Changes in functional properties of the caffeine-sensitive Ca2+ store during differentiation of human SH-SY5Y neuroblastoma cells

We have used single cell fluorescence imaging techniques to examine how functional properties of the caffeine-sensitive Ca(2+) store change during differentiation of a sub-population of caffeine-sensitive SH-SY5Y cells. Application of caffeine (30 mM) 1-10.5 min after a 'priming' depolarisation pulse of 55 mM K(+) revealed that the caffeine-sensitive store in undifferentiated cells remained replete, whereas that in 9-cis retinoic acid (9cRA)-differentiated cells spontaneously dissipated with a t(1/2) of 2.8 min, and was essentially completely depleted approximately 10 min after priming. In 9cRA-differentiated cells that were stimulated with methacholine (10 microM) 1 min after priming, the amplitude, rate of rise and propagation velocity of the Ca(2+) wave in the neurites were all constant, whereas these kinetic parameters all progressively decreased as the wave travelled along the neurites in cells that were stimulated 10 min after priming. Use-dependent block with ryanodine inhibited the global Ca(2+) signal in 9cRA-differentiated cells stimulated with methacholine 1 min after priming (71+/-8%) but not 10 min after priming. Depolarisation was more effective at priming the caffeine-sensitive Ca(2+) store in 9cRA-differentiated cells, which lack a functional store-operated Ca(2+) entry pathway. We conclude that differentiation of caffeine-sensitive SH-SY5Y cells is accompanied by an increase in lability of the caffeine-sensitive Ca(2+) store, and that spontaneous dissipation of Ca(2+) from the store limits the time course of its molecular 'memory' during which it can amplify the hormone-induced Ca(2+) signal by Ca(2+)-induced Ca(2+) release.     

Coincident signalling between the Gi/Go-coupled delta-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells

In SH-SY5Y cells, activation of delta-opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 microM) did not alter the intracellular free Ca(2+) concentration [Ca(2+)](i). However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca(2+)](i). The DPDPE-evoked increase in [Ca(2+)](i) was abolished when the carbachol-sensitive intracellular Ca(2+) store was emptied. There was a marked difference between the concentration-response relationship for the elevation of [Ca(2+)](i) by carbachol (EC(50) 13 microM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta-opioid receptor-mediated elevation of [Ca(2+)] (EC(50) 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca(2+)](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta-opioid receptor activation did not stimulate phospholipase C. Furthermore, delta-opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the delta-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca(2+)](i) in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed delta-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca(2+) release signalling pathway at a step after the activation of phospholipase C.     

Caffeine-stimulated GTH-II release involves Ca(2+) stores with novel properties

Modulation of Ca(2+) stores with 10 mM caffeine stimulates robust secretion of gonadotropin (GTH-II) from goldfish gonadotropes. Although both endogenous forms of gonadotropin-releasing hormone (GnRH) utilize a common intracellular Ca(2+) store, sGnRH, but not cGnRH-II, uses an additional caffeine-sensitive mechanism. We examined caffeine signaling by using Ca(2+) imaging, electrophysiology, and cell-column perifusion. Although caffeine inhibited K+ channels, this action appeared to be unrelated to caffeine-induced GTH-II release, because the latter was insensitive to tetraethylammonium. The effects of caffeine also were not mediated by the cAMP/protein kinase A pathway. Instead, caffeine-evoked GTH-II responses were Ca(2+) signal dependent because they were abolished by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid loading. Caffeine generated localized Ca(2+) signals that began near secretory granules. Surprisingly, caffeine-stimulated GTH-II release was insensitive to 100 microM ryanodine and, unlike GnRH action, was unaffected by inhibitors of voltage-gated Ca(2+) channels or sarco(endo)plasmic reticulum Ca(2+)-ATPases. Collectively, these data indicate that caffeine-stimulated GTH-II release is not mediated by typical agonist-sensitive Ca(2+) stores found in endoplasmic reticulum.     

