Influence of magnesium on chloride-induced calcium release in skinned muscle fibers

Chloride-induced Ca release in skinned muscle fibers was studied by measuring isometric force transients and 45Ca loss from fiber to washout solutions. Skinned fibers prepared from muscles soaked in normal Ringer solution made large force transients in 120 mM Cl solution with 5 mM ATP and 1 mM Mg, but 3 mM Mg was inhibitory. Mg inhibition was antagonized by low temperature and by Cd, agents which slow active Ca uptake by the sarcoplasmic reticulum (SR). In low Mg++, Cl stimulated rapid 45Ca release from the SR in sufficient amounts to account for the force response. The increased 45Ca release was inhibited by EGTA, suggesting that release requires free Ca under these conditions. The 45Ca initially released was partially reaccumulated later. Reaccumulation was increased in higher Mg++. These results provide additional evidence that the Ca uptake rate is an important determinant of net release, and suggest that Mg++ acts primarily on this mechanism. Skinned fibers prepared from muscles soaked in low Cl solutions could give force responses to Cl solutions with 3 mM and 6 mM Mg. This observation suggests that the Cl stimulus varies with the [Cl] gradient across the internal membranes, and supports the hypothesis that applied Cl causes membrane depolarization.     

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.     

Regulation by magnesium of intracellular calcium movement in skinned muscle fibers

The effect of Mg on Ca movement between the sarcoplasmic reticulum (SR) and myofilament space (MFS) was studied in skinned muscle fibers by using isometric force as an indicator of MFS Ca. In Ca-loaded fibers at 20 degrees C, the large force spike induced by Ca in 1 mM Mg (5 mM ATP) was strongly inhibited in 3 mM Mg, and force development was extremely slow. After a brief Ca stimulus in 1 mM Mg, relaxation in Ca-free solution was significantly faster in 3 mM Mg. These changes were due to altered Ca movements, since the effect of 3 mM Mg on steady force in CaEGTA solutions was small. Changes in Mg alone induced force transients apparently due to altered Ca movement. In relaxed fibers, decreasing the Mg to 0.25 mM caused phasic force development. In contracting fibers in Ca solutions, increasing the Mg caused a large transient relaxation. The effects of increased Mg were antagonized by 0.5 mM Cd, an inhibitor of the SR Ca transport system. The results indicate that active Ca uptake by the SR in situ is stimulated by Mg, and that it can affect local MFS [Ca++] in the presence of a substantial Ca source. These results provide evidence that an increased rate of Ca uptake in 3 mM Mg could account for inhibition of the large force spike associated with Ca-induced Ca release in skinned fibers.     

Excitation of skinned muscle fibers by imposed ion gradients. II. Influence of quercetin and ATP removal on the Ca2+-insensitive component of stimulated 45Ca efflux

Ionic gradients imposed by choline Cl replacement of K methanesulfonate (Mes) at constant [K][Cl] product stimulate 45Ca efflux from skinned muscle fibers; a small, sustained Ca2+-insensitive efflux component, observed in EGTA, appears to grade a much larger Ca2+-dependent component responsible for contractile activation and is likely to reflect intermediate steps in excitation-contraction coupling. The present studies examined ATP-related effects on the Ca2+-insensitive stimulation. 45Ca efflux was measured on segments of frog semitendinosus muscle skinned by microdissection, with isometric force monitored continuously. The Ca2+-insensitive component was potentiated by quercetin, a flavonoid thought to inhibit the sarcoplasmic reticulum (SR) Ca pump by stabilizing a phosphorylated intermediate. Quercetin increased the stimulated net 45Ca release in the absence of EGTA, as expected from inhibition of reaccumulation, but its effectiveness in EGTA indicated potentiation of unidirectional efflux as such. Quercetin also increased unstimulated (control) 45Ca efflux in EGTA, to a smaller extent; potentiation appeared to be a function of efflux, with stimulation above control loss increased approximately 2.6-fold. ATP removal before stimulation, which led to rigor force and increased stiffness, prevented all quercetin effects in EGTA. ATP removal by itself inhibited ionic stimulation of the Ca2+-insensitive component, with little residual increase above the parallel control loss. Addition of the nonhydrolyzable ATP analogue AMP-PCP ([adenylyl-beta,gamma-methylene]diphosphate) (0.8 mM) after ATP removal gave similar results to ATP-free solution, which suggests that adenine nucleotide binding alone does not support stimulation by choline Cl. These results imply a fundamental role for ATP in the excitation of skinned fibers by imposed diffusion potentials; they also suggest that ATP regulates the SR Ca efflux channel, in a manner that could provide the positive feedback in Ca2+-dependent Ca release.     

