Inositol triphosphate-induced Ca2+ release from human platelet membranes

Inositol (1,4,5) triphosphate (IP3) was observed to induce release of sequestered Ca2+ from crude human platelet membranes. This activity was also shown to be present in purified membranes enriched in Ca2+-ATPase activity. Maximal Ca2+ release occurred at 8 microM IP3 and half maximal activity was at 0.4 microM. Release was quite rapid and was complete by 40 s. Released Ca2+ was pumped back into the membrane vesicles and the rate of this reuptake was increased by the presence of phosphate. These results demonstrate that internal platelet membranes that possess an active Ca2+-pump will release sequestered Ca2+ in the presence of the second messenger IP3. IP3 did not induce release of Ca2+ from skeletal muscle sarcoplasmic reticulum when ATP was present.     

Heavy metal-induced Ca2+ release from sarcoplasmic reticulum

Two distinct forms of Ca2+ release from isolated sarcoplasmic reticulum vesicles in response to additions of heavy metals (silver and mercurials) are described. One form of heavy metal-induced Ca2+ release involves the ruthenium red-sensitive Ca2+ release channel localized in terminal cisternae. The other form of heavy metal-induced Ca2+ release appears to involve all portions of the sarcoplasmic reticulum and is insensitive to ruthenium red. This latter form of Ca2+ release occurs over a similar range of heavy metal concentrations as inhibition of the sarcoplasmic reticulum Ca2+ pump but does not appear to be a result solely of such pump inhibition. Both forms of Ca2+ release are inhibited by glutathione, an endogenous constituent of muscle fibers, and by dithiothreitol, agents which prevent sulfhydryl oxidation. To assess the role of any sulfhydryl oxidation in sarcoplasmic reticulum Ca2+ release physiologically, dithiothreitol and glutathione were introduced inside muscle fibers and effects on excitation-contraction coupling examined. The results strongly suggest that sulfhydryl oxidation plays no essential role in skeletal muscle excitation-contraction coupling.     

Ruthenium red and caffeine affect the Ca2+-ATPase of the sarcoplasmic reticulum

Effects of ruthenium red and caffeine (a Ca2+ release blocker and an inducer, respectively) on Ca2+ uptake by sarcoplasmic reticulum (SR) vesicles and formation of the phosphorylated intermediate (EP) of the Ca2+-ATPase were studied using fast-kinetic techniques. Ruthenium red increased the rate and the maximum level of EP formation, while caffeine decreased both. Similarly, ruthenium red accelerated rapid Ca2+ uptake, while caffeine inhibited it. These drugs affected EP formation also with detergent solubilized Ca2+-ATPase. The concentrations required for half maximal effects on these functions (0.2 microM ruthenium red, 1.0 mM caffeine) are about the same as those for altering Ca2+ release. These results indicate that these reagents affect both the Ca2+-pump as well as the Ca2+ release mechanism, suggesting that the Ca2+-pump and Ca2+ release have some mechanisms in common.     

Inositol polyphosphates regulate Ca2+ efflux in a cardiac membrane subtype distinct from junctional sarcoplasmic reticulum

