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.     

Evidence for an alteration in the microsomal Ca2+ release mechanism in senescent rat parotid acinar cells

Previously, we have shown that Ca2+ mobilization following an alpha 1-adrenergic receptor stimulus is reduced in parotid acinar cells from senescent rats as a result of an altered ability of inositol 1,4,5-trisphosphate (IP3) to induce Ca2+ release from a non-mitochondrial, intracellular Ca2+ store (Ishikawa, Y., et al. Biochim. Biophys. Acta 968, 203-210). We have used this model to examine the IP3-induced Ca2+ release mechanism in these cells. 45Ca2+ efflux, after exposure to (-) epinephrine, from cells of young adult (3-6 months) rats was approx. 2-fold that observed from cells from older animals (approx. 24 months) either in the presence or absence of extracellular Ca2+. Similarly, cytosolic Ca2+ levels were greater in cells of young adult rats under these same incubation conditions. However, microsomal membrane preparations, from both age groups displayed similar IP3 binding sites (Kd approximately 90 nM, Bmax approximately 850 fmol/mg protein) and ATP-dependent Ca2+ transport ability (approx. 8 nmol/mg protein.min -1). These data suggest that there is an alteration in the IP3-induced Ca2+ release mechanism in microsomal membranes of parotid glands from senescent rats which may account for the decreased Ca2+ release seen after agonist stimulation of this tissue.     

Mechanisms of activated Ca2+ entry in the rat pancreatoma cell line, AR4-2J.

The characteristics of Ca2+ entry activated by surface receptor agonists and membrane depolarization were studied in the rat pancreatoma cell line, AR4-2J. Ca2+ mobilization activated by substance P, bombesin, or muscarinic receptor stimulation was found to involve both Ca2+ release and entry. In addition, depolarization of the surface membrane of AR4-2J cells with elevated concentrations of K+ activated Ca2+ entry. Ca2+ entry induced by membrane depolarization was inhibited by the L-channel antagonist, nimodipine, while that due to surface receptor agonists was not inhibited by this agent. The microsomal Ca(2+)-ATPase inhibitor, thapsigargin, caused both depletion of the agonist-sensitive intracellular Ca2+ pool and sustained Ca2+ influx indistinguishable from that produced by bombesin or methacholine. These results confirm that, unlike the pancreatic acinar cells from which they are presumably derived, AR4-2J cells express voltage-sensitive, dihydropyridine-inhibitable Ca2+ channels. However, in contrast to previous reports with this cell line, in the AR4-2J cells in use in our laboratory, and under our experimental conditions, surface receptor agonists (including substance P) do not cause Ca2+ influx through voltage-sensitive Ca2+ channels. Instead, we conclude that agonist-activated Ca2+ mobilization is initiated by (1,4,5)IP3-mediated intracellular Ca2+ release and that Ca2+ influx is regulated primarily, if not exclusively, by the state of depletion of the (1,4,5)IP3-sensitive intracellular Ca2+ pool.     

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.     

Heterogeneity of microsomal Ca2+ stores in chicken Purkinje neurons.

Chicken cerebellum microsomes were subfractionated on isopycnic, linear sucrose (15-50%) density gradients. The distribution of four markers of intracellular, rapidly-exchanging Ca2+ stores, i.e. the Ca2+ pump, the receptors for inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry), and calsequestrin (CS, an intralumenal, high capacity Ca2+ binding protein) was investigated biochemically and immunologically. In the cerebellum, high levels of these markers are expressed by one of the cell types, the Purkinje neuron. Heavy subfractions were enriched in both CS and Ry receptor, intermediate subfractions in the IP3 receptor, while the Ca2+ pump was present in both intermediate and heavy subfractions. Intact cells and pelleted subfractions were examined by conventional and immuno-electron microscopy (immunogold labeling of ultrathin cryosections with anti-CS and anti-IP3 receptor antibodies). Of the strongly CS-labeled, moderately dense-cored vacuoles (calciosomes) recently described in chicken Purkinje neurons only partly exhibited labeling for the IP3 receptor as well, and the rest appeared negative. The latter were enriched in a heavy subfraction of the gradient where Ry receptors were also concentrated, whereas the CS-rich vacuoles in an intermediate subfraction were almost always IP3 receptor-positive. The population of CS-rich calciosomes of chicken Purkinje neurons appears therefore to be molecularly heterogeneous, with a part responsive to IP3 and the rest possibly sensitive to Ry.     

