Acetylcholine induces Ca2+ oscillations via m3/m4 muscarinic receptors in the mouse oocyte

Changes in intracellular Ca²⁺ concentration are required for the activation of mammalian oocytes. They are caused mainly by Ca²⁺ release from the endoplasmic reticulum (ER) via InsP ₃ receptors (InsP ₃R). Several studies have reported that acetylcholine (ACh) is capable of triggering early activation events in mouse oocytes over-expressed with the m1 muscarinic ACh receptor (m1AChR). Here we examined which subtypes of the mAChR (m1 to m4) are involved in the generation of Ca²⁺ oscillations in native mouse oocytes. ACh (10 M) elicited regular Ca²⁺ oscillations similar to those induced by sperm in their temporal characteristics. The Ca²⁺ oscillations were abolished by application with atropine, the mAChR inhibitor. Within 1 min after treatment of ACh, intracellular Fluo-3 fluorescence intensity increased from 794±119 to 2023±755 (increase to 250% of original value), indicating a strong rise of cytosolic Ca²⁺ concentration. 4-DAMP mustard and Tropicamide, specific antagonists of m3AChR and m4AChR, completely abolished ACh-induced Ca²⁺ oscillations. In the ovulated oocytes, the expression of m3/m4 AChR was clearly detected by RT-PCR analysis. Furthermore, ACh-induced Ca²⁺ oscillations were also abolished or decreased by PLC inhibitors (U73122 or D609) and an InsP ₃-receptor antagonist (xestospongin C), confirming that ACh generates Ca²⁺ oscillations via the PLC-InsP ₃ (PI) pathway. These results strongly suggest that m3/m4AChR is coupled to the generation of Ca²⁺ oscillations mainly via the PI pathway in mouse oocytes.     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca²⁺ ([Ca²⁺]_i). The increases of [Ca²⁺]_i evoked by TG was associated with significant changes of plasma membrane Ca²⁺ permeability, with [Ca²⁺]_i values following changes in extracellular [Ca²⁺]. Plasma membrane Ca²⁺ extrusion is activated rapidly as a consequence of the rise in [Ca²⁺]_i evoked by TG and the rate of extrusion is linearly dependent on [Ca²⁺]_i up to 1 μM Ca²⁺. In contrast, the activation of the Ca²⁺ entry pathway is delayed and the apparent rate of Ca²⁺ entry is independent of [Ca²⁺]_i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP₃), the increase of [Ca²⁺]_i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca²⁺]_i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca²⁺]_i increase (from 12.3±1.0 to 6.1±0.6 nM Ca²⁺/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca²⁺]_i. The amplitude of the [Ca²⁺]_i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La³⁺, which is known to inhibit the plasma membrane Ca²⁺-activated adenosine triphosphatase, induced a much higher peak of [Ca²⁺]_i. This increase was associated with an augmentation of the apparent rate of [Ca²⁺]_i increase (from 12.3±1.2 to 16.1±1.9 nM Ca²⁺/s). The inhibitory effect of caffeine, as well as the increase in [Ca²⁺]_i observed on caffeine removal was not affected by the presence of 1 mM La³⁺. These data indicate that an important component of the TG-evoked [Ca²⁺]_i increase is due to InsP₃-sensitive Ca²⁺ release which is probably mediated by the resting levels of InsP₃.     

2-Aminoethoxydiphenyl borate inhibits agonist-induced Ca2+ signals by blocking inositol trisphosphate formation in acutely dissociated mouse pancreatic acinar cells

Evidence suggests that 2-aminoethoxydiphenyl borate (2-APB) modulates intracellular Ca²⁺ signals in a complex manner. 2-APB inhibits or potentiates intracellular Ca²⁺ signals in different cell types, perhaps through different mechanisms. Here, we report a novel mechanism underlying 2-APB-induced inhibition of agonist-activated Ca²⁺ oscillations in mouse pancreatic acinar cells, using patch-clamp and biochemical techniques. Pre-treatment of the cells with 100 µM 2-APB completely abolished ACh- but not inositol trisphosphate (InsP₃)-induced Ca²⁺ oscillations, suggesting that the mechanism of inhibition occurs between cytoplasmic receptors and InsP₃ receptor activation. In addition, 100 µM 2-APB significantly inhibited ACh-induced phospholipase C (PLC) activation. These findings indicate that, in mouse pancreatic acinar cells, in addition to modulating InsP₃ receptors and blocking the store-operated Ca²⁺ pathway, high concentrations of 2-APB also inhibit agonist-induced Ca²⁺ signals by reducing InsP₃ formation.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Stimulatory and inhibitory actions of VIP and cyclic AMP on cytoplasmic Ca2+ signal generation in pancreatic acinar cells

