Calcium uptake and Ca2+-ATPase activity in goat spermatozoa membrane vesicles do not require Mg2+

Microsomal membrane vesicles isolated from goat spermatozoa contain Ca²⁺-ATPase, and exhibit Ca²⁺ transport activities that do not require exogenous Mg²⁺ .The enzyme activity is inhibited by calcium-channel inhibitors,e.g. verapamil and diltiazem, like the well known Ca²⁺ , Mg²⁺-ATPase. The uptake of calcium is ATP (energy)-dependent and the accumulated Ca²⁺ can be completely released by the Ca²⁺ ionophore A23187, suggesting that a significant fraction of the vesicles are oriented inside out     

Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins

Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, including transient expression in COS cells, acute injection of recombinant protein into Xenopus oocytes and (45)Ca(2+) flux from permeabilised COS cells, we demonstrated that CaBPs decrease the sensitivity of InsP(3)-induced Ca(2+) release (IICR). In addition, we found a Ca(2+)-independent interaction between CaBP1 and the NH(2)-terminal 159 amino acids of the type 1 InsP(3)R. This interaction resulted in decreased InsP(3) binding to the receptor reminiscent of that observed for CaM. Unlike CaM, however, CaBPs do not inhibit ryanodine receptors, have a higher affinity for InsP(3)Rs and more potently inhibited IICR. We also show that phosphorylation of CaBP1 at a casein kinase 2 consensus site regulates its inhibition of IICR. Our data suggest that CaBPs are endogenous regulators of InsP(3)Rs tuning the sensitivity of cells to InsP(3).     

Suppression of Ca2+ oscillations in cultured rat hepatocytes by chemical hypoxia

The model of "chemical hypoxia" with KCN plus iodoacetic acid mimics the ATP depletion and reductive stress of hypoxia. Here, we examined the effects of chemical hypoxia on cytosolic free Na+ and Ca2+ in single cultured rat hepatocytes by multiparameter digitized video microscopy and ratio imaging of sodium-binding furan indicator (SBFI) and Fura-2. Intracellular Na+ increased from about 10 mM to more than 100 mM after 20 min of chemical hypoxia, whereas cytosolic free Ca2+ remained virtually unchanged. In normoxic hepatocytes, phenylephrine (50 microM) and Arg-vasopressin (20-40 nM) induced Ca2+ oscillations in 70 and 40% of cells, respectively. These Ca2+ oscillations were suppressed after one spike following the onset of chemical hypoxia. Phenylephrine and vasopressin also increased inositol phosphate formation by 22 and 147%, respectively. This effect was suppressed by KCN plus iodoacetate. Intracellular acidosis is characteristic of chemical hypoxia. Intracellular acidosis induced by 40 mM Na-acetate suppressed Ca2+ oscillations but did not inhibit hormone-induced inositol phosphate formation. Cytosolic alkalinization also suppressed Ca2+ oscillations. However, prevention of intracellular acidosis with monensin (10 microM) did not prevent suppression of Ca2+ oscillations during chemical hypoxia. Mitochondrial depolarization with uncoupler did not change free Ca2+ levels during chemical hypoxia, indicating that mitochondria do not regulate free Ca2+ during chemical hypoxia. From these results, we conclude: 1) chemical hypoxia does not block Na+ influx across the plasma membrane; 2) Chemical hypoxia inhibits hormone-stimulated Ca2+ flux pathways across cellular membranes by two different mechanisms: (a) by ATP depletion, which disrupts hormone-myo-inositol 1,4,5-triphosphate coupling, and (b) by intracellular acidosis, which inhibits myo-inositol 1,4,5-triphosphate-stimulated Ca2+ release from intracellular stores; 3) during ATP depletion by chemical hypoxia, mitochondria do not take up Ca2+ to maintain cytosolic free Ca2+ at low concentrations.     

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3-dependent Ca2+ release in mouse brain endothelial cells

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the target cells, which activates the Ca²⁺/Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca²⁺]_i and NO production. The current study assessed whether and how glutamate drives Ca²⁺-dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca²⁺]_i, which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca²⁺ oscillations were triggered by rhythmic endogenous Ca²⁺ mobilization and maintained over time by extracellular Ca²⁺ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca²⁺ release was mediated by InsP₃-sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca²⁺ entry mediated Ca²⁺ entry during ongoing Ca²⁺ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca²⁺ signals. Of note, glutamate induced Ca²⁺-dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca²⁺ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.     

