Regulation of Ca2+ release from internal stores in cardiac and skeletal muscles

It is widely accepted that Ca2+ is released from the sarcoplasmic reticulum by a specialized type of calcium channel, i.e., ryanodine receptor, by the process of Ca2+-induced Ca2+ release. This process is triggered mainly by dihydropyridine receptors, i.e., L-type (long lasting) calcium channels, directly or indirectly interacting with ryanodine receptor. In addition, multiple endogenous and exogenous compounds were found to modulate the activity of both types of calcium channels, ryanodine and dihydropyridine receptors. These compounds, by changing the Ca2+ transport activity of these channels, are able to influence intracellular Ca2+ homeostasis. As a result not only the overall Ca2+ concentration becomes affected but also spatial distribution of this ion in the cell. In cardiac and skeletal muscles the release of Ca2+ from internal stores is triggered by the same transport proteins, although by their specific isoforms. Concomitantly, heart and skeletal muscle specific regulatory mechanisms are different.     

Sphingosine releases Ca2+ from intracellular stores via the ryanodine receptor in sea urchin egg homogenates

Various reports have demonstrated that the sphingolipids sphingosine and sphingosine-1-phosphate are able to induce Ca2+ release from intracellular stores in a similar way to second messengers. Here, we have used the sea urchin egg homogenate, a model system for the study of intracellular Ca2+ release mechanisms, to investigate the effect of these sphingolipids. While ceramide and sphingosine-1-phosphate did not display the ability to release Ca2+, sphingosine stimulated transient Ca2+ release from thapsigargin-sensitive intracellular stores. This release was inhibited by ryanodine receptor blockers (high concentrations of ryanodine, Mg2+, and procaine) but not by pre-treatment of homogenates with cADPR, 8-bromo-cADPR or blockers of other intracellular Ca2+ channels. However, sphingosine rendered the ryanodine receptor refractory to cADPR. We propose that, in the sea urchin egg, sphingosine is able to activate the ryanodine receptor via a mechanism distinct from that used by cADPR.     

Metabotropic glutamate receptor activation and intracellular cyclic ADP-ribose release Ca2+ from the same store in cultured DRG neurones

The whole cell patch clamp technique has been used to record Ca(2+)-activated cation and chloride conductances evoked by release of Ca2+ from intracellular stores of cultured neonatal dorsal root ganglion neurones. The aim of this study was to investigate metabotropic glutamate receptor (mGluR) mechanisms and evaluate a possible role for cyclic ADP-ribose as an intracellular signalling molecule. Glutamate and the metabotropic glutamate receptor agonist (1S, 3R)-ACPD-evoked transient depolarizations, Ca(2+)-activated inward currents and rises in intracellular Ca2+. The (1S, 3R)-ACPD-activated currents were insensitive to InsP3 signalling inhibitors, heparin and pentosan polysulphate. Intracellular application of ryanodine alone activated currents in this study and proved a difficult tool to use as a potential inhibitor of cyclic ADP-ribose-mediated responses. However, intracellular dantrolene did attenuate both (1S, 3R)-ACPD and cyclic ADP-ribose responses. Intracellular photo-release of cGMP and cyclic ADP-ribose mimicked the responses to mGluR receptor activation. Intracellular application of nicotinamide and W7 inhibited the responses to photo-released cGMP but did not prevent responses to mGluR activation. The cyclic ADP-ribose receptor antagonist 8-amino cyclic ADP-ribose attenuated responses to (1S, 3R)-ACPD, cGMP and cyclic ADP-ribose, but some Ca(2+)-activated inward currents were still observed in the presence of this antagonist. In conclusion, mGluR receptor activation, cGMP and cyclic ADP-ribose release Ca2+ from intracellular stores. Some evidence suggests that pharmacologically related pathways are involved.     

