Spatial and temporal organization of calcium signalling in hepatocytes.

Treatment of hepatocytes with agonists which act via the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), results in increases of cytosolic free Ca2+ [( Ca2+]i) which are manifest as a series of discrete [Ca2+]i transients or oscillations. With increasing agonist dose [Ca2+]i oscillation frequency increases and the initial latent period decreases, but the amplitude of the [Ca2+]i oscillations remains constant. Studies of these [Ca2+]i oscillations at the subcellular level have indicated that the [Ca2+]i changes do not occur synchronously throughout the cell, but initiate at a specific subcellular domain, adjacent to a region of the plasma membrane, and then propagate through the cell as a [Ca2+]i wave. For a given ceil, the locus of [Ca2+]i wave initiation is constant for every oscillation in a series and is also identical when the cell is sequentially stimulated with different agonists or when the phospholipase C-linked G protein is activated directly using AIF4-. The kinetics of the [Ca2+]i waves indicate that a Ca(2+)-activated mechanism is involved in propagating the oscillatory [Ca2+]i increases throughout the cell, and the data appear to be most consistent with a process of Ca(2+)-induced Ca2+ release. It is proposed that the ability to propagate [Ca2+]i oscillations into regions of the cell distal to the region in which the signal transduction apparatus is localized could serve an important function in allowing all parts of the cell to respond to the stimulus.     

The role of extracellular Ca2+ in the response of the hepatocyte to Ca2+-dependent hormones

The influence of extracellular Ca2+ on hormone-mediated increases of cytosolic free Ca2+ [( Ca2+]i) and phosphorylase activity was studied in isolated hepatocytes. In the presence of 1.3 mM extracellular Ca2+, the stimulation of phosphorylase activity produced by vasopressin or phenylephrine was maintained for 20-30 min. In contrast, the change in [Ca2+]i under these conditions was more transient and declined within 3-4 min to steady state values only 70 +/- 8 nM above the resting [Ca2+]i. Removal of the hormone from its receptor with specific antagonists caused a decline in [Ca2+]i back to the original resting values. Subsequent addition of a second hormone elicited a further Ca2+ transient. If the antagonist was omitted, the second hormone addition did not increase [Ca2+]i indicating that the labile intracellular Ca2+ pool remains depleted during receptor occupation. When extracellular Ca2+ was omitted, both the changes of [Ca2+]i and phosphorylase a caused by vasopressin were transient and returned exactly to resting values within 3-4 min. The subsequent readdition of Ca2+ to these cells produced a further increase of [Ca2+]i and phosphorylase activity which was larger than the changes observed upon Ca2+ addition to untreated cells. This reactivation of phosphorylase showed saturation kinetics with respect to extracellular [Ca2+], was maximally stimulated within 1 min of vasopressin addition and was inhibited by high concentration of diltiazem. We conclude that entry of extracellular Ca2+ into the cell is required in order to obtain a sustained hormonal stimulation of phosphorylase activity and is responsible for the maintenance of a small steady state elevation of [Ca2+]i.     

Coordinated intercellular calcium waves induced by noradrenaline in rat hepatocytes: dual control by gap junction permeability and agonist.

Calcium-mobilizing agonists induce intracellular Ca2+ concentration ([Ca2+]i) changes thought to trigger cellular responses. In connected cells, rises in [Ca2+]i can propagate from cell to cell as intercellular Ca2+ waves, the mechanisms of which are not elucidated. Using fura2-loaded rat hepatocytes, we studied the mechanisms controlling coordination and intercellular propagation of noradrenaline-induced Ca2+ signals. Gap junction blockade with 18 alpha-glycyrrhetinic acid resulted in a loss of coordination between connected cells. We found that second messengers and [Ca2+]i rises in one hepatocyte cannot trigger Ca2+ responses in connected cells, suggesting that diffusion across gap junctions, while required for coordination, is not sufficient by itself for the propagation of intercellular Ca2+ waves. In addition, our experiments revealed functional differences between noradrenaline-induced Ca2+ signals in connected hepatocytes. These results demonstrate that intercellular Ca2+ signals in multicellular systems of rat hepatocytes are propagated and highly organized through complex mechanisms involving at least three factors. First, gap junction coupling ensures coordination of [Ca2+]i oscillations between the different cells; second, the presence of hormone at each hepatocyte is required for cell-cell Ca2+ signal propagation; and third, functional differences between adjacent connected hepatocytes could allow a 'pacemaker-like' intercellular spread of Ca2+ waves.     