Excitation of skinned muscle fibers by imposed ion gradients. IV. Effects of stretch and perchlorate ion

Depolarizing ion gradients stimulate 45Ca release in skeletal muscle fibers skinned by microdissection. Several lines of indirect evidence suggest that sealed transverse (T) tubules rather than sarcoplasmic reticulum (SR) are the locus of such stimulatory depolarization. Two implications of this hypothesis were tested. (a) A requirement for signal transmission was evaluated from the stimulation of 45Ca efflux in fibers that had been highly stretched, an intervention that can impair the electrical stimulation of intact fibers. Length was increased over approximately 95-115 s, after loading with 45Ca and rinsing at normal length; prestimulus 45Ca loss due to stretch itself was very small. In the first study, stimulation of 45Ca release by KCl replacement of K propionate was inhibited completely in fibers stretched to twice slack length, compared with fibers at 1.05-1.1 times slack length. Identical protocols did not alter 45Ca release stimulated by caffeine or Mg2+ reduction, implying that SR Ca release per se was fully functional and inhibition was selective for a preceding step in ionic stimulation. In a second study, stimulation by choline Cl replacement of K methanesulfonate, at constant [K+] [Cl-] product, was inhibited strongly; total 45Ca release decreased 69%, and stimulation above control loss decreased 78%, in segments stretched to twice the length at which sarcomere spacing had been 2.2 micron, compared with paired controls from the same fibers kept at 2.3 micron. (b) Perchlorate potentiation of T tubule activation was evaluated in fibers stimulated at constant [K+] [Cl-] at normal length (2.3 micron); this anion shifts the voltage dependence of intramembrane charge movement and contractile activation in intact fibers. Perchlorate (8 mM) potentiated both submaximal stimulation of Ca2+-dependent 45Ca release by partial choline Cl replacement of K methanesulfonate and the small Ca2+-insensitive 45Ca efflux component stimulated by nearly full replacement in the presence of 5 mM EGTA. These results provide independent support for the hypothesis that the T tubules are the locus of stimulation by depolarizing ion gradients, with junctional transmission of this signal causing SR 45Ca release.     

Excitation of skinned muscle fibers by imposed ion gradients. I. Stimulation of 45Ca efflux at constant [K][Cl] product

45Ca efflux from skinned muscle fibers is stimulated transiently, by a highly Ca2+-dependent mechanism, by KCl replacement of K propionate. In the present studies, Cl replaced the much less permeant anion methanesulfonate (Mes) either (a) at constant [K], in which increased [K][Cl] permits net KCl and water flux across internal membranes, or (b) at constant [K][Cl] (choline substitution), in which the imposed gradients and diffusion potentials should dissipate slowly. 45Ca efflux and isometric force were measured simultaneously on segments of frog semitendinosus fibers skinned by microdissection. EGTA was applied to chelate released 45Ca either (a) shortly after high [Cl] (interrupted response), to minimize reaccumulation, (b) before high [Cl] (pretreated response), to evaluate Ca2+ dependence, or (c) under control conditions in KMes. KCl replacement of KMes stimulated release of 65% fiber 45Ca within 1 min in interrupted responses; EGTA pretreatment was only moderately inhibitory with substantial residual stimulation. In contrast, choline Cl replacement of KMes induced release of 26-35% fiber 45Ca in interrupted responses; EGTA pretreatment was strongly inhibitory, but release significantly exceeded control with a small, sustained increase in Ca2+-insensitive efflux. These differences in 45Ca release and EGTA inhibition suggest that Cl replacement of Mes at constant [K] stimulates efflux by osmotic effects as well as imposed diffusion potentials; at least half the stimulated 45Ca loss (above control) in interrupted KCl responses is attributable to an osmotic component with low Ca2+ sensitivity. In the highly Ca2+-sensitive stimulation at constant [K][Cl], 45Ca release (above control) in interrupted responses correlated well with that in the pretreated responses of segments from the same fiber, with a slope of 8.4. This relationship suggests that imposed diffusion potentials stimulate a small Ca2+-insensitive component that gradates a much larger Ca2+-dependent efflux. The Ca2+-insensitive component apparently reflects intermediate steps in the excitation-contraction coupling that require positive feedback to result in sufficient Ca release for contraction.     

