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.     

Multiple modes of calcium-induced calcium release in sympathetic neurons I: attenuation of endoplasmic reticulum Ca2+ accumulation at low [Ca2+](i) during weak depolarization.

Many cells express ryanodine receptors (RyRs) whose activation is thought to amplify depolarization-evoked elevations in cytoplasmic Ca2+ concentration [Ca2+](i) through a process of Ca2+ -induced Ca2+ release (CICR). In neurons, it is usually assumed that CICR triggers net Ca2+ release from an ER Ca2+ store. However, since net ER Ca 2+ transport depends on the relative rates of Ca2+ uptake and release via distinct pathways, weak activation of a CICR pathway during periods of ER Ca accumulation would have a totally different effect: attenuation of Ca2+ accumulation. Stronger CICR activation at higher [Ca2+](i) could further attenuate Ca2+ accumulation or trigger net Ca2+ release, depending on the quantitative properties of the underlying Ca2+ transporters. This and the companion study (Hongpaisan, J., N.B. Pivovarova, S.L. Colgrove, R.D. Leapman, and D.D. Friel, and S.B. Andrews. 2001. J. Gen. Physiol. 118:101-112) investigate which of these CICR "modes" operate during depolarization-induced Ca2+ entry in sympathetic neurons. The present study focuses on small [Ca2+](i) elevations (less than approximately 350 nM) evoked by weak depolarization. The following two approaches were used: (1) Ca2+ fluxes were estimated from simultaneous measurements of [Ca2+](i) and I(Ca) in fura-2-loaded cells (perforated patch conditions), and (2) total ER Ca concentrations ([Ca](ER)) were measured using X-ray microanalysis. Flux analysis revealed triggered net Ca2+ release during depolarization in the presence but not the absence of caffeine, and [Ca2+](i) responses were accelerated by SERCA inhibitors, implicating ER Ca2+ accumulation, which was confirmed by direct [Ca](ER) measurements. Ryanodine abolished caffeine-induced CICR and enhanced depolarization-induced ER Ca2+ accumulation, indicating that activation of the CICR pathway normally attenuates ER Ca2+ accumulation, which is a novel mechanism for accelerating evoked [Ca2+](i) responses. Theory shows how such a low gain mode of CICR can operate during weak stimulation and switch to net Ca2+ release at high [Ca2+](i), a transition demonstrated in the companion study. These results emphasize the importance of the relative rates of Ca2+ uptake and release in defining ER contributions to depolarization-induced Ca2+ signals.     

Ca2+ -induced Ca2+ release through localized Ca2+ uncaging in smooth muscle

Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca(2+) release or Ca(2+) sparks and, in some spiking tissues, as Ca(2+) release that is triggered by the activation of sarcolemmal Ca(2+) channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca(2+) (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca(2+) activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca(2+) release in the form of Ca(2+) sparks and Ca(2+) waves that were distinguishable from increases in Ca(2+) associated with Ca(2+) uncaging, unequivocally demonstrating that Ca(2+) release occurs subsequent to a localized rise in [Ca(2+)](i). TPFP-triggered Ca(2+) release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca(2+) sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca(2+) release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca(2+)](i) through inositol trisphosphate (InsP(3)) receptors (InsP(3)Rs). We conclude that CICR activated by localized Ca(2+) release bears essential similarities to those observed by the activation of I(Ca) (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca(2+) release through InsP(3)R can occur at high local [Ca(2+)](i).     