Inositol trisphosphate is required for the propagation of calcium waves in Xenopus oocytes.

Stimuli which act through the second messenger inositol 1,4,5-trisphosphate (InsP3) often increase free intracellular Ca2+ concentration ([Ca2+]i) in a localized subcellular area. Actively propagated Ca2+ waves then extend this focal Ca2+ signal to other parts of the cell. To understand how cells may control the spatial distribution of Ca2+, we investigated the mechanism by which Ca2+ waves propagate through the cytoplasm of Xenopus oocytes. Heparin, which inhibits the binding of InsP3 to its receptor, prevented the migration of Ca2+ waves induced by a poorly metabolized InsP3 (InsP3S3). This result suggested that Ca2+ waves move through the cell via the serial release of Ca2+ from InsP3-sensitive stores. Interventions which caused a localized increase in [Ca2+]i without elevations of InsP3 did not trigger Ca2+ waves. In the presence of a Ins-P3S3, however, endogenously released or locally injected Ca2+ elicited Ca2+ waves. A cooperative interaction between Ca2+ and InsP3 may therefore be responsible for the propagation of Ca2+ waves.     

Characterization of cytosolic calcium oscillations induced by phenylephrine and vasopressin in single fura-2-loaded hepatocytes

Changes of cytosolic free Ca2+ [( Ca2+]i) in response to receptor activation were studied at the single cell level by using digital imaging fluorescence microscopy of fura-2-loaded primary cultured hepatocytes. In response to phenylephrine and vasopressin, individual hepatocytes displayed dose-dependent oscillations of [Ca2+]i similar to those observed in aequorin-injected hepatocytes by Woods et al. (Woods, N. M., Cuthbertson, K. S. R., and Cobbold, P. H. (1986) Nature 329, 719-721). With increasing agonist concentration, the frequency of oscillations increased and the latent period decreased. For a given cell, peak [Ca2+]i was independent of applied agonist concentration. However, there was considerable variation from cell to cell in the absolute value of peak [Ca2+]i. There was also marked intercellular heterogeneity in the latency, frequency, and overall pattern of the Ca2+ responses. Such asynchronous responses can be explained in part by the apparent differential agonist sensitivity of individual cells for latency and frequency. At high doses, phenylephrine maintained an oscillatory pattern, whereas vasopressin produced a complex mixture of spiking and sustained [Ca2+]i responses. Vasopressin and phenylephrine also displayed differently shaped [Ca2+]i oscillations at submaximal doses, due primarily to a slower rate of decay with vasopressin. Despite the large cell-cell variation in the patterns of [Ca2+]i oscillations, successive readditions of the same agonist elicited identical cell-specific patterns of oscillation. In the absence of extracellular Ca2+ the frequency but not the magnitude of [Ca2+]i oscillations was decreased. Buffering of [Ca2+]i by increasing the fura-2 load of single hepatocytes also decreased the frequency of oscillations without affecting the peak Ca2+ level. These data provide further support for the importance of frequency modulation in agonist-induced Ca2+ responses and suggest that Ca2+ itself plays an important role in regulating the frequency of [Ca2+]i oscillations. Furthermore, the data demonstrate a broad heterogeneity in hepatocyte [Ca2+]i oscillations which may underlie the nonoscillatory responses of cell populations.     