Vectorial Ca2+ release via ryanodine receptors contributes to Ca2+ extrusion from freshly isolated rabbit aortic endothelial cells

In this study, we identified ryanodine receptors (RyRs) as a component of a cytosolic Ca(2+) removal pathway in freshly isolated rabbit aortic endothelial cells. In an earlier article, we reported that the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and Na(+)/Ca(2+) exchanger (NCX) function in series to extrude cytosolic Ca(2+) to the extracellular space. Here we employed caffeine and ryanodine as modulators of RyR and showed that they act as the linkage between SERCA and NCX in removing Ca(2+) from the cytoplasm. Our data indicate that both 15 mM caffeine and 1 microM ryanodine facilitated Ca(2+) extrusion by activating RyRs while 100 microM ryanodine had the opposite effect by blocking RyRs. A further attempt to investigate RyR pharmacology revealed that in the absence of extracellular Ca(2+), ryanodine at 1 microM, but not 100 microM, stimulated Ca(2+) loss from the endoplasmic reticulum (ER). Blockade of RyR had no effect on the Ca(2+) removal rate when NCX had been previously blocked. In addition, the localization of RyR was determined using confocal microscopy of BODIPY TR-X fluorescent staining. Taken together, our findings suggest that in freshly isolated endothelial cells Ca(2+) is removed in part by transport through SERCA, RyR, and eventually NCX, and that RyR and NCX are in close functional proximity near the plasma membrane. After blockade of this component, Ca(2+) extrusion could be further inhibited by carboxyeosin, indicating a parallel contribution by the plasmalemmal Ca(2+)-ATPase (PMCA).     

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.     

Ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by calmodulin

The effects of calmodulin (CaM) and CaM antagonists on microsomal Ca(2+) release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. When caffeine (10 mM) was added after a steady state of ATP-dependent (45)Ca(2+) uptake into the microsomal vesicles, the caffeine-induced (45)Ca(2+) release was significantly increased by pretreatment with ryanodine (10 microM). The presence of W-7 (60 microM), a potent inhibitor of CaM, strongly inhibited the release, while W-5 (60 microM), an inactive CaM antagonist, showed no inhibition. Inhibition of the release by W-7 was observed at all caffeine concentrations (5-30 mM) tested. The presence of exogenously added CaM (10 microg/ml) markedly increased the caffeine (5-10 mM)-induced (45)Ca(2+) release and shifted the dose-response curve of caffeine-induced (45)Ca(2+) release to the left. Cyclic ADP-ribose (cADPR, 2 microM)-induced (45)Ca(2+) release was enhanced by the presence of ryanodine (10 microM). cADPR (2 microM)- or ryanodine (500 microM)-induced (45)Ca(2+) release was also inhibited by W-7 (60 microM), but not by W-5 (60 microM), and was stimulated by CaM (10 microg/ml). These results suggest that the ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by CaM.     

Ryanodine Inhibits Caffeine-Evoked Ca2+ Mobilization and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

The effects of ryanodine, a selective inhibitor of the Ca(2+)-induced Ca2+ release mechanism, on caffeine-evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5-40 mM) caused a concentration-dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg(2+)-free medium containing 0.2 mM EGTA. Ryanodine (5 x 10(-5) M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 x 10(-7) - 5 x 10(-5) M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2+/Mg(2+)-free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin-permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.     

Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor

Recent studies have suggested a central role for Ca(2+) in the signaling pathway of apoptosis and certain anti-apoptotic effects of Bcl-2 family of proteins have been attributed to changes in intracellular Ca(2+) homeostasis. Here we report that depletion of Ca(2+) from endoplasmic reticulum (ER) leads to apoptosis in Chinese hamster ovary cells. Stable expression of ryanodine receptor (RyR) in these cells enables rapid and reversible changes of both cytosolic Ca(2+) and ER Ca(2+) content via activation of the RyR/Ca(2+) release channel by caffeine and ryanodine. Sustained depletion of the ER Ca(2+) store leads to apoptosis in Chinese hamster ovary cells, whereas co-expression of Bcl-xL and RyR in these cells prevents apoptotic cell death but not necrotic cell death. The anti-apoptotic effect of Bcl-xL does not correlate with changes in either the Ca(2+) release process from the ER or the capacitative Ca(2+) entry through the plasma membrane. The data suggest that Bcl-xL likely prevents apoptosis of cells at a stage downstream of ER Ca(2+) release and capacitative Ca(2+) entry.     