Ca2+-induced Ca2+ release from the endoplasmic reticulum amplifies the Ca2+ signal mediated by activation of voltage-gated L-type Ca2+ channels in pancreatic beta-cells

Stimulus-secretion coupling in pancreatic beta-cells involves membrane depolarization and Ca(2+) entry through voltage-gated L-type Ca(2+) channels, which is one determinant of increases in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). We investigated how the endoplasmic reticulum (ER)-associated Ca(2+) apparatus further modifies this Ca(2+) signal. When fura-2-loaded mouse beta-cells were depolarized by KCl in the presence of 3 mm glucose, [Ca(2+)](i) increased to a peak in two phases. The second phase of the [Ca(2+)](i) increase was abolished when ER Ca(2+) stores were depleted by thapsigargin. The steady-state [Ca(2+)](i) measured at 300 s of depolarization was higher in control cells compared with cells in which the ER Ca(2+) pools were depleted. The amount of Ca(2+) presented to the cytoplasm during depolarization as estimated from the integral of the increment in [Ca(2+)](i) over time (integralDelta[Ca(2+)](i).dt) was approximately 30% higher compared with that in the Ca(2+) pool-depleted cells. neo-thapsigargin, an inactive analog, did not affect [Ca(2+)](i) response. Using Sr(2+) in the extracellular medium and exploiting the differences in the fluorescence properties of Ca(2+)- and Sr(2+)-bound fluo-3, we found that the incoming Sr(2+) triggered Ca(2+) release from the ER. Depolarization-induced [Ca(2+)](i) response was not altered by, an inhibitor of phosphatidylinositol-specific phospholipase C, suggesting that stimulation of the enzyme by Ca(2+) is not essential for amplification of Ca(2+) signaling. [Ca(2+)](i) response was enhanced when cells were depolarized in the presence of 3 mm glucose, forskolin, and caffeine, suggesting involvement of ryanodine receptors in the amplification process. Pretreatment with ryanodine (100 microm) diminished the second phase of the depolarization-induced increase in [Ca(2+)](i). We conclude that Ca(2+) entry through L-type voltage-gated Ca(2+) channels triggers Ca(2+) release from the ER and that such a process amplifies depolarization-induced Ca(2+) signaling in beta-cells.     

Ca(2+) dynamics in the lumen of the endoplasmic reticulum in sensory neurons: direct visualization of Ca(2+)-induced Ca(2+) release triggered by physiological Ca(2+) entry

In cultured rat dorsal root ganglia neurons, we measured membrane currents, using the patch-clamp whole-cell technique, and the concentrations of free Ca(2+) in the cytosol ([Ca(2+)](i)) and in the lumen of the endoplasmic reticulum (ER) ([Ca(2+)](L)), using high- (Fluo-3) and low- (Mag-Fura-2) affinity Ca(2+)-sensitive fluorescent probes and video imaging. Resting [Ca(2+)](L) concentration varied between 60 and 270 microM. Activation of ryanodine receptors by caffeine triggered a rapid fall in [Ca(2+)](L) levels, which amounted to only 40--50% of the resting [Ca(2+)](L) value. Using electrophysiological depolarization, we directly demonstrate the process of Ca(2+)-induced Ca(2+) release triggered by Ca(2+) entry through voltage-gated Ca(2+) channels. The amplitude of Ca(2+) release from the ER lumen was linearly dependent on I(Ca).     

Interplay between ER Ca2+ uptake and release fluxes in neurons and its impact on [Ca2+] dynamics

In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca(2+)-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+](I)-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.     

Mitochondria efficiently buffer subplasmalemmal Ca2+ elevation during agonist stimulation