Magnesium effects on activation of skinned fibers from striated muscle

The intracellular Ca movements that control contraction and relaxation of striated muscle are regulated by the membrane potential and influenced by Mg2+. In skinned fibers, the internal composition can be manipulated directly by Ca movements estimated from isometric force transients, net changes in sarcoplasmic reticulum (SR) Ca, and 45Ca flux between fiber and bath. Stimulated Ca release, unlike unstimulated 45Ca efflux at low external [Ca2+], is highly [Mg2+]-sensitive at 20 C. Force and tracer measurements indicate three major sites of Mg2+-Ca2+ interaction in situ: Mg2+ can stimulate the SR active Ca transport system, inhibit a Ca2+-dependent Ca efflux pathway of SR, and shift the force-[Ca2+] relation, presumably by reducing Ca2+ binding to myofilament regulatory sites. These mechanisms constrain the resting Ca flux and are adaptive during relaxation. However, analysis of CI-stimulated 45Ca release and reaccumulation suggests that the depolarization process may inhibit Mg2+-dependent Ca influx, the membrane potential controlling both efflux and influx; recent studies on voltage-clamped cut fibers support this hypothesis. The Ca2+ and Mg2+ dependence of caffeine-stimulated 45Ca efflux suggests that Mg2+ inhibition of the Ca2+-dependent efflux pathway is small during rapid Ca2+ efflux. Therefore, both Mg2+ mechanisms, which minimize net release, may be reversed during normal activation.     

The role of free calcium ion in calcium release in skinned muscle fibers

Major questions in excitation--contraction coupling of fast skeletal muscle concern the mechanism of signal transmission between sarcolemma and sarcoplasmic reticulum (SR), the mechanism of SR Ca release, and operation of the SR active transport system during excitation. Intracellular Ca movement can be studied in skinned muscle fibers with more direct control, analysis of 45Ca flux, and simultaneous isometric force measurements. Ca release can be stimulated by bath Ca2+ itself, ionic "depolarization," Mg2+ reduction, or caffeine. The effectiveness of bath Ca2+ has suggested a possible role for Ca2+ in physiological release, but this response is difficult to analyze and evaluate. Related evidence emerged from analysis of other responses: with all agents studied, stimulation of 45Ca efflux is highly Ca2+-dependent. The presence of a Ca chelator prevents detectable stimulation by ionic "depolarization" or Mg2+ reduction and inhibits the potent caffeine stimulus; inhibition is graded with chelator concentration and caffeine concentration, and is synergistic with inhibition by increased Mg2+. The results indicate that a Ca2+-dependent pathway mediates most or all of stimulated 45Ca efflux in skinned fibers, and has properties compatible with a function in physiological Ca release.     

