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Department of Physiology, University College London.
1. Fluorescence measurements have been made in single, isolated rat ventricular myocytes using the Ca2(+)-sensitive indicators Fura-2 and Indo-1. In Fura-2-loaded cells, the application of caffeine (2-20 mM) produced a change of fluorescence indicating an increase of [Ca2+]i which then spontaneously decayed to control levels. These changes of [Ca2+]i were accompanied by a contracture. 2. In contrast, in Indo-1-loaded cells, in addition to the changes of fluorescence expected for the transient increase of [Ca2+]i produced by caffeine, there was a maintained decrease of fluorescence. 3. Measurements in vitro showed that caffeine quenches the fluorescence of Indo-1 (but not of Fura-2) in a [Ca2+]-and wavelength-independent manner. Caffeine therefore had no effect on the ratio of Indo-1 fluorescence measured at two wavelengths. This inhibition by caffeine could be described by an apparent Ki of 4 mM. In the cell the Ki was considerably larger (18 mM). 4. We have separated the Indo-1 fluorescence changes into caffeine- and [Ca2+]i-dependent components. The time course of change of intracellular caffeine was calculated. When [caffeine]o was rapidly increased, [caffeine]i changed with a rate constant of 8 s-1 giving an apparent permeability to caffeine of 2 x 10(-3) cm s-1. 5. This method was used to measure [caffeine]i and [Ca2+]i simultaneously during caffeine-induced contractures. The shape of the caffeine contracture was found to depend on both the speed of application of caffeine and the concentration applied. If caffeine was applied quickly then the contracture developed within 1 s to a maximum level and then relaxed to a lower maintained level. With slower application, there was a more complete relaxation of the initial contraction followed by a slower redevelopment of contraction. 6. Despite the difference in contraction time course, irrespective of the flow rate, [Ca2+]i decayed monotonically. The slow secondary development of contraction has the same time course as the increase of [caffeine]i. The caffeine contracture can be reproduced by a model in which both [Ca2+]i and [caffeine]i affect contraction. 7. The increase of [Ca2+]i is not greatly affected by altering the caffeine concentration from 2.5 to 50 mM. In contrast the maintained level of contraction increases over this range showing that the Ca2(+)-independent effects of caffeine on the myofilaments have a low affinity for caffeine.
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