Showing papers by "David A. Eisner published in 1995"

Comparison of subsarcolemmal and bulk calcium concentration during spontaneous calcium release in rat ventricular myocytes

Abstract: 1. The aim of these experiments was to compare the time course of changes in intracellular Ca2+ concentration ([Ca2+]i) measured in the bulk cytoplasm with those estimated to occur near the sarcolemma. Sarcolemmal Na(+)-Ca2+ exchange current and [Ca2+]i were measured in single, voltage-clamped ventricular myocytes. 2. Spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) resulted in a transient inward current. This current developed and decayed more quickly than the accompanying changes in [Ca2+]i (measured with indo-1) resulting in a hysteresis between [Ca2+]i and current. A similar hysteresis was also observed if [Ca2+]i was elevated with caffeine and was removed if the current was low pass filtered with a time constant of 132 ms. 3. Digital video imaging (using fluo-3 or calcium green-1 to measure [Ca2+]i) allowed measurement of [Ca2+]i at all points in the cell during the wave of spontaneous Ca2+ release. The hysteresis between [Ca2+]i and current remained, even after allowing for the spatial and temporal properties of this wave. 4. The hysteresis can be accounted for if there is a barrier to diffusion of Ca2+ ions separating the bulk cytoplasm from the space under the sarcolemma (into which Ca2+ is released from the sarcoplasmic reticulum). The calculated subsarcolemmal [Ca2+] rises and falls more quickly (and reaches a higher peak) than does the bulk [Ca2+]. The delay introduced by this barrier is equivalent to a time constant of 133 ms. 5. The subsarcolemmal space described in this paper may be equivalent to the 'fuzzy space' previously suggested to be important in controlling SR Ca2+ release.

Estimate of net calcium fluxes and sarcoplasmic reticulum calcium content during systole in rat ventricular myocytes.

Abstract: 1. The experiments were performed on voltage-clamped cells in which intracellular calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo-1 (acetoxymethyl ester (AM) loading). When cells were stimulated with a short (100 ms) depolarizing pulse, following a rest, the magnitude of the first systolic calcium transient was greater than that in the steady state (rest potentiation) and decayed to its steady level over a few stimuli. If a longer pulse (800 ms) was used then the systolic calcium transient was either unaffected or increased in magnitude following a rest. During constant stimulation, if the length of the pulse is decreased, then the magnitude of the calcium transient decreased reversibly over several beats. 2. The calcium entry into the cell was measured from the integral of the inward calcium current and the efflux from the Na(+)-Ca2+ exchange current on repolarization. During the negative staircase the calcium current was approximately constant whilst the Na(+)-Ca2+ exchange current decayed in parallel with the systolic calcium transient. A net loss of calcium from the cell can be calculated from the extra Na(+)-Ca2+ exchange current following the initial pulses. 3. The application of caffeine produces a transient increase of both [Ca2+]i and an inward Na(+)-Ca2+ exchange current. The integral of this current can be used to estimate the caffeine-releasable calcium content of the sarcoplasmic reticulum (SR), which decreases following stimulation with short compared to long pulses. This difference in SR calcium content is quantitatively similar to that estimated from the sarcolemmal currents. 4. At a given membrane potential, the relationship between [Ca2+]i and current during the caffeine exposure can be used to estimate the Na(+)-Ca2+ exchange flux from the measured [Ca2+]i and thence the Na(+)-Ca2+ exchange flux during depolarization. 5. For a long depolarizing pulse the extrusion of calcium from the cell on Na(+)-Ca2+ exchange is comparable to the entry on the calcium current. In contrast, for short pulses the extrusion of calcium on the Na(+)-Ca2+ exchange immediately after the pulse is greater than the entry during the pulse on the calcium current. 6. These results show that rest potentiation can be correlated with changes in the amount of calcium stored in the SR and this, in turn, can be accounted for by sarcolemmal fluxes.

Propagating calcium waves initiated by local caffeine application in rat ventricular myocytes.