Ca2+ dependence of transverse tubule-mediated calcium release in skinned skeletal muscle fibers

Isometric force and 45Ca efflux from the sarcoplasmic reticulum were measured at 19 degrees C in frog skeletal muscle fibers skinned by microdissection. After Ca2+ loading, application of the ionophores monensin, an Na+(K+)/H+ exchanger, or gramicidin D, an H+ greater than K+ greater than Na+ channel-former, evoked rapid force development and stimulated release of approximately 30% of the accumulated 45Ca within 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+-specific ionophore, did not. When monensin was present in all bathing solutions, i.e., before and during Ca2+ loading, subsequent application failed to elicit force development and to stimulate 45Ca efflux. 5 min pretreatment of the skinned fibers with 50 microM digitoxin, a permeant glycoside that specifically inhibits the Na+,K+ pump, inhibited monensin and gramicidin D stimulation of 45Ca efflux; similar pretreatment with 100 microM ouabain, an impermeant glycoside, was ineffective. Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that: monensin and gramicidin D stimulate Ca2+ release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na+,K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin; the transverse tubule-mediated stimulation of 45Ca efflux appears to be entirely Ca2+ dependent.     

Mechanism of chloride-dependent release of Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle.

We investigated the effect of Cl- on the Ca2+ permeability of rabbit skeletal muscle junctional sarcoplasmic reticulum (SR) using 45Ca2+ fluxes and single channel recordings. In 45Ca2+ efflux experiments, the lumen of the SR was passively loaded with solutions of 150 mM univalent salt containing 5 mM 45Ca2+. Release of 45Ca2+ was measured by rapid filtration in the presence of extravesicular 0.4-0.8 microM free Ca2+ and 150 mM of the same univalent salt loaded into the SR lumen. The rate of release was 5-10 times higher when the univalent salt equilibrated across the SR-contained Cl- (Tris-Cl, choline-Cl, KCl) instead of an organic anion or other halides (gluconate-, methanesulfonate-, acetate-, HEPES-, Br-, I-). Cations (K+, Tris+) could be interchanged without a significant effect on the release rate. To determine whether Cl- stimulated ryanodine receptors, we measured the stimulation of release by ATP (5 mM total) and caffeine (20 mM total) and the inhibition by Mg2+ (0.8 mM estimated free) in Cl(-)-free and Cl(-)-containing solutions. The effects of ATP, caffeine, and Mg2+ were the largest in K-gluconate and Tris-gluconate, intermediate in KCl, and notably poor or absent in choline-Cl and Tris-Cl. Procaine (10 mM) inhibited the caffeine-stimulated release measured in K-gluconate, whereas the Cl- channel blocker clofibric acid (10 mM) but not procaine inhibited the caffeine-insensitive release measured in choline-Cl. Ruthenium red (20 microM) inhibited release in all solutions. In SR fused to planar bilayers we identified a nonselective Cl- channel (PCl: PTris: PCa = 1:0.5:0.3) blocked by ruthenium red and clofibric acid but not by procaine. These conductive and pharmacological properties suggested the channel was likely to mediate Cl(-)-dependent SR Ca2+ release. The absence of a contribution of ryanodine receptors to the Cl(-)-dependent release were indicated by the lack of an effect of Cl- on the open probability of this channel, a complete block by procaine, and a stimulation rather than inhibition by clofibric acid. A plug model of Cl(-)-dependent release, whereby Cl- removed the inhibition of the nonselective channel by large anions, was formulated under the assumption that nonselective channels and ryanodine receptor channels operated separately from each other in the terminal cisternae. The remarkably large contribution of Cl- to the SR Ca2+ permeability suggested that nonselective Cl- channels may control the Ca2+ permeability of the SR in the resting muscle cell.     

Silver ions trigger Ca2+ release by acting at the apparent physiological release site in sarcoplasmic reticulum

