Showing papers by "David A. Eisner published in 1999"

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes

Abstract: This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca2+]i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca2+]i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114+/-18 micromol l-1 and Kd of 0.59+/-0.17 microM (n=8). During the systolic rise of [Ca2+]i, the measured changes of [Ca2+]i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca.

The role of sarcolemmal Ca2+‐ATPase in the regulation of resting calcium concentration in rat ventricular myocytes

Abstract: The aim of this work was to investigate the role of sarcolemmal Ca2+-ATPase in rat ventricular myocytes. We have measured intracellular Ca2+ concentration ([Ca2+]i) using indo-1. The actions of the ATPase inhibitor carboxyeosin were studied. Carboxyeosin increased resting [Ca2+]i and the magnitude of the systolic Ca2+ transient and slowed the rate of its relaxation by 5%. Carboxyeosin increased the magnitude of the caffeine-evoked increase in [Ca2+]i and slowed its relaxation by 20%. Removal of extracellular Na+ slowed the rate constant by 80%. When Na+ was removed in a carboxyeosin-treated cell, the caffeine-evoked increase in [Ca2+]i did not decay. Carboxyeosin increased the integral of the Na+-Ca2+ exchange current activated by caffeine. This is, in part, due to an increase in sarcoplasmic reticulum Ca2+ content. When extracellular Na+ was removed, there was a transient increase in [Ca2+]i which then decayed. The rate of this decay was slowed by carboxyeosin by a factor of about four. The residual decay could be abolished with caffeine. In the absence of extracellular Na+, increasing extracellular Ca2+ concentration ([Ca2+]o) elevated [Ca2+]i. In carboxyeosin-treated cells, [Ca2+]i was much more sensitive to [Ca2+]o. These results demonstrate the role of a carboxyeosin-sensitive Ca2+-ATPase in the control of resting [Ca2+]i and the reduction in [Ca2+]i following an increase in [Ca2+]i. In cardiac cells, as in many other cells, there is a large electrochemical gradient favouring Ca2+ entry into the cell from the extracellular fluid. Two sarcolemmal mechanisms are known to extrude Ca2+ from the cytoplasm: sarcolemmal Ca2+-ATPase and Na+-Ca2+ exchange. The role of Na+-Ca2+ exchange in Ca2+ homeostasis has been extensively characterized in cardiac muscle (Chapman & Tunstall, 1981; Allen et al. 1984; Crespo et al. 1990). In contrast, much less is known about Ca2+-ATPase. A sarcolemmal Ca2+-ATPase has been demonstrated in the heart (Caroni & Carafoli, 1981). It has been suggested that Ca2+-ATPase has a higher affinity and lower maximum velocity than Na+-Ca2+ exchange and may therefore be more important for control of resting [Ca2+]i than for removing larger Ca2+ loads produced by stimulation (DiPolo & Beauge, 1979) although recent work has questioned this functional distinction (Lamont & Eisner, 1996). Most previous work studying the role of Ca2+-ATPase in cardiac muscle has been performed under conditions that inhibit Na+-Ca2+ exchange to reveal any potential contribution of ATPase. Specifically, the following observations have led to the suggestion that something other than Na+-Ca2+ exchange, perhaps sarcolemmal Ca2+-ATPase, may have a significant functional role in Ca2+ homeostasis, at least under experimental conditions. (1) When caffeine is applied to release Ca2+ from the sarcoplasmic reticulum (SR), inhibition of Na+-Ca2+ exchange slows but does not abolish the decay of [Ca2+]i (O'Neill et al. 1991; Bassani et al. 1992; Negretti et al. 1993; Bassani et al. 1994). It has been suggested that the Na+-Ca2+ exchange-independent component of the decay of [Ca2+]i is due to a combination of sarcolemmal Ca2+-ATPase and mitochondrial sequestration. Separation of mitochondrial and sarcolemmal Ca2+-ATPase components has been achieved by inhibiting the former with uncouplers or the latter by elevating extracellular Ca2+ (Bassani et al. 1992; Negretti et al. 1993). However, these approaches are both rather non-specific. (2) Removal of extracellular Na+ produces an increase in [Ca2+]i as Ca2+ enters the cell via Na+-Ca2+ exchange. However, [Ca2+]i then falls to levels which are only slightly elevated above the control. This secondary relaxation of [Ca2+]i may reflect the activity of a sarcolemmal Ca2+-ATPase. (3) In the absence of extracellular Na+, [Ca2+]i can still be regulated and respond to changes in extracellular Ca2+ concentration (Lamont & Eisner, 1996) and, again, this may reflect the activity of a sarcolemmal Ca2+-ATPase. Evidence for the role of sarcolemmal Ca2+-ATPase would be much stronger if there was a specific inhibitor which could be applied to dissect out its contribution. It has been shown that ATPase is specifically inhibited by carboxyeosin (Gatto & Milanik, 1993). In rabbit and ferret ventricular myocytes, carboxyeosin has been used to show that Ca2+-ATPase makes an appreciable contribution to the decay of [Ca2+]i during a caffeine-evoked response (Bassani et al. 1995). Using carboxyeosin, in the present paper we show that sarcolemmal Ca2+-ATPase has significant effects on cell function even under physiological conditions. Furthermore it provides the major component of the secondary decay of [Ca2+]i following Na+ removal and is essential for [Ca2+]i homeostasis in Na+-free solutions.

