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Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224.
1. Intracellular [Ca2+] ([Ca2+]i) transients, monitored by the fluorescent Ca2+ indicator, indo-1, and twitch contractions elicited by action potentials, by voltage clamp pulses or by rapid, brief pulses of caffeine, were measured in guinea-pig single ventricular myocytes. Experiments were designed to determine whether and to what extent the trans-sarcolemmal Ca2+ influx is immediately sequestered by the sarcoplasmic reticulum (SR). 2. Rapid, brief (100-200 ms) pulses of caffeine onto a rested myocyte elicited a [Ca2+]i transient and a contraction. Following exposure to specific SR inhibitors, ryanodine (100 nM) or thapsigargin (200 nM), the rapid application of caffeine onto a rested myocyte failed to elicit changes in [Ca2+]i or in cell length, indicating that caffeine increases [Ca2+]i by specifically discharging Ca2+ from the SR. In the absence of these inhibitors, a second pulse of caffeine, within 3 min following a prior pulse, failed to elicit a [Ca2+]i transient or contraction, indicating that a caffeine pulse depletes the SR releasable Ca2+ pool. 3. Following Ca2+ depletion of the SR by double caffeine pulses at rest, an electrical stimulation elicited a slow increase in [Ca2+]i, and, after a delay, a small, slow twitch contraction. The simultaneous application of caffeine and electrical stimulation of cells in which the SR was Ca2+ depleted elicited [Ca2+]i transients with an increased rate of rise and a larger amplitude (53 +/- 8 and 63 +/- 9% respectively; mean +/- S.E.M., n = 21) than those elicited by electrical stimulation alone. 4. Whether caffeine affected the L-type calcium current (ICa) elicited by electrical stimulation was determined under whole-cell voltage clamp. A caffeine pulse delivered at the onset of a depolarizing voltage clamp step also increased the rates of rise and the amplitudes of the [Ca2+]i transients and twitch contractions in cells in which the SR was depleted of Ca2+. However, Ca2+ influx via ICa decreased when caffeine was pulsed in conjunction with the voltage clamp, as the peak ICa was either unchanged or decreased while its inactivation was consistently accelerated. 5. Because the stimulation-dependent trans-sarcolemmal Ca2+ influx via ICa is not increased by a caffeine pulse, the augmentation of the rates of rise and the amplitudes of the electrically stimulated [Ca2+]i transients by caffeine pulsed in conjunction with the electrical stimulation in cells in which the SR had been depleted of Ca2+ indicates that a portion of Ca2+ influx during depolarization in the absence of caffeine is rapidly buffered by the SR.(ABSTRACT TRUNCATED AT 400 WORDS)
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