Effect of ryanodine receptor mutations on interleukin-6 release and intracellular calcium homeostasis in human myotubes from malignant hyperthermia-susceptible individuals and patients affected by central core disease

In this study we report for the first time the functional properties of human myotubes isolated from patients harboring the native RYR1 I4898T and R4893W mutations linked to central core disease. We examined two aspects of myotube physiology, namely excitation-contraction and excitation-secretion coupling. Our results show that upon activation of the ryanodine receptor (RYR), myotubes release interleukin-6 (IL-6); this was dependent on de novo protein synthesis and could be blocked by dantrolene and cyclosporine. Myotubes from the two patients affected by central core disease showed a 4-fold increase in the release of the inflammatory cytokine IL-6, compared with cells derived from control or malignant hyperthermia susceptible individuals. All tested myotubes released calcium from intracellular stores upon stimulation via surface membrane depolarization or direct RYR activation by 4-chloro-m-cresol. The functional impact on calcium release of RYR1 mutations linked to central core disease or malignant hyperthermia is different: human myotubes carrying the malignant hyperthermia-linked RYR1 mutation V2168M had a shift in their sensitivity to the RYR agonist 4-chloro-m-cresol to lower concentrations, whereas human myotubes harboring C-terminal mutations linked to central core disease exhibited reduced [Ca2+]i increase in response to 4-chloro-m-cresol, caffeine, and KCl. Taken together, these results suggest that abnormal release of calcium via mutated RYR enhances the production of the inflammatory cytokine IL-6, which may in turn affect signaling pathways responsible for the trophic status of muscle fibers.     

A recessive ryanodine receptor 1 mutation in a CCD patient increases channel activity

Ryanodine receptors plays a crucial role in skeletal muscle excitation–contraction coupling by releasing calcium ions required for muscle contraction from the sarcoplasmic reticulum. At least three phenotypes associated with more than 100 RYR1 mutations have been identified; in order to elucidate possible pathophysiological mechanisms of RYR1 mutations linked to neuromuscular disorders, it is essential to define the mutation class by studying the functional properties of channels harbouring clinically relevant amino acid substitutions. In the present report we investigated the functional effects of the c.7304G > T RYR1 substitution (p.Arg2435Leu) found in a patient affected by central core disease. Both parents were heterozygous for the substitution while the proband was homozygous. We characterized Ca²⁺ homeostasis in myoD transduced myotubes from controls, the heterozygous parents and the homozygous proband expressing the endogenous mutation. We also expressed the recombinant mutant channels in heterologous cells and characterized their [³H]ryanodine binding and single channel properties. Our results show that the p.Arg2435Leu substitution affects neither the resting [Ca²⁺], nor the sensitivity of the ryanodine receptor to pharmacological activators, but rather reduces the release of Ca²⁺ from intracellular stores induced by pharmacological activators as well as by KCl via the voltage sensing dihydropyridine receptor.     

Ca2+ entry via AMPA-type glutamate receptors triggers Ca2+-induced Ca2+ release from ryanodine receptors in rat spiral ganglion neurons

Ryanodine receptor (RyR)-gated Ca2+ stores have recently been identified in cochlear spiral ganglion neurons (SGN) and likely contribute to Ca2+ signalling associated with auditory neurotransmission. Here, we identify an ionotropic glutamate receptor signal transduction pathway which invokes RyR-gated Ca2+ stores in SGN via Ca2+-induced Ca2+ release (CICR). Ca2+ levels were recorded in SGN in situ within rat cochlear slices (postnatal day 0-17) using the Ca2+ indicator fluo-4. RyR-gated Ca2+ stores were confirmed by caffeine-induced increases in intracellular Ca2+ which were blocked by ryanodine (100 microM) and were independent of external Ca2+. Glutamate evoked comparable increases in intracellular Ca2+, but required the presence of external Ca2+. Ca2+ influx via the glutamate receptor was found to elicit CICR via RyR-gated Ca2+ stores, as shown by the inhibition of the response by prior depletion of the Ca2+ stores with caffeine, the SERCA inhibitor thapsigargin, or ryanodine. The glutamate analogue AMPA (alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid) elicited Ca2+ responses that could be inhibited by caffeine. Glutamate- and AMPA-mediated Ca2+ responses were eliminated with the AMPA/Kainate receptor antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione). These data demonstrate functional coupling between somatic AMPA-type glutamate receptors and intracellular Ca(2+) stores via RyR-dependent CICR in primary auditory neurons.     