In pancreatic acinar cells the effects of vasoactive intestinal polypeptide (VIP) and dibutyryl cyclic AMP (dbcAMP) alone and during stimulation with acetylcholine (ACh) or internally applied inositol trisphosphate (InsP₃) were investigated utilizing the patch-clamp whole-cell current recording configuration to assess changes in cytoplasmic Ca²⁺ concentration by measurement of Ca²⁺ dependent Cl^– current. VIP (1 nM) and dbcAMP (0.5 mM) each evoked repetitive pulses of Ca²⁺-dependent Cl^– current. A high VIP concentration (100 nM) acutely and reversibly inhibited the responses evoked by 1 nM VIP and also acutely inhibited ACh-evoked responses. DbcAMP (2 mM) also reversibly inhibited the ACh-evoked responses. Neither VIP nor dbcAMP were able to inhibit InsP₃-evoked repetitive Ca²⁺ pulses. VIP in a low concentration can generate cytosolic Ca²⁺ oscillations via cyclic AMP, but high VIP concentrations inhibit both ACh and VIP-evoked Ca²⁺ signals and this effect is mediated by high intracellular cyclic AMP levels.     

[Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange

Transient inward currents (I _ti) during oscillations of intracellular [Ca²⁺] ([Ca²⁺]_i) in ventricular myocytes have been ascribed to Na/Ca exchange. We have investigated whether other Ca²⁺-dependent membrane currents contribute to I _ti in single guinea-pig ventricular myocytes, by examining membrane currents during [Ca²⁺]_i oscillations and during caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum in the absence of Na⁺. Membrane currents were recorded during whole-cell voltage clamp and [Ca²⁺]_i measured simultaneously with fura-2. In the absence of Na/Ca exchange, i.e., with Li⁺, Cs⁺ or N-methyl-D-glucamine (NMDG⁺) substituted for Na⁺, the cell could be loaded with Ca²⁺ by repetitive depolarizations to +10 mV, resulting in spontaneous [Ca²⁺]_i oscillations. During these oscillations, no inward currents were seen, but instead spontaneous Ca²⁺ release was accompanied by a shift of the membrane current in the outward direction at potentials between –40 mV and +60 mV. This [Ca²⁺]_i-dependent outward current shift was not abolished when NMDG⁺ was substituted for internal monovalent cations, nor was it sensitive to substitution of external Cl^–. It was however, sensitive to the blockade of I_Ca by verapamil. These results suggest that the transient outward current shift observed during spontaneous Ca²⁺ release represents [Ca²⁺]_idependent transient inhibition of I _Ca. Similarly, during the [Ca²⁺]_i transients induced by brief caffeine (10 mM) applications, we could not detect membrane currents attributable to a Ca²⁺-activated nonselective cation channel, or to a Ca²⁺-activated Cl^– channel; however, transient Ca²⁺-dependent inhibition of I _Ca was again observed. We conclude that neither the Ca²⁺-activated nonselective cation channel nor the Ca²⁺-activated Cl^– channel contribute significantly to the membrane currents during spontaneous [Ca²⁺]_i oscillations in guineapig ventricular myocytes. However, in the voltage range between –40 mV and +60 mV Ca²⁺-dependent transient inhibition of I _Ca will contribute to the oscillations of the membrane current.     

