Zymosan-induced release of inositol phosphates at resting cytosolic Ca2+ concentrations in macrophages.

Hydrolysis of polyphosphoinositides by phosphodiesterase has been demonstrated to be involved in the control of cytosolic Ca2+ concentrations. The stimulation of Ca2+ ionophores of the release of inositol phosphates in macrophages, and other cells, together with the Ca2+ requirements for zymosan-induced phospholipase C activation, make unclear the relationship between Ca2+ mobilization and polyphosphoinositide hydrolysis. The results in the present paper strongly suggest that, for zymosan-induced phospholipase C activation, a previous increase in cytosolic Ca2+ is not a required event. These results also show that zymosan-activated release of inositol phosphates may be mediated by a guanine-nucleotide-binding protein.     

Measurement of cytosolic free Ca2+ in individual pancreatic acini

The kinetics of changes in cytosolic free Ca2+ ([Ca2+]i) were determined in individual rat pancreatic acini by microfluorimetry. Three major findings are reported. First, at maximal stimulatory concentrations for amylase release, both caerulein and bombesin induced an initial rise in [Ca2+]i followed by prolonged secondary oscillations of smaller amplitude. The latter effect was not observed with supramaximal doses of caerulein. Second, these cyclic changes were dependent, at least in part, on extracellular Ca2+. Finally, comparison of the threshold doses for [Ca2+]i mobilization and enzyme discharge demonstrated that pathways independent of an elevation of [Ca2+]i control the secretory activity of pancreatic acini at low, picomolar agonist concentrations.     

A comparison of cytosolic free Ca2+ in resting feline and rat ventricular myocytes

The Ca2+-sensitive dye quin-2 was used to measure the cytosolic free Ca2+ (Cai2+) in suspensions of ventricular myocytes isolated from cat and rat ventricles. Following an isolation procedure that was similar for both species, the cells were loaded with quin-2 AM (25 microM) for 30 min at 37 degrees C. After two washes to remove extracellular dye, the cells were resuspended for fluorescence measurements. Extracellular Ca2+ was 2.0 mM. Resting Cai2+ in the rat (121 +/- 11 nM) was found to be significantly higher than in the cat (57 +/- 4 nM). These results are discussed in terms of known differences in excitation-contraction coupling between these two species.     

Effect of thapsigargin on cytosolic Ca2+ level and amylase release in rat parotid acinar cells.

At concentrations greater than 0.01 microM, thapsigargin (ThG) dose-dependently caused an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in rat parotid acinar cells, as measured by the fluorescent Ca(2+)-indicator fura-2. In the absence of extracellular Ca2+, a transient increase in [Ca2+]i by ThG was observed, and subsequent addition of carbachol (CCh) did not produce a further [Ca2+]i response, suggesting that ThG released Ca2+ from the CCh-sensitive intracellular Ca2+ pool. Since ThG did not stimulate formation of inositol phosphates, the ThG-induced Ca2+ mobilization is independent of phosphoinositide breakdown. High concentrations (greater than 0.1 microM) of ThG induced amylase release from rat parotide acini, but the effect was very poor as compared with that of CCh or the protein kinase C activator, PMA (phorbol 12-myristate 13-acetate). Combined addition of ThG and PMA modestly potentiated amylase release induced by PMA alone. These results support the view that amylase release by muscarinic stimulation is mediated mainly by activation of protein kinase C rather than a rise in [Ca2+]i, although Ca2+ may modulate the secretory response.     

Muscarinic receptor-induced phosphoinositide hydrolysis at resting cytosolic Ca2+ concentration in PC12 cells

In PC12 cells, cultured in the presence of nerve growth factor to increase their complement of muscarinic receptors, treatment with carbachol induces muscarinic receptor-dependent rises in free cytosolic Ca2+ as well as hydrolysis of membrane phosphoinositides. Experiments were carried out to clarify the relationship between these two receptor-triggered events. In particular, since inositol-1,4,5-trisphosphate (the hydrophilic metabolite produced by the hydrolysis of phosphatidylinositol-4,5-bisphosphate) is believed to mediate intracellularly the release of Ca2+ from nonmitochondrial store(s), it was important to establish whether it can be generated at resting cytoplasmic concentration of Ca2+ (approximately 0.1 microM). Cells incubated in Ca2+-free medium were depleted of their cytoplasmic Ca2+ stores by pretreatment with ionomycin. When these cells were then treated with carbachol, their cytosolic concentration of Ca2+ remained at the resting level, whereas inositol-1,4,5-trisphosphate generation was still markedly stimulated. Our results demonstrate that an increase in the concentration of cytosolic Ca2+ is not a necessary intermediate between receptor activation and phosphoinositide hydrolysis, and therefore support the second-messenger role of inositol-1,4,5-trisphosphate.     

Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation.

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.     

Effects of Ca ionophore A23187 and Ca deficiency on pancreatic phospholipids and amylase release in vitro

The Ca²⁺ ionophore A23187 elicits a transient increase in pancreatic amylase release in vitro, and this is accompanied by a transient decrease in phosphatidyl inositol concentration. Effects of ionophore A23187 and carbachol on amylase release and phosphatidylinositol breakdown are dependent on medium Ca²⁺. These results suggest that major secretagogue-induced, pancreatic phospholipid changes follow, rather than precede, changes in Ca²⁺ in the pancreas.     

Luminal Ca2+ controls the activation of the inositol 1,4,5-trisphosphate receptor by cytosolic Ca2+.

Luminal Ca2+ controls the sensitivity of the intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Ins(1,4,5)P3-induced Ca2+ release is also controlled by cytosolic Ca2+; low concentrations of Ca2+ stimulate the release. The aim of this work was to investigate whether luminal Ca2+ would affect the stimulation of the Ins(1,4,5)P3 receptor by cytosolic Ca2+ in permeabilized A7r5 smooth muscle cells. We also report that the Ins(1,4,5)P3 receptor in A7r5 cells is activated by low concentrations of cytosolic Ca2+. Cytoplasmic Ca2+ increases the Ins(1,4,5)P3 sensitivity without affecting the cooperativity. The increase in Ins(1,4,5)P3 sensitivity becomes relatively more pronounced when the Ca2+ content of the stores decreases. This modulatory effect of luminal Ca2+ on the responsiveness to cytosolic Ca2+ is an intrinsic property of the Ins(1,4,5)P3 receptor.     

Direct demonstration of increases in cytosolic free Ca2+ during stimulation of pancreatic enzyme secretion

The Ca²⁺-activated photoprotein aequorin has been incorporated into intact, isolated rat pancreatic acini by a hypotonic swelling method. The isolated acini retained normal secretory responses after loading with aequorin. Increases in cytosolic Ca²⁺ concentration in response to a physiological secretagogue, carbamylcholine, and to divalent-cation ionophore A23187 have been demonstrated. Simultaneous measurement of the dynamics of enzyme secretion and changes in cytosolic Ca²⁺ concentration has been achieved using a newly developed apparatus.     

Regulation of cardiac muscle Ca2+ release channel by sarcoplasmic reticulum lumenal Ca2+.

The cardiac muscle sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor) is a ligand-gated channel that is activated by micromolar cytoplasmic Ca2+ concentrations and inactivated by millimolar cytoplasmic Ca2+ concentrations. The effects of sarcoplasmic reticulum lumenal Ca2+ on the purified release channel were examined in single channel measurements using the planar lipid bilayer method. In the presence of caffeine and nanomolar cytosolic Ca2+ concentrations, lumenal-to-cytosolic Ca2+ fluxes >/=0.25 pA activated the channel. At the maximally activating cytosolic Ca2+ concentration of 4 microM, lumenal Ca2+ fluxes of 8 pA and greater caused a decline in channel activity. Lumenal Ca2+ fluxes primarily increased channel activity by increasing the duration of mean open times. Addition of the fast Ca2+-complexing buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cytosolic side of the bilayer increased lumenal Ca2+-activated channel activities, suggesting that it lowered Ca2+ concentrations at cytosolic Ca2+-inactivating sites. Regulation of channel activities by lumenal Ca2+ could be also observed in the absence of caffeine and in the presence of 5 mM MgATP. These results suggest that lumenal Ca2+ can regulate cardiac Ca2+ release channel activity by passing through the open channel and binding to the channel's cytosolic Ca2+ activation and inactivation sites.     

Insulin release independent of a rise in cytosolic free Ca2+ by forskolin and phorbol ester

The role of cytosolic free Ca2+ in insulin release was evaluated using isolated rat pancreatic islets permeabilized with digitonin and incubated in Ca-EGTA buffers to fix free Ca2+ concentration at arbitrary levels. Ca2+ induced insulin release in a concentration-dependent manner with the threshold being between 0.1 and 1 microM. The hormone release was increased by forskolin and 12-O-tetradecanoyl phorbol-13-acetate (TPA), a potent activator of adenylate cyclase and that of protein kinase C, respectively. The findings suggest that activation of both protein kinase A and protein kinase C modulate insulin release without a concomitant increase in cytosolic free Ca2+.     