The membrane location and mechanism of inositol 1,3,4,5-tetrakisphosphate (InsP4)-regulated Ca2+ uptake in cardiac membrane vesicles was investigated. In canine and rat membranes separated by sucrose density gradient centrifugation, InsP4-regulated Ca2+ uptake was slightly more enriched in low density than in higher density membranes. Membranes supporting InsP4-regulated Ca2+ uptake were correspondingly enriched in type 1 InsP3 receptors. Junctional sarcoplasmic reticulum (J-SR), enriched in sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and ryanodine receptors, separated predominantly with higher density membranes. In membranes supporting InsP4-regulated Ca2+ uptake, Ca2+ uptake was facilitated by a high Ca2+ affinity carrier that was insensitive to thapsigargin. Ca2+ uptake in J-SR was mediated by thapsigargin-sensitive SERCA2a. Net Ca accumulation was enhanced by oxalate in both SR subtypes. Although Ca2+-carrier-mediated Ca2+ uptake was ATP independent, ATP indirectly regulated net Ca2+ accumulation by modifying Ca2+ efflux via a Ca2+ channel with properties of type 1 InsP3 receptors. In the presence of < or = 0.1 mM ATP, InsP4 enhanced Ca2+ accumulation whereas InsP4 inhibited Ca2+ uptake at higher ATP concentrations. In the presence of 0.15 mM ATP, InsP4 stimulated Ca2+ efflux from vesicles preloaded with Ca. Several other InsP4 isomers and 1,3,4-InsP3 also stimulated Ca2+ efflux but with slightly less potency than 1,3,4,5-InsP4. Ruthenium red enhanced net Ca accumulation by the Ca2+ carrier and reduced the potency of ATP, InsP4, and InsP3 to stimulate Ca2+ efflux in vesicles. In summary, this investigation shows that a Ca2+ carrier facilitates Ca loading in a sarcoplasmic reticulum subtype distinct from J-SR. InsP4 and InsP3 are proposed to regulate Ca2+ efflux in low density SR by acting on an ATP-modulated Ca2+ channel with properties of type 1 InsP3 receptors.     

Rapid filtration studies of Ca2+-induced Ca2+ release from skeletal sarcoplasmic reticulum. Role of monovalent ions

We have developed a rapid filtration technique for the measurement of Ca2+ release from isolated sarcoplasmic reticulum vesicles. Using this technique, we have studied the Ca2+-induced Ca2+ release of sarcoplasmic reticulum vesicles from rabbit skeletal muscle passively loaded with 5 mM Ca2+. The effect of known effectors (adenine nucleotides and caffeine) and inhibitors (Mg2+ and ruthenium red) of this release were investigated. In a medium composed of 100 mM KCl buffered at pH 6.8 with 20 mM K/3-(N-morpholino)propanesulfonic acid the Ca2+ release rate was maximal (500 nmol of Ca2+ released.(mg of protein)-1.s-1) at 1 micron external Ca2+ and 5 mM ATP. We also observed a rapid Ca2+ release induced by micromolar Ag+ in the presence of ATP (at 1 nM Ca2+). The Ag+-induced Ca2+ release was totally inhibited by 5 micron ruthenium red. We have also investigated the effect of monovalent ions on the Ca2+ release elicited by Ca2+ or Ag+. We show that the Ca2+ release rate: 1) was dependent upon the presence of K+ or Na+ in the release medium and 2) was influenced by a K+ gradient created across the sarcoplasmic reticulum membrane. These results directly support the idea of the involvement of an influx of K+ (through K+ channels) during the Ca2+ release and allow to reconsider a possible influence of the membrane potential of the sarcoplasmic reticulum on the Ca2+ release.     

Ca2+ transport in human platelet membranes. Kinetics of active transport and passive release

Active Ca2+ transport and passive release were characterized in crude and purified human platelet membranes to facilitate comparison with skeletal muscle sarcoplasmic reticulum. Endoplasmic reticulum markers were enriched from 3- to 14-fold in the purified membranes, while surface membrane antigens were reduced 4-fold and mitochondrial contamination was completely eliminated. The pH optimum for active Ca2+ transport in platelet membranes was 7.6, and the optimum for Ca2+-ATPase activity ranged from 7.6 to 8.0. Upon addition of MgATP there was a burst in active Ca2+ transport activity. In the absence of phosphate, steady state was reached within 20 s; added phosphate promoted continued uptake for greater than 1 h. The maximum pump stoichiometry was 2.0 Ca2+/ATP. The Ca2+ ionophore A23187 caused rapid release of 90% of the sequestered Ca2+ in the presence of phosphate. The dependence of Ca2+ transport on MgATP was biphasic with apparent Km values of 0.6 mM and 9.5 microM. Kinetic measurements with varied external Ca2+ yielded a single Km of 0.1 microM. Mg2+ stimulated Ca2+ transport and Ca2+-ATPase activities. Results with crude and purified membranes were similar, and comparison with the Ca2+ pump from sarcoplasmic reticulum revealed nearly identical enzymatic properties. In contrast to the results of comparing active Ca2+ transport, the characteristics of Ca2+ release from platelet membranes were quite different from those of sarcoplasmic reticulum. External Ca2+ did not promote release of sequestered Ca2+ from platelet membranes in contrast to sarcoplasmic reticulum. In addition, spontaneous release of Ca2+ from platelet membranes did not occur after ATP depletion. Inositol trisphosphate induced rapid partial release of Ca2+ from platelet membranes but had no effect on sarcoplasmic reticulum under identical conditions. Thus active Ca2+ transport is quite similar in internal membranes of platelet and skeletal muscle, but the mechanism of Ca2+ release appears to be entirely different.     

