The effects of caffeine on sodium transport, membrane potential, mechanical tension and ultrastructure in barnacle muscle fibres.

1. The effects of graded concentrations of caffeine on the Na efflux were investigated. External application of 10 mM caffeine usually caused a biphasic response, viz. a fall, followed by a rise in the Na efflux. 1 and 5 mM caffeine usually caused stimulation. Only the stimulatory phase of this response depended on the presence of external Ca(2+).2. Internal application of 100 mM caffeine caused a small rise in the Na efflux, the magnitude of which was independent of external Ca(2+) and comparable to that obtained with external application of 1 mM caffeine. This action, however, could be greatly augmented by pre-treating the fibre with 5 x 10(-5)M ouabain.3. The rise in Na efflux caused by external application of 10 mM caffeine could be greatly augmented by pre-treating the fibre with 5 x 10(-5)M ouabain. The observed stimulatory response was biphasic, more so in the absence of external Ca(2+). Restoration of external Ca(2+) following the onset of the second stimulatory phase resulted in further rise of the Na efflux. Measurements of the Na efflux during treatment with graded concentrations of ouabain and 10 mM caffeine showed that the rate coefficient for Na efflux varied with the ouabain concentration in the range 10(-8)-10(-4)M. Measurements of the ouabain-insensitive Na efflux before and during treatment with 10 mM caffeine in bathing media containing varying concentrations of Ca, disclosed the existence of two Ca(2+)-thresholds, one in the 0-2.5 mM range and the other in the 12.5-15 mM range.4. Comparisons were made between the effects on the Na efflux of 10 mM caffeine followed by external acidification, and external acidification, followed by 10 mM caffeine. The magnitude of the response of the ouabain-insensitive Na efflux to external acidification before treatment with 10 mM caffeine was greater than that found when external acidification followed external application of the alkaloid. It also was considerably greater than that of the response to external application of 10 mM caffeine before external acidification.5. External application of 10 mM procaine prevented 10 mM caffeine from stimulating the Na efflux, and from inducing contractures. Internal application of 100 mM-EGTA reduced the response of the Na efflux to 10 mM caffeine, and also prevented the fibre from contracting. External application of 10(-4)M diphenylhydantoin reduced the response of the Na efflux to 10 mM caffeine but failed to prevent the development of contractures.6. Internal application of 0.05 M-cGMP, cAMP or its dibutyryl derivative caused a large rise in the Na efflux. The magnitude of the effects observed in ouabain-poisoned fibres was often greater than that in unpoisoned fibres. Internal application of 2.5 units/ml. phosphodiesterase beforehand failed to reduce the magnitude of the stimulatory response to injected cyclic nucleotides. Injected phosphodiesterase also failed to reduce the response of the Na efflux to 10 mM caffeine.7. External application of 10 mM caffeine to unpoisoned and ouabain-poisoned fibres caused a fall of approximately 10 mV in the membrane potential. In unpoisoned fibres this effect was transitory. The response of the membrane potential to internal application of graded concentrations of CaCl(2) was biphasic. When low concentrations of CaCl(2) were used the membrane potential underwent a small rise but when high concentrations were used the opposite was found. These results could not be repeated with graded concentrations of MgCl(2).8. The effects of graded concentrations of caffeine on tension development were also studied. Strong contractures were observed with caffeine concentrations as low as 4 mM, while peak tetanus tension was usually exceeded with 7-8 mM concentrations. The tension-external Ca(2+) curve was sigmoidal in shape.9. Electron microscopic studies showed that 10 mM caffeine in ASW caused little or no distension and disorganization of cisternal fine structure. Such structural changes, however, were far more pronounced in fibres suspended in Ca(2+)-free ASW and then treated with 10 mM caffeine in Ca(2+)-free ASW. Fibres soaked in Ca(2+)-free ASW had ruptured mitochondria and mitoplasts, whereas those additionally treated with 10 mM caffeine had relatively intact mitochondria.10. The main conclusions drawn from this work are: (i) that caffeine stimulates the ouabain-insensitive Na efflux (and inhibits the transport enzyme) by raising the internal free Ca(2+) concentration; (ii) that in the presence of inhibition of the transport enzyme, the magnitude of the stimulatory response to 10 mM caffeine depends not only on the external Ca(2+) concentration but mainly on the residual level of activity of the transport enzyme; (iii) that the Ca(2+)-sensitive and CO(2)-sensitive components of the ouabain-insensitive Na efflux, though not the same, may overlap at the level of the plasma membrane or share a common metabolic step away from the membrane; (iv) that cyclic nucleotides participate in the control of the magnitude of the ouabain-insensitive Na efflux, and that the phosphodiesterase system under the present experimental conditions does not seem to be involved in the mechanism underlying the stimulatory action of caffeine; (v) that the membrane potential changes caused by caffeine fail to explain the stimulatory response of the Na efflux, as well as contracture of these fibres; (vi) that the contractile machinery in these fibres is considerably more sensitive to caffeine than that in vertebrate muscle and the muscles of other arthropods, and (vii) that the Ca(2+) released by the sarcoplasmic reticulum which leads to a biphasic response of the Na efflux is the result of cisternal disorganization caused by caffeine.