Caffeine and the contraction of frog heart.

The thesis reports an investigation of the response of frog heart muscle to the methylxanthines, with special emphasis on caffeine. Contractile and electrical responses were recorded from perfused, regularly stimulated preparations of both the ventricle and atrium, which respond rapidly to changes in the ionic composition of the bathing medium. In addition, an investigation of the ultrastructure of the atrial trabecula was made by electronmicroscopy. Twitch tension in the sub-optimally beating ventricle is enhanced by caffeine (c. 0.1 to 10 mM plus) and the other methylxanthines with an accompanying increase in the maximum rate of rise and fall of tension and reduction in time to peak tension. The change in time to peak tension was shown to result from a reduction in the duration of the action potential and was not causally related to the increase in twitch tension. The time course of peak twitch tension changes could be fitted by a single exponential component when the reduction in time to peak tension was small enough to render the evaluation significant. The half-time for this process was some ten times slower (at 20 C) than that of exchange in the extracellular space in these preparations and was independent of drug concentration, calcium concentration, twitch tension and stimulus frequency. The response of the ventricle to a reduction of [Ca] can be fitted by three exponential components in time, the middle time constant has a value very close to that for the response to caffeine. Enhancement of twitch tension by caffeine is shown in several ways not to result from an increase in calcium influx. It was deduced, therefore, that as in skeletal muscle, caffeine releases calcium ions from an internal site. When contracture has been evoked by high-K or zero-Na Ringer's solution in atrial trabeculae, tension spontaneously relaxes even in the continued presence of the contracture solution. Caffeine will then produce a redevelopment of tension which is similarly transient. The dose-dependence of the enhancement of contractility (both for contracture production and twitch-tension increase) is consistent with caffeine having a single site of action. Under zero-Na conditions at least, the contracture response to caffeine is independent of the external calcium concentration and will occur in effective absence of ionised calcium ([Ca] less than 10-7 to 10-8 M). However, the caffeine contracture requires the previous occurrence of the contracture induced by high-K or zero-Na Ringer and does depend upon the calcium concentration existing before caffeine is added. It is suggested that, in the process of relaxation, internal sites become replete with calcium which can subsequently be released by caffeine. A preliminary investigation of the ultrastructure of the atrial trabecula shows that sarcoplasmic reticulum exists in amounts which may be sufficient to provide the internal site for the action of caffeine. The second part of the thesis concerns a study of the activity of the other methylxanthines in relation to the results for caffeine from the first part f the work. A simple molecular feature is found to differentiate which of the methylxanthines are able to enhance contractility. All active methylxanthines (e.g. theophylline, theobromine, paraxanthine, and caffeine) are similar in their potency. Inactive methylxanthines are alkylated at the 9-position nitrogen atom which appears in the imidazole ring of the xanthine base. A test of the hypothesis derived from these results was made by experiments with imidazole-based compounds (e.g. Histamine). The production of responses comparable to methylxanthine-induced contractures confirmed the hypothesis that an uncombined ring nitrogen atom in the 'lone-pair' configuration is the feature which confers inotropic activity to these compounds. Direct and indirect evidence is also presented that 3'5' cyclic-AMP is not involved in the responses of frog heart to the methylxanthines.