Calcium-induced calcium release in neurosecretory insect neurons: fast and slow responses

The dynamics of intracellular free Ca(2+)([Ca(2+)](i)) changes were investigated in dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana. Activation of voltage-gated Ca(2+) channels caused a steep increase in [Ca(2+)](i). Depolarizations lasting for < 100ms led to Ca(2+) release from intracellular stores as is indicated by the finding that the rise of [Ca(2+)](i) was greatly reduced by the antagonists of ryanodine receptors, ryanodine and ruthenium red. There is a resting Ca(2+)current which is potentiated on application of a neuropeptide, Neurohormone D (NHD), a member of the adipokinetic hormone family. Ca(2+) influx enhanced in this way again caused a rise of [Ca(2+)](i) sensitive to ryanodine and ruthenium red. Such rises developed and relaxed much more slowly than the depolarization-induced signals. Ca(2+)responses similar to those induced by NHD were obtained with the ryanodine receptor agonists caffeine (20mM) and cADP-ribose (cADPR, 100nM). These Ca(2+) responses, however, varied considerably in size and kinetics, and part of the cells did not respond at all to caffeine or cADPR. Such cells, however, produced Ca(2+) rises after having been treated with NHD. Thus, the variability of Ca(2+) signals might be caused by different filling states of Ca(2+) stores, and the resting Ca(2+) current seems to represent a source to fill empty Ca(2+) stores. In line with this notion, block of the endoplasmic Ca(2+) pump by thapsigargin (1 microM) produced either no or largely varying Ca(2+) responses. The Ca(2+) signals induced by caffeine and cADPR displayed different sensitivity to ryanodine receptor blockers. cADPR failed to elicit any response when ryanodine or ruthenium red were present. By contrast, the response to caffeine, in the presence of ryanodine, was only reduced by about 50% and, in the presence of ruthenium red, it was not at all reduced. Thus, there may be different types of Ca(2+) release channels. Block of mitochondrial Ca(2+) uptake with carbonyl cyanide m -chlorophenylhydrazone (CCCP, 1 microM) completely abolished cADPR-induced Ca(2+) signals, but it did not affect the caffeine-induced signals. Taken together our findings seem to indicate that there are different stores using different Ca(2+) uptake pathways and that some of these pathways involve mitochondria.     

Regulation of muscarinic cationic current in myocytes from guinea-pig ileum by intracellular Ca2+ release: a central role of inositol 1,4,5-trisphosphate receptors

The dynamics of carbachol (CCh)-induced [Ca(2+)](i) changes was related to the kinetics of muscarinic cationic current (mI(cat)) and the effect of Ca(2+) release through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs) on mI(cat) was evaluated by fast x-y or line-scan confocal imaging of [Ca(2+)](i) combined with simultaneous recording of mI(cat) under whole-cell voltage clamp. When myocytes freshly isolated from the longitudinal layer of the guinea-pig ileum were loaded with the Ca(2+)-sensitive indicator fluo-3, x-y confocal imaging revealed CCh (10 microM)-induced Ca(2+) waves, which propagated from the cell ends towards the myocyte centre at 45.9 +/- 8.8 microms(-1) (n = 13). Initiation of the Ca(2+) wave preceded the appearance of any measurable mI(cat) by 229 +/- 55 ms (n = 7). Furthermore, CCh-induced [Ca(2+)](i) transients peaked 1.22 +/- 0.11s (n = 17) before mI(cat) reached peak amplitude. At -50 mV, spontaneous release of Ca(2+) through RyRs, resulting in Ca(2+) sparks, had no effect on CCh-induced mI(cat) but activated BK channels leading to spontaneous transient outward currents (STOCs). In addition, Ca(2+) release through RyRs induced by brief application of 5 mM caffeine was initiated at the cell centre but did not augment mI(cat) (n = 14). This was not due to an inhibitory effect of caffeine on muscarinic cationic channels (since application of 5 mM caffeine did not inhibit mI(cat) when [Ca(2+)](i) was strongly buffered with Ca(2+)/BAPTA buffer) nor was it due to an effect of caffeine on other mechanisms possibly involved in the regulation of Ca(2+) sensitivity of muscarinic cationic channels (since in the presence of 5 mM caffeine, photorelease of Ca(2+) upon cell dialysis with 5 mM NP-EGTA/3.8 mM Ca(2+) potentiated mI(cat) in the same way as in control). In contrast, IP(3)R-mediated Ca(2+) release upon flash photolysis of "caged" IP(3) (30 microM in the pipette solution) augmented mI(cat) (n = 15), even though [Ca(2+)](i) did not reach the level required for potentiation of mI(cat) during photorelease of Ca(2+) (n = 10). Intracellular calcium stores were visualised by loading of the myocytes with the low-affinity Ca(2+) indicator fluo-3FF AM and consisted of a superficial sarcoplasmic reticulum (SR) network and some perinuclear formation, which appeared to be continuous with the superficial SR. Immunostaining of the myocytes with antibodies to IP(3)R type 1 and to RyRs revealed that IP(3)Rs are predominant in the superficial SR while RyRs are confined to the central region of the cell. These results suggest that IP(3)R-mediated Ca(2+) release plays a central role in the modulation of mI(cat) in the guinea-pig ileum and that IP(3) may sensitise the regulatory mechanisms of the muscarinic cationic channels gating to Ca(2+).     