Ryanodine receptors in liver

The ryanodine receptor has been mainly regarded as the Ca2+ release channel from sarcoplasmic reticulum controlling skeletal and cardiac muscle contraction. However, many studies have shown that it is widely expressed, with functions not restricted to muscular contraction. This study examined whether ryanodine receptor plays a role in calcium signaling in the liver. RT-PCR analysis of isolated hepatocytes showed expression of a truncated type 1 ryanodine receptor, but no type 2 or type 3 message was detected. We also detected binding sites for [3H]ryanodine in the microsomal cellular fraction and in permeabilized hepatocytes. This binding was displaced by caffeine and dantrolene, but not by ruthenium red, heparin or cyclic ADP-Ribose. Ryanodine, by itself, did not trigger Ca2+ oscillations in either primary cultured hepatocytes or hepatocytes within the intact perfused rat liver. In both preparations, however, ryanodine significantly increased the frequency of the cytosolic free [Ca2+] oscillations evoked by an alpha1 adrenergic receptor agonist. Experiments in permeabilized hepatocytes showed that both ryanodine and cyclic ADP-ribose evoked a slow Ca2+ leak from intracellular stores and were able to increase the Ca2+-released response to a subthreshold dose of inositol 1,4,5-trisphosphate. Our findings suggest the presence of a novel truncated form of the type 1 ryanodine receptor in rat hepatocytes. Ryanodine modulates the pattern of cytosolic free [Ca2+] oscillations by increasing oscillation frequency. We propose that the Ca2+ released from ryanodine receptors on the endoplasmic reticulum provides an increased pool of Ca2+ for positive feedback on inositol 1,4,5-trisphosphate receptors.     

Ion channels in smooth muscle: regulators of intracellular calcium and contractility

Smooth muscle (SM) is essential to all aspects of human physiology and, therefore, key to the maintenance of life. Ion channels expressed within SM cells regulate the membrane potential, intracellular Ca2+ concentration, and contractility of SM. Excitatory ion channels function to depolarize the membrane potential. These include nonselective cation channels that allow Na+ and Ca2+ to permeate into SM cells. The nonselective cation channel family includes tonically active channels (Icat), as well as channels activated by agonists, pressure-stretch, and intracellular Ca2+ store depletion. Cl--selective channels, activated by intracellular Ca2+ or stretch, also mediate SM depolarization. Plasma membrane depolarization in SM activates voltage-dependent Ca2+ channels that demonstrate a high Ca2+ selectivity and provide influx of contractile Ca2+. Ca2+ is also released from SM intracellular Ca2+ stores of the sarcoplasmic reticulum (SR) through ryanodine and inositol trisphosphate receptor Ca2+ channels. This is part of a negative feedback mechanism limiting contraction that occurs by the Ca2+-dependent activation of large-conductance K+ channels, which hyper polarize the plasma membrane. Unlike the well-defined contractile role of SR-released Ca2+ in skeletal and cardiac muscle, the literature suggests that in SM Ca2+ released from the SR functions to limit contractility. Depolarization-activated K+ chan nels, ATP-sensitive K+ channels, and inward rectifier K+ channels also hyperpolarize SM, favouring relaxation. The expression pattern, density, and biophysical properties of ion channels vary among SM types and are key determinants of electrical activity, contractility, and SM function.     

