Local oscillations of frog skeletal muscle sarcomeres induced by subthreshold concentration of caffeine.

Different intracellular processes are selectively controlled by a signalling system based on transient rises or oscillations of cytoplasmic calcium concentration, which transmit extracellular signals at subcellular level. When treated with a subthreshold concentration of caffeine, skeletal muscle cells provide a suitable preparation to study mechanisms which generate repetitive calcium transients. Based on optical diffraction measurements of local contractions of individual sarcomeres, we have shown substantial enhancement of spontaneous repetitive calcium release in the presence of subthreshold caffeine concentration. Calcium release propagates to neighbor calcium sources and forms slow contraction waves. A power spectra density analysis has revealed parameters of the time course of these events. However, substantial amounts of calcium released in sarcomeres are not synchronized.     

A realistic model of biphasic calcium transients in electrically nonexcitable cells.

In many electrically nonexcitable cells, the release of calcium from internal stores is followed by a much slower phase in which the intracellular calcium concentration decreases gradually to a sustained value higher than the concentration before stimulation. This elevated calcium plateau has been shown to be the result of calcium influx. The model presented in this work describes a system consisting of a cytoplasmic calcium store and a plasma membrane calcium channel, both excitable by a membrane receptor; a fast cytoplasmic calcium buffer; and calcium pumps in both the calcium store and cellular membranes. Inherent difficulties in the numerical evaluation of the model, caused by very large calcium fluxes across the store membrane, were overcome by analytically separating the fast processes of calcium release from the slower processes of calcium cycling across the plasma membrane. This enabled the simulation of realistic biphasic calcium transients similar to those observed experimentally. The model predicted 1) a strong correlation between the rate of calcium cycling across the plasma membrane and the rate of calcium decay; and 2) a dependence on the level of cell excitation of the maximum rise in cytoplasmic calcium concentration, the level of the elevated calcium plateau, and the rate of calcium decay. Using the model, we simulated the washout of agonist from the bathing solution and the depletion of the calcium store by a pharmacological agent (such as thapsigargin) under several experimental conditions.     

Cytoplasmic free calcium concentration in porcine platelets: Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1·10^?7 M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thromin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.     

Cytoplasmic free calcium concentration in porcine platelets. Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1 X 10(-7) M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thrombin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.     

Rapidly exchanging Ca2+ stores: Role in the control of Ca2+ homeostasis and [Ca2+] in oscillations

The rapidly exchanging intracellular calcium stores play an important role in control of cytoplasmic calcium homeostasis and in generation of intracellular calcium signals. These stores are specific intracellular compartments which are able to accumulate and release calcium in response to appropriate stimuli. Two types of stores can be distinguished in nonmuscle cells based on substances discharging these stores: (1) Ca²⁺-sensitive and (2) inositol-1,4,5-trisphosphate-sensitive intracellular depots. These two depots can be either separate intracellular compartments or a single compartment that shares both releasing mechanisms. The state of the art of our understanding of the cytoplasmic calcium release is the focus of this review.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 9–15, January–February, 1994.     

Small molecular weight heat shock-related protein, HSP20, exhibits an anti-platelet activity by inhibiting receptor-mediated calcium influx

We have shown that Hsp20, one of small molecular weight heat shock protein, which is present at a high concentration both in vascular smooth muscle cells and in circulating blood in patient with vascular disease, strongly inhibits platelet aggregation in vitro and ex vivo. To clarify the mechanism, we investigated the effect of Hsp20 on free calcium concentration in human platelet cytoplasm using fura 2. Hsp20 inhibited thrombin-induced calcium influx without affecting calcium release from intracellular calcium stores. The degree of inhibition is well-correlated with that of suppression of thrombin-induced platelet aggregation by this substance. Hsp20 also inhibited the elevation of cytoplasmic free calcium level triggered by collagen, but not that by A-23187. In contrast, Hsp28, another type of small molecular weight Hsp, failed to affect the cytoplasmic free calcium level. These findings suggest that Hsp20 inhibits the receptor-mediated calcium influx of platelets without affecting calcium release from intracellular calcium stores, leading to its anti-platelet activity.     

Pharmacology of capacitative calcium entry

The versatility of Ca(2+) as a messenger in multiple signaling events requires that the concentration of calcium ions within the cytoplasm be highly regulated. In particular, the release of calcium from intracellular stores must often be linked to calcium influx across the cell membrane. Capacitative calcium entry, whereby the depletion of intracellular Ca(2+) stores induces the influx of extracellular calcium, is a crucial element of concerted calcium signaling. Investigations into the phenomenon are contributing to a new appreciation for the organized cytoplasmic framework that supports calcium signaling.     