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor. The brain ryanodine receptor has been purified 6700-fold with no change in [3H]ryanodine binding affinity and shown to be a homotetramer composed of an approximately 500 kd protein subunit, which is identified by anti-peptide antibodies against the skeletal and cardiac muscle ryanodine receptors. Our results demonstrate that the brain ryanodine receptor functions as a caffeine-sensitive Ca2+ release channel and thus is the likely gating mechanism for intracellular caffeine-sensitive Ca2+ pools in neurons.     

Comparison of Ca2+ mobilizing activities of cyclic ADP-ribose and inositol trisphosphate.

We have previously shown that a metabolite of NAD+ generated by an enzyme present in sea urchin eggs and mammalian tissues can mobilize intracellular Ca2+ in the eggs. Structural determination established it to be a cyclized ADP-ribose, and the name cyclic ADP-ribose (cADPR) has been proposed. In this study, Ca2+ mobilizations induced by cADPR and inositol trisphosphate (IP3) in sea urchin egg homogenates were monitored with Ca2+ indicators and Ca2(+)-specific electrodes. Both methods showed that cADPR can release Ca2+ from egg homogenates. Evidence indicated that it did not act as a nonspecific Ca2(+)-ionophore or as a blocker of the microsomal Ca2(+)-transport; instead, it was likely to be operating through a specific receptor system. This was supported by its half-maximal effective concentration of 18 nM, which was 7 times lower than that of IP3. The receptor for cADPR appeared to be different from that of IP3 because heparin, an inhibitor of IP3 binding, had no effect on the cADPR action. The Ca2+ releases induced by cADPR and IP3 were not additive and had an inverse relationship, indicating overlapping stores were mobilized. Microinjection of cADPR into intact eggs induced transient intracellular Ca2+ changes and activated the cortical reaction. The in vivo effectiveness of cADPR was directly comparable with IP3 and neither required external Ca2+. In addition, both were effective in activating the eggs to undergo multiple nuclear cycles and DNA synthesis. These results suggest that cADPR could function as a second messenger in sea urchin eggs.     

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.     

Inositol 1,4,5-trisphosphate receptors. Localization in epithelial tissue.

Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.     

Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors.

Elevation of intracellular Ca2+ by platelet-derived growth factor (PDGF) and other growth factors involves both release of Ca2+ from intracellular Ca2+ stores and Ca2+ entry from the extracellular medium. Release from intracellular stores is believed to be mediated by inositol 1,4,5-trisphosphate (IP3) and the heparin-sensitive IP3 receptor. We studied the mechanism by which entry of extracellular Ca2+ is induced by PDGF. Intracellular free Ca2+ (Ca2+i) was measured in single cultured rat vascular smooth muscle cells using fura 2 microspectrofluorometry. In nominally Ca2(+)-free medium, PDGF (recombinant BB, 10 ng/ml) raised intracellular Ca2+ transiently (less than 5 min); addition of 2 mM Ca2+ to the bathing medium after 5 min caused a second, prolonged increase in intracellular Ca2+. Repeated changes in extracellular Ca2+ from 0 to 2 mM over 90 min caused rapid, parallel changes in Ca2+i of approximately 200 nM. This change in Ca2+i in response to changes in extracellular Ca2+ was virtually undetectable in control or thrombin-treated cells. The intracellular response to changes in medium Ca2+ after PDGF was completely blocked by 10 mM CoCl2, but not by 10(-7) M nicardipine. Microinjection of monoclonal antibodies to phosphatidylinositol 4,5-bisphosphate (PIP2) (kt 10, 2 mg/ml) totally abolished both mobilization of intracellular Ca2+ stores and entry of extracellular Ca2+. Consistent with this finding, maintenance of Ca2+ entry required ongoing receptor occupancy, since displacement of PDGF from its receptor with suramin (1 mM) eradicated extracellular Ca2+ entry in less than 5 min. To determine whether extracellular Ca2+ entry involves the heparin-sensitive IP3 receptor, cells were microinjected with heparin (4 mg/ml) prior to addition of PDGF. Heparin, but not chondroitin sulfate, prevented mobilization of intracellular Ca2+ stores but did not affect extracellular Ca2+ entry. We PDGF requires ongoing receptor occupancy and involves PIP2 or PIP2 metabolism. However, the signal which mediates PDGF-induced Ca2+ entry does not require the heparin-sensitive IP3 receptor.     