The role of the sarcoplasmic reticulum as a Ca2+ sink in rat uterine smooth muscle cells.

Abstract: The mechanisms responsible for removing calcium ions from the cytoplasm were investigated in single rat uterine myocytes using indo-1. Trains of depolarizing voltage-clamp pulses increased [Ca2+]i. The rate of decay of [Ca2+]i was slowed by inhibition of the sarcoplasmic reticulum (SR) Ca2+-ATPase with cyclopiazonic acid (CPA). However, if the sarcolemmal Na+-Ca2+ exchanger and Ca2+-ATPase were inhibited then recovery of [Ca2+]i was abolished showing that the SR Ca2+-ATPase alone cannot produce decay of [Ca2+]i. In another series of experiments, Ca2+ release from the SR was induced with carbachol in a Ca2+-free solution. Under these conditions responses to repeated applications of carbachol could be obtained. In the presence of CPA, however, only the first application was effective. This suggests that the SR Ca2+-ATPase sequesters a significant amount of Ca2+ into the SR. CPA slowed the rate of decay of [Ca2+]i following carbachol addition by > 50%. Again, however, after a brief transient fall, decay was abolished when the Na+-Ca2+ exchanger and sarcolemmal Ca2+-ATPase were inhibited. These data show that, although the SR Ca2+-ATPase contributes to the decay of [Ca2+]i, it cannot function effectively in the absence of Ca2+ removal from the cell. These data are discussed in the context of the superficial buffer barrier model in which Ca2+ is taken up into the SR and then released very close to sarcolemmal Ca2+ extrusion sites, i.e. the SR acting in series with the surface membrane extrusion mechanisms. We also suggest that the amount of filling of the SR influences the rate of Ca2+ removal. Contraction of smooth muscle requires an increase of [Ca2+]i. This is derived from Ca2+ entry from the extracellular fluid, as well as Ca2+ release from the sarcoplasmic reticulum (SR). Correspondingly, relaxation is initiated by the removal of intracellular Ca2+ either to the extracellular fluid or to the SR. Transport of Ca2+ out of the cell is an active process that depends on the activity of the plasma membrane Ca2+-ATPase (PMCA) and the Na+-Ca2+ exchanger. Both have been found in uterus of different species (Kosterin et al. 1994), but their quantitative importance for Ca2+ extrusion from the uterine cell has only recently been examined (Shmigol et al. 1998a). The above studies suggested that both mechanisms made a significant contribution to, and together were entirely responsible for, Ca2+ extrusion. An additional more complex mechanism of Ca2+ extrusion has, however, been proposed (van Breemen et al. 1986). It was suggested that the SR may act as a ‘superficial buffer barrier’, by taking up a fraction of the Ca2+ that enters the cell through the plasmalemma before it reaches the contractile machinery. In order for the SR to buffer calcium on a steady-state basis, Ca2+ must be translocated from the SR lumen to the extracellular space. This translocation is thought to be mediated by the release of accumulated Ca2+ from the SR into the narrow space between the SR and plasmalemma (referred to as ‘vectorial Ca2+ release’), from where the Na+-Ca2+ exchanger and PMCA complete the extrusion process (Moore et al. 1993). It is therefore implied that sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) functions in series with the sarcolemmal Na+-Ca2+ exchanger closely opposed to the peripheral SR, thereby contributing significantly to Ca2+ extrusion (Moore et al. 1993; Villa et al. 1993). Evidence supporting such a ‘superficial buffer barrier’ hypothesis has been obtained in vascular smooth muscle cells (Chen & van Breemen, 1993; Nazer & van Breemen, 1998; Rembold & Cheng, 1998) and in gastric fundus (Petkov & Boev, 1996). It is, however, unclear whether or not the SR of uterine smooth muscle cells contributes to the extrusion of Ca2+ by means of vectorial calcium release (Taggart & Wray, 1997). Recently, we have shown that the PMCA and Na+-Ca2+ exchanger play an important role in the decay of depolarization-induced [Ca2+]i transients in uterine smooth muscle cells (Shmigol et al. 1998a,c). When these two mechanisms were inhibited simultaneously, there was no [Ca2+]i decay after a train of depolarizing pulses. This could be taken as evidence that no other mechanisms contribute to the decay of [Ca2+]i. However, since membrane depolarization greatly increases the total calcium content of the cell, the intracellular calcium accumulating systems may saturate, due to their finite capacity. This saturation could account for the lack of [Ca2+]i transient decay under the inhibition of calcium extrusion. Further insight into the mechanisms responsible for [Ca2+]i transient decay can be obtained by investigating the decay of agonist-induced [Ca2+]i transients, since the release of Ca2+ from the SR does not increase the total calcium content of the cell. Our aim in this study was therefore to evaluate the role of the SR in Ca2+ extrusion from uterine smooth muscle cells. In particular we wished to answer the following questions: (i) does the SR contribute to the decay of the depolarization- and agonist-induced [Ca2+]i transients? and (ii) how is the SR Ca2+ pump mechanism related to the plasmalemmal Ca2+ extrusion mechanisms?

