the cytoplasmic side increased the mean channel opening probability 2-12-fold in the presence and absence of physiological Mg2+ and ATP. From flux studies, PIP2-induced Ca2+ release, occurring through the ryanodine-sensitive Ca2+ release channel, displayed saturation kinetics. The rate of Ca2+ release induced by PIP2 was approximately greater than 50% slower than the rates induced by other agents (e.g. caffeine, Ca2+, ATP). PIP2, and not IP3, effectively elicited Ca2+ release from terminal cisternae. On the contrary, IP3, and not PIP2, specifically mediated Ca2+ release from dog brain cerebellum microsomes, where IP3 receptors are known to be found. The PIP2-induced Ca2+ release from muscle membranes was not dependent on medium [Ca2+] (from less than 10(-9) to approximately 10(-4) M). However, IP3 could activate the terminal cisternae Ca2+ channel in the bilayer when there was low Ca2+ (less than 10(-7) M). The data suggest that the ionic microenvironment around the Ca2+ channel may be different for observing the two phosphoinositide actions.     

Characterization of the inositol 1,4,5-trisphosphate-induced calcium release from permeabilized endocrine cells and its inhibition by decavanadate and p-hydroxymercuribenzoate.

The inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ compartment of endocrine cells was studied with alpha-toxin- and digitonin-permeabilized rat insulinoma (RINA2) and rat pheochromocytoma (PC12) cells. The Ca2+ uptake was ATP-dependent, and submicromolar concentrations of IP3 specifically released the stored Ca2+. Half-maximal Ca2+ release was observed with 0.25-0.5 mumol of IP3/l, and the amount of Ca2+ released due to IP3 could be enhanced by additional loading of the Ca2+ compartment. Consecutive additions of the same concentration of IP3 for 1-2 h always released the same amount of Ca2+ without desensitization, providing an ideal basis to further characterize the IP3-induced Ca2+ release. Here we describe for the first time a reversible inhibitory effect of decavanadate on the IP3-induced Ca2+ release. Among the vanadium species tested (decavanadate, oligovanadate and monovanadate), only decavanadate was inhibitory, with a half-maximal effect at 5 mumol/l in both cell types. The effect of decavanadate could be overcome by increasing the amount of sequestered Ca2+ or added IP3. Decavanadate did not affect the ATP-driven Ca2+ uptake but oligovanadate was inhibitory on Ca2+ uptake. p-Hydroxymercuribenzoate (pHMB) at concentrations between 10 and 30 mumol/l also inhibited the Ca2+ release due to IP3. Thiol compounds such as dithiothreitol (DTT; 1 mmol/l) added before pHMB removed all its inhibitory effect on the IP3-induced Ca2+ release, whereas the inhibition caused by decavanadate was unaffected by DTT. Thus, the decavanadate-dependent inhibition functions by a distinctly different mechanism than pHMB and could serve as a specific tool to analyse various aspects of the IP3-induced Ca2+ release within endocrine cells.     

Multiple transduction mechanisms are likely involved in calcium-mediated exocrine secretory events in rat parotid cells

The parotid gland of the aged rat provides an example of an altered alpha 1-adrenergic physiologic response (K+ efflux) resulting from a postreceptor perturbation in signal transduction mechanisms (Ito, H., Baum, B. J., Uchida, T., Hoopes, M. T., Bodner, L. & Roth, G. S. (1982) J. Biol. Chem. 257, 9532-9538). This alteration in gland function can be completely circumvented by eliciting K+ efflux via the Ca2+-ionophore, A23187, at several Ca2+ concentrations (ibid.). Since Ca2+ is purported to mediate other secretory events in the rat parotid, we have probed neurotransmitter regulated Ca2+ mobilization and secretory mechanisms in this tissue by employing an aging paradigm. The responses studied were alpha-adrenergic- and muscarinic-cholinergic-mediated K+ efflux, 45Ca2+ release, and amylase secretion. No differences were detected between young (3 months) and old (24 months) cell preparations for any muscarinic-cholinergic agonist-induced response studied. Following alpha-adrenergic stimulation, K+ efflux and 45Ca2+ release from old cell preparations were reduced markedly, while no changes were found for the amylase secretion response. These results suggest that 1) alpha-adrenergic and cholinergic signal transduction mechanisms for K+ efflux and 45Ca2+ release are dissociated in cells of the rat parotid gland, and 2) following alpha 1-adrenergic stimulation, signal transduction likely proceeds by at least two pathways, one which is apparently involved in protein excytosis (intact in cells from old rats) and the other which is apparently involved in K+ efflux and 45Ca2+ release (perturbed in old cells).     