Mobilization of Ca2+ stores in individual pancreatic beta-cells permeabilized or not with digitonin or alpha-toxin

The concentration of free Ca2+ in the cytoplasm and organelles of individual mouse pancreatic beta-cells was estimated with dual wavelength microfluorometry and the indicators Fura-2 and furaptra. Measuring the increase of cytoplasmic Ca2+ resulting from intracellular mobilization of the ion in ob/ob mouse beta-cells, most organelle calcium (92%) was found in acidic compartments released when combining the Ca2+ ionophore Br-A23187 with a protonophore. Only 3-4% of organelle calcium was recovered from a pool sensitive to the Ca(2+)-ATPase inhibitor thapsigargin. Organelle Ca2+ was also measured directly in furaptra-loaded beta-cells after controlled plasma membrane permeabilization. The permeabilizing agent alpha-toxin was superior to digitonin in preserving the integrity of intracellular membranes, but digitonin provided more reproducible access to intracellular sites. After permeabilization, the thapsigargin-sensitive fraction of Ca2+ detected by furaptra was as high as 90%, suggesting that the indicator essentially measures Ca2+ in endoplasmic reticulum (ER). Both alpha-toxin- and digitonin-permeabilized cells exhibited ATP-dependent uptake of Ca2+ into thapsigargin-sensitive stores with half-maximal and maximal filling at 6-11 microM and 1 mM ATP respectively. Most of the thapsigargin-sensitive Ca2+ was mobilized by inositol 1,4,5-trisphosphate (IP3), whereas caffeine, ryanodine, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate lacked effects both in beta-cells from ob/ob mice and normal NMRI mice. Mobilization of organelle Ca2+ by 4-chloro-3-methylphenol was attributed to interference with the integrity of the ER rather than to activation of ryanodine receptors. The observations emphasize the importance of IP3 for Ca2+ mobilization in pancreatic beta-cells, but question a role for ryanodine receptor agonists.     

Store depletion by caffeine/ryanodine activates capacitative Ca(2+) entry in nonexcitable A549 cells

Capacitative Ca(2+) entry is essential for refilling intracellular Ca(2+) stores and is thought to be regulated primarily by inositol 1, 4,5-trisphosphate (IP(3))-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca(2+) release in the absence of extracellular Ca(2+) similar to that induced by thapsigargin (Tg), and Ca(2+) entry occurs upon the readdition of extracellular Ca(2+). The channels thus activated are also permeable to Mn(2+). The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca(2+) concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca(2+) release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca(2+) release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP(3) receptor. These results suggest that in A549 cells, (i) capacitative Ca(2+) entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP(3), probably via Ca(2+)-induced Ca(2+) release.     

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor. The brain ryanodine receptor has been purified 6700-fold with no change in [3H]ryanodine binding affinity and shown to be a homotetramer composed of an approximately 500 kd protein subunit, which is identified by anti-peptide antibodies against the skeletal and cardiac muscle ryanodine receptors. Our results demonstrate that the brain ryanodine receptor functions as a caffeine-sensitive Ca2+ release channel and thus is the likely gating mechanism for intracellular caffeine-sensitive Ca2+ pools in neurons.     

Ca2+ release via ryanodine receptors and Ca2+ entry: major mechanisms in NAADP-mediated Ca2+ signaling in T-lymphocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing nucleotide essentially involved in T cell activation. Using combined microinjection and single cell calcium imaging, we demonstrate that co-injection of NAADP and the D-myo-inositol 1,4,5-trisphosphate antagonist heparin did not inhibit Ca2+ mobilization. In contrast, co-injection of the ryanodine receptor antagonist ruthenium red efficiently blocked NAADP induced Ca2+ signalling. This pharmacological approach was confirmed using T cell clones stably transfected with plasmids expressing antisense mRNA targeted specifically against ryanodine receptors. NAADP induced Ca2+ signaling was strongly reduced in these clones. In addition, inhibition of Ca2+ entry by SK&F 96365 resulted in a dramatically decreased Ca2+ signal upon NAADP injection. Gd3+, a known blocker of Ca2+ release activated Ca2+ entry, only partially inhibited NAADP mediated Ca2+ signaling. These data indicate that in T cells (i) ryanodine receptor are the major intracellular Ca2+ release channels involved in NAADP induced Ca2+ signals, and that (ii) such Ca2+ release events are largely amplified by Ca2+ entry.     