AlF 4 − induces Ca2+ oscillations in guinea-pig ileal smooth muscle

The effects of different compounds that inhibit the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase on the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and on the corresponding force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. F^–, in the presence of Al³⁺, induced an increase of the resting force and of the amplitude of the superimposed phasic contractions. The increase of resting force was associated with an increased level of basal [Ca²⁺]_i while the phasic contractions were accompanied by concomitant oscillations in [Ca²⁺]_i. Comparable contractions could be induced by vanadate and the calmodulin antagonist calmidazolium. The oscillations of [Ca²⁺]_i and of force elicited by AlF ₄ ^– were not modified by adrenergic or cholinergic blocking agents but were inhibited by verapamil. These phasic contractions were not affected by depleting the intracellular Ca²⁺ stores with ryanodine. This finding excludes a cytosolic origin of these oscillations. However, hyperpolarization and complete depolarization of the cells inhibited the oscillations. It is concluded that AlF ₄ ^– , vanadate and calmidazolium induce cytoplasmic Ca²⁺ oscillations possibly by acting at the plasma membrane. Indeed all these substances affect by different mechanisms the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase. The generation of membrane-linked Ca²⁺ oscillations could therefore be related to an inhibition of the plasma-membrane Ca²⁺ pump resulting in an increase of [Ca²⁺]_i. This change in [Ca²⁺]_i could be responsible for the pronounced changes of the electrical and mechanical activity of this tissue.     

Mechanisms responsible for quantal Ca2+ release from inositol trisphosphate-sensitive calcium stores

Activation of cells by hormones, growth factors or neurotransmitters leads to an increased production of inositol trisphosphate (InsP₃) and, after activation of the InsP₃ receptor (InsP₃R), to Ca²⁺ release from intracellular Ca²⁺ stores. The release of intracellular Ca²⁺ is characterised by a graded response when submaximal doses of agonists are used. The basic phenomenon, called “quantal Ca²⁺ release”, is that even the maintained presence of a submaximal dose of agonist or of InsP₃ for long time periods (up to 20 min) provokes only a partial release of Ca²⁺. This partial, or quantal, release phenomenon is due to the fact that the initially very rapid InsP₃-induced Ca²⁺ release eventually develops into a much slower release phase. Physiologically, quantal release allows the Ca²⁺ stores to function as increment detectors and to induce local Ca²⁺ responses. The basic mechanism for quantal release of Ca²⁺ is presently not known. Possible mechanisms to explain the quantal behaviour of InsP₃- induced Ca²⁺ release include the presence of InsP₃Rs with varying sensitivities for InsP₃, heterogeneous InsP₃R distribution, intrinsic inactivation of the InsP₃Rs, and regulation of the InsP₃Rs by Ca²⁺ store content. This article reviews critically the evidence for the various mechanisms and evaluates their functional importance. A Ca²⁺-mediated conformational change of the InsP₃R is most likely the key feature of the mechanism for quantal Ca²⁺ release, but the exact mode of operation remains unclear. It should also be pointed out that in intact cells more than one mechanism can be involved.     

Histamine-evoked Ca2+ oscillations in HeLa cells are sensitive to methylxanthines but insensitive to ryanodine

The relative contribution of inositol-trisphosphate(InsP ₃)-sensitive and InsP ₃-insensitive Ca²⁺ stores to the agonist-evoked oscillatory release of Ca²⁺ in HeLa cells was investigated using fura-2 cytosolic Ca²⁺ measurements and whole-cell recordings of Ca²⁺-activated K⁺ currents [K(Ca²⁺)]. The experimental approach chosen consisted in studying the effects on Ca²⁺ oscillations of a variety of pharmacological agents such as ryanodine, ruthenium red, caffeine and theophylline, which are known to affect the Ca²⁺ channels responsible for Ca²⁺-induced Ca²⁺ release (CICR) in excitable cells. The results obtained essentially indicate (a) that neither ryanodine nor ruthenium red affects the generation of periodic K(Ca²⁺) current pulses in whole-cell experiments, and (b) that histamine-induced Ca²⁺ oscillations are inhibited by caffeine and theophylline in a dose-dependent manner. However, these methylxanthines were unable, at concentrations ranging from 0.1 mM to 10 mM, either to mobilize Ca²⁺ from internal stores or to block the initial Ca²⁺ rise evoked by histamine. In addition, both methylxanthines showed at high concentrations (10–20 mM) a moderate inhibitory action on the production of InsP ₃ induced by histamine. This effect was not essential to the action of caffeine on the oscillatory release of Ca²⁺, since an inhibition by caffeine of InsP ₃-induced Ca²⁺ oscillations was still observed in whole-cell experiments where the InsP ₃ concentration was kept constant. The results also show (c) that the application of either caffeine or theophylline during histamine stimulation leads systematically to an increased Ca²⁺ sequestration in InsP ₃-sensitive Ca²⁺ pools, the effect observed with theophylline being stronger than that resulting from the application of caffeine, and finally (d) that the action of caffeine and theophylline is not related to an increase in cAMP concentration since neither forskolin (10–50 M) nor 8-Br-cAMP (1 mM) caused an inhibition of the InsP ₃-induced Ca²⁺ oscillations. It is concluded on the basis of these results that the agonist-evoked Ca²⁺ oscillations in HeLa cells do not involve directly or indirectly a ryanodine-sensitive Ca²⁺-release channel with CICR properties, but rather arise from a control by Ca²⁺ of the InsP ₃ Ca²⁺-release process.     