In endothelial cells, local Ca(2+) release from superficial endoplasmic reticulum (ER) activates BK(Ca) channels. The resulting hyperpolarization promotes capacitative Ca(2+) entry (CCE), which, unlike BK(Ca) channels, is inhibited by high Ca(2+). To understand how the coordinated activation of plasma membrane ion channels with opposite Ca(2+) sensitivity is orchestrated, the individual contribution of mitochondria and ER in regulation of subplasmalemmal Ca(2+) concentration ([Ca(2+)](pm)) was investigated. For organelle visualization, cells were transfected with DsRed and yellow cameleon targeted to mitochondria and ER. The patch pipette was placed far from any organelle (L1), close to ER (L3), or mitochondria (L2) and activity of BK(Ca) channels was used to estimate local [Ca(2+)](pm). Under standard patch conditions (130 mm K(+) in the bath), histamine increased [Ca(2+)](pm) at L1 and L3 to approximately 1.6 microm, whereas close to mitochondria [Ca(2+)](pm) remained unchanged. If mitochondria moved apart from the pipette or in the presence of carbonyl cyanide-4-trifluoromethoxyphenylhyrazone, [Ca(2+)](pm) at L2 increased in response to histamine. Under standard patch conditions Ca(2+) entry was negligible due to cell depolarization. Using a physiological patch approach (5.6 mm K(+) in the bath), changes in [Ca(2+)](pm) to histamine could be monitored without cell depolarization and, thus, in conditions where Ca(2+) entry occurred. Here, histamine induced an initial transient Ca(2+) elevation to > or =3.5 microm followed by a long lasting plateau at approximately 1.2 microm in L1 and L3, whereas mitochondria kept neighboring [Ca(2+)](pm) low during stimulation. Thus, superficial mitochondria and ER generate local domains of low and high Ca(2+) allowing simultaneous activation of BK(Ca) and CCE, despite their opposite Ca(2+) sensitivity.     

Mitochondrial Ca2+ uptake requires sustained Ca2+ release from the endoplasmic reticulum

We analyzed the role of inositol 1,4,5-trisphosphate-induced Ca(2+) release from the endoplasmic reticulum (ER) (i) in powering mitochondrial Ca(2+) uptake and (ii) in maintaining a sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)). For this purpose, we expressed in HeLa cells aequorin-based Ca(2+)-sensitive probes targeted to different intracellular compartments and studied the effect of two agonists: histamine, acting on endogenous H(1) receptors, and glutamate, acting on co-transfected metabotropic glutamate receptor (mGluR1a), which rapidly inactivates through protein kinase C-dependent phosphorylation and thus causes transient inositol 1,4,5-trisphosphate production. Glutamate induced a transient [Ca(2+)](c) rise and drop in ER luminal [Ca(2+)] ([Ca(2+)](er)), and then the ER refilled with [Ca(2+)](c) at resting values. With histamine, [Ca(2+)](c) after the initial peak stabilized at a sustained plateau, and [Ca(2+)](er) decreased to a low steady-state value. In mitochondria, histamine evoked a much larger mitochondrial Ca(2+) response than glutamate ( approximately 15 versus approximately 65 microm). Protein kinase C inhibition, partly relieving mGluR1a desensitization, reestablished both the [Ca(2+)](c) plateau and the sustained ER Ca(2+) release and markedly increased the mitochondrial Ca(2+) response. Conversely, mitochondrial Ca(2+) uptake evoked by histamine was drastically reduced by very transient ( approximately 2-s) agonist applications. These data indicate that efficient mitochondrial Ca(2+) uptake depends on the preservation of high Ca(2+) microdomains at the mouth of ER Ca(2+) release sites close to mitochondria. This in turn depends on continuous Ca(2+) release balanced by Ca(2+) reuptake into the ER and maintained by Ca(2+) influx from the extracellular space.     

Dissection of mitochondrial Ca2+ uptake and release fluxes in situ after depolarization-evoked [Ca2+](i) elevations in sympathetic neurons