Receptor-activated Ca2+ influx. Two independently regulated mechanisms of influx stimulation coexist in neurosecretory PC12 cells.

Receptor-activated Ca2+ influx was investigated in PC12 cells clones loaded with fura-2. Cells were stimulated in a Ca(2+)-free medium and studied after reintroduction of the cation or addition of Mn2+ into the medium. A first influx component, independent of receptor activation and sustained by depletion of the intracellular inositol 1,4,5-trisphosphate sensitive Ca2+ store (store-dependent Ca2+ influx, SDCI), was identified by experiments with carbachol followed by atropine and with agents that induce store discharge without polyphosphoinositide hydrolysis: thapsigargin, an inhibitor of Ca(2+)-ATPase activity; ryanodine and caffeine, activators of the ryanodine receptor. A second component of Ca2+ influx, induced by carbachol and rapidly blocked by atropine, relies on receptor-effector coupling via G protein(s) different from that (those) involved in phospholipase C activation. SDCI and receptor-coupled influx are similar in their voltage dependence and insensitivity to forskolin and phorbol esters but they differ with respect to their Mn2+ permeability and their sensitivity to the SC 38249 imidazole blocker. The two components might play different roles. SDCI might act as a safety device to prevent Ca2+ store depletion whereas receptor-dependent influx might control physiological functions such as secretion and growth.     

Functional expression of recombinant type 1 ryanodine receptor in insect cells

We have investigated the biochemical properties of the rabbit ryanodine receptor type 1 (RyR1) from skeletal muscle functionally expressed in insect sf 21 cells infected with recombinant baculovirus. Equilibrium [3H]ryanodine binding assays applied to total membrane fractions from sf 21 cells expressing recombinant RyR1 showed a non-hyperbolic saturation curve (Hill coefficient = 2.1). The [3H]ryanodine binding was enhanced by 1 mM AMP-PCP and 10 mM caffeine, whereas 10 mM Mg(2+) and 5 microM ruthenium red reduced the specific binding. The dependence of [3H]ryanodine binding on ionic strength showed positive cooperativity (Hill coefficient = 2.2) with a plateau at 1 M KCl. The recombinant RyR1 showed a bell-shaped [3H]ryanodine binding curve when free [Ca(2+)] was increased, with an optimal concentration around 100 microM.Confocal microscopy studies using the Ca(2+) ATPase selective inhibitor, thapsigargin coupled to fluorescein and ryanodine coupled to Texas red demonstrated that the recombinant RyR1 and the Ca(2+) ATPase co-localize to the same intracellular membrane. No significant RyR1 fluorescence was observed at the plasma membrane.Fluo-4-loaded sf 21 cells expressing recombinant RyR1 responded to activating-low ryanodine concentrations (100 nM) or caffeine (10 mM) with a sharp rise in intracellular Ca2 followed by a sustained phase, in contrast, sf 21 cells expressing the human bradykinin type 2 receptor did not respond to ryanodine or caffeine.These results demonstrate the expression of recombinant RyR1 in sf 21 cells with functional properties similar to what has been previously reported for native RyR1 in mammalian tissues, however, some differences were observed in [3H]ryanodine binding assays compared to native rabbit RyR1. Hence, the baculovirus expression system provides a generous source of protein to accomplish structure-function studies and an excellent model to assess functional properties of wild type and mutant RyR1.     