Ca2+ signalling pathways activated by acetylcholine in mouse C2C12 myotubes

In mouse C2C12 myotubes acetylcholine (ACh) elevates the concentration of myoplasmic Ca²⁺ ([Ca²⁺]_i) by inducing Ca²⁺ influx through transmittergated and voltage-gated channels, and by mobilizing Ca²⁺ from internal stores. The relative contribution of each of these ACh-activated sources to the global [Ca²⁺]_i elevation was estimated. We found that Ca²⁺ entry through voltage- and ACh-gated channels accounts for roughly 80% of the total [Ca²⁺]_i increment, while mobilization from internal caffeine-sensitive and inositol-trisphosphate (InsP ₃ ^–) sensitive stores contributes the remaining 20% to the maximal [Ca²⁺]_i increment. Furthermore, we found that ACh-induced mobilization from InsP ₃-sensitive stores also develops in embryonic chick myotubes. The differential importance of the Ca²⁺ signalling pathways activated by ACh during myogenesis is discussed.     

Capacitative Ca2+ entry contributes to the Ca2+ influx induced by thyrotropin-releasing hormone (TRH) in GH3 pituitary cells

Treatment of GH₃ cells with either hypothalamic peptide thyrotropin-releasing hormone (TRH), the endomembrane Ca²⁺-ATPase inhibitor thapsigargin or the Ca²⁺ ionophore ionomycin mobilized, with different kinetics, essentially all of the Ca²⁺ pool from the intracellular Ca²⁺ stores. Any of the above-described treatments induced a sustained increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), which was dependent on extracellular Ca²⁺ and was prevented by Ni²⁺ but not by dihydropyridines (DHPs), suggesting that it was due to capacitative Ca²⁺ entry via activation of a plasma membrane pathway which opened upon the emptying of the intracellular Ca²⁺ stores. The increase of the plasma membrane permeability to Ca²⁺ correlated negatively with the filling degree of the intracellular Ca²⁺ stores and was reversed by refilling of the stores. The mechanism of capacitative Ca²⁺ entry into GH₃ cells differed from similar mechanisms described in several types of blood cells in that the pathway was poorly permeable to Mn²⁺ and not sensitive to cytochrome P₄₅₀ inhibitors. In GH₃ cells, TRH induced a transient [Ca²⁺]_i increase due to Ca²⁺ release from the stores (phase 1) followed by a sustained [Ca²⁺]_i increase due to Ca²⁺ entry (phase 2). At the single-cell level, phase 2 was composed of a DHP-insensitive sustained [Ca²⁺]_i increase, due to activation of capacitative Ca²⁺ entry, superimposed upon which DHP-sensitive [Ca²⁺]_i oscillations took place. The two components of the TRH-induced Ca²⁺ entry differed also in that [Ca²⁺]_i oscillations remained for several minutes after TRH removal, whereas the sustained [Ca²⁺]_i increase dropped quickly to prestimulatory levels, following the same time course as the refilling of the stores. The drop was prevented when the refilling was inhibited by thapsigargin. It is concluded that, even though the mechanisms of capacitative Ca²⁺ entry may show differences from cell to cell, it is also present and may contribute to the regulation of physiological functions in excitable cells such as GH₃. There, capacitative Ca²⁺ entry cooperates with voltage-gated Ca²⁺ channels to generate the [Ca²⁺]_i increase seen during phase 2 of TRH action. This contribution of capacitative Ca²⁺ entry may be relevant to the enhancement of prolactin secretion induced by TRH.     