Rapid Ca2+ release from Ca2+ -loaded sarcoplasmic reticulum vesicles (SR) was previously shown to occur upon the addition of micromolar concentrations of heavy metals, and the extent of Ca2+ release was dependent on the binding affinity of the metal to sulfhydryl group(s) on an SR protein (Abramson, J.J., Weden, L., Trimm, J.L., and Salama, G. (1982) Biophys. J. 37, 134a; Abramson, J.J., Trimm, J.L., Weden, L., and Salama, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1526). The nature of this Ca2+ release site was examined further and found to be predominantly distributed in heavy SR (HSR) rather than light SR fractions. Ag+ -induced Ca2+ release from heavy SR was blocked by local anesthetics and ruthenium red which are known to inhibit Ca2+ release in skeletal fibers and in heavy SR, respectively. The rate of Ca2+ efflux from SR triggered by Ag+ was dependent on pH, Mg2+, and ionic strength of the medium. Efflux rates increased by a factor of 4 from pH 6.0 to 7.0 and then decreased in more alkaline reaction mixtures. Efflux rates from actively or passively loaded SR increased by a factor of 2.5 with increasing Mg2+ from 0 to 1 mM and then decreased in the range of 1 to 10 mM Mg2+. ATP-dependent Ca2+ uptake by SR was similar in 100 mM KCl and in 200 mM sucrose solutions, but the extent and rate of Ca2+ efflux induced by Ag+ were dramatically reduced with decreasing ionic strength of the medium. In solutions containing 5 mM Mg2+, the rate of Ca2+ efflux from heavy SR averaged over the first 1.5 s after the addition of Ag+ was 58 nmol of Ca2+/mg of SR/s, a value comparable to the fast initial rate of ATP-dependent Ca2+ uptake. The maximum initial rate of Ag+ -induced Ca2+ efflux from heavy SR in 1 mM Mg2+ may be comparable to the rate of Ca2+ release and tension development in muscle fibers. Our data indicate that Ag+ reacts with a protein or proteins in the SR, probably not the (Ca2+, Mg2+)-ATPase, to induce a rapid release of Ca2+, possibly from the physiological Ca2+ release site.     

Calcium and EDTA fluxes in dialyzed squid axons

Ca efflux in dialyzed squid axons was measured with 45Ca as a function of internal ionized Ca in the range 0.005-10 muM. Internal Ca stores were depleted by treatment with CN and dialysis with media free of high energy compounds. The [Ca]iota was stabilized with millimolar concentrations of EDTA, EGTA, or DTPA. Nonspecific leak of chelated Ca was measured with [14C]-EDTA and found to be 0.02 pmol/cm2s/mM EDTA. Correction of the measured Ca efflux for this leak of chelated calcium was made when appropriate. Ca efflux was roughly linear with internal free Ca in the range 0.005-0.1 muM. Above 0.1 muM, efflux was less than proportional to concentration but did not saturate at the highest concentration studied. Ca efflux was reduced about 50% by replacement of external Na with Li at Caiota approximately 1 muM, but was insensitive to such replacement for Ca less than 0.1 muM. Ca efflux was insensitive to internal Mg in the range 0-4 mM, indicating that the Ca pump favors Ca over Mg by a factor of about 10(6). Ca efflux was reduced about 60% by increasing internal Na from 1 to 80 mM. This effect could represent weak interference of a Ca carrier by Na or a loss of driving force because of a reduction in ENa - Em occasioned by an increase in Naiota. A few measurements were made of Ca influx in intact and in dialyzed fibers. In both cases, Ca influx increased when external Na was replaced by Li.     

Control of mitochondrial Mg++-efflux]

Inorganic phosphate stimulates the release of Mg++ from liver mitochondria, depending on concentration; a concentration as low as 0.1 mM phosphate is already effective. The process is dependent on the electron transfer of the respiratory chain, and its rate is highest under conditions of endogenous respiration and with ascorbate and TMPD as substrates, respectively. The phosphate stimulated release of Mg++ is followed, with a pronounced delay, by a Ca++ efflux and a swelling of mitochondria. Addition of EGTA strongly reduced the rate of Mg++ liberation in the presence and absence of inorganic phosphate. Exogenous Ca++ is able to abolish the EGTA effect. ADP and ATP inhibit the phosphate stimulated release of Mg++. Phosphoenol pyruvate and free fatty acids enhance the rate of Mg++ and Ca++ efflux from the mitochondria. The results permit the conclusion that inorganic phosphate, Ca++ and various metabolites of the cell metabolism influence the Mg++ distribution between the extra- and intramitochondrial space, thus controlling the permeability of the mitochondrial inner membrane for monovalent cations.     