Role of Intracellular Calcium Stores on the Effect of Metabotropic Glutamate Receptors on Phosphorylation of Glial Fibrillary Acidic Protein in Hippocampal Slices from Immature Rats

Phosphorylation of glial fibrillary acidic protein (GFAP) in slices from immature rats is stimulated by glutamate via a group II metabotropic glutamate receptor (mGluR II) and by absence of external Ca2+ in reactions that are not additive (Wofchuk and Rodnight, Neurochem. Int. 24:517-523, 1994). These observations suggested that glutamate, via an mGluR, inhibits Ca(2+)-entry through L-type Ca2+ channels and down-regulates a Ca(2+)-dependent dephosphorylation event coupled to GFAP. Because ryanodine receptors are present on internal Ca2+ stores and are associated with L-type Ca(2+)-channels, we investigated the possibility that the glutamatergic modulation of GFAP phosphorylation involves internal Ca2+ stores regulated by ryanodine receptors and whether the Ca2+ originating from these stores acts in a similar manner to external Ca2+. The results showed that the ryanodine receptor-agonists, caffeine and ryanodine and thapsigargin, all of which in appropriate doses increase cytoplasmic Ca2+, reversed the stimulation of GFAP phosphorylation given by 1S,3R-ACPD, an mGluR II agonist.     

Rapidly exchanging Ca2+ stores of non-muscle cells

The rapid and transient redistribution of calcium from intracellular stores is a key event of cell activation. The nature and molecular composition of intracellular Ca2+ stores of non-muscle cells are the object of intense investigation. In this paper, we review: (a) the experimental evidence in favor of the existence of intracellular, membrane-bound compartments specialized for uptake, storage and release of calcium, (b) the main protein components of rapidly exchanging Ca2+ stores, i.e. Ca2+ pump, intralumenal Ca2+ binding proteins (calsequestrin, calreticulin, etc.) and Ca2+ channels sensitive to either inositol 1,4,5-trisphosphate or Ca2+, caffeine and ryanodine, and (c) the relationship between Ca2+ stores and the endoplasmic reticulum.     

Expression of Ca(2+)-induced Ca2+ release channel activity from cardiac ryanodine receptor cDNA in Chinese hamster ovary cells.

We constructed an expression plasmid (pMAMCRR51) that carried the entire protein-coding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the dexamethasone-inducible mouse mammary tumor virus promoter and Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt). Chinese hamster ovary (CHO) cells were transfected with pMAMCRR51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular Ca2+ transients were selected. Immunoprecipitation with a monoclonal antibody against the canine cardiac ryanodine receptor revealed that the cell clones thus selected exhibited Ca(2+)-dependent [3H]ryanodine binding activity, which was stimulated by 5 mM ATP or 1 M KCl. The apparent dissociation constant (Kd) for [3H]ryanodine was 6.6 nM in 1 M KCl, which was similar to the Kd obtained with cardiac microsomes. Immunoprecipitation also demonstrated that these cell clones expressed a protein indistinguishable in M(r) from the ryanodine receptor in canine cardiac microsomes. The ryanodine binding activity expressed in CHO cells increased significantly after dexamethasone induction. In saponin-skinned CHO cells transfected with pMAMCRR51, micromolar Ca2+ or millimolar caffeine evoked rapid Ca2+ release from the intracellular Ca2+ stores. In skinned control CHO cells, we did not observe such Ca2+ release activity. These results clearly demonstrate that the cardiac ryanodine receptor is stably expressed in internal membranes of CHO cells and functions as Ca(2+)-induced Ca2+ release channels.     

Ca(2+)-activated K+ currents underlying the afterhyperpolarization in guinea pig vagal neurons: a role for Ca(2+)-activated Ca2+ release.