ATP depletion inhibits Ca2+ release, influx and extrusion in pancreatic acinar cells but not pathological Ca2+ responses induced by bile

Here, we describe novel mechanisms limiting a toxic cytosolic Ca²⁺ rise during adenosine 5′-triphosphate (ATP) depletion. We studied the effect of ATP depletion on Ca²⁺ signalling in mouse pancreatic acinar cells. Measurements of ATP in isolated cells after adenovirus-mediated expression of firefly luciferase revealed that the cytosolic ATP concentration fell from approximately 1 mM to near zero after treatment with oligomycin plus iodoacetate. ATP depletion resulted in the inhibition of Ca²⁺ extrusion, which was accompanied by a remarkably synchronous inhibition of store-operated Ca²⁺ influx. Alternative inhibition of Ca²⁺ extrusion by carboxyeosin had a much smaller effect on Ca²⁺ influx. The coordinated metabolic inhibition of Ca²⁺ influx and extrusion suggests the existence of a common ATP-dependent master regulator of both processes. ATP-depletion also suppressed acetylcholine (ACh)-induced Ca²⁺ oscillations, which was due to the inhibition of Ca²⁺ release from internal stores. This could be particularly important for limiting Ca²⁺ toxicity during periods of hypoxia. In contrast, metabolic control of Ca²⁺ influx and Ca²⁺ release from internal stores spectacularly failed to prevent large toxic Ca²⁺ responses induced by bile acids—activators of acute pancreatitis (a frequent and often fatal disease of the exocrine pancreas). The bile acids taurolithocholic acid 3-sulphate (TLC-S), taurochenodeoxycholic acid (TCDC) and taurocholic acid (TC) were used in our experiments. Neither Ca²⁺ release from internal stores nor Ca²⁺ influx triggered by bile acids were inhibited by ATP depletion, emphasising the danger of these pathological mechanisms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inositol trisphosphate receptor Ca2+ release channels in neurological diseases

The modulation of cytoplasmic Ca²⁺ concentration by release from internal stores through the inositol trisphosphate receptor (InsP₃R) Ca²⁺ release channel is a ubiquitous signaling system involved in the regulation of numerous processes. Because of its ubiquitous expression and roles in regulating diverse cell physiological processes, it is not surprising that the InsP₃R has been implicated in a number of disease states. However, relatively few mutations in InsP₃R genes have been identified to date. Here, I will discuss mutations in the type 1 InsP₃R that have been discovered by analyses of human patients and mice with neurological disorders. In addition, I will highlight diseases caused by mutations in other genes, including Huntington’s and Alzheimer’s diseases and some spinocerebellar ataxias, where the mutant proteins have been found to exert strong influences on InsP₃R function that may link InsP₃R to disease pathogenesis.     

Induction of Ca2+ oscillations by selective,U73122-mediated,depletion of inositol-trisphosphate-sensitive Ca2+ stores in rabbit pancreatic acinar cells

The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca²⁺ sequestering and releasing properties of internal Ca²⁺ stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca²⁺ uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P ₃]-sensitive, but not the Ins(1,4,5)P ₃-insensitive, Ca²⁺ store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca²⁺ indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca²⁺ concentration ([Ca²⁺]_i,av), which was largely independent of external Ca²⁺. Addition of U73122 to cell suspensions prestimulated with either cholecystokinin octapeptide or JMV-180 revealed an inverse relationship in size between the U73122- and the agonistevoked [Ca²⁺]_i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca²⁺-ATPase activity resulted in a [Ca²⁺]_i,av transient, the size of which was not different following maximal prestimulation with either U73122 or agonist. These observations suggest that U73122 selectively affects the Ins(1,4,5)P ₃- casu quo agonist-sensitive internal Ca²⁺ store, whereas thapsigargin affects both the Ins(1,4,5)P ₃-sensitive and -insensitive Ca²⁺ store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca²⁺]_i. The U73122-evoked oscillations were abolished in the absence of external Ca²⁺. The ability of U73122 to generate external Ca²⁺-dependent Ca²⁺ oscillations suggests that depletion of the agonistsensitive store leads to an increase in Ca²⁺ permeability of the plasma membrane and that the Ins(1,4,5)P ₃-insensitive Ca²⁺ pool is necessary for the Ca²⁺ oscillations.     