Synaptic activity augments muscarinic acetylcholine receptor-stimulated inositol 1,4,5-trisphosphate production to facilitate Ca2+ release in hippocampal neurons

Intracellular Ca2+ store release contributes to activity-dependent synaptic plasticity in the central nervous system by modulating the amplitude, propagation, and temporal dynamics of cytoplasmic Ca2+ changes. However, neuronal Ca2+ stores can be relatively insensitive to increases in the store-mobilizing messenger inositol 1,4,5-trisphosphate (IP3). Using a fluorescent biosensor we have visualized M1 muscarinic acetylcholine (mACh) receptor signaling in individual hippocampal neurons and observed increased IP3 production in the absence of concurrent Ca2+ store release. However, coincident glutamate-mediated synaptic activity elicited enhanced and oscillatory IP3 production that was dependent upon ongoing mACh receptor stimulation and S-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid receptor activation of Ca2+ entry. Moreover, the enhanced levels of IP3 now mobilized Ca2+ from intracellular stores that were refractory to the activation of mACh receptors alone. We conclude that convergent ionotropic and metabotropic receptor inputs can facilitate Ca2+ signaling by enhancing IP3 production as well as augmenting release by Ca2+-induced Ca2+ release.     

Inositol trisphosphate induces calcium release from nonmitochondrial stores i sea urchin egg homogenates

This study presents evidence that inositol trisphosphate (IP3) releases Ca2+ from intracellular stores in sea urchin eggs. First, high voltage discharge was used to transiently permeabilize eggs and introduce IP3; the resultant induction of cortical reactions (a well characterized Ca2+-dependent event) provided indirect evidence that IP3 released Ca2+ from intracellular stores. Next, Ca2+ uptake and release from egg homogenates and homogenate fractions were monitored by both Ca2+ minielectrodes and the fluorescent Ca2+ indicator, quin-2. Both assay methods showed Ca2+ release upon IP3 addition, with a half-maximal response at 50-60 nM IP3 and maximal Ca2+ release at approximately 1 microM IP3. Homogenates were 300-fold more sensitive to IP3 than IP2, and Ca2+ release was 95% inhibited by the Ca2+ antagonist TMB-8 (3 mM). Fractionation by density gradient centrifugation showed that activities for Ca2+ sequestration and IP3 responsiveness co-purified with endoplasmic reticulum microsomes. Following an initial IP3 addition, homogenates were refractory (desensitized) to additional IP3. However, if homogenates were centrifuged and the vesicles resuspended in media lacking IP3, they would respond to added IP3, therefore, showing that desensitization is most likely due to the presence of IP3. This study also shows that the mechanism of IP3 action is inherent to the microsomes and ions present in the medium used, with no cytoplasmic factors being required. The stability of this microsome preparation and the purification obtained with density gradient centrifugation make this a promising system with which to further characterize the mechanism of IP3 action.     

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.     

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.     

MgATP-dependent glucose 6-phosphate-stimulated Ca2+ accumulation in liver microsomal fractions. Effects of inositol 1,4,5-trisphosphate and GTP

Ca2+ release triggered by inositol 1,4,5-trisphosphate (IP3) and/or GTP has been studied with rough and smooth microsomes isolated from rat liver. Microsomes were loaded with Ca2+ in the presence of MgATP and in the presence or in the absence of glucose 6-phosphate (glucose-6-P) which markedly stimulated the MgATP-dependent Ca2+ accumulation in rough and smooth microsomes (5- and 10-fold, respectively). Upon addition of IP3 (5 microM), rough and smooth microsomes rapidly release a part (not exceeding 20%) of the Ca2+ previously accumulated both in the absence and in the presence of glucose-6-P. Under the same experimental conditions, inositol 1,3,4,5-tetrakisphosphate was ineffective in triggering any Ca2+ release. Upon addition of GTP (10 microM) both the microsomal fractions progressively release the Ca2+ previously accumulated in the presence of glucose-6-P, when 3% polyethylene glycol was also present. In the absence of polyethylene glycol, GTP released Ca2+ from rough microsomes only, and GTP plus IP3 caused a Ca2+ release which was the sum of the Ca2+ releases caused by GTP and IP3 independently. Both IP3 and GTP, added to microsomes at the beginning of the glucose-6-P-stimulated Ca2+ uptake, reduced the Ca2+ accumulation into rough and smooth microsomes without modifying the initial rate (3 min) of Ca2+ uptake. Also in these conditions, the effects of GTP and IP3 were merely additive. These results indicate that both rough and smooth liver microsomes are responsive to IP3 and GTP with respect to Ca2+ release and that IP3 and GTP likely act independently.     