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 microM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) could not replace GTP but prevented the action of GTP. The effects of GTP and GTP gamma S were reversible. Neither GTP nor GTP gamma S induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 microM free Ca2+, a half-maximal Ca2+ no Ca2+ release was observed with 0.1 microM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 microM) were required to evoke Ca2+ release. At 8 microM free Ca2+, even 0.25 microM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 microM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.     

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.     

2-Aminoethoxydiphenyl borate affects the inositol 1,4,5-trisphosphate receptor, the intracellular Ca2+ pump and the non-specific Ca2+ leak from the non-mitochondrial Ca2+ stores in permeabilized A7r5 cells

2-Aminoethoxydiphenyl borate (2APB) is a membrane-permeable blocker of the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release in bi-directional Ca2+ -flux conditions. We have now studied the effects of 2APB on the 45Ca2+ uptake into, and on the basal and IP(3)-stimulated unidirectional 45Ca2+ efflux from the non-mitochondrial Ca2+ stores in permeabilized A7r5 smooth-muscle cells. 2APB inhibited the IP3 -induced Ca2+ release, with a half maximal inhibition at 36 microM 2APB, without affecting [3H]IP3 binding to the receptor. This inhibition did not depend on the IP3, ATP or free Ca2+ concentration. The Ca2+ pumps of the non-mitochondrial Ca2+ stores were half-maximally inhibited at 91microM 2APB. Higher concentrations of 2APB increased the non-specific leak of Ca2+ from the stores. We conclude that 2APB can not be considered as a selective blocker of the IP3 -induced Ca2+ release. Our results can explain the various effects of 2APB observed in intact cells.     

Ca2+ release triggered by NAADP in hepatocyte microsomes

NAADP (nicotinic acid-adenine dinucleotide phosphate) is fast emerging as a new intracellular Ca2+-mobilizing messenger. NAADP induces Ca2+ release by a mechanism that is distinct from IP3 (inositol 1,4,5-trisphosphate)- and cADPR (cADP-ribose)-induced Ca2+ release. In the present study, we demonstrated that micromolar concentrations of NAADP trigger Ca2+ release from rat hepatocyte microsomes. Cross-desensitization to IP3 and cADPR by NAADP did not occur in liver microsomes. We report that non-activating concentrations of NAADP can fully inactivate the NAADP-sensitive Ca2+-release mechanism in hepatocyte microsomes. The ability of thapsigargin to block the NAADP-sensitive Ca2+ release is not observed in sea-urchin eggs or in intact mammalian cells. In contrast with the Ca2+ release induced by IP3 and cADPR, the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ concentration and pH (in the range 6.4-7.8). The NAADP-elicited Ca2+ release cannot be blocked by the inhibitors of the IP3 receptors and the ryanodine receptor. On the other hand, verapamil and diltiazem do inhibit the NAADP- (but not IP3- or cADPR-) induced Ca2+ release.     