Effect of halothane on Ca2+-induced Ca2+ release from sarcoplasmic reticulum vesicles isolated from rat skeletal muscle

Halothane induces the release of Ca2+ from a subpopulation of sarcoplasmic reticulum vesicles that are derived from the terminal cisternae of rat skeletal muscle. Halothane-induced Ca2+ release appears to be an enhancement of Ca2+-induced Ca2+ release. The low-density sarcoplasmic reticulum vesicles which are believed to be derived from nonjunctional sarcoplasmic reticulum lack the capability of both Ca2+-induced and halothane-induced Ca2+ release. Ca2+ release from terminal cisternae vesicles induced by halothane is inhibited by Ruthenium red and Mg2+, and require ATP (or an ATP analogue), KCl (or similar salt) and extravesicular Ca2+. Ca2+-induced Ca2+ release has similar characteristics.     

Differential functional properties of Ca2+ stores in pulmonary arterial conduit and resistance myocytes

In smooth muscle cells, oscillations of intracellular Ca2+ concentration ([Ca2+]i) are controlled by inositol 1,4,5-trisphosphate (InsP3) and ryanodine (Ry) receptors on the sarcoplasmic reticulum (SR). Here we show that these Ca2+ oscillations are regulated differentially by InsP3 and Ry receptors in cells dispersed from the main trunk of the pulmonary artery (conduit myocytes) or from tertiary and quaternary arterial branches (resistance myocytes). Ry receptor antagonists inhibit either spontaneous or ATP-induced Ca2+ oscillations in resistance myocytes but they do not affect the oscillations in most conduit myocytes. In contrast, agents that inhibit InsP3 production or activation of InsP3 receptors do not alter the oscillations is resistance myocytes but block them in conduit myocytes. We have also examined the degree of overlap of Ry- and InsP3-sensitive stores in myocytes along the pulmonary arterial tree. In conduit myocytes, depletion of Ry-sensitive stores with repeated application of caffeine in the presence of Ry or in Ca2+ free solutions did not prevent the ATP-induced Ca2+ release from InsP3-dependent stores. However, responsiveness to ATP was completely abolished in resistance myocytes subjected to the same experimental protocol. Thus, InsP3- and Ry-dependent stores appear to be separated in conduit myocytes but joined in resistance myocytes. These data demonstrate for the first time differential properties of intracellular Ca2+ stores and receptors in myocytes distributed along the pulmonary arterial tree and help to explain the distinct functional responses of large and small pulmonary vessels to vasoactive agents.     

Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca(2+)-induced Ca2+ release and Ca2+ oscillations in hamster eggs.

The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.     

