Spontaneous Ca Release Research Articles

Scattered light intensity fluctuation in the canine ventricular myocardium: correlation with inotropic drug effect

Scattered light intensity fluctuation (SLIF) of coherent light by a strip of ventricular muscle during diastole is believed to be due to asynchronous cellular motion within the myocyte as a result of spontaneous release of Ca from the sacoplamic reticulum. Previous studies have shown a correlation between inotropic agents, such as ouabain and elevated extracellular Ca or decreased extracellular Na, and SLIF. The purpose of this study was to see if this correlation could be extended to other inotropic agents. The digitalis genin, ouabagenin, produces inotropy by increasing intracellular free Ca. In toxic concentrations the drug produces abnormal aftercontractions by spontaneous Ca release from the sarcoplasmic reticulum. On the other hand, the Ca channel agonist BAY k 8644 is also positively inotropic, but its effect is associated with a decrease in Ca release from the sarcoplasmic reticulum, manifested by conversion of "rest potentiation" to "rest depression." The effects of these inotropic agents on the power spectra of SLIF were dissimilar. Both frequency and amplitude of SLIF were increased after ouabagenin (1 microM), but these changes were most marked after the onset of toxicity, at which time contractility was decreased, rather than during the positive inotropic response. In contrast, BAY k 8644 (1 microM) decreased SLIF at all levels of inotropic response. The beta-adrenoceptor stimulant drug, dobutamine, and the adenylate cyclase activator, forskolin, produced minimal increase in SLIF at inotropic concentrations but caused a large increase in SLIF only after the onset of toxicity. These results suggest that SLIF is a better indicator of intracellular Ca overload and toxic oscillatory contractions in the presence of an inotrope and not of increased inotropy, per se.

Calcium compartmentation in isolated renal tubules in suspension

Substantial increases of total cell Ca 2+ have been observed in suspensions of isolated rabbit proximal tubules subjected to hypoxic injury or treated with exogenous ATP followed by apparent recovery with reoxygenation of the hypoxic tubules or continued incubation of ATP-treated tubules. Ca 2+ compartmentation studies using digitonin and metabolic inhibitors were done to clarify the basis for these changes. Digitonin, 40–90 μg/mg tubule protein, rapidly permeabilized the tubule cells and did not impair mitochondrial Ca 2+ sequestration. Most of the increases of tubule cell Ca 2+ produced by hypoxia and ATP were accounted for by pools which could be rapidly removed by exposure of tubules to EGTA and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone without concomitant use of digitonin, suggesting that the changes of Ca 2+ predominantly reflect sequestration by mitochondria in severely damaged cells or mitochondria already released to the medium from them. The time course of uptake followed by spontaneous release of mitochondrial Ca 2+ from tubule cells deliberately permeabilized with digitonin, then incubated for prolonged periods, indicated that the decreases of tubule cell Ca 2+ during reoxygenation of hypoxic suspensions and prolonged incubation of ATP-treated tubules were likely to be attributable to loss of Ca 2+ from free mitochondria and those in damaged cells rather than to extrusion by intact cells.

Synchronous occurrence of spontaneous localized calcium release from the sarcoplasmic reticulum generates action potentials in rat cardiac ventricular myocytes at normal resting membrane potential.

Under certain conditions, spontaneous release of Ca2+ from the sarcoplasmic reticulum occurs in resting mammalian myocardium. In single rat ventricular myocytes, such spontaneous Ca2+ release appears localized rather than homogeneous. When the increase in cytosolic Ca2+ is present in a single locus within a cell, it causes a small depolarization, which, at the normal resting potential, is subthreshold for generating an action potential. However, when spontaneous Ca2+ release occurs simultaneously at more than a single discrete locus, the resultant sarcolemmal depolarization is augmented to levels that can induce an action potential, even when this depolarization begins at the normal resting membrane potential. Thus, the synchronous occurrence of multifocal localized increases in cytosolic Ca2+ due to spontaneous Ca2+ release from the sarcoplasmic reticulum within ventricular myocytes is a mechanism for "abnormal automaticity."

A model for the uptake and release of Ca2+ by sarcoplasmic reticulum.

On addition of ATP to vesicles derived from the sarcoplasmic reticulum (SR) of skeletal muscle, Ca2+ is accumulated from the external medium. Following uptake, spontaneous release of Ca2+ occurs in the presence or in the absence of ATP. These processes of Ca2+ uptake and release were simulated by using the models derived for ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227; Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730] and for Ca2+ release from passively loaded vesicles [McWhirter, Gould, East & Lee (1987) Biochem. J. 245, 713-722]. The simulations are consistent with measurements of the effects of pH, K+, Ca2+ and Mg2+ on uptake and release of Ca2+. The increase in maximal Ca2+ accumulation observed in the presence of maleate is explained in terms of complexing of Ca2+ and maleate within the SR. The calculated concentration of ADP generated by hydrolysis of ATP has a large effect on the simulations. The effects of an ATP-regenerating system on the measured Ca2+ uptake is explained in terms of both removal of ADP and precipitation of Ca3(PO4)2 within the vesicles. It is concluded that both the process of Ca2+ uptake and the process of Ca2+ release seen with SR vesicles can be interpreted quantitatively in terms solely of the properties of the Ca2+ + Mg2+-activated ATPase.

Mg inhibits voltage and tension oscillation but potentiates twitch in depolarized myocardium.