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.     

Store depletion by caffeine/ryanodine activates capacitative Ca(2+) entry in nonexcitable A549 cells

Capacitative Ca(2+) entry is essential for refilling intracellular Ca(2+) stores and is thought to be regulated primarily by inositol 1, 4,5-trisphosphate (IP(3))-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca(2+) release in the absence of extracellular Ca(2+) similar to that induced by thapsigargin (Tg), and Ca(2+) entry occurs upon the readdition of extracellular Ca(2+). The channels thus activated are also permeable to Mn(2+). The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca(2+) concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca(2+) release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca(2+) release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP(3) receptor. These results suggest that in A549 cells, (i) capacitative Ca(2+) entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP(3), probably via Ca(2+)-induced Ca(2+) release.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

Ca2+-dependent potentiation of muscarinic receptor-mediated Ca2+ elevation

Muscarinic receptor-mediated increases in Ca(2+) in SH-SY5Y neuroblastoma cells consist of an initial fast and transient phase followed by a sustained phase. Activation of voltage-gated Ca(2+) channels prior to muscarinic stimulation resulted in a several-fold potentiation of the fast phase. Unlike the muscarinic response under control conditions, this potentiated elevation of intracellular Ca(2+) was to a large extent dependent on extracellular Ca(2+). In potentiated cells, muscarinic stimulation also activated a rapid Mn(2+) entry. By using known organic and inorganic blockers of cation channels, this influx pathway was easily separated from the known Ca(2+) influx pathways, the store-operated pathway and the voltage-gated Ca(2+) channels. In addition to the Ca(2+) influx, both IP(3) production and Ca(2+) release were also enhanced during the potentiated response. The results suggest that a small increase in intracellular Ca(2+) amplifies the muscarinic Ca(2+) response at several stages, most notably by unravelling an apparently novel receptor-activated influx pathway.     

Identification of cyclic ADP-ribose-dependent mechanisms in pancreatic muscarinic Ca(2+) signaling using CD38 knockout mice

We showed that muscarinic acetylcholine (ACh)-stimulation increased the cellular content of cADPR in the pancreatic acinar cells from normal mice but not in those from CD38 knockout mice. By monitoring ACh-evoked increases in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) using fura-2 microfluorimetry, we distinguished and characterized the Ca(2+) release mechanisms responsive to cADPR. The Ca(2+) response from the cells of the knockout mice (KO cells) lacked two components of the muscarinic Ca(2+) release present in wild mice. The first component inducible by the low concentration of ACh contributed to regenerative Ca(2+) spikes. This component was abolished by ryanodine treatment in the normal cells and was severely impaired in KO cells, indicating that the low ACh-induced regenerative spike responses were caused by cADPR-dependent Ca(2+) release from a pool regulated by a class of ryanodine receptors. The second component inducible by the high concentration of ACh was involved in the phasic Ca(2+) response, and it was not abolished by ryanodine treatment. Overall, we conclude that muscarinic Ca(2+) signaling in pancreatic acinar cells involves a CD38-dependent pathway responsible for two cADPR-dependent Ca(2+) release mechanisms in which the one sensitive to ryanodine plays a crucial role for the generation of repetitive Ca(2+) spikes.     