Calcium signaling in liver

In hepatocytes, hormones linked to the formation of the second messenger inositol 1,4,5-trisphosphate (InsP3) evoke transient increases or spikes in cytosolic free calcium ([Ca2+]i), that increase in frequency with the agonist concentration. These oscillatory Ca2+ signals are thought to transmit the information encoded in the extracellular stimulus to down-stream Ca2+-sensitive metabolic processes. We have utilized both confocal and wide field fluorescence microscopy techniques to study the InsP3-dependent signaling pathway at the cellular and subcellular levels in the intact perfused liver. Typically InsP3-dependent [Ca2+]i spikes manifest as Ca2+ waves that propagate throughout the entire cytoplasm and nucleus, and in the intact liver these [Ca2+]i increases are conveyed through gap junctions to encompass entire lobular units. The translobular movement of Ca2+ provides a means to coordinate the function of metabolic zones of the lobule and thus, liver function. In this article, we describe the characteristics of agonist-evoked [Ca2+]i signals in the liver and discuss possible mechanisms to explain the propagation of intercellular Ca2+ waves in the intact organ.     

Mitochondria suppress local feedback activation of inositol 1,4, 5-trisphosphate receptors by Ca2+.

The concerted action of inositol 1,4,5-trisphosphate (IP3) and Ca2+ on the IP3 receptor Ca2+ release channel (IP3R) is a fundamental step in the generation of cytosolic Ca2+ oscillations and waves, which underlie Ca2+ signaling in many cells. Mitochondria appear in close association with regions of endoplasmic reticulum (ER) enriched in IP3R and are particularly responsive to IP3-induced increases of cytosolic Ca2+ ([Ca2+]c). To determine whether feedback regulation of the IP3R by released Ca2+ is modulated by mitochondrial Ca2+ uptake, the interactions between ER and mitochondrial Ca2+ pools were examined by fluorescence imaging of compartmentalized Ca2+ indicators in permeabilized hepatocytes. IP3 decreased luminal ER Ca2+ ([Ca2+]ER), and this was paralleled by an increase in mitochondrial matrix Ca2+ ([Ca2+]m) and activation of Ca2+-sensitive mitochondrial metabolism. Remarkably, the decrease in [Ca2+]ER evoked by submaximal IP3 was enhanced when mitochondrial Ca2+ uptake was blocked with ruthenium red or uncoupler. Moreover, subcellular regions that were relatively deficient in mitochondria demonstrated greater sensitivity to IP3 than regions of the cell with a high density of mitochondria. These data demonstrate that Ca2+ uptake by the mitochondria suppresses the local positive feedback effects of Ca2+ on the IP3R, giving rise to subcellular heterogeneity in IP3 sensitivity and IP3R excitability. Thus, mitochondria can play an important role in setting the threshold for activation and establishing the subcellular pattern of IP3-dependent [Ca2+]c signaling.     

Relationship between asynchronous Ca2+ waves and force development in intact smooth muscle bundles of the porcine trachea

Fluctuations in intracellular calcium concentration ([Ca2+]i) constitute the main link in excitation-contraction coupling (E-C coupling) in airway smooth muscle cells (ASMC). It has recently been reported that ACh induces asynchronous recurring Ca2+ waves in intact ASMC of murine bronchioles. With the use of a novel technique allowing us to simultaneously measure subcellular [Ca2+]i and force generation in ASMC located within an intact tracheal muscle bundle, we examined a similar pattern of Ca2+ signaling in the trachea. We found that application of ACh resulted in the generation of recurring intracellular Ca2+ waves progressing along the longitudinal axis of the ribbon-shaped intact ASMC. These Ca2+ waves were not synchronized between neighboring cells, and induction of wave-like [Ca2+]i oscillations was temporally associated with development of force by the tracheal muscle bundle. By comparing the concentration dependence of force generation and the parameters characterizing the [Ca2+]i oscillations, we found that the concentration-dependent increase in ACh-induced force development by the tracheal smooth muscle bundle is achieved by differential recruitment of intact ASMC to initiate Ca2+ waves and by enhancement in the frequency of [Ca2+]i oscillations and elevation of interspike [Ca2+]i once the cells are recruited. Our findings demonstrate that asynchronous recurring Ca2+ waves underlie E-C coupling in ACh-induced contraction of the intact tracheal smooth muscle bundle. Furthermore, in contrast to what was reported in enzymatically dissociated ASMC, Ca2+ influx through the L-type voltage-gated Ca2+ channel was not an obligatory requirement for the generation of [Ca2+]i oscillations and development of force in ACh-stimulated intact ASMC.     

Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition.

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [( Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.     