Reversible block of the calcium release channel/ryanodine receptor by protamine, a heparin antidote

Channel activity of the calcium release channel from skeletal muscle, ryanodine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity was measured after incorporating channels into planar lipid bilayers. Optimally and suboptimally calcium-activated calcium release channels were inactivated by the application of protamine to the cytoplasmic side of the channel. Recovery of channel activity was not observed while protamine was present. The addition of protamine bound to agarose beads did not change channel activity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentration of the medium. The block of channel activity by protamine could be reversed either by removal by perfusion with buffer or by the addition of heparin to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C(2)C(12) mouse muscle cells prevented caffeine-induced intracellular calcium release. The results suggest that protamine acts on the ryanodine receptor in a similar but opposite manner from heparin and that protamine can be used as a potent, reversible inhibitor of ryanodine receptor activity.     

Ionophore-induced apoptosis: role of DNA fragmentation and calcium fluxes

Two ionophores specific for K+, valinomycin and beauvericin, induce a type of cell death very similar to apoptosis due to tumor necrosis factor (TNF alpha). Both ionophores cause cytolysis accompanied by internucleosomal DNA fragmentation of the dying cell into units of 200 base pairs. Morphologically, the cell death appears to consist of a mixture of nuclear apoptotic changes and cytoplasmic necrotic changes. As in the case for TNF alpha-mediated death, metabolic inhibitors have no effect on the course of cell death, but DNA fragmentation and cytolysis are decreased by the endonuclease inhibitor, zinc. Beauvericin and valinomycin trigger an increase in the cytoplasmic calcium concentration, most likely due to release of calcium from intracellular stores, and chelation of cytoplasmic calcium with quin-2 inhibits DNA fragmentation. Thus, these ionophores set off apoptosis through a calcium-activatable endonuclease, suggesting that other nonphysiological toxins might also cause apoptosis through their ability to indirectly elevate the cytoplasmic calcium concentration, without the need to invoke specific surface receptors.     

The versatility of mitochondrial calcium signals: from stimulation of cell metabolism to induction of cell death

Both the contribution of mitochondria to intracellular calcium (Ca(2+)) signalling and the role of mitochondrial Ca(2+) uptake in shaping the cytoplasmic response and controlling mitochondrial function are areas of intense investigation. These studies rely on the appropriate use of emerging techniques coupled with judicious data interpretation to a large extent. The development of targeted probes based on the molecular engineering of luminescent proteins has allowed the specific measurement of Ca(2+) concentration ([Ca(2+)]) and adenosine trisphosphate concentration ([ATP]) in intracellular organelles or cytoplasmic subdomains. This approach has given novel information on different aspects of mitochondrial homeostasis.     

Calcium, mitochondria and apoptosis studied by fluorescence measurements

Among the many unsolved problems of calcium signalling, the role of calcium elevations in apoptotic and necrotic cell death has been a focus of research in recent years. Evidence has been presented that calcium oscillations can effectively trigger apoptosis under certain conditions and that dysregulation of calcium signalling is a common cause of cell death. These effects are regularly mediated through calcium signal propagation to the mitochondria and the ensuing mitochondrial membrane permeabilization and release of pro-apoptotic factors from mitochondria to the cytoplasm. The progress in this area depended on the development of (1) fluorescent/luminescent probes, including fluorescent proteins that can be genetically targeted to different intracellular locations and (2) the digital imaging technology, fluorescence-activated cell sorting and fluorescent high throughput approaches, which allowed dynamic measurements of both [Ca2+] in the intracellular compartments of interest and the downstream processes. Fluorescence single cell imaging has been the only possible approach to resolve the cell-to-cell heterogeneity and the complex subcellular spatiotemporal organization of the cytoplasmic and mitochondrial calcium signals and downstream events. We outline here fluorometric and fluorescence imaging protocols that we set up for the study of calcium in the context of apoptosis.     