Effect of thapsigargin and caffeine on Ca2+ homeostasis in HeLa cells: implications for histamine-induced Ca2+ oscillations

Several studies have already established that the stimulation of H1 receptors by exogenous histamine induces intracellular Ca²⁺ oscillations in HeLa cells. The molecular mechanism underlying this oscillatory process remains, however, unclear. A series of fura-2 experiments was undertaken in which the nature of the Ca²⁺ pools involved in the histamine-induced Ca²⁺ oscillations was investigated using the tumour promoter agent thapsigargin (TG) and the Ca²⁺-induced Ca²⁺-release promoter, caffeine. The results obtained indicate first that TG causes a gradual increase in cytosolic Ca²⁺ without inducing internal Ca²⁺ oscillations, and second that TG and histamine share common internal Ca²⁺ storage sites. The latter conclusion was derived from experiments performed in the absence of external Ca²⁺, where the addition of TG before histamine resulted in a total inhibition of the Ca²⁺ response linked to H1 receptor stimulation, whereas the addition of histamine before TG decreased by more than 90% the TG-induced Ca²⁺ release. Finally, TG was found to inhibit irreversibly histamine-induced Ca²⁺ oscillations when added to the bathing medium during the oscillatory process. The effect of caffeine at concentrations ranging from 1 mM to 10 mM on intracellular Ca²⁺ homeostasis was also investigated. The results obtained show that caffeine does not affect systematically the internal Ca²⁺ concentration in resting and TG-stimulated HeLa cells, but increases the Ca²⁺ sequestration ability of inositol-trisphosphate (InsP ₃)-related Ca²⁺ stores. These results suggest either that TG acts on InsP ₃-sensitive as well as InsP ₃-insensitive Ca²⁺ pools, so that no final conclusion on the nature of the pools involved in Ca²⁺ spike generation can be currently drawn, or that the contribution of an InsP ₃-insensitive Ca²⁺-induced Ca²⁺-release process is not essential to the Ca²⁺ oscillation machinery in these cells. It is also concluded that a release of Ca²⁺ by caffeine may not be directly accessible to fura-2 measurements in this cellular preparation, but that the inhibitory effect of caffeine on the Ca²⁺ mobilization process triggered by InsP ₃ can be clearly documented using this experimental approach.     

Mechanisms of Ca2+ handling in zebrafish ventricular myocytes

The zebrafish serves as a promising transgenic animal model that can be used to study cardiac Ca²⁺ regulation. However, mechanisms of sarcoplasmic reticulum (SR) Ca²⁺ handling in the zebrafish heart have not been systematically explored. We found that in zebrafish ventricular myocytes, the action potential-induced Ca²⁺ transient is mainly (80 %) mediated by Ca²⁺ influx via L-type Ca²⁺ channels (LTCC) and only 20 % by Ca²⁺ released from the SR. This small contribution of the SR to the Ca²⁺ transient was not the result of depleted SR Ca²⁺ load. We found that the ryanodine receptor (RyR) expression level in zebrafish myocytes was ～72 % lower compared to rabbit myocytes. In permeabilized myocytes, increasing cytosolic [Ca²⁺] from 100 to 350 nM did not trigger SR Ca²⁺ release. However, an application of a low dose of caffeine activated Ca²⁺ sparks. These results show that the zebrafish cardiac RyR has low sensitivity to the mechanism of Ca²⁺-induced Ca²⁺ release. Activation of protein kinase A by forskolin increased phosphorylation of the RyR in zebrafish myocardium. In half of the studied cells, an increased Ca²⁺ transient by forskolin was entirely mediated by augmentation of LTCC current. In the remaining myocytes, the forskolin action was associated with an increase of both LTCC and SR Ca²⁺ release. These results indicate that the mechanism of excitation–contraction coupling in zebrafish myocytes differs from the mammalian one mainly because of the small contribution of SR Ca²⁺ release to the Ca²⁺ transient. This difference is due to a low sensitivity of RyRs to cytosolic [Ca²⁺].     

Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N)

Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1–100 M) on the intracellular free calcium ion concentration ([Ca²⁺]_i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca²⁺]_i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca²⁺]_i transients. Overall, the pattern of response was concentration related. In general, 0.1–10 M acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca²⁺, whilst at higher concentrations the responses consisted of a rapid rise in [Ca²⁺]_i followed by a smaller more sustained increase. Without external Ca²⁺, 100 M acetylcholine caused only a transient rise in [Ca²⁺]_i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca²⁺]_i oscillations. Acetylcholine-evoked Ca²⁺ signals were abolished by atropine (1–10 M), verapamil (100 M) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca²⁺]_i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca²⁺]_i. Ryanodine (50–500 nM) and caffeine (1–20 mM) did not increase basal Ca²⁺ levels, but the Ca²⁺-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca²⁺]_i levels. These data demonstrate for the first time cytosolic Ca²⁺ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca²⁺ influx and Ca²⁺ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.     