Microsomal Ca2+ flux modulation as an indicator of heavy metal toxicity

Inositol 1,4,5-trisphosphatee (IP3), an intracellular messenger, releases Ca2+ from microsomes. Ca2+ plays a major role in regulating various cellular events like neural transmission and regulation of hormones and growth factors. Aluminum (Al), lead (Pb) and mercury (Hg) were reported to alter Ca(2+)-regulated events thereby causing neurotoxicity. Hence, an attempt was made characterize IP3 mediated Ca2+ release from rat brain microsomes under the influence of Al, Pb and Hg. Different concentrations of metals were tested over a designated time scale and their effects on IP3 mediated Ca2+ release from microsomes were monitored using Fura-2 technique. All the three metals inhibited IP3 mediated Ca2+ release, Pb being more potent. The order of potency of these three metals was Pb>Hg>Al. Except for Al, both Hg and Pb independently released Ca2+ from microsomes. Re-uptake of Ca2+ into microsomes was inhibited by all the three metals, Pb being more potent. Microsomal Ca(2+)-ATPase activity was also inhibited by all the three metals. These results suggest that neurotoxicity exerted by Al, Pb and Hg may be due to the interference of these metals with IP3 mediated calcium release and also interfering with the microsomal Ca2+ sequestration mechanism. Differential effects of heavy metal induced changes in Ca2+ flux can be used as an index of relative toxicity.     

H2O2 directly activates inositol 1,4,5-trisphosphate receptors in endothelial cells

The mechanisms of H2O2-induced Ca2+ release from intracellular stores were investigated in human umbilical vein endothelial cells. It was found that U73122, the selective inhibitor of phospholipase C, could not inhibit the H2O2-induced cytosolic Ca2+ mobilization. No elevation of inositol 1,4,5-trisphosphate (IP3) was detected in cells exposed to H2O2. By loading mag-Fura-2, a Ca2+ indicator, into intracellular store, the H2O2-induced Ca2+ release from intracellular calcium store was directly observed in the permeabilized cells in a dose-dependent manner. This release can be completely blocked by heparin, a well-known antagonist of IP3 receptor, indicating a direct activation of IP3 receptor on endoplasmic reticulum (ER) membrane by H2O2. It was also found that H2O2 could still induce a relatively small Ca2+ release from internal stores after the Ca2+-ATPase on ER membrane and the Ca2+ uptake to mitochondria were simultaneously inhibited by thapsigargin and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The later observation suggests that a thapsigargin-insensitive non-mitochondrial intracellular Ca2+ store might be also involved in H2O2-induced Ca2+ mobilization.     

CCK-JMV-180, an analog of cholecystokinin, releases intracellular calcium from an inositol trisphosphate-independent pool in rat pancreatic acini.

In pancreatic acinar cells cholecystokinin and its analogs, caerulein and CCK-JMV-180, stimulate an increase in intracellular free [Ca2+] by releasing Ca2+ from non-mitochondrial intracellular pools. It is generally believed that the caerulein-induced release of Ca2+ is mediated by phospholipase C-catalyzed production of 1,4,5-inositol triphosphate (IP3). In this study we have investigated the source and mechanism of Ca2+ release induced by CCK-JMV-180 using streptolysin O-permeabilized pancreatic acinar cells. Caerulein-stimulated release of Ca2+ was completely blocked by either neomycin, an inhibitor of phospholipase C, or by heparin, an IP3 receptor antagonist. These observations are compatible with the conclusion that caerulein releases Ca2+ from an IP3-sensitive pool. In contrast to caerulein, however, CCK-JMV-180-stimulated release of Ca2+ was not blocked by either neomycin or by heparin, indicating that CCK-JMV-180 releases Ca2+ by mechanisms which do not involve the generation or action of IP3. CCK-JMV-180 stimulated the release of Ca2+ even after the IP3-sensitive pool had been completely emptied by prior exposure to a supramaximally stimulating concentration of IP3 (40 microM). Prestimulation of permeabilized acini with 20 mM caffeine did not abolish the CCK-JMV-180-induced Ca2+ release. These results indicate that CCK-JMV-180 stimulates release of Ca2+ from a hitherto uncharacterized intracellular storage pool which is insensitive to either IP3 or caffeine.     