D-galactosamine induced hepatocyte apoptosis is inhibited in vivo and in cell culture by a calcium calmodulin antagonist, chlorpromazine, and a calcium channel blocker, verapamil.

Studies were conducted in C57BL/6N Crj male mice and in cultured hepatocytes to clarify the relationship between galactosamine (GaIN) induced apoptosis and [Ca2+]i kinetics. Chlorpromazine (CPZ), a Ca(2+)-calmodulin antagonist, and verapamil (VR), a Ca(2+)-channel blocker each inhibited GaIN-induced DNA fragmentation and the appearance of apoptotic bodies. The kinetics of calcium uptake were evaluated using a calcium analyzer with the acetoxymethyl ester of fura-PE3 (fura-PE3/AM, 2.5 microM) as the calcium reporter. An increase in [Ca2+]i was detected in the cultured hepatocytes within 3 hours after treatment with 20 mM GaIN; this increase was inhibited by pretreatment with either 20 microM CPZ or 30 microM VR. Ca2+ imaging by confocal laser scanning microscopy showed that increase in [Ca2+]i after treatment with GaIN was initially localized around nuclei, while [Ca2+]i signals were later diffuse and observed throughout the cytoplasm. The activities of lactate dehydrogenase (LDH) and serum glutamate-pyruvate transaminase (sGPT), used as indicators of plasma membrane damage and leakage, however, were not reduced by pretreatment with CPZ or VR. From these findings, we infer that the DNA fragmentation in GaIN-induced hepatocyte apoptosis is associated with an elevation in the perinuclear concentration of Ca2+, but GaIN-induced necrotic cell death is triggered through pathway(s) that are insensitive to blockage of Ca2+ influx and therefore appear to occur independently of elevation in [Ca2+]i. These results help to clarify the role of calcium flux in hepatocyte apoptosis and necrosis induced by exposure to hepatotoxins in vivo and in vitro.     

Mobilization of intracellular calcium by alpha 1-adrenergic receptor activation in muscle cell monolayers

The fluorescent chelating agent quin 2 has been employed to monitor alterations of intracellular free Ca2+ concentrations ([Ca2+]i) in response to alpha 1-adrenergic receptor activation in adherent BC3H-1 cells. To correlate the kinetics of [Ca2+]i changes with transmembrane fluxes of this ion, continuous monitoring of [Ca2+]i has been undertaken on a monolayer of cells. Previous measurements of the transmembrane efflux of Ca2+ show a distinct lag in the response over a range of phenylephrine concentrations. By contrast, the elevation of [Ca2+]i is rapid (t1/2 approximately 2 s) and maintained for 30 s before it begins to decline to basal concentrations. The differences in kinetics indicate that the temporal delay in cellular Ca2+ efflux results from either activation of the transport system for Ca2+ extrusion or translocation of free Ca2+ to the transport site. The decline of [Ca2+]i with continued agonist exposure parallels both the efflux kinetics from the cell and the decline of total cellular Ca2+. At a time when free [Ca2+]i approaches the resting concentration, total cellular Ca2+ is reduced to a steady state value of 60% of that seen prior to stimulation. The Kact for phenylephrine-stimulated elevation in [Ca2+]i on the monolayer is 0.51 microM, which is similar to the Kact of 0.90 microM observed for phenylephrine-activated 45Ca2+ efflux. Addition of phentolamine subsequent to phenylephrine addition immediately reverses the agonist-stimulated Ca2+ mobilization, initiating a rapid return of [Ca2+]i to resting levels. A comparison of the kinetics of Ca2+ mobilization with its transmembrane flux suggests that the agonist augments the rate of recycling of intracellular Ca2+ between the free and bound states rather than causing release as a single bolus from the bound stores.     

Thimerosal enhances agonist-specific differences between [Ca2+]i oscillations induced by phenylephrine and ATP in single rat hepatocytes.