We examined the possibility that Ca2+ released from intracellular stores could activate K+ currents underlying the afterhyperpolarization (AHP) in neurons. In neurons of the dorsal motor nucleus of the vagus, the current underlying the AHP had two components: a rapidly decaying component that was maximal following the action potential (GkCa,1) and a slower component that had a distinct rising phase (GkCa,2). Both components required influx of extracellular Ca2+ for their activation, and neither was blocked by extracellular TEA (10 mM). GkCa,1 was selectively blocked by apamin, whereas GkCa,2 was selectively reduced by noradrenaline. The time course of GkCa,2 was markedly temperature sensitive. GkCa,2 was selectively blocked by application of ryanodine or sodium dantrolene, or by loading cells with ruthenium red. These results suggest that influx of Ca2+ directly gates one class of K+ channels and leads to release of Ca2+ from intracellular stores, which activates a different class of K+ channel.     

Intracellular Ca2+ signals evoked by stimulation of nicotinic acetylcholine receptors in SH-SY5Y cells: contribution of voltage-operated Ca2+ channels and Ca2+ stores

Neuronal nicotinic acetylcholine receptors (nAChR) can regulate several neuronal processes through Ca2+-dependent mechanisms. The versatility of nAChR-mediated responses presumably reflects the spatial and temporal characteristics of local changes in intracellular Ca2+ arising from a variety of sources. The aim of this study was to analyse the components of nicotine-evoked Ca2+ signals in SH-SY5Y cells, by monitoring fluorescence changes in cells loaded with fluo-3 AM. Nicotine (30 microm) generated a rapid elevation in cytoplasmic Ca2+ that was partially and additively inhibited (40%) by alpha7 and alpha3beta2* nAChR subtype selective antagonists; alpha3beta4* nAChR probably account for the remaining response (60%). A substantial blockade (80%) by CdCl2 (100 microm) indicates that voltage-operated Ca2+ channels (VOCC) mediate most of the nicotine-evoked response, although the alpha7 selective antagonist alpha-bungarotoxin (40 nm) further decreased the CdCl2- resistant component. The elevation of intracellular Ca2+ levels provoked by nicotine was sustained for at least 10 min and required the persistent activation of nAChR throughout the response. Intracellular Ca2+ stores were implicated in both the initial and sustained nicotine-evoked Ca2+ responses, by the blockade observed after ryanodine (30 microm) and the inositoltriphosphate (IP3)-receptor antagonist, xestospongin-c (10 microm). Thus, nAChR subtypes are differentially coupled to specific sources of Ca2+: activation of nAChR induces a sustained elevation of intracellular Ca2+ levels which is highly dependent on the activation of VOCC, and also involves Ca2+ release from ryanodine and IP3-dependent intracellular stores. Moreover, the alpha7, but not alpha3beta2* nAChR, are responsible for a fraction of the VOCC-independent nicotine-evoked Ca2+ increase that appears to be functionally coupled to ryanodine sensitive Ca2+ stores.     

Simultaneous measurement of Ca2+ release and influx into smooth muscle cells in response to caffeine. A novel approach for calculating the fraction of current carried by calcium

Activation of ryanodine receptors on the sarcoplasmic reticulum of single smooth muscle cells from the stomach muscularis of Bufo marinus by caffeine is accompanied by a rise in cytoplasmic [Ca2+] ([Ca2+]i), and the opening of nonselective cationic plasma membrane channels. To understand how each of these pathways contributes to the rise in [Ca2+]i, one needs to separately monitor Ca2+ entry through them. Such information was obtained from simultaneous measurements of ionic currents and [Ca2+]i by the development of a novel and general method to assess the fraction of current induced by an agonist that is carried by Ca2+. Application of this method to the currents induced in these smooth muscle cells by caffeine revealed that approximately 20% of the current passing through the membrane channels activated following caffeine application is carried by Ca2+. Based on this information we found that while Ca2+ entry through these channels rises slowly, release of Ca2+ from stores, while starting at the same time, is much faster and briefer. Detailed quantitative analysis of the Ca2+ release from stores suggests that it most likely decays due to depletion of Ca2+ in those stores. When caffeine was applied twice to a cell with only a brief (30 s) interval in between, the amount of Ca2+ released from stores was markedly diminished following the second caffeine application whereas the current carried in part by Ca2+ entry across the plasma membrane was not significantly affected. These and other studies described in the preceding paper indicate that activation of the nonselective cation plasma membrane channels in response to caffeine was not caused as a consequence of emptying of internal Ca2+ stores. Rather, it is proposed that caffeine activates these membrane channels either by direct interaction or alternatively by a linkage between ryanodine receptors on the sarcoplasmic reticulum and the nonselective cation channels on the surface membrane.     