In the presence of 1-5 X 10(-8) M isoproterenol, guinea pig ventricular muscle, depolarized by K-rich (27 mM) solution, exhibits a biphasic twitch tension, after contraction (AC), and oscillatory afterpotentials (OAP) in response to 1-Hz stimulation. We obtained data suggesting that OAP and AC are due to a spontaneous oscillatory release of Ca from the sarcoplasmic reticulum (SR) and that the initial component (C1) of the biphasic twitch, which produces a large and early peak in tension, reflects the excitation-coupled Ca release from SR. Elevation of extracellular Mg concentration ([Mg]o) to 3-6 mM suppressed AC and OAP. However, C1 was markedly potentiated by high [Mg]o. When single extrasystoles with various coupling intervals were interposed during a regular stimulation at 0.2 Hz, C1 in the extrasystole, which was large with a short coupling interval, gradually decreased with lengthening of the coupling interval. Elevation of [Mg]o markedly reduced the rate of this decrease suggesting that the decrease in available Ca in the SR during diastole was slowed by high [Mg]o. Thus, in depolarized ventricle, high Mg solution seems to depress the spontaneous oscillatory Ca release from SR and subsequently enhances the Ca release coupled to membrane excitation. The latter enhancement may be related to stabilization of the Ca in SR during diastole.

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.

Control of intracellular ionized calcium concentration by sarcolemmal and intracellular mechanisms

The regulation of the resting intracellular ionized calcium concentration [( Ca2+]i) has been studied in ferret papillary muscle using the photoprotein aequorin to measure [Ca2+]i. Elevating [Ca2+]o produced an initial rapid increase of [Ca2+]i and tension which then decayed to a steady level. This secondary fall of [Ca2+]i is attributed to a secondary decrease of Ca entry on Na-Ca exchange produced by the known fall of [Na+]i. Replacing external Na by K produced a large transient increase of both [Ca2+]i and tension which then decayed spontaneously to near the resting level. If Na was removed after metabolic inhibition with cyanide and deoxyglucose then neither tension nor [Ca2+]i recovered. The addition of the mitochondrial uncoupler FCCP to a muscle in Na-free solution produced a gradual rise of tension but only elevated [Ca2+]i after a delay of many minutes. Similarly caffeine did not elevate [Ca2+]i. These experiments do not support the hypothesis that the regulation of resting [Ca2+]i in Na-free solutions depends solely on intracellular sequestration of [Ca2+]i. The first twitch elicited in Na-containing solutions after exposure to Na-free solution was much larger than control and was associated with a large Ca transient attributed to increased loading of the sarcoplasmic reticulum with Ca in the Na-free solution. The elevation of [Ca2+]i in Na-free solutions was accompanied by spontaneous fluctuations of both [Ca2+]i and tension with a frequency of about 3 Hz. These fluctuations were abolished by drugs such as caffeine or ryanodine which interfere with sarcoplasmic reticulum function. These results provide direct evidence for the spontaneous release of Ca from the sarcoplasmic reticulum inferred from previous, less direct, work.

Fatty acid effects on calcium influx and efflux in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

Low concentrations of fatty acids inhibited initial Ca uptake by sarcoplasmic reticulum vesicles, the extent of inhibition varying with chain length and unsaturation in a series of C 14–C 20 fatty acids. Oleic acid was a more potent inhibitor of initial Ca uptake than stearic acid at 25°C, whereas at 5°C there was less difference between the inhibitory effects of low concentrations of these fatty acids. When the fatty acids were added later, during the phase of spontaneous Ca release that follows Ca uptake in reactions carried out at 25°C, 1–4 μM oleic and stearic acids caused Ca content to increase. This effect was due to marked inhibition of Ca efflux and slight stimulation of Ca influx. At concentrations of >4 μM, both fatty acids inhibited the Ca influx that occurs during spontaneous Ca release; in the case of oleic acid, this inhibition resembled that of initial Ca uptake at 5°C. The different effects of fatty acids at various times during Ca uptake reactions may be explained in part if alterations in the physical state of the membranes occur during the transition from the phase of initial Ca uptake to that of spontaneous Ca release.

An electrogenic Na +/Ca 2+ antiporter in addition to the Ca 2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na + + K +)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca 2+ transport. The Ca 2+ pump was stimulated 1.7- and 2.1-fold by external Na + and K +, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca 2+ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca 2+ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca 2+ in a medium containing 160 mM NaCl. Ca 2+ movements were also studied in the absence of ATP and Mg 2+. Vesicles containing an outwardly directed Na + gradient showed the highest Ca 2+ uptake activity. These findings suggested the operation of a Ca 2+/Na + antiporter in addition to the active Ca 2+ pump in these sarcolemmal vesicles. A valinomycin-induced inward K +-diffusion potential stimulated the Na +- Ca 2+ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg 2+ the transmembrane Na i +/Na o + gradient exceeded 160/15 mM concentrations, Ca 2+ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na + gradient-induced Ca 2+ uptake to be a translocation of Ca 2+ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na +-Ca 2+ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.

Effect of unsaturated fatty acids and monovalent cations on calcium efflux from kidney cortex mitochondria

1. 1. The effect of exogenously added, or endogenously produced, unsaturated fatty acids (UFA) on the uptake and release of Ca 2+ by rat kidney mitochondria has been studied. 2. 2. Linolenic acid decreases the duration of Ca 2+ steady state maintenance in mitochondria, and promotes its release. 3. 3. The spontaneous release of Ca 2+ observed at high temperatures is related to the activation of membrane-bound phospholipases. 4. 4. The effect of endogenously produced, or exogenously added, UFA depends on the cationic composition of the medium, and is strongly inhibited by Na + and Li. 5. 5. Evidence is also presented that the releasing effect of UFA is not linked to non-specific alterations in the permeability characteristics of the inner mitochondrial membrane.