Modulation of free Ca oscillations in single hepatocytes by changes in extracellular K+, Na+ and Ca2+

Single rat hepatocytes, microinjected with the calcium-sensitive photoprotein aequorin, when stimulated with either phenylephrine or arg8-vasopressin exhibit agonist-specific oscillations in cytosolic free calcium levels (free Ca). In the majority of the cells examined adding excess potassium chloride, sodium chloride or choline chloride abolished transient behaviour. However, in cells that continued to oscillate the transient parameters were subtly modified by these treatments. In experiments using phenylephrine as the agonist, adding excess potassium chloride to the superfusate significantly reduced transient length, increased the rate of transient rise and reduced the smoothed peak free Ca level without significantly altering the intertransient resting free Ca level or the falling time constant. The possible mechanisms by which these alterations may occur are discussed.     

Spatio-temporal modelling explains the effect of reduced plasma membrane Ca2+ efflux on intracellular Ca2+ oscillations in hepatocytes

In many non-excitable eukaryotic cells, including hepatocytes, Ca²⁺ oscillations play a key role in intra- and intercellular signalling, thus regulating many cellular processes from fertilisation to death. Therefore, understanding the mechanisms underlying these oscillations, and consequently understanding how they may be regulated, is of great interest. In this paper, we study the influence of reduced Ca²⁺ plasma membrane efflux on Ca²⁺ oscillations in hepatocytes. Our previous experiments with carboxyeosin show that a reduced plasma membrane Ca²⁺ efflux increases the frequency of Ca²⁺ oscillations, but does not affect the duration of individual transients. This phenomenon can be best explained by taking into account not only the temporal, but also the spatial dynamics underlying the generation of Ca²⁺ oscillations in the cell. Here we divide the cell into a grid of elements and treat the Ca²⁺ dynamics as a spatio-temporal phenomenon. By converting an existing temporal model into a spatio-temporal one, we obtain theoretical predictions that are in much better agreement with the experimental observations.     

Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.     

The inositol (1,4,5)-trisphosphate 3-kinase of Xenopus oocyte is activated by CaMKII and involved in the regulation of InsP3-mediated Ca2+ release

The effect of Ca2+ on inositol (1,4,5)-trisphosphate 3-kinase (3-kinase) activity was measured on Xenopus oocyte cytosolic extracts. The Ca2+-evoked elevation in 3-kinase activity appeared to be mediated by calmodulin (CaM) and the calmodulin-dependent protein kinase II (CaMKII). The results observed in vitro were totally retrieved in intact oocytes and tend to demonstrate the involvement of a CaMKII-mediated phosphorylation in the regulation of 3-kinase activity. Finally, electrophysiological recordings of InsP3-elicited chloride current transients in the presence of CaM/CaMKII inhibitors allowed to postulate an involvement of 3-kinase activity in the regulation of InsP3-mediated Ca2+ release.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca²⁺ release by photolysis of caged InsP₃ have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca²⁺-activated Cl⁻ and K⁺ currents. Photolytic release of InsP₃ from caged InsP₃ at 100 Joules caused transient inward (V_H = 60 mV) and outward (V_H = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl⁻ concentration was decreased, and the outward current was suppressed by K⁺ channel blockers, indicating that they were carried by Cl⁻ and K⁺, respectively. Intracellular pre-loading of the InsP₃ receptor antagonist heparin or the Ca²⁺ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca²⁺-dependent Cl⁻ and K⁺ currents underlies the inward and the outward currents. At low flash intensities, InsP₃ caused Ca²⁺ release which normally activated the K⁺ and Cl⁻ currents in a mono-transient manner. At higher intensities, however, InsP₃ induced an additional delayed outward K⁺ current (I_K(delay)). I_K(delay) was independent of the initial K⁺ current, independent of extracellular Ca²⁺, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca²⁺ from caged Ca²⁺ did not mimic the I_K(delay). It is suggested that Ca²⁺ releases from the InsP₃-sensitive pools in an InsP₃ concentration-dependent manner. Low concentrations of InsP₃ induce the transient Ca²⁺-dependent Cl⁻ and K⁺ currents, which reflects the local Ca²⁺ release, whereas high concentrations of InsP₃ induce a delayed Ca²⁺-dependent K⁺ current, which may reflect the Ca²⁺ wave propagation. J. Cell. Physiol. 174:387–397, 1998. © 1998 Wiley-Liss, Inc.     