Lanthanum increases the rat thymocyte cytoplasmic free calcium concentration by enhancing calcium influx

In the present study, I have examined the effect of lanthanum (La3+) on cytoplasmic free calcium concentration in isolated rat thymocytes employing the quin2 technique. As with its effect on 15Ca accumulation in rat thymocytes (Segal, J. and Ingbar, S.H. (1984) Endocrinology, 115, 160-166), La3+ produced a concentration-related increase in thymocyte cytoplasmic free calcium concentration. This effect of La3+ was very prompt in onset, evident within about 30 s from the time of addition of La3+. The lowest effective concentration of La3+ was 6 microM (+22.7% above control), and it increased progressively to reach maximal values at 25 microM (+100% above control). La3+ added to quin2-loaded thymocytes suspended in a calcium-free medium was without effect. In addition, La3+ had no significant effect on 45Ca efflux, and La3+ did not inhibit calcium-ATPase activity in the rat thymocytes. These results demonstrate that in rat thymocytes La3+ increases cytoplasmic free calcium concentration by increasing the extracellular calcium influx into the cell rather than the release of calcium from an intracellular pool.     

Adenophostin A induces spatially restricted calcium signaling in Xenopus laevis oocytes.

The activation of intracellular calcium release and calcium entry across the plasmalemma in response to intracellular application of inositol 2,4,5-trisphosphate and adenophostin A, two metabolically stable agonists for inositol 1,4,5-trisphosphate receptors, was investigated using Xenopus laevis oocytes and confocal imaging. Intracellular injection of inositol 2,4,5-trisphosphate induced a rapidly spreading calcium signal associated with regenerative calcium waves; the calcium signal filled the peripheral regions of the cell in 1-5 min. Injection of high concentrations of adenophostin A (250 nM) similarly induced rapidly spreading calcium signals. Injection of low concentrations of adenophostin A resulted in calcium signals that spread slowly (>1 h). With extremely low concentrations of adenophostin A (approximately 10 pM), stable regions of Ca2+ release were observed that did not expand to peripheral regions. When the adenophostin A-induced calcium signal was restricted to central regions, compartmentalized calcium oscillations were sometimes observed. Restoration of extracellular calcium caused a rise in cytoplasmic calcium restricted to the region of adenophostin A-induced calcium mobilization. The limited diffusion of adenophostin A provides an opportunity to examine calcium signaling processes under spatially restricted conditions and provides insights into mechanisms of intracellular calcium oscillations and capacitative calcium entry.     

Biphasic stimulation of cellular calcium concentration by 3,5,3'-triiodothyronine in rat thymocytes

3,5,3'-Triiodothyronine (T3) produced a rapid and transient increase in 45Ca uptake and cytoplasmic free calcium concentration in rat thymocytes, which is the most rapid effect of T3 in this system. This effect was manifested in cells suspended in medium containing 1 mM calcium. The T3 effect on 45Ca uptake was evident at 15-30 s, reached maximum at 30-60 s, and returned to control values at 5 min. The T3 effect on cytoplasmic free calcium concentration was seen after 30 s, reached maximum at 7 min, and returned to control values after 24 min. In cells suspended in Ca2+-free medium, T3 produced a similar rapid increase in 45Ca uptake, which was sustained for at least 60 min, but T3 failed to change cytoplasmic free calcium concentration. Alprenolol (10 microM) blocked the stimulatory effects of T3 on these two functions in a similar fashion. From these results, I suggest that in rat thymocytes T3 influences cellular calcium economy through a biphasic mechanism in which T3 first increases calcium uptake which, in turn, is followed by a release of calcium from intracellular pool(s), resulting in a further increase in cytoplasmic free calcium concentration and the activation of Ca2+ -regulated systems. Moreover, the present study provides further support for the postulate that in the rat thymocyte calcium serves as the first messenger for the plasma membrane-mediated stimulatory effects of T3 on several metabolic functions.     

Calcium gradients and the Golgi

Changes in intracellular free calcium regulate many intracellular processes. With respect to the secretory pathway and the Golgi apparatus, changes in calcium concentration occurring either in the adjacent cytosol or within the lumen of the Golgi act to regulate Golgi function. Conversely, the Golgi sequesters calcium to shape cytosolic calcium signals as well as initiate them by releasing calcium via inositol-1,4,5-triphosphate (IP(3)) receptors, located on Golgi membranes. Local calcium transients juxtaposed to the Golgi (arising from release by the Golgi or other organelles) can activate calcium dependent signalling molecules located on or around the Golgi. This review focuses on the reciprocal relationship between the cell biology of the Golgi apparatus and intracellular calcium homeostasis.     