Oscillations in cytosolic free Ca2+, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets

Insulin secretion in the intact organism, and by the perfused pancreas and groups of isolated perifused islets, is pulsatile. We have proposed a metabolic model of glucose-induced insulin secretion in which oscillations in the ATP/ADP ratio drive alterations in metabolic and electrical events that lead to insulin release. A key prediction of our model is that metabolically driven Ca2+ oscillations will also occur. Using the fluorescent Ca2+ probe, fura 2, digital image analysis, and sensitive O2 electrodes, we investigated cytosolic free Ca2+ responses and O2 consumption in perifused rat islets that had been maintained in culture for 1-4 days. We found that elevated ambient glucose increased the average cytosolic free Ca2+ level, the ATP/ADP ratio, and oxygen consumption, as previously found in freshly isolated islets. Oscillatory patterns were obtained for Ca2+, O2 consumption, and insulin secretion in the presence of 10 and 20 mM glucose. Very low amplitude oscillations in cytosolic free Ca2+ were observed at 3 mM nonstimulatory glucose levels. Evaluation of the Ca2+ responses of a large series of individual islets, monitored by digital image analysis and perifused at both 3 and 10 mM glucose, indicated that the rise in glucose concentration caused more than a doubling of the average cytosolic free Ca2+ value and a 4-fold increase in the amplitude of the oscillations with little change in period. The pattern of Ca2+ change within the islets was consistent with recruitment of responding cells. The coexistence of oscillations with similar periods in insulin secretion, oxygen consumption, and cytosolic free Ca2+ is consistent with the model of metabolically driven pulsatile insulin secretion.     

Role of free cytosolic calcium in secretagogue-stimulated amylase release from dispersed acini from guinea pig pancreas

To determine the role of free cytosolic calcium ([Ca+2]i) in stimulated enzyme secretion from exocrine pancreas, we determined the effects of various pancreatic secretagogues on [Ca+2]i and amylase release in dispersed acini from the guinea pig pancreas. Cholecystokinin-octapeptide (CCK-OP), carbachol, and bombesin, but not vasoactive intestinal peptide, stimulated rapid increases in [Ca+2]i from 100 to 600-800 nM that were independent of extracellular calcium. The increases in [Ca+2]i were transient (lasting less than 5 min) and correlated with an initial rapid phase of amylase release. After 5 min, secretagogue-stimulated amylase release occurred at basal [Ca+2]i. Carbachol pretreatment of the acini abolished the effects of CCK-OP and bombesin on [Ca+2]i and the initial rapid phase of amylase release. 4 beta-phorbol 12-myristate 13-acetate (PMA) had no effect on [Ca+2]i but stimulated an increase in amylase release. The addition of CCK-OP or A23187 to PMA-stimulated acini caused an increase in [Ca+2]i and PMA-stimulated amylase release only during the first 5 min after addition of these agents. These results indicate that CCK-OP, carbachol, and bombesin release calcium from an intracellular pool, resulting in a transient increase in [Ca+2]i and that this increase in [Ca+2]i mediates enzyme secretion during the first few minutes of incubation. The results with PMA suggest that secretagogue-stimulated secretion not mediated by increased [Ca+2]i (sustained secretion) is mediated by 1,2-diacylglycerol.     

Melatonin modulates Ca2+ mobilization and amylase release in response to cholecystokinin octapeptide in mouse pancreatic acinar cells

In the present work, we have evaluated the effect of an acute addition of melatonin on cholecystokinin octapeptide (CCK-8)-evoked Ca²⁺ signals and amylase secretion in mouse pancreatic acinar cells. For this purpose, freshly isolated mouse pancreatic acinar cells were loaded with fura-2 to study intracellular free Ca²⁺ concentration ([Ca²⁺]_c). Amylase release and cell viability were studied employing colorimetric methods. Our results show that CCK-8 evoked a biphasic effect on amylase secretion, finding a maximum at a concentration of 0.1 nM and a reduction of secretion at higher concentrations. Pre-incubation of cells with melatonin (1 μM–1 mM) significantly attenuated enzyme secretion in response to high concentrations of CCK-8. Stimulation of cells with 1 nM CCK-8 led to a transient increase in [Ca²⁺]_c, followed by a decrease towards a constant level. In the presence of 1 mM melatonin, stimulation of cells with CCK-8 resulted in a smaller [Ca²⁺]_c peak response, a faster rate of decay of [Ca²⁺]_c and lower values for the steady state of [Ca²⁺]_c, compared with the effect of CCK-8 alone. Melatonin also reduced the oscillatory pattern of Ca²⁺ mobilization evoked by a physiological concentration of CCK-8 (20 pM), and completely inhibited Ca²⁺ mobilization induced by 10 pM CCK-8. On the other hand, Ca²⁺ entry from the extracellular space was not affected in the presence of melatonin. Finally, melatonin alone did not change cell viability. We conclude that melatonin, at concentrations higher than those found in blood, might regulate exocrine pancreatic function via modulation of Ca²⁺ signals.     