Activation of Ca2+-activated Cl- channels by store-operated Ca2+ entry in arterial smooth muscle cells does not require reverse-mode Na+/Ca2+ exchange

The main purpose of this study was to characterize the stimulation of Ca(2+)-activated Cl(-) (Cl(Ca)) by store-operated Ca(2+) entry (SOCE) channels in rabbit pulmonary arterial smooth muscle cells (PASMCs) and determine if this process requires reverse-mode Na(+)/Ca(2+) exchange (NCX). In whole-cell voltage clamped PASMCs incubated with 1 μmol/L nifedipine (Nif) to inhibit Ca(2+) channels, 30 μmol/L cyclopiazonic acid (CPA), a SERCA pump inhibitor, activated a nonselective cation conductance permeable to Na(+) (I(SOC)) during an initial 1-3 s step, ranging from-120 to +60 mV, and Ca(2+)-activated Cl(-) current (I(Cl(Ca))) during a second step to +90 mV that increased with the level of the preceding hyperpolarizing step. Niflumic acid (100 μmol/L), a Cl(Ca) channel blocker, abolished I(Cl(Ca)) but had no effect on I(SOC), whereas the I(SOC) blocker SKF-96365 (50 μmol/L) suppressed both currents. Dual patch clamp and Fluo-4 fluorescence measurements revealed the appearance of CPA-induced Ca(2+) transients of increasing magnitude with increasing hyperpolarizing steps, which correlated with I(Cl(Ca)) amplitude. The absence of Ca(2+) transients at positive potentials following a hyperpolarizing step combined with the observation that SOCE-stimulated I(Cl(Ca)) was unaffected by the NCX blocker KB-R7943 (1 μmol/L) suggest that the SOCE/Cl(Ca) interaction does not require reverse-mode NCX in our conditions.     

Characterization of Ca2+ transients induced by intracellular photorelease of InsP3 in mouse ovarian oocytes

Ca2+ transients (measured with Fluo-3) were induced in single mouse ovarian oocytes by photolytic liberation of InsP3. The time course of cytosolic Ca2+ changes induced in this way is composed of distinct phases: upstroke, fast decline, slow declining plateau and fast decline to rest level. All the phases reflect mainly intracellular redistributions of the ion and not influx, since they are not strongly dependent on external Ca2+ or on changes in transmembrane potential. Often sustained Ca2+ oscillations followed the first InsP3-induced Ca2+ transient. These persisted for several minutes in the absence of external Ca2+. The initial rate of Ca2+ rise and the delay between the InsP3 stimulus and Ca2+ upstroke are correlated with the amount of liberated InsP3. A second InsP3 stimulation, applied during the plateau, causes only small Ca2+ elevations, lacking the upstroke phase. A second, full sized, transient could be elicited only after a complete return to the basal level. Vanadate, applied intracellularly, appeared to inhibit the re-uptake phase into the stores, stabilizing the plateau level. The present observations suggest that in mouse oocytes the InsP3-sensitive stores provide only a small and graded Ca2+ release which may then act as a trigger for a more substantial Ca(2+)-induced Ca2+ release (CICR) process.     

Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3 receptor channel gating

Mutations in presenilins (PS) are the major cause of familial Alzheimer's disease (FAD) and have been associated with calcium (Ca2+) signaling abnormalities. Here, we demonstrate that FAD mutant PS1 (M146L)and PS2 (N141I) interact with the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel and exert profound stimulatory effects on its gating activity in response to saturating and suboptimal levels of InsP3. These interactions result in exaggerated cellular Ca2+ signaling in response to agonist stimulation as well as enhanced low-level Ca2+signaling in unstimulated cells. Parallel studies in InsP3R-expressing and -deficient cells revealed that enhanced Ca2+ release from the endoplasmic reticulum as a result of the specific interaction of PS1-M146L with the InsP3R stimulates amyloid beta processing,an important feature of AD pathology. These observations provide molecular insights into the "Ca2+ dysregulation" hypothesis of AD pathogenesis and suggest novel targets for therapeutic intervention.     

NCX activity generates spontaneous Ca2+ oscillations in the astrocytic leaflet microdomain

The synergy between synaptic Glu release and astrocytic Glu-Na⁺ symport is essential to the signalling function of the tripartite synapse. Here we used kinetic data of astrocytic Glu transporters (EAAT) and the Na⁺/Ca²⁺ exchanger (NCX) to simulate Glu release, Glu uptake and subsequent Na⁺ and Ca²⁺ dynamics in the astrocytic leaflet microdomain following single release event. Model simulations show that Glu-Na⁺ symport differently affect intracellular [Na⁺] in synapses with different extent of astrocytic coverage. Surprisingly, NCX activity alone has been shown to generate markedly stable, spontaneous Ca²⁺ oscillation in the astrocytic leaflet. These on-going oscillations appear when NCX operates either in the forward or reverse direction. We conjecture that intrinsic NCX activity may play a prominent role in the generation of astrocytic Ca²⁺ oscillations.     

Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca²⁺] ([Ca²⁺]_i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP₃-evoked Ca²⁺ release. The kinetics of both processes were studied with flash photolytic release of InsP₃ and time resolved measurements of [Ca²⁺]_i in single cells. InsP₃ evoked Ca²⁺ flux into the cytosol was measured as d[Ca²⁺]_i/dt, and the kinetics of Ca²⁺ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP₃ concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP₃ suggests that metabolism of InsP₃ is important in determining the minimal concentration needed to produce Ca²⁺ release. A distinct latency or delay of several hundred milliseconds after release of low InsP₃ concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca²⁺]_i, suggesting a cooperative action of Ca²⁺ in InsP₃ receptor activation. InsP₃-evoked flux and peak [Ca²⁺]_i increased with InsP₃ concentration up to 5–10 μM, with large variation from cell to cell at each InsP₃ concentration. The duration of InsP₃-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca²⁺]_i/dt and much less cell to cell variation than the dependence of flux on InsP₃ concentration, suggesting that the rate of termination of the Ca²⁺ flux depends on the free Ca²⁺ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca²⁺ flux, up to 50 μM · s⁻¹ and in Purkinje neurons at high flux up to 1,400 μM · s⁻¹. Experiments in which [Ca²⁺]_i was controlled at resting or elevated levels support a mechanism in which InsP₃-evoked Ca²⁺ flux is inhibited by Ca²⁺ inactivation of closed receptor/channels due to Ca²⁺ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP₃-evoked Ca²⁺ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP₃ receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP₃ receptor density promotes fast rising, rapidly inactivating InsP₃-evoked [Ca²⁺]_i transients.     

InsP3 signaling induces pulse-modulated Ca2+ signals in the nucleus of airway epithelial ciliated cells

The phenomenology of nuclear Ca(2+) dynamics has experienced important progress revealing the broad range of cellular processes that it regulates. Although several agonists can mobilize Ca(2+) from storage in the nuclear envelope (NE) to the intranuclear compartment (INC), the mechanisms of Ca(2+) signaling in the nucleus still remain uncertain. Here we report that the NE/INC complex can function as an inositol-1,4,5-trisphosphate (InsP(3))-controlled Ca(2+) oscillator. Thin optical sectioning combined with fluorescent labeling of Ca(2+) probes show in cultured airway epithelial ciliated cells that ATP can trigger periodic oscillations of Ca(2+) in the NE ([Ca(2+)](NE)) and corresponding pulses of Ca(2+) release to the INC. Identical results were obtained in InsP(3)-stimulated isolated nuclei of these cells. Our data show that [Ca(2+)](NE) oscillations and Ca(2+) release to the INC result from the interplay between the Ca(2+)/K(+) ion-exchange properties of the intralumenal polyanionic matrix of the NE and two Ca(2+)-sensitive ion channels-an InsP(3)-receptor-Ca(2+) channel and an apamin-sensitive K(+) channel. A similar Ca(2+) signaling system operating under the same functional protocol and molecular hardware controls Ca(2+) oscillations and release in/to the endoplasmic reticulum/cytosol and in/to the granule/cytosol complexes in airway and mast cells. These observations suggest that these intracellular organelles share a remarkably conserved mechanism of InsP(3)-controlled frequency-encoded Ca(2+) signaling.     

Ca2+ dependence of inositol 1,4,5-trisphosphate 3-kinase activity in the cytosol fraction of pig coronary artery

1. The activity of inositol 1,4,5-trisphosphate 3-kinase in subcellular fractions of smooth muscles of the pig coronary artery was examined. 2. Incubation of [3H]inositol 1,4,5-trisphosphate (IP3) with muscle homogenates produced more polar 3H-radioactivity (probably as inositol 1,3,4,5-tetrakisphosphate, IP4) than IP3, in the Mg2+- and ATP-dependent manner, thereby indicating the presence of IP3 3-kinase activity in homogenates of the muscle. 3. Most of the kinase activity was present in the cytosol fraction. The enzyme activity was reversibly activated by Ca2+ with a half-maximal effective concentration of 2.5 x 10(-7) M. 4. The calmodulin antagonists, W-7 and chlorpromazine inhibited the Ca2+-activated enzyme activity.     