We studied how mitochondrial Ca2+ transport influences [Ca2+](i) dynamics in sympathetic neurons. Cells were treated with thapsigargin to inhibit Ca2+ accumulation by SERCA pumps and depolarized to elevate [Ca2+(i); the recovery that followed repolarization was then examined. The total Ca2+ flux responsible for the [Ca2+](i) recovery was separated into mitochondrial and nonmitochondrial components based on sensitivity to the proton ionophore FCCP, a selective inhibitor of mitochondrial Ca2+ transport in these cells. The nonmitochondrial flux, representing net Ca2+ extrusion across the plasma membrane, has a simple dependence on [Ca2+](i), while the net mitochondrial flux (J(mito)) is biphasic, indicative of Ca+) accumulation during the initial phase of recovery when [Ca2+](i) is high, and net Ca2+ release during later phases of recovery. During each phase, mitochondrial Ca2+ transport has distinct effects on recovery kinetics. J(mito) was separated into components representing mitochondrial Ca2+ uptake and release based on sensitivity to the specific mitochondrial Na(+)/Ca2+ exchange inhibitor, CGP 37157 (CGP). The CGP-resistant (uptake) component of J(mito) increases steeply with [Ca2+](i), as expected for transport by the mitochondrial uniporter. The CGP-sensitive (release) component is inhibited by lowering the intracellular Na(+) concentration and depends on both intra- and extramitochondrial Ca2+ concentration, as expected for the Na(+)/Ca2+ exchanger. Above approximately 400 nM [Ca2+](i), net mitochondrial Ca2+ transport is dominated by uptake and is largely insensitive to CGP. When [Ca2+](i) is approximately 200-300 nM, the net mitochondrial flux is small but represents the sum of much larger uptake and release fluxes that largely cancel. Thus, mitochondrial Ca2+ transport occurs in situ at much lower concentrations than previously thought, and may provide a mechanism for quantitative control of ATP production after brief or low frequency stimuli that raise [Ca(2+)](i) to levels below approximately 500 nM.     

Calreticulin modulates capacitative Ca2+ influx by controlling the extent of inositol 1,4,5-trisphosphate-induced Ca2+ store depletion

Calreticulin (CRT) is a highly conserved Ca(2+)-binding protein that resides in the lumen of the endoplasmic reticulum (ER). We overexpressed CRT in Xenopus oocytes to determine how it could modulate inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) influx. Under conditions where it did not affect the spatially complex elevations in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) due to InsP(3)-induced Ca(2+) release, overexpressed CRT decreased by 46% the Ca(2+)-gated Cl(-) current due to Ca(2+) influx. Deletion mutants revealed that CRT requires its high capacity Ca(2+)-binding domain to reduce the elevations of [Ca(2+)](i) due to Ca(2+) influx. This functional domain was also required for CRT to attenuate the InsP(3)-induced decline in the free Ca(2+) concentration within the ER lumen ([Ca(2+)](ER)), as monitored with a "chameleon" indicator. Our data suggest that by buffering [Ca(2+)](ER) near resting levels, CRT may prevent InsP(3) from depleting the intracellular stores sufficiently to activate Ca(2+) influx.     

Fura-2 antagonises calcium-induced calcium release

Calcium-induced calcium release (CICR) from the endoplasmic reticulum (ER) takes place through ryanodine receptors (RyRs) and it is often revealed by an increase of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by caffeine. Using fura-2-loaded cells, we find such an effect in bovine adrenal chromaffin cells, but not in cerebellar granule neurones or in HEK-293 cells. In contrast, a caffeine-induced [Ca(2+)](c) increase was clearly visible with either fluo-3 or cytosolic aequorin. Simultaneous loading with fura-2 prevented the [Ca(2+)](c) increase reported by the other Ca(2+) probes. Caffeine-induced Ca(2+) release was also measured by following changes of [Ca(2+)] inside the ER ([Ca(2+)](ER)) with ER-targeted aequorin in HEK-293 cells. Fura-2 loading did not modify Ca(2+) release from the ER. Thus, fura-2, but not fluo-3, antagonises the generation of the cytosolic Ca(2+) signal induced by activation of RyRs. Cytosolic Ca(2+) buffering and/or acceleration of Ca(2+) diffusion through the cytosol may contribute to these actions. Both effects may interfere with the generation of microdomains of high [Ca(2+)](c) near the ER release channels, which are essential for the propagation of the Ca(2+) wave through the cytosol. In any case, our results caution the use of fura-2 to study CICR.     