GTP and GDP will stimulate platelet cytosolic phospholipase C independently of Ca2+

The hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) by cytosolic phospholipase C from human platelets was determined. Cytosolic fractions were prepared from platelets that had or had not been preactivated with thrombin. Thrombin pretreatment did not affect cytosolic phospholipase C activity. In both cytosolic fractions, phospholipase C was activated by GTP and GTP gamma S. This action is observed in the presence of 2 mM EGTA. GDP was as effective as GTP in stimulating cytosolic phospholipase C in the presence of Ca2+ or EGTA. Partially purified phospholipase C obtained from platelet cytosol is activated by GTP, but not by GTP gamma S, in the presence of 2 mM EGTA. However, in the presence of 6 microM Ca2+, both GTP and GTP gamma S stimulated the partially purified phospholipase C. Our present information indicates that GTP and GDP have a direct effect on the cytosolic phospholipase C.     

Intermittent increases in cytosolic Ca2+ stimulate mitochondrial biogenesis in muscle cells

Muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle to identify which of the many signals generated by contractions are responsible for stimulating mitochondrial biogenesis. One purpose of this study was to evaluate the usefulness of L6 myotubes, which do not contract, for studying mitochondrial biogenesis. A second purpose was to evaluate further the possibility that increases in cytosolic Ca2+ can stimulate mitochondrial biogenesis. Continuous exposure to 1 microM ionomycin, a Ca2+ ionophore, for 5 days induced an increase in mitochondrial enzymes but also caused a loss of myotubes, as reflected in an approximately 40% decrease in protein per dish. However, intermittent (5 h/day) exposure to ionomycin, or to caffeine or W7, which release Ca2+ from the sarcoplasmic reticulum, did not cause a decrease in protein per dish. Raising cytosolic Ca2+ intermittently with these agents induced significant increases in mitochondrial enzymes. EGTA blocked most of this effect of ionomycin, whereas dantrolene, which blocks Ca2+ release from the sarcoplasmic reticulum, largely prevented the increases in mitochondrial enzymes induced by W7 and caffeine. These findings provide evidence that intermittently raising cytosolic Ca2+ stimulates mitochondrial biogenesis in muscle cells.     

Effects of acidosis on resting cytosolic and mitochondrial Ca2+ in mammalian myocardium

Acidosis increases resting cytosolic [Ca2+], (Cai) of myocardial preparations; however, neither the Ca2+ sources for the increase in Cai nor the effect of acidosis on mitochondrial free [Ca2+], (Cam) have been characterized. In this study cytosolic pH (pHi) was monitored in adult rat left ventricular myocytes loaded with the acetoxymethyl ester (AM form) of SNARF-1. A stable decrease in the pHi of 0.52 +/- 0.05 U (n = 16) was obtained by switching from a bicarbonate buffer equilibrated with 5% CO2 to a buffer equilibrated with 20% CO2. Electrical stimulation at either 0.5 or 1.5 Hz had no effect on pHi in 5% CO2, nor did it affect the magnitude of pHi decrease in response to hypercarbic acidosis. Cai was measured in myocytes loaded with indo- 1/free acid and Cam was monitored in cells loaded with indo-1/AM after quenching cytosolic indo-1 fluorescence with MnCl2. In quiescent intact myocytes bathed in 1.5 mM [Ca2+], hypercarbia increased Cai from 130 +/- 5 to 221 +/- 13 nM. However, when acidosis was effected in electrically stimulated myocytes, diastolic Cai increased more than resting Cai in quiescent myocytes, and during pacing at 1.5 Hz diastolic Cai was higher (285 +/- 17 nM) than at 0.5 Hz (245 +/- 18 nM; P < 0.05). The magnitude of Cai increase in quiescent myocytes was not affected either by sarcoplasmic reticulum (SR) Ca2+ depletion with ryanodine or by SR Ca2+ depletion and concomitant superfusion with a Ca(2+)-free buffer. In unstimulated intact myocytes hypercarbia increased Cam from 95 +/- 12 to 147 +/- 19 nM and this response was not modified either by ryanodine and a Ca(2+)-free buffer or by 50 microM ruthenium red in order to block the mitochondrial uniporter. In mitochondrial suspensions loaded either with BCECF/AM or indo-1/AM, acidosis produced by lactic acid addition decreased both intra- and extramitochondrial pH and increased Cam. Studies of mitochondrial suspensions bathed in indo- 1/free acid-containing solution showed an increase in extramitochondrial Ca2+ after the addition of lactic acid. Thus, in quiescent myocytes, cytoplasmic and intramitochondrial buffers, rather than transsarcolemmal Ca2+ influx or SR Ca2+ release, are the likely Ca2+ sources for the increase in Cai and Cam, respectively; additionally, Ca2+ efflux from the mitochondria may contribute to the raise in Cai. In contrast, in response to acidosis, diastolic Cai in electrically stimulated myocytes increases more than resting Cai in quiescent cells; this suggests that during pacing, net cell Ca2+ gain contributes to enhance diastolic Cai.     