Calmodulin inhibits inositol trisphosphate-induced Ca2+ mobilization from the endoplasmic reticulum of islets

IP3-induced Ca2+ release from the endoplasmic reticulum (ER) of islets is believed to be a key intracellular event in glucose-induced insulin secretion. Calmodulin was shown to increase ATP-dependent Ca2+ steady-state and inhibit by 57.2% IP3-induced Ca2+ mobilization from the ER. Conversely, the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide (W-7), induced Ca2+ release from the ER. The combination of W-7 (100 microM) and IP3 (10 microM), resulted in a greater release of Ca2+ from the ER than either W-7 or IP3 alone. W-7 was shown not to affect the structural integrity of the ER. Our results suggest that IP3-induced Ca2+ release from the ER is regulated by a calmodulin-dependent process.     

Modulation of rat parotid cell alpha-adrenergic responsiveness at a step subsequent to receptor activation

Parotid gland acinar cells, prepared from 12- and 24-month-old rats, show decreased physiological responsiveness to alpha-adrenergic stimulation in vitro compared to cells from 3-month-old rats. K+ efflux, an index of water and electrolyte secretion, was approximately 35% lower with 12- and 24-month-old parotid cells. No loss of alpha-adrenergic receptors, their binding affinity for specific alpha-adrenergic ligands, or their relative subtype distribution, accompanied the diminished exocrine function. Conversely, a significant reduction in alpha-adrenergic-mediated phospholipid turnover in, and 45Ca2+ efflux from, parotid cells of older rats was observed. These changes in phospholipid metabolism and Ca2+ flux were parallel to changes seen in K+ efflux as judged by dose-response studies. When the alpha-adrenergic receptor was by-passed by using the Ca2+-ionophore A-23187 to elicit K+ efflux, young and old parotid cells were equally responsive. In aggregate the findings suggest that parotid gland cells from older rats display an altered alpha-adrenergic signal transduction mechanism at a site between the receptor and phospholipid turnover/Ca2+ mobilization.     

Effects of adenine nucleotides on inositol 1,4,5-trisphosphate-induced calcium release in vascular smooth muscle cells

Effects of adenine nucleotides on the inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanism were studied in smooth muscle cells of the guinea pig portal vein. A microfluorometry method of fura-2 was used to measure Ca release from saponin-skinned thin muscle strips (width approximately 200 microns, thickness 50-70 microns, length 2-3 mm). About 80% of ionomycin-releasable Ca store was sensitive to IP3, of which approximately 20% was also sensitive to caffeine. The rate of Ca release by 0.1 microM IP3 depended biphasically on ATP concentration in the absence of Mg2+; it was dose-dependently enhanced by ATP up to approximately 0.5 mM, and above this concentration the enhancement became smaller. However, the decline of enhancement of the IICR at the higher ATP concentrations was absent at IP3 concentrations greater than 1 microM. This suggests competitive antagonism between IP3 and ATP. Clear effects of ATP were observed not only at pCa 7 or 8, where the Ca-induced Ca release was not activated, but after a ryanodine treatment to excise the functional compartment that possessed the Ca-induced Ca release mechanism. ATP had no effect on the rate of Ca leakage in the absence of IP3 even at pCa 5.5 after the ryanodine treatment. Therefore, ATP has direct biphasic effects on the IP3-induced Ca release mechanism. The Ca release induced by 0.1 microM IP3 at pCa 7 was potentiated not only by ATP, but by 0.5 mM ADP, AMP, or beta, gamma-methyleneadenosine 5'-triphosphate. 0.5 mM GTP had only a little effect on the IP3-induced Ca release. These results extend the functional similarities between Ca- and IP3-induced Ca release mechanisms in that adenine nucleotides enhance Ca release. Millimolar concentration of ATP, which is present physiologically, will shift the dose-response relation of IP3 toward the higher IP3 concentration and enhance the maximal effect of IP3. Thus, ATP is expected to assist the Ca release by higher concentrations of IP3 while having less effect on the Ca release by low levels of IP3. These effects of ATP may be important in the switching of Ca release from the intracellular Ca store by IP3.