Activation of the nicotinic acetylcholine receptor mobilizes calcium from caffeine-insensitive stores in C2C12 mouse myotubes

In cultured mouse C2C12 myotubes, digital Ca²⁺ imaging fluorescence microscopy using the acetoxymethyl ester of Fura-2, Fura-2-AM, showed that, in the absence of extracellular Ca²⁺, acetylcholine (ACh) and nicotine, but not muscarine, raised the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) by about tenfold. AChinduced Ca²⁺ mobilization was prevented by thapsigargin, a drug known to deplete inositol 1,4,5-trisphosphate (InsP₃)-sensitive stores, and was concomitant with InsP₃ accumulation. Caffeine, which releases Ca²⁺ from the ryanodine-sensitive stores of the sarcoplasmic reticulum, did not interfere with the ACh-induced [Ca²⁺]_i increase. Ca²⁺ mobilization was also inhibited when myotubes were depolarized by high K⁺, or when extracellular Na⁺ was omitted. Nicotinic ACh receptor (nAChR) stimulation lowered intracellular pH with a time course slower than the [Ca²⁺]_i increase. Possible mechanisms linking the current flowing through the nAChR pore to [Ca²⁺]_i increase are discussed.     

Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca²⁺ oscillations from an inositol 1,4,5-trisphosphate-sensitive cytoplasmic Ca²⁺ store. In this study, Ca²⁺ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca²⁺ video imaging and the Fura-2/Mn²⁺ quenching technique. Oscillations were dependent on extracellular Ca²⁺ concentration and were inhibited by extracellularly applied La³⁺, Co²⁺ and Ni²⁺. Depolarization of the cell membrane with high K⁺ concentrations and the L-type Ca²⁺ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca²⁺ channels. Mn²⁺ quenching experiments disclosed significant Ca²⁺ influx into MDCK-F cells. The rate of this influx was constant between Ca²⁺ spikes, but markedly increased during the spontaneous Ca²⁺ spikes. Similar transient increases in Ca²⁺ entry could be mimicked by agents triggering intracellular Ca²⁺ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca²⁺ permeability permitting Ca²⁺ entry into the cytoplasm. The rate of Ca²⁺ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca²⁺ concentration.     

Pacing-Induced Non-Uniform Ca2+ Dynamics in Rat Atria Revealed by Rapid-Scanning Confocal Microscopy

Intracellular Ca²⁺ ([Ca²⁺]_i) dynamics in isolated myocytes differ between the atria and ventricles due to the distinct t-tubular distributions. Although cellular aspects of ventricular [Ca²⁺]_i dynamics in the heart have been extensively studied, little is known about those of atrial myocytes in situ. Here we visualized precise [Ca²⁺]_i dynamics of atrial myocytes in Langendorff-perfused rat hearts by rapid-scanning confocal microscopy. Of 16 fluo-4-loaded hearts imaged during pacing up to 4-Hz, five hearts showed spatially uniform Ca²⁺ transients on systole among individual cells, whereas no discernible [Ca²⁺]_i elevation developed during diastole. In contrast, the remaining hearts showed non-uniform [Ca²⁺]_i dynamics within and among the cells especially under high-frequency (4 Hz) excitation, where subcellular cluster-like [Ca²⁺]_i rises or wave-like [Ca²⁺]_i propagation occurred on excitation. Such [Ca²⁺]_i inhomogeneity was more pronounced at high-frequency pacing, showing beat-to-beat Ca²⁺ transient alternans. Despite such non-uniform dynamics, cessation of burst pacing of the atria was not followed by emergence of spontaneous Ca²⁺ waves, indicating minor Ca²⁺-releasing potentials of the sarcoplasmic reticulum (SR). In summary, rat atria display a propensity to show non-uniform [Ca²⁺]_i dynamics on systole due to impaired Ca²⁺-release from the SR and paucity of t-tubules. Our results provide an important basis for understanding atrial pathophysiology.