Single rat hepatocytes, microinjected with the Ca(2+)-sensitive photoprotein aequorin, respond to agonists acting through the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]i). The duration of [Ca2+]i transients generated is characteristic of the stimulating agonist; the differences lie in the rate of fall of [Ca2+]i from its peak. We considered that differential sensitivity of the InsP3 receptor may underlie agonist specificity. The thiol reagent, thimerosal, is known to increase the sensitivity of the Ca2+ stores to InsP3 by increasing the affinity of the InsP3 receptor for InsP3 in rat hepatocytes. We show here that a low dose of thimerosal (1 microM), insufficient alone to elevate [Ca2+]i, potentiates [Ca2+]i oscillations induced by phenylephrine or ATP in single, aequorin-injected, rat hepatocytes. Moreover, thimerosal enhances both the frequency and amplitude of phenylephrine-induced oscillations, whereas, in contrast, ATP-induced oscillations undergo an increase in the duration of the falling phase of individual [Ca2+]i transients. Thimerosal, therefore, enhances, rather than eliminates, agonist-specific differences in the hepatocyte [Ca2+]i oscillator.     

Time course of the initial [Ca2+]i response to extracellular ATP in smooth muscle depends on [Ca2+]e and ATP concentration.

In response to extracellular application of 50 microM ATP, all individual porcine aortic smooth muscle cells respond with rapid rises from basal [Ca2+]i to peak [Ca2+]i within 5 s. The time from stimulus to the peak of the [Ca2+]i response increases with decreasing concentration of ATP. At ATP concentrations of 0.5 microM and below, the time to the [Ca2+]i peak varies more significantly from cell to cell than at higher concentrations, and each cell shows complicated initiation and decay kinetics. For any individual cell, the lag phase before a response decreases with increasing concentration of ATP. An increase in lag time with decreasing ATP concentration is also observed in the absence of extracellular Ca2+, but the lag phase is more pronounced, especially at concentrations of ATP below 0.5 microM. Whole-cell patch-clamp electrophysiology shows that in porcine aortic smooth muscle cells, ATP stimulates an inward current carried mainly by Cl- ion efflux with a time course similar to the [Ca2+]i changes and no detectable current from an ATP-gated cation channel. A simple signal cascade initiation kinetics model, starting with nucleotide receptor activation leading to IP3-mediated Ca2+ release from IP3-sensitive internal stores, fits the data and suggests that the kinetics of the Ca2+ response are dominated by upstream signal cascade components.     

G protein-dependent Ca2+ signaling complexes in polarized cells

Polarized cells signal in a polarized manner. This is exemplified in the patterns of [Ca2+]i waves and [Ca2+]i oscillations evoked by stimulation of G protein-coupled receptors in these cells. Organization of Ca(2+)-signaling complexes in cellular microdomains, with the aid of scaffolding proteins, is likely to have a major role in shaping G protein-coupled [Ca2+]i signal pathways. In epithelial cells, these domains coincide with sites of [Ca2+]i-wave initiation and local [Ca2+]i oscillations. Cellular microdomains enriched with Ca(2+)-signaling proteins have been found in several cell types. Microdomains organize communication between Ca(2+)-signaling proteins in the plasma membrane and internal Ca2+ stores in the endoplasmic reticulum through the interaction between the IP3 receptors in the endoplasmic reticulum and Ca(2+)-influx channels in the plasma membrane. Ca2+ signaling appears to be controlled within the receptor complex by the regulators of G protein-signaling (RGS) proteins. Three domains in RGS4 and related RGS proteins contribute important regulatory features. The RGS domain accelerates GTP hydrolysis on the G alpha subunit to uncouple receptor stimulation from IP3 production; the C-terminus may mediate interaction with accessory proteins in the complex; and the N-terminus acts in a receptor-selective manner to confer regulatory specificity. Hence, RGS proteins have both catalytic and scaffolding function in Ca2+ signaling. Organization of Ca(2+)-signaling proteins into complexes within microdomains is likely to play a prominent role in the localized control of [Ca2+]i and in [Ca2+]i oscillations.     

Imaging [Ca2+]i dynamics during signal transduction

The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca2+]i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca2+]i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca2+]i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca2+]i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca2+]i as well as the interactions between the various subcellular components which impart the delicate control of [Ca2+]i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca2+]i changes found in this biological system will be given.     