Calcium release in HSY cells conforms to a steady-state mechanism involving regulation of the inositol 1,4,5-trisphosphate receptor Ca2+ channel by luminal [Ca2+]

In many cell types, low concentrations of inositol 1,4,5-trisphosphate (IP3) release only a portion of the intracellular IP3-sensitive Ca2+ store, a phenomenon known as "quantal" Ca2+ release. It has been suggested that this effect is a result of reduced activity of the IP3- dependent Ca2+ channel with decreasing calcium concentration within the IP3-sensitive store ([Ca2+]s). To test this hypothesis, the properties of IP3-dependent Ca2+ release in single saponin-permeabilized HSY cells were studied by monitoring [Ca2+]s using the Ca(2+)-sensitive fluorescent dye mag-fura-2. In permeabilized cells, blockade of the sarco/ER Ca(2+)-ATPase pump in stores partially depleted by IP3 induced further Ca2+ release via an IP3-dependent route, indicating that Ca2+ entry via the sarco/ER Ca(2+)-ATPase pump had been balanced by Ca2+ loss via the IP3-sensitive channel before pump inhibition. IP3- dependent Mn2+ entry, monitored via quenching of luminal mag-fura-2 fluorescence, was readily apparent in filled stores but undetectable in Ca(2+)-depleted stores, indicating markedly reduced IP3-sensitive channel activity in the latter. Also consistent with reduced responsiveness of Ca(2+)-depleted stores to IP3, the initial rate of refilling of these stores was unaffected by the presence of 0.3 microM IP3, a concentration that was clearly effective in eliciting Ca2+ release from filled stores. Analysis of the rate of Ca2+ release at various IP3 concentrations indicated a significant shift of the IP3 dose response toward higher [IP3] with decreasing [Ca2+]s. We conclude that IP3-dependent Ca2+ release in HSY cells is a steady-state process wherein Ca2+ efflux via the IP3 receptor Ca2+ channel is regulated by [Ca2+]s, apparently via changes in the sensitivity of the channel to IP3.     

Characterization of inositol 1,4,5-trisphosphate receptors and calcium mobilization in a hepatic plasma membrane fraction

The distribution of hepatic binding sites for the calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (IP3), was analyzed in subcellular fractions of the rat liver by binding studies with [32P]IP3 and compared with the Ca2+ release elicited by IP3 in each fraction. Three major subcellular fractions enriched in plasma membrane, mitochondria, and endoplasmic reticulum were characterized for their 5'-nucleotidase, glucose-6-phosphatase, succinate reductase, and angiotensin II binding activities. The fraction enriched in plasma membrane showed 7- and 20-fold increases in IP3 binding capacity over those enriched in endoplasmic reticulum and mitochondria, respectively, and contained a single class of high-affinity binding sites with Kd of 1.7 +/- 1.0 nM and concentration of 239 +/- 91 fmol/mg protein. IP3 binding reached equilibrium in 30 min at 0 degrees C, and the half-time of dissociation was about 15 min. The specificity of the IP3 binding sites was indicated by their markedly lower affinities for inositol 1-phosphate, phytic acid, fructose 1,6-bisphosphate, 2,3-bisphosphoglycerate, and inositol 1,3,4,5-tetrakisphosphate. The Ca2+-releasing activity of IP3 in the subcellular fractions was monitored with the fluorescent indicator, Fura-2. All three fractions showed ATP-dependent Ca2+ uptake and rapidly released Ca2+ in response in IP3. The fraction enriched in plasma membrane was the most active in this regard, releasing 174 +/- 67 pmol Ca2+/mg of protein compared to 45 +/- 10 and 48 +/- 7 pmol/mg protein for the fractions enriched in endoplasmic reticulum and mitochondria, respectively. These data suggest that the [32P]IP3 binding sites represent specific intracellular receptors through which IP3 mobilizes Ca2+ from a storage site associated (or co-purifying) with the plasma membrane of the rat liver. It is likely that a specialized vesicular system (to which IP3 can bind and trigger the release of Ca2+) is located in close proximity with the plasma membrane and is thus adjacent to the site at which IP3 is produced during stimulation of the hepatocyte by Ca2+-mobilizing hormones.