45Ca and (14C)EDTA efflux from dialyzed barnacle muscle fibers.

45Ca and 14C-labeled ethylenediamine-N, N'-tetraacetic acid (EDTA) effluxes were measured in internally dialyzed barnacle muscle fibers. In 45Ca experiments the internal ionized 45Ca was fixed at 0.2 muM with ethyleneglycolbis-(beta-aminoethylether)-N, N'-tetraacetic acid(EGTA). The 45Ca efflux was found to increase with internal CaEGTA from 0.05 pmol/cm2.s(CaEGTA equal to 0.02 mM) to 5.0 pmol/cm2.s(CaEGTAequal to 9.6 mM). To determine whether or not most of this increase in efflux was due to the exit of undissociated CaEGTA, comparable experiments were performed with Ca-[14-C]EDTA. Over the same range of internal calcium as studied in the 45Ca experiments, the Ca-[14-C]EDTA efflux was no more than 12% of the 45Ca efflux. We conclude that the exit of undissociated 45Ca cannot account for most of the 45Ca efflux nor can it account for the dependence of 45Ca efflux on internal CaEGTA. The experiments also demonstrated the existence of an endogenous pool of calcium, of 0.43 mmol/kg (about half the total calcium), which remained unexchanged during dialysis.     

Does endothelin mobilize calcium from intracellular store sites in rat aortic vascular smooth muscle cells in primary culture?

In the presence of endothelin, there was a rapid increase in the 45Ca++ efflux from primary cultured rat vascular smooth muscle cells, both in physiological salt solution and in calcium free medium containing 2 mM EGTA. The 45Ca++ influx was not affected. The endothelin-induced, transient increase in cytosolic calcium concentration is probably mainly due to release of calcium from the intracellular store in vascular smooth muscle cells.     

Excitation of skinned muscle fibers by imposed ion gradients. III. Distribution of permeant ions in unstimulated and stimulated fibers

Ion gradients imposed across an internal membrane system stimulate skinned muscle fibers; to evaluate the sarcoplasmic reticulum (SR) as the primary target site, SR polarization under resting and stimulatory conditions was assessed from fiber uptake of permeant probe ions. Solvent spaces were estimated from simultaneous [14C]urea (U) or [3H]deoxyglucose (DOG) uptake in segments of fibers from bullfrog semitendinosus muscle, skinned by microdissection. The distribution spaces, i.e., virtual solvent volumes at bath concentrations (Vu and VDOG), of these uncharged probes correlated well with the protein content of the same segments, which validated the tracer methodology for volume normalization. The membrane-bounded volume fraction (Vm), derived from the difference between total solvent volume (Vs) and the non-membrane-bounded solvent volume (Vc), was sufficient to detect appreciable SR ion accumulation. The Vm estimated from the difference between VU and VDOG assayed simultaneously with 2 or 5-6 min exposures was 10-11%, which is consistent with the morphometric volume fraction (mostly SR) in frog fibers; however, the change in this difference after membrane permeabilization corresponded to Vm only 5%. The change in permeant ion distribution space caused by member permeabilization was used to assess SR membrane polarization, assuming the free ions distribute across the intact membrane according to the Nernst ratio. Resting polarization (SR lumen positive) was assessed from [14C]SCN- or [14C]propionate- distribution spaces in unstimulated fibers, expressed relative to VDOG (assayed simultaneously). The ratios for (a) [14C]SCN- space (carrier 2 mM) and (b) [14C]propionate- space (carrier 120 mM) were not decreased by membrane permeabilization. This indicated that anion distribution was independent of membrane integrity and did not reflect an SR transmembrane potential, although a was more and b was less than 1. Polarization under stimulatory conditions (lumen negative) was assessed from 86Rb+ distribution, before and after an imposed ion gradient (choline Cl replacement of K methanesulfonate (KMes) at constant [K+] [Cl-]) that theoretically could generate a 48-fold transmembrane cation ratio; Ca release was minimized by EGTA. The ratio of 86Rb+ space to VU, greater than 1 in KMes (120 mM K, the effective carrier), was higher in choline Cl (2.5 mM K) but not decreased by membrane permeabilization; this indicated that 86Rb+ distribution did not reflect an SR transmembrane potential. Similar results in the presence of valinomycin ruled out the possibility of inadequate 86Rb+ equilibration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphatidylinositol 4,5-bisphosphate enhances calcium release from sarcoplasmic reticulum of skeletal muscle