Regulation of ryanodine receptor opening by lumenal Ca(2+) underlies quantal Ca(2+) release in PC12 cells

Graded or "quantal" Ca(2+) release from intracellular stores has been observed in various cell types following activation of either ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (InsP(3)R). The mechanism causing the release of Ca(2+) stores in direct proportion to the strength of stimulation is unresolved. We investigated the properties of quantal Ca(2+) release evoked by activation of RyR in PC12 cells, and in particular whether the sensitivity of RyR to the agonist caffeine was altered by lumenal Ca(2+). Quantal Ca(2+) release was observed in cells stimulated with 1 to 40 mM caffeine, a range of caffeine concentrations giving a >10-fold change in lumenal Ca(2+) content. The Ca(2+) load of the caffeine-sensitive stores was modulated by allowing them to refill for varying times after complete discharge with maximal caffeine, or by depolarizing the cells with K(+) to enhance their normal steady-state loading. The threshold for RyR activation was sensitized approximately 10-fold as the Ca(2+) load increased from a minimal to a maximal loading. In addition, the fraction of Ca(2+) released by low caffeine concentrations increased. Our data suggest that RyR are sensitive to lumenal Ca(2+) over the full range of Ca(2+) loads that can be achieved in an intact PC12 cell, and that changes in RyR sensitivity may be responsible for the termination of Ca(2+) release underlying the quantal effect.     

An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility.

Hyperactivated motility, a swimming pattern displayed by mammalian sperm in the oviduct around the time of ovulation, is essential to fertilization. Ca(2+) has been shown to be crucial for the initiation and maintenance of hyperactivated motility. Nevertheless, how Ca(2+) reaches the axoneme in the core of the flagellum to switch on hyperactivation is unknown. Ca(2+)-releasing agents were used to determine whether an intracellular store provides Ca(2+) to the axoneme. Hyperactivation was induced immediately in bull sperm by thapsigargin, caffeine, and thimerosal. The responses were dose-dependent and were induced in both capacitated and uncapacitated sperm. When external Ca(2+) was buffered below 50 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the response to caffeine was significantly reduced; however, the responses to thapsigargin and thimerosal were not affected. This indicates caffeine-induced hyperactivation depends on external Ca(2+) influx, whereas hyperactivation by thapsigargin and thimerosal do not. Acrosome reactions were not induced by these treatments; therefore, an acrosomal store was probably not involved. Indirect immunofluorescence labeling showed type I inositol 1,4,5-trisphosphate receptors (IP(3)R) in the acrosome and neck region, but no ryanodine receptors (RyR) were found using anti-RyR antibodies or BODIPY FL-X ryanodine. These data indicate that there is an IP(3)R-gated Ca(2+) store in the neck region of sperm that regulates hyperactivated motility.     

Role of extracellular Ca2+ in diaphragmatic contraction: effects of ouabain, monensin, and ryanodine.

The effects of zero extracellular Ca2+ on the contractility of rat diaphragmatic strips in vitro were studied in conjunction with various pharmacological agents known to influence the intracellular Ca2+ concentration: the Na+ ionophore, monensin, and the Na(+)-K+ pump inhibitor, ouabain, which enhance [Ca2+]i, caffeine, which induces Ca2+ release from the sarcoplasmic reticulum (SR), and ryanodine, which prevents Ca2+ retention by the SR. The effect of increasing [Ca2+]i on diaphragmatic contraction was assessed by comparing contractions induced by 120 mM K+ in the small muscle strips before and after the addition of ouabain or monensin. Monensin (20 microM) and ouabain (1-100 microM) augmented contractions up to threefold. Treatment of diaphragm strips with 3 nM ryanodine increased baseline tension 360% above the original resting tension but only if the diaphragm was electrically stimulated concurrently; 100 microM ryanodine induced contracture in quiescent tissue. High K+ contractures were of greater magnitude in the presence of ryanodine compared with control, and relaxation time was prolonged by greater than 200%. Ca(2+)-free conditions ameliorated these actions of ryanodine. Ryanodine reduced contractions induced by 10 mM caffeine and nearly abolished them in Ca(2+)-free solution. The data demonstrate that extracellular Ca2+ is important in certain types of contractile responses of the diaphragm and suggest that the processes necessary to utilize extracellular Ca2+ are present in the diaphragm.