Involvement of two-pore channels in hydrogen peroxide-induced increase in the level of calcium ions in the cytoplasm of human umbilical vein endothelial cells

Hydrogen peroxide at concentrations below cytotoxic ones causes an increase in the cytoplasmic calcium concentration in human umbilical vein endothelial cells as a result of calcium release from intracellular stores. Two-pore calcium channel blocker trans-NED19 partially suppresses the increase in the level of calcium ions in the cells in response to the addition of hydrogen peroxide. The staining of endothelial cells with the fluorescent stereoisomer cis-NED19 and LysoTracker confirmed the localization of two-pore calcium channels in lysosomes and endolysosomal vesicles.     

Assessment of the role of Ca2+ mobilization from intracellular pool(s), using dantrolene, in the glycogenolytic action of alpha-adrenergic stimulation in perfused rat liver

To identify the role of Ca2+ mobilization from intracellular pool(s) in the action of alpha-adrenergic agonist, the effects of dantrolene on phenylephrine-induced glycogenolysis were investigated in perfused rat liver. Dantrolene (5 X 10(-5) M) inhibited both glycogenolysis and 45Ca efflux induced by 5 X 10(-7) M phenylephrine. The inhibition by dantrolene was observed in the presence and absence of perfusate calcium. In contrast, dantrolene did not inhibit glycogenolysis induced by glucagon. To confirm the specificity of dantrolene action on calcium release in liver, experiments were also carried out using isolated hepatocytes. Dantrolene did not affect phenylephrine-induced production of inositol 1,4,5-trisphosphate. The compound did inhibit a rise in cytoplasmic Ca2+ concentration induced by phenylephrine both in the presence and absence of extracellular Ca2+. Thus, these results suggest that calcium release from an intracellular pool is essential for the initiation of alpha-adrenergic stimulation of glycogenolysis in the perfused rat liver.     

Synthetic diacylglycerols trigger an increase of intracellular free calcium in promyelocytic HL60 cells

Phosphoinositide turnover is known to play an important role in intracellular free calcium homeostasis through the inositol trisphophate-mediated release of calcium from intracellular stores. We find that the other product of phosphoinositide turnover, 1,2-diacylglycerol, elicits an increase in intracellular free calcium in HL60 cells which is due, at least in part, to release of calcium from intracellular stores. This effect is specific for calcium, since intracellular sodium and potassium levels and cellular volume were unaffected. Concomitant with the intracellular calcium increase, we find an increase in cellular inositol trisphosphate levels, suggesting that the effect of diacylglycerol on calcium may be mediated by inositol trisphosphate. Diacylglycerols also stimulate calcium efflux. This stimulation is not simply due to the increase in intracellular calcium. These effects appear not to be mediated through stimulation of a phorbol ester-activatable protein kinase C (Ca2+/phospholipid-dependent enzyme) since phorbol esters do not elicit an increase in cytoplasmic free calcium or an increase in calcium efflux.     

Characterization of the Ca2+ influx into embryonic cells after stimulation of the embryonic muscarinic receptor.

Embryonic cells transiently express an embryonic muscarinic system during morphogenesis. Stimulation of the embryonic muscarinic receptor results in biphasic intracellular Ca2+ mobilization: an initial "peak" due to Ca2+ release from intracellular stores is followed by a sustained "plateau" of enhanced cytoplasmic Ca2+ due to influx of extracellular Ca2+. In the present investigation, we characterized the Ca2+ influx by measuring the cytoplasmic free Ca2+ concentration [Ca2+]i using the Ca2+ indicator fura-2: 1. The increase of [Ca2+]i during the plateau depended linearly on the logarithm of the extracellular calcium concentration whereas the initial peak was almost independent from extracellular calcium. 2. The organic Ca2+ entry blockers verapamil, gallopamil, nifedipine, nitrendipine and the inorganic blockers Mn2+, Mg2+ and La3+ were without effect on both phases of Ca2+ mobilization. Only Ni2+ at concentrations above 1 mM was able to reduce the influx without affecting the intracellular Ca2+ release. 3. Substitution of extracellular Na+ by guanidine+, choline+ or tris+ and membrane depolarisation by increasing the extracellular K+ concentration had no effect on either phase of Ca2+ mobilization. We conclude that a non-voltage dependent, receptor-operated influx mechanism, probably a "second messenger operated Ca2+ channel", is responsible for the Ca2+ influx after stimulation of the embryonic muscarinic receptor.