Effect of dimethyl sulfoxide on cytosolic ionized calcium concentration and cytoskeletal organization of hepatocytes in a primary culture

The addition of dimethyl sulfoxide (DMSO) to a chemically defined, serum free medium prolonged hepatocytes survival in primary culture. DMSO exposure had a remarkable effect on morphological change and F-actin filaments distribution of hepatocytes. When hepatocytes were cultured in a medium containing 2% DMSO, the cells showed a compact and cubical shape and intracellular F-actin filaments were mainly observed in a ring-like fashion around the intercellular space. After exposure to DMSO, fibronectin fibers in the interspace between cell and substratum were not apparent. Exposing the hepatocytes to DMSO also caused a sharp increase in cytosolic free ionized calcium ([Ca2+]). The initial increase in [Ca2+]i following the addition of DMSO was not attenuated by the chelation of extracellular Ca2+ with EGTA. The Ca2+ signal in the absence of extracellular Ca2+ was transient and returned to the basal levels within 1-2 min, while it was maintained at a high steady state in the presence of extracellular Ca2+. These results suggest that DMSO may be able to increase [Ca2+]i by two mechanisms, by the release of the ion from intracellular pools and, by the stimulation of influx across the plasma membrane. The increase in [Ca2+]i induced by DMSO treatment may play a role in prolonging hepatocyte survival in culture, since [Ca2+]i is one of the most important dynamic second messengers in various cellular metabolic processes.     

Mechanisms of subcellular cytosolic Ca2+ signaling evoked by stimulation of the vasopressin V1a receptor.

Receptor activation may result in distinct subcellular patterns of Ca2+ release. To define the subcellular distribution of Ca2+i signals induced by stimulation of the vasopressin V1a receptor, we expressed the cloned receptor in Xenopus oocytes. Oocytes were then loaded with fluo-3 and observed using confocal microscopy. Vasopressin induced a single concentric wave of increased Ca2+ that radiated inward from the plasma membrane. With submaximal stimulation, however, regions of the Ca2+ wave spontaneously reorganized into repetitive (oscillatory) waves. Focal stimulation of a small part of the plasma membrane resulted in a Ca2+ wave which began at the point of stimulation, radiated toward the center of the cell, then reorganized into multiple foci of repetitive, colliding waves and spirals of increased Ca2+i. The pattern of Ca2+ signaling induced by focal or global stimulation was not altered in Ca(2+)-free medium, although signals did not propagate as fast. Finally, subcellular Ca2+ signaling patterns induced by vasopressin were inhibited by caffeine, while neither vasopressin nor microinjection of inositol trisphosphate blocked caffeine-induced increases in cytosolic Ca2+. Thus, stimulation of the V1a receptor in this cell system induces a complex pattern of Ca2+ signaling which is influenced by (1) the magnitude of the stimulus, (2) the distribution of the surface receptors that are stimulated, and (3) mobilization of Ca2+ from the extracellular space as well as from two distinct endogenous Ca2+ pools. The manner in which a single type of receptor is activated may represent an important potential mechanism for subcellular Ca2+i signaling.     

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.     

Dynamic regulation of intracellular calcium signals through calcium release channels

After the seminal work of Ebashi and coworkers which established the essential role of the intracellular Ca2+ concentration ([Ca2+]i) in the regulation of skeletal muscle contraction, we have witnessed an explosive elongation of the list of cell functions that are controlled by the [Ca2+]i. In numerous instances, release of intracellular Ca2+ stores plays important roles in Ca2+ signalling which displays significant variation in spatio-temporal pattern. There are two families of Ca2+ release channels, ryanodine receptors and inositol 1,4,5-trisphosphate (IP3) receptors. These Ca2+ release channels are structurally and functionally similar. In particular, the activity of both types of channels is regulated by the [Ca2+]i. The [Ca2+]i dependence of the Ca2+ release channel activity provides both types of channels with properties of a Ca2+ signal amplifier. This function of the ryanodine receptor is important in striated muscle excitation-contraction coupling, whereas that of the IP3 receptor seems to be the basis of the generation of Ca2+ waves. Thus the wide variety of Ca2+ signalling patterns seem to be critically dependent on the [Ca2+]i dependence of the Ca2+ release channels.