SERCA pump optimizes Ca2+ release by a mechanism independent of store filling in smooth muscle cells

Thapsigargin-sensitive sarco/endoplasmic reticulum Ca(2+) pumps (SERCAs) are involved in maintaining and replenishing agonist-sensitive internal stores. Although it has been assumed that release channels act independently of SERCA pumps, there are data suggesting the opposite. Our aim was to study the relationship between SERCA pumps and the release channels in smooth muscle cells. To this end, we have rapidly blocked SERCA pumps with thapsigargin, to avoid depletion of the internal Ca(2+) stores, and induced Ca(2+) release with either caffeine, to open ryanodine receptors, or acetylcholine, to open inositol 1,4,5-trisphosphate receptors. Blocking SERCA pumps produced smaller and slower agonist-induced [Ca(2+)](i) responses. We determined the Ca(2+) level of the internal stores both indirectly, measuring the frequency of spontaneous transient outward currents, and directly, using Mag-Fura-2, and demonstrated that the inhibition of SERCA pumps did not produce a reduction of the sarco/endoplasmic reticulum Ca(2+) levels to explain the decrease in the agonist-induced Ca(2+) responses. It appears that SERCA pumps are involved in sustaining agonist-induced Ca(2+) release by a mechanism that involves the modulation of Ca(2+) availability in the lumen of the internal stores.     

Biophysical re-equilibration of Ca2+ fluxes as a simple biologically plausible explanation for complex intracellular Ca2+ release patterns

Physiological regulation of Ca(2+) release from the endoplasmic reticulum (ER) is critical for cell function. Recent direct measurements of free [Ca(2+)] inside the ER ([Ca(2+)](ER)) revealed that [Ca(2+)](ER) itself is a key regulator of ER Ca(2+) handling. However, the role of this new regulatory process in generating various patterns of Ca(2+) release remains to be elucidated in detail. Here, we incorporate the recently quantified experimental correlations between [Ca(2+)](ER) and Ca(2+) movements across the ER membrane into a mathematical model ER Ca(2+) handling. The model reproduces basic experimental dynamics of [Ca(2+)](ER). Although this was not goal in model design, the model also exhibits mechanistically unclear experimental phenomena such as "quantal" Ca(2+) release, and "store charging" by increasing resting cytosolic [Ca(2+)]. While more complex explanations cannot be ruled out, on the basis of our data we propose that "quantal release" and "store charging" could be simple re-equilibration phenomena, predicted by the recently quantified biophysical dynamics of Ca(2+) movements across the ER membrane.     

Ca(2+)-induced Ca(2+) release via inositol 1,4,5-trisphosphate receptors is amplified by protein kinase A and triggers exocytosis in pancreatic beta-cells

Hormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca(2+)-induced Ca(2+) release (CICR) in pancreatic beta-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca(2+) spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca(2+) spiking at low or even in the absence of depolarization-dependent elevation of [Ca(2+)](i). The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca(2+), failed to mobilize intracellular Ca(2+). On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Therefore, the cell-permeable IP(3)R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic beta-cells were followed by [Ca(2+)](i) spikes in neighboring human erythroleukemia cells, used to report secretory events in the beta-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP(3)Rs is part of the mechanism by which cAMP amplifies insulin release.     

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.     

Multiple modes of calcium-induced calcium release in sympathetic neurons II: a [Ca2+](i)- and location-dependent transition from endoplasmic reticulum Ca accumulation to net Ca release.