Distinct occurrence of phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release and inositol 1,4,5-triphosphate-induced release in ATP-dependent Ca2+-transporting platelet microsomes

The effects of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and inositol 1,4,5-triphosphate(InsP3) on the Ca2+ release from ATP-dependent Ca2+-transporting microsomes prepared from ox platelets were investigated. Under optimal conditions, both PtdInsP2 and InsP3 released Ca2+ from the microsomes in a similar dose-dependent manner. However, the maximal amount of Ca2+ released by InsP3 was almost one-fourth of that released by PtdInsP2. Neither PtdInsP2 nor InsP3 appeared to act as a Ca2+ ionophore since they showed no effect on the Ca2+ content of liposomes prepared from platelet microsomal lipids. InsP3-induced but not PtdInsP2-induced Ca2+ release was decreased with increasing extravesicular Ca2+ from 0.1 microM to 10 microM and it was completely inhibited by 10 microM Ca2+. PtdInsP2-induced but not InsP3-induced Ca2+ release was markedly inhibited by Mg2+, ruthenium red and neomycin. In addition, InsP3 could induce no additional Ca2+ release after the accumulated Ca2+ had been maximally released by PtdInsP2. These results indicate that PtdInsP2 releases Ca2+ from platelet microsomes more effectively than InsP3 by a mechanism distinct from that of InsP3-induced release, and further that InsP3-sensitive microsomes are included within the population of PtdInsP2-sensitive microsomes.     

Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release.

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).     

Release of Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle induced by heparin. Relation between the Ca2+ release caused by Ca2+ ions and caffeine

Using a Ca2+-selective electrode and Quin 2 and chlortetracycline fluorescence, a Ca2+ release from terminal cysterns of skeletal muscle sarcoplasmic reticulum under effects of heparin, caffeine and Ca2+ has been studied. It was shown that Ca2+ release induced by heparin is insensitive to the blockers of Mg2+-dependent system of Ca2+-induced Ca2+ release, i.e., Mg2+, tetracaine and dimethylsulfoxide. Preliminary release of Ca2+ in the presence of caffeine, which activates Mg2+-dependent Ca2+ release, does not prevent the heparin-induced Ca2+ release. At the same time, after Ca2+ release caused by Ca2+ in a Mg2+-independent system, heparin cannot cause additional efflux of Ca2+. It has been shown that the heparin-induced release of Ca2+ diminishes with a decrease in a decrease in Ca2+ concentration. This effect is less pronounced in the presence of Na+ than with K+. The data obtained suggest that sarcoplasmic reticulum terminal cysterns contain two systems of Ca2+-induced release of Ca2+, i.e., a Mg2+-dependent, caffeine-sensitive and a Mg2+-independent heparin-sensitive ones. The mechanism of activation of both systems by caffeine and heparin consists, in all probability, in their increased affinity for Ca2+.     

Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling

In order to test the physiological significance of inositol 1,4,5-trisphosphate (InsP3) in pharmacomechanical coupling, we have utilized two near-physiological systems, in which relatively high molecular weight solutes can be applied intracellularly and receptor coupling is retained: beta-escin permeabilization and reversible permeabilization. We showed that in smooth muscle permeabilized with beta-escin, one of the saponin esters, alpha 1-adrenergic (phenylephrine) and muscarinic (carbachol) agonists, as well as caffeine and InsP3, cause contractions mediated by Ca2+ release. These contractions were calmodulin-dependent and blocked by depletion of Ca2+ stored in the sarcoplasmic reticulum. Intracellular heparin (Mr = about 5000), a blocker of InsP3 binding to its receptor and a specific inhibitor of InsP3-induced Ca2+ release in smooth muscles, inhibited the responses to the agonists and to InsP3, but not those to caffeine, nor did it block the enhanced contractile response to cytoplasmic Ca2+ induced by agonists and by GTP gamma S. Neomycin blocked Ca2+ release induced by carbachol, but not by caffeine. In reversibly permeabilized ileum smooth muscle cells, loaded with Fura-2 acid and heparin, the intracellular heparin inhibited Ca2+ release and contractions induced by carbachol in Ca2+-free, high K+ solution. Heparin did not inhibit the high K+ contractions (with 1.2 mM Ca2+) and had no significant inhibitory effects on carbachol-induced responses in the presence of extracellular Ca2+. These results, obtained under near-physiological conditions, support the conclusion that InsP3 is the major physiological messenger of the Ca2+ release component of pharmacomechanical coupling, but not of the components mediated by Ca2+ influx or by potentiation of the contractile response to Ca2+.     

