Inotropic effect of cyclic AMP in calf ventricular muscle studied by a cut end method.

Abstract

Cyclic[c]AMP was introduced into ventricular muscle by a cut-end method. Trabecular bundles were pulled through a partition which divided the preparation into a loading region and a test region. The loading region was exposed to Ca-free solution, cut transversely near the partition and then briefly exposed to cAMP. The test region was continually superfused with Tyrode solution. In preliminary experiments, cell-to-cell movements were studied in long bundles by including [3H]cAMP in the loading procedure and allowing redistribution to occur. After suitable test periods, the bundles were removed, frozen and sliced into segments. Segment radioactivity was plotted against distance and fitted by a theoretical diffusion curve. The results showed longitudinal redistribution of label over many cell lengths with an average effective diffusivity of 8 .times. 10-7 cm2/s. This value did not appear sensitive to the length of the test period or to the presence of a phosphodiesterase inhibitor. The metabolic fate of cAMP introduced by the cut-end method was determined by chromatographic separation of [3H]cAMP and its break-down products. Most of the cAMP was metabolized, but the results suggest the cell-to-cell movements of cAMP contribute to the overall redistribution of label. The cut-end method was used to study the influence of cAMP on the contractile activity in the test region. Introduction of cAMP evoked a delayed increase in twitch tension, about 25% above control. The inotropic effect peaked about 50 min after the end of the loading procedure, a delay which seemed compatible with slow longitudinal diffusion into the test region. In control experiments, the cut-end procedure was repeated with 5''AMP (the immediate break-down product of cAMP) instead of cAMP. No delayed increase in twitch tension was observed. Introduction of dibutyryl cAMP increased twitch amplitude by 130%, with a delayed time course similar to that found for cAMP. The results using the cut-end procedure provide new evidence that cAMP helps mediate adrenergic effects on the strength of contraction.