CICR from an intracellular store, here directly characterized as the ER, usually refers to net Ca(2)+ release that amplifies evoked elevations in cytosolic free calcium [Ca2+](i). However, the companion paper (Albrecht, M.A., S.L. Colegrove, J. Hongpaisan, N.B. Pivovarova, S.B. Andrews, and D.D. Friel. 2001. J. Gen. Physiol. 118:83-100) shows that in sympathetic neurons, small [Ca2+](i) elevations evoked by weak depolarization stimulate ER Ca accumulation, but at a rate attenuated by activation of a ryanodine-sensitive CICR pathway. Here, we have measured depolarization-evoked changes in total ER Ca concentration ([Ca](ER)) as a function of [Ca2+](i), and found that progressively larger [Ca2+](i) elevations cause a graded transition from ER Ca accumulation to net release, consistent with the expression of multiple modes of CICR. [Ca](ER) is relatively high at rest (12.8 +/- 0.9 mmol/kg dry weight, mean +/- SEM) and is reduced by thapsigargin or ryanodine (5.5 +/- 0.7 and 4.7 +/- 1.1 mmol/kg, respectively). [Ca](ER) rises during weak depolarization (to 17.0 +/- 1.6 mmol/kg over 120s, [Ca2+](i) less than approximately 350 nM), changes little in response to stronger depolarization (12.1 +/- 1.1 mmol/kg, [Ca2+](i) approximately 700 nM), and declines (to 6.5 +/- 1.0 mmol/kg) with larger [Ca2+](i) elevations (>1 microM) evoked by the same depolarization when mitochondrial Ca2+ uptake is inhibited (FCCP). Thus, net ER Ca2+ transport exhibits a biphasic dependence on [Ca2+](i). With mitochondrial Ca2+ uptake enabled, [Ca](ER) rises after repolarization (to 16.6 +/- 1.8 mmol/kg at 15 min) as [Ca2+](i) falls within the permissive range for ER Ca accumulation over a period lengthened by mitochondrial Ca2+ release. Finally, although spatially averaged [Ca](ER) is unchanged during strong depolarization, net ER Ca2+ release still occurs, but only in the outermost approximately 5-microm cytoplasmic shell where [Ca2+](i) should reach its highest levels. Since mitochondrial Ca accumulation occurs preferentially in peripheral cytoplasm, as demonstrated here by electron energy loss Ca maps, the Ca content of ER and mitochondria exhibit reciprocal dependencies on proximity to sites of Ca2+ entry, possibly reflecting indirect mitochondrial regulation of ER Ca(2)+ transport.     

Phosphatidylinositol 3-kinase facilitates bile acid-induced Ca(2+) responses in pancreatic acinar cells

Bile acids are known to induce Ca(2+) signals in pancreatic acinar cells. We have recently shown that phosphatidylinositol 3-kinase (PI3K) regulates changes in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) elicited by CCK by inhibiting sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). The present study sought to determine whether PI3K regulates bile acid-induced [Ca(2+)](i) responses. In pancreatic acinar cells, pharmacological inhibition of PI3K with LY-294002 or wortmannin inhibited [Ca(2+)](i) responses to taurolithocholic acid 3-sulfate (TLC-S) and taurochenodeoxycholate (TCDC). Furthermore, genetic deletion of the PI3K gamma-isoform also decreased [Ca(2+)](i) responses to bile acids. Depletion of CCK-sensitive intracellular Ca(2+) pools or application of caffeine inhibited bile acid-induced [Ca(2+)](i) signals, indicating that bile acids release Ca(2+) from agonist-sensitive endoplasmic reticulum (ER) stores via an inositol (1,4,5)-trisphosphate-dependent mechanism. PI3K inhibitors increased the amount of Ca(2+) in intracellular stores during the exposure of acinar cells to bile acids, suggesting that PI3K negatively regulates SERCA-dependent Ca(2+) reloading into the ER. Bile acids inhibited Ca(2+) reloading into ER in permeabilized acinar cells. This effect was augmented by phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), suggesting that both bile acids and PI3K act synergistically to inhibit SERCA. Furthermore, inhibition of PI3K by LY-294002 completely inhibited trypsinogen activation caused by the bile acid TLC-S. Our results indicate that PI3K and its product, PIP(3), facilitate bile acid-induced [Ca(2+)](i) responses in pancreatic acinar cells through inhibition of SERCA-dependent Ca(2+) reloading into the ER and that bile acid-induced trypsinogen activation is mediated by PI3K. The findings have important implications for the mechanism of acute pancreatitis since [Ca(2+)](i) increases and trypsinogen activation mediate key pathological processes in this disorder.