Silver ions trigger Ca2+ release by interaction with the (Ca2+-Mg2+)-ATPase in reconstituted systems

It has been suggested that vesicles derived from the sarcoplasmic reticulum of skeletal muscle contain Ca2+ channels which can be opened by interaction with sulfhydryl reagents such as Ag+ or Hg2+. We show that, in reconstituted vesicles containing the (Ca2+-Mg2+)-ATPase purified from sarcoplasmic reticulum as the only protein, the ATPase can act as a pathway for Ca2+ efflux and that Ag+ induces a rapid release of Ca2+ from such reconstituted vesicles. We also show that Ag+ has a marked inhibitory effect on the ATPase activity of the purified ATPase. We suggest that the (Ca2+-Mg2+)-ATPase can act as a pathway for rapid Ca2+ release from sarcoplasmic reticulum.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: a comparison with skinned muscle fiber studies

Uptake and release of Ca2+ in heavy and light fractions of fragmented sarcoplasmic reticulum (FSR) isolated from frog and rabbit skeletal muscle was studied under conditions similar to those employed in skinned muscle fiber experiments, where ATP and Mg2+ concentrations were considered to be physiological and free Ca2+ concentration was kept constant during the Ca2+ uptake and release. Ca2+ level in FSR monotonously approached a steady state level which depended only on the final experimental conditions. Heavy fractions, but not light fractions, exhibited characteristics similar to those of Ca2+-induced Ca2+ release reported in skinned fiber studies: i) the rate and steady state level of Ca2+ uptake increased with increase in free Ca2+ concentration in the reaction medium up to 10(-6) M. With further increase in free Ca2+ concentration, the steady state level of Ca2+ taken up decreased while the Ca2+ uptake rate increased. ii) The steady state Ca2+ level was decreased by caffeine but increased by procaine or ruthenium red. Parallel measurement of Ca2+-ATPase activity clearly showed that these drugs modify the Ca2+ efflux but hardly affect the Ca2+-pump activity. It was concluded that the Ca2+-induced Ca2+ release mechanism was in operation at as low as 10(-6) M free Ca2+ concentration. Treatment of FSR with 0.6 M KCl did not have any significant effect.     

Structure, function and subcellular localization of a human platelet Ca2+-ATPase

Human platelets contain a Ca2+-ATPase in internal membranes that is essential for Ca2+ homeostasis. This Ca2+ pump has enzymatic properties quite similar to the sarcoplasmic reticulum (SR) Ca2+ pumps. Antibodies against the SR Ca2+ pump crossreact with the human platelet protein. However, the platelet Ca2+-ATPase is approximately 10 kD larger than the SR pumps and exhibits a larger mRNA coding for the protein in a megakaryocyte tumor cell line. In addition, the platelet Ca2+-pump may be localized in specialized internal membrane structures that function in Ca2+ uptake and release. These results suggest that the platelet Ca2+-ATPase may represent a new class of internal membrane Ca2+-pumps.     

Release of Ca2+ ions from the sarcoplasmic reticulum of skeletal muscles after treatment with caffeine

Using a Ca2+-selective electrode and Quin 2 and chlortetracycline fluorescence spectra, a comparative study of caffeine- and Ca2+-induced release of Ca2+ from the terminal cisterns of rabbit fast skeletal muscle sarcoplasmic reticulum was carried out. It was shown that the caffeine-induced release of Ca2+ depends on Ca2+ and Mg2+ concentration in the medium; Mg2+ inhibit, while Ca2+ stimulate this process. The caffeine-induced transport of Ca2+ is blocked by ruthenium red, tetracaine and dimethylsulfoxide. The Ca2+ release induced by Ca2+ was shown to occur in two ways, i. e., via Mg2+-dependent (inhibited by Mg2+ and caffeine blockers) and Mg2+-independent (insensitive to caffeine inhibitors, including Mg2+) routes. It was assumed that caffeine stimulates the Mg2+-dependent, Ca2+-induced release of Ca2+. The sensitivity of Ca2+ transport to caffeine testifies to the fact that about 80% of the total Ca2+ transport activity of fast skeletal muscle homogenates belongs to terminal cisterns. The total amount of sarcoplasmic reticulum membranes in the muscle makes up to 15-20 mg of protein/g of tissue.     