Influence of ryanodine and inositol trisphosphate receptors inhibitions on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP

The influence of ryanodine and inositol triphosphate receptors inhibitors on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP was investigated using fluorescent dye chlortetracycline. Porcine oocytes were isolated from ovaries with yellow body. Ca2+ exit from intracellular stores of porcine oocytes activated by prolactin (5 and 50 ng/ml) in calcium free medium was decreased after treatment of oocytes by heparin (inhibitor of inositol triphosphate receptors) and was not changed after treatment of oocytes by ruthenium red (inhibitor of ryanodine receptors). Inhibition of protein kinase C did not affect on the Ca2+ exit stimulated by prolactin. GTP did not stimulate Ca2+ exit from intracellular stores of pig oocytes, and inhibitors of both calcium channels and proteinkinase C had no influence on this process. The joint action of prolactin and GTP did not result in additional Ca2+ exit from intracellular stores of oocytes after both pretreatment and untreatment by the inhibitor of protein kinase C. The data obtained testify to activation of IP3-sensitive receptors under effect of prolactin and in the absence of GTP influence on these receptors.     

Cross-signaling between L-type Ca2+ channels and ryanodine receptors in rat ventricular myocytes

Calcium-mediated cross-signaling between the dihydropyridine (DHP) receptor, ryanodine receptor, and Na(+)-Ca2+ exchanger was examined in single rat ventricular myocytes where the diffusion distance of Ca2+ was limited to < 50 nm by dialysis with high concentrations of Ca2+ buffers. Dialysis of the cell with 2 mM Ca(2+)- indicator dye, Fura-2, or 2 mM Fura-2 plus 14 mM EGTA decreased the magnitude of ICa-triggered intracellular Ca2+ transients (Cai-transients) from 500 to 20-100 nM and completely abolished contraction, even though the amount of Ca2+ released from the sarcoplasmic reticulum remained constant (approximately 140 microM). Inactivation kinetics of ICa in highly Ca(2+)-buffered cells was retarded when Ca2+ stores of the sarcoplasmic reticulum (SR) were depleted by caffeine applied 500 ms before activation of ICa, while inactivation was accelerated if caffeine- induced release coincided with the activation of ICa. Quantitative analysis of these data indicate that the rate of inactivation of ICa was linearly related to SR Ca(2+)-release and reduced by > 67% when release was absent. Thapsigargin, abolishing SR release, suppressed the effect of caffeine on the inactivation kinetics of ICa. Caffeine- triggered Ca(2+)-release, in the absence of Ca2+ entry through the Ca2+ channel (using Ba2+ as a charge carrier), caused rapid inactivation of the slowly decaying Ba2+ current. Since Ba2+ does not release Ca2+ but binds to Fura-2, it was possible to calibrate the fluorescence signals in terms of equivalent cation charge. Using this procedure, the amplification factor of ICa-induced Ca2+ release was found to be 17.6 +/- 1.1 (n = 4). The Na(+)-Ca2+ exchange current, activated by caffeine- induced Ca2+ release, was measured consistently in myocytes dialyzed with 0.2 but not with 2 mM Fura-2. Our results quantify Ca2+ signaling in cardiomyocytes and suggest the existence of a Ca2+ microdomain which includes the DHP/ ryanodine receptors complex, but excludes the Na(+)- Ca2+ exchanger. This microdomain appears to be fairly inaccessible to high concentrations of Ca2+ buffers.