Spontaneous [Ca2+]i fluctuations in rat chromaffin cells do not require inositol 1,4,5-trisphosphate elevations but are generated by a caffeine- and ryanodine-sensitive intracellular Ca2+ store

A considerable fraction (65%) of single rat chromaffin cells loaded with the fluorescent [Ca2+]i indicator fura-2 exhibited spontaneous rhythmic fluctuations with an average period of approximately 100 s. Parallel patch clamp experiments as well as fura-2 experiments carried out in Ca2(+)-free and other modified media in the presence of Ca2+ and Na+ channel blockers indicated an origin from intracellular stores. Appropriate concentrations of agonists (bradykinin and histamine) for receptors (B2 and H1) that trigger generation of inositol 1,4,5-trisphosphate induced increased fluctuation frequency, recruitment of silent cells, and large [Ca2+]i changes at high doses. These effects were blocked by cell pretreatment with neomycin, a drug that inhibits inositol 1,4,5-trisphosphate generation. In contrast, spontaneous fluctuations and the effects of another drug, caffeine, which also induced increased frequency and recruitment, were unaffected by neomycin. Ryanodine caused first a prolongation and then (approximately 10 min) a block of both spontaneous fluctuations and caffeine effects, where the single transients after bradykinin and histamine were maintained. Caffeine and ryanodine are known to affect selectively the process of calcium-induced Ca2+ release; this is the first demonstration of [Ca2+]i fluctuation activity arising from Ca2(+)-induced Ca2+ release in nonmuscle cells with no strict requirement for inositol 1,4,5-trisphosphate involvement.     

Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells.

Oscillations of free intracellular Ca2+ concentration ([Ca2+]i) are known to occur in many cell types during physiological cell signaling. To identify the basis for the oscillations, we measured both [Ca2+]i and extracellular Ca2+ concentration ([Ca2+]o) to follow the fate of Ca2+ during stimulation of [Ca2+]i oscillations in pancreatic acinar cells. [Ca2+]i oscillations were initiated by either t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Nle-Asp-2-phenylethyl ester (CCK-J), which mobilized Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-insensitive pool, or low concentration of cholecystokinin octapeptide (CCK-OP), which mobilized Ca2+ from the IP3-sensitive internal pool. Little Ca2+ efflux occurred during the oscillations triggered by CCK-J or CCK-OP in spite of a large average increase in [Ca2+]i. When internal store Ca2+ pumps were inhibited with thapsigargin (Tg) during [Ca2+]i oscillations, a rapid Ca2+ efflux occurred similar to that measured in intensely stimulated, nonoscillatory cells. Tg also stimulated 45Ca efflux from internal pools of cells stimulated with CCK-J or a low concentration of CCK-OP. Hence, a large fraction of the Ca2+ released during each spike is reincorporated by the internal store Ca2+ pumps. Surprisingly, when the increase in [Ca2+]i during stimulation of oscillations was prevented by loading the cells with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, a persistent activation of Ca2+ release and Ca2+ efflux occurred. This was reflected as a persistent increase in [Ca2+]o in cells suspended at low [Ca2+]o or persistent efflux of 45Ca from internal stores of cells maintained at high [Ca2+]o. Since agonist-stimulated Ca2+ release evidently remains activated when [Ca2+]i is highly buffered, the primary mechanism determining Ca2+ oscillations must include an inhibition of Ca2+ release by [Ca2+]i. Loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no apparent effect on the levels or kinetics of IP3 formation in agonist-stimulated cells. This suggests that [Ca2+]i regulated the oscillation by inhibition of Ca2+ release independent of its possible effects on cellular levels of IP3.     

Xestospongin C empties the ER calcium store but does not inhibit InsP3-induced Ca2+ release in cultured dorsal root ganglia neurones

The action of Xestospongin C (XeC) on calcium concentration in the cytosol ([Ca2+]i) and within the lumen of endoplasmic reticulum (ER) ([Ca2+]L) was studied using cultured dorsal root ganglia (DRG) neurones. Application of 2.5 microM of XeC triggered a slow [Ca2+]i transient as measured by Fura-2 video-imaging. The kinetics and amplitude of XeC-induced [Ca2+]i response was similar to that triggered by 1 microM thapsigargin (TG). The [Ca2+]L was monitored in cells loaded with low-affinity Ca2+ indicator Mag-Fura-2. The cytosolic portion of Mag-Fura-2 was removed by permeabilisation of the plasmalemma with saponin. Application of XeC to these permeabilised neurones resulted in a slow depletion of the ER Ca2+ store. XeC, however, failed to inhibit inositol 1,4,5-trisphosphate (InsP3)-induced [Ca2+]L responses. We conclude that XeC is a potent inhibitor of sarco(endo)plasmic reticulum calcium ATPase, and it cannot be regarded as a specific inhibitor of InsP3 receptors in cultured DRG neurones.