In the course of our study on the function of sarcoplasmic reticulum (SR) in skeletal muscle, the stimulatory action of phosphatidylinositol 4,5-bisphosphate (PIP2) on the Ca2+ release from SR was demonstrated by using chemically skinned fibers and fragmented SR vesicles. PIP2 induced a tension spike followed by sustained contraction in skinned fibers. PIP2 enhanced the caffeine-induced Ca2+ release from SR vesicles at low concentrations and triggered Ca2+ release by itself at high concentrations. PIP2 also enhanced 45Ca2+ efflux from SR vesicles. However, inositol 1,4,5-triphosphate never produced these effects. The Ca2+-releasing action of PIP2 was only weakly affected by ruthenium red or procaine. These observations suggest that PIP2 activates an SR Ca2+ release channel whose properties are different from those of the Ca2+-induced Ca2+ release channel.     

Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.     

Hg2+-induced contracture in mechanically skinned fibers of frog skeletal muscle

In mechanically skinned fibers of the semitendinosus muscle of bullfrogs, we examined the role of membrane sulfhydryl groups on Ca2+ release from the sarcoplasmic reticulum (SR). Hg2+, a sulfhydryl reagent (20-100 microM), induced a repetitive contracture of skinned fibers, and this contracture did not occur in skinned fibers in which the SR had been disrupted by treatment with a detergent (Brij 58). Procaine (10 mM), Mg2+ (5 mM), or dithiothreitol (1 mM) blocked the Hg2+-induced contracture. Ag+ or p-chloromercuribenzenesulfonic acid produced similar contractures to that induced by Hg2+. We conclude that Hg2+ releases Ca2+ from SR of a skinned fiber by modifying sulfhydryl groups on the SR membrane, and suggest that the Ca2+ released by Hg2+ may trigger a greater release of Ca2+ from SR to develop tension.     

ATP-dependent interaction of propranolol and local anaesthetic with sarcoplasmic reticulum. Stimulation of Ca2+ efflux.

Preincubation of sarcoplasmic reticulum (SR) with propranolol or tetracaine inhibits Ca2+ accumulation and stimulates ATPase activity by more than 2-fold. This effect is obtained only when the preincubation is carried out in the presence of ATP or other nucleoside triphosphates. The (ATP + drug)-induced inhibition of Ca2+ accumulation is pH-dependent, increasing as the pH rises above 7.5. The presence of micromolar concentrations of Ca2+ or Mg2+ during the preincubation prevents the inhibitory effect of ATP plus drug on Ca2+ accumulation or ATPase activity. The (ATP + drug) modification of SR vesicles resulted in stimulation of a rapid Ca2+ efflux from passively loaded vesicles. The ATP-dependent inhibition of Ca2+ accumulation by the drug is obtained with other local anaesthetics. The drug concentration required for 50% inhibition was 0.15 mM for dibucaine and 0.4 mM for both propranolol and tetracaine, whereas it was 5 mM, 8 mM and greater than 10 mM for lidocaine, benzocaine and procaine respectively. The heavy SR vesicles were only slightly affected by the incubation with propranolol or tetracaine in the presence of ATP, but their sensitivity increased markedly after storage at 0 degrees C for 24-48 h. These results suggest that propranolol and some local anaesthetics, in the presence of ATP, stimulate Ca2+ efflux by modifying a protein factor(s) rather than the phospholipid bilayer.