Abstract: 1. Caffeine was applied locally to one region of a resting cell via an extracellular pipette while simultaneously imaging the concentrations of intracellular calcium ([Ca2+]i) and intracellular caffeine ([caffeine]i). 2. Local application of caffeine produced a rise of [caffeine]i which was confined to the region of the cell near the pipette. There was also a local increase of [Ca2+]i which then, in most resting cells, propagated along the cell as a linear Ca2+ wave. The initial magnitude of the rise of [Ca2+]i was greater than that of the electrically stimulated Ca2+ transient. 3. As the wave of increase of [Ca2+]i propagated along the cell it decreased in both amplitude and velocity in cells that had not been treated to elevate the cellular Ca2+ load. 4. In some cells the caffeine response did not propagate significantly. In these cases an increase of the cellular Ca2+ load enabled caffeine-induced Ca2+ wave propagation along the entire cell length without significant decay in amplitude and velocity. 5. Previous work has shown that an electrically evoked local systolic Ca2+ transient does not propagate. The fact that the caffeine-evoked response does propagate and the correlation between decay of amplitude and velocity suggest that the transient has to be a certain size before it can propagate. It is suggested that one of the factors which favour propagation of waves under conditions of elevated sarcoplasmic reticulum Ca2+ content is the increased release of Ca2+.

Factors controlling changes in intracellular Ca2+ concentration produced by noradrenaline in rat mesenteric artery smooth muscle cells.

Abstract: 1. The intracellular Ca2+ concentration ([Ca2+]i) was measured in mesenteric artery smooth muscle cells using the fluorescent indicator indo-1. 2. Noradrenaline (1-10 microM) produced a transient increase in [Ca2+]i. This response was unaffected by the removal of external calcium suggesting that the bulk of the increase in [Ca2+]i produced by noradrenaline is due to release from an intracellular store. 3. The maintained application of caffeine (10 mM) produced a transient rise in [Ca2+]i. The rate of relaxation was slower than that of the noradrenaline response. If caffeine was removed at the peak of the rise in [Ca2+]i then [Ca2+]i recovered more quickly than was the case in both the maintained response to noradrenaline and that to caffeine. 4. In the presence of noradrenaline, caffeine or thapsigargin elevated [Ca2+]i. However, if thapsigargin or caffeine was added first, the subsequent application of noradrenaline did not increase [Ca2+]i, suggesting that only part of the caffeine-sensitive store is sensitive to noradrenaline. 5. The recovery of [Ca2+]i during the application of caffeine was unaffected by the removal of external sodium suggesting that Na+-Ca2+ exchange is not important in the reduction in [Ca2+]i. The addition of lanthanum (1 mM) did, however, greatly slow [Ca2+]i recovery. 6. We conclude that the three major factors responsible for removing Ca2+ ions from the cytoplasm are: (i) a caffeine- and noradrenaline-sensitive store (43%), (ii) a caffeine-sensitive but noradrenaline-insensitive store (36%), and (iii) a sarcolemmal Ca(2+)-ATPase (16%). Finally, a 5% contribution remains to be accounted for.

Changes of pH affect calcium currents but not outward potassium currents in rat myometrial cells

Abstract: Spontaneous contraction of uterine smooth muscle is enhanced by alkalinization and depressed by acidification. We have investigated the ionic currents responsible for this in single myometrial cells. Intracellular acidification (20 mM butyrate) at constant external pH depressed the magnitude of the calcium current to 58±6% of control, but had little effect on outward currents. Similar but slower effects were also observed when the extracellular pH was lowered to 6.9 (56±9% of control). Correspondingly, when the intracellular or extracelluar pH was elevated (20 mM NH4Cl or pH 7.9 respectively) the calcium current magnitude increased (165±15 % in NH4Cl; 136±2 % at pH 7.9) and there was, again, no effect on the outward currents. These observations are consistent with the effects of pH on spontaneous contractile activity being due to an effect on the membrane calcium current.