The effects of inhibition of the sarcolemmal Ca-ATPase on systolic calcium fluxes and intracellular calcium concentration in rat ventricular myocytes.

Abstract: The effects of carboxyeosin, an inhibitor of the sarcolemmal Ca-ATPase, were studied on intracellular Ca and membrane currents in isolated rat ventricular myocytes. In the absence of carboxyeosin, 150-ms-duration depolarizing pulses from –40 to 0 mV resulted in an L-type Ca current on depolarization and a Na-Ca exchange ”tail” current on repolarization. The calculated entry of Ca on the L-type current was 1.3 times greater than the efflux via the Na-Ca exchange. The addition of carboxyeosin (20 µM) resulted in either an increase of the Na-Ca exchange current or a decrease of the L-type Ca current such that the Ca entry and efflux were exactly equal. These results suggest that, under control conditions, a carboxyeosin-sensitive Ca-ATPase contributes about 24% of the total Ca efflux from the cell and, therefore, that the sarcolemmal Ca-ATPase has a significant role in regulation of sarcolemmal Ca fluxes.

Strophanthidin-induced gain of Ca2+ occurs during diastole and not systole in guinea-pig ventricular myocytes.

Abstract: We have investigated the effects of inhibiting the Na-K pump with strophanthidin on the intracellular Ca2+ concentration ([Ca2+]i), sarcoplasmic reticulum (s.r.) Ca2+ content and membrane currents. s.r. Ca2+ content was measured by integrating the Na-Ca exchange current resulting from application of 10 mM caffeine. The application of strophanthidin increased both diastolic and systolic [Ca2+]i. This was accompanied by an increase of s.r. Ca2+ content from a resting value of 17.9±1.5 µmol/l to 36.9±3.3 µmol/l (n=16) after 5 min. Systolic fluxes of Ca2+ into and out of the cell before and during strophanthidin application were also measured. Ca2+ efflux (measured as the integral of the Na-Ca exchange tail current) rose steadily in the presence of strophanthidin, while Ca2+ influx (the integral of the L-type Ca2+ current) was reduced. In spite of this, s.r. Ca2+ content rose substantially. In the presence of Cd2+ (100 µM), which inhibits the L-type Ca2+ current, strophanthidin had negligible effects on current suggesting that Ca2+ influx via Na-Ca exchange during depolarization does not account for the increase of s.r. Ca2+ content. This suggests that changes of Ca2+ flux during systole are not responsible for the strophanthidin-induced increase of s.r. Ca2+. We conclude that the primary mechanism by which the cardiac cell gains Ca2+ when the Na-K pump is inhibited is by a net influx during diastole.