Nicotinic receptors that bind alpha-bungarotoxin on neurons raise intracellular free Ca2+.

Many populations of vertebrate neurons have a membrane component that binds alpha-bungarotoxin and cholinergic ligands. Despite the abundance of this component and its similarities to nicotinic receptors, its function has remained controversial. Using a fluorescence assay, we show here that activation of the component elevates the intracellular concentration of free Ca2+, demonstrating a receptor function for the toxin-binding component. Whole-cell voltage-clamp and intracellular recordings did not detect a significant current resulting from receptor activation, possibly because the currents were small or the receptors rapidly desensitized. The rise in intracellular free Ca2+ caused by the receptor was prevented by Ca2+ channel blockers. This suggests a signaling cascade likely to have important regulatory consequences for the neuron.     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

The free [Ca2+] in endoplasmic/sarcoplasmic reticulum Ca2+ stores regulates excitability of Ca2+ release by stimulating the Ca2+ release channels. Just how the stored Ca2+ regulates activation of these channels is still disputed. One proposal attributes luminal Ca2+-activation to luminal facing regulatory sites, whereas another envisages Ca2+ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca2+ activation for single cardiac ryanodine receptors (RyR2) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR2 involves three modes of action associated with Ca2+ sensors in different parts of the molecule; a luminal activation site (L-site, 60 microM affinity), a cytoplasmic activation site (A-site, 0.9 microM affinity), and a novel cytoplasmic inactivation site (I2-site, 1.2 microM affinity). RyR activation by luminal Ca2+ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca2+ feedthrough. Ca2+ binding to the L-site initiates brief openings (1 ms duration at 1-10 s(-1)) allowing luminal Ca2+ to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR2 mutations on a range of Ca2+-mediated physiological and pathological processes.     

Stimulation of inositol trisphosphate formation in hepatocytes by vasopressin, adrenaline and angiotensin II and its relationship to changes in cytosolic free Ca2+.

At maximally effective concentrations, vasopressin (10(-7) M) increased myo-inositol trisphosphate (IP3) in isolated rat hepatocytes by 100% at 3 s and 150% at 6 s, while adrenaline (epinephrine) (10(-5) M) produced a 17% increase at 3 s and a 30% increase at 6 s. These increases were maintained for at least 10 min. Both agents increased cytosolic free Ca2+ [( Ca2+]i) maximally by 5 s. Increases in IP3 were also observed with angiotensin II and ATP, but not with glucagon or platelet-activating factor. The dose-responses of vasopressin and adrenaline on phosphorylase and [Ca2+]i showed a close correspondence, whereas IP3 accumulation was 20-30-fold less sensitive. However, significant (20%) increases in IP3 could be observed with 10(-9) M-vasopressin and 10(-7) M-adrenaline, which induce near-maximal phosphorylase activation. Vasopressin-induced accumulation of IP3 was potentiated by 10mM-Li+, after a lag of approx. 1 min. However the rise in [Ca2+]i and phosphorylase activation were not potentiated at any time examined. Similar data were obtained with adrenaline as agonist. Lowering the extracellular Ca2+ to 30 microM or 250 microM did not affect the initial rise in [Ca2+]i with vasopressin but resulted in a rapid decline in [Ca2+]i. Brief chelation of extracellular Ca2+ for times up to 4 min also did not impair the rate or magnitude of the increase in [Ca2+]i or phosphorylase a induced by vasopressin. The following conclusions are drawn from these studies. IP3 is increased in rat hepatocytes by vasopressin, adrenaline, angiotensin II and ATP. The temporal relationships of its accumulation to the increases in [Ca2+]i and phosphorylase a are consistent with it playing a second message role. Influx of extracellular Ca2+ is not required for the initial rise in [Ca2+]i induced by these agonists, but is required for the maintenance of the elevated [Ca2+]i.