Effect of caffeine on kinetics of accumulation and release of Ca2+ by vesicles of the sarcoplasmic reticulum of skeletal muscle

The action of caffeine was studied on the heavy sarcoplasmic reticulum fraction enriched by vesicles derived from terminal cisterns. Caffeine lowers the ATP-dependent accumulation of Ca2+ by vesicles and enhances the first rapid phase of the Ci2+ release from vesicles. The action of caffeine was transient, reversed, Ca2+-dependent. The data obtained suggest that the reduction of ATP-dependent calcium accumulation and enhancement of calcium release by caffeine are mediated by the mechanism of Ca2+-induced Ca2+ release and support the view that caffeine may regulate the equilibrium between open and closed states of Ca2+-channel by increasing the affinity of Ca2+-receptor site of the channel.     

Ca2+ release in the endoplasmic reticulum of guinea pig peritoneal macrophages

Passive permeability of the endoplasmic reticulum of saponin-treated macrophages to Ca2+ was studied by the filtration method using 45Ca. The Ca2+ release from the endoplasmic reticulum of macrophages was enhanced by the presence of submicromolar concentrations of Ca2+ in the medium. The Ca2+ release was enhanced by caffeine, and suppressed by MgCl2. These phenomena are similar to the Ca2+-induced Ca2+ release reported for the sarcoplasmic reticulum of skeletal muscle. On the other hand, adenine suppressed the Ca2+ release from the endoplasmic reticulum, while it reportedly enhanced the Ca2+-induced Ca2+ release of the skeletal muscle. The threshold concentration of Ca2+ for the Ca2+-induced Ca2+ release was approximately 10(-8) M in the presence of 0.95 mM MgCl2 in macrophages. The spontaneous spreading of macrophages and spontaneous migration of macrophages were inhibited by adenine, and also by caffeine in spite of the enhancement of the Ca2+-induced Ca2+ release.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds

The caffeine-sensitive Ca2+ release pathway in skeletal muscle was identified and characterized by studying the release of 45Ca2+ from heavy sarcoplasmic reticulum (SR) vesicles and by incorporating the vesicles or the purified Ca2+ release channel protein complex into planar lipid bilayers. First-order rate constants for 45Ca2+ efflux of 1 s-1 were obtained in the presence of 1-10 microM free Ca2+ or 2 X 10(-9) M free Ca2+ plus 20 mM caffeine. Caffeine- and Ca2+-induced 45Ca2+ release were potentiated by ATP and Mg.ATP, and were both inhibited by Mg2+. Dimethylxanthines were similarly (3,9-dimethylxanthine) or more (1,7-, 1,3-, and 3,7-dimethylxanthine) effective than caffeine in increasing the 45Ca2+ efflux rate. 1,9-Dimethylxanthine and 1,3-dimethyluracil (which lacks the imidazole ring) did not appreciably stimulate 45Ca2+ efflux. Recordings of calcium ion currents through single channels showed that the Ca2+- and ATP-gated SR Ca2+ release channel is activated by addition of caffeine to the cis (cytoplasmic) and not the trans (lumenal) side of the channel in the bilayer. The single channel measurements further revealed that caffeine activated Ca2+ release by increasing the number and duration of open channel events without a change of unit conductance (107 pS in 50 mM Ca2+ trans). These results suggest that caffeine exerts its Ca2+ releasing effects in muscle by activating the high-conductance, ligand-gated Ca2+ release channel of sarcoplasmic reticulum.