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Effects of isoproterenol and forskolin on tension, cyclic AMP levels and cyclic AMP-dependent protein kinase activity in bovine coronary artery Vegesna, Venkata Krishnam Raju


The effects of isoproterenol and forskolin on tension, cyclic AMP (cAMP) levels, and cyclic AMP-dependent protein kinase (cA kinase) activity were compared in helical strips of bovine coronary artery. Isoproterenol and forskolin produced time-dependent and dose-dependent increases in cAMP levels and both compounds relaxed potassium-contracted arteries. Relaxation and cAMP elevation appeared to be well correlated in the isoproterenol experiments. However, in contrast to the results with isoproterenol, cAMP elevation and relaxation were not well correlated at lower concentrations of forskolin. For example, 0.1 μM forskolin increased cAMP levels in the arteries by approximately 5.5 fold but did not relax the muscles. A smaller elevation of cAMP produced by 1.0 μM isoproterenol, on the other hand, was accompanied by an almost complete relaxation of arteries. Physiological processes which are thought to be mediated by cAMP are assumed to be a consequence of selective activation of cyclic AMP-dependent protein kinase (cA kinase). It is possible that low doses of forskolin may elevate cyclic AMP in a compartment which does not have access to these kinases, whereas, higher doses may increase cAMP levels in all compartments. In order to investigate this possibility, optimal conditions were established in our laboratory for the measurement of cA kinase activity in coronary arteries. It has been suggested that addition of 0.5 M NaCl to the buffer used for homogenization of coronary arteries is essential in order to demonstrate hormonal effects on cA kinase in this tissue. However, under our experimental conditions, increasing the ionic strength in the homogenization buffer significantly inhibited total cA kinase activity. Interestingly, in agreement with previous reports in other tissues, an apparent translocation of cA kinase activity from the cytosol to the particulate fraction was observed under conditions associated with high tissue levels of cAMP. For example, relaxation caused by isoproterenol was accompanied by increased cA kinase activity in the particulate fraction and decreased activity in the soluble fraction of the coronary arteries. A similar shift in the distribution of the kinase was caused by various concentrations of forskolin, irrespective of whether the arteries were relaxed or not. Thus, as was observed with cAMP levels, activation of cA kinase did not always correlate well with relaxation in this tissue. Other workers have suggested that low doses of forskolin can potentiate hormonally-induced effects in several tissues. It was suggested that such a potentiation would constitute evidence in favour of a role for cAMP in the hormonal response. However, in the present experiments in coronary arteries, it was found that low doses of forskolin, which significantly increased cAMP levels, did not potentiate isoproterenol-induced relaxation or elevation of cAMP. Only higher doses of forskolin (≥ 1 μM) potentiated isoproterenol-induced cAMP generation. These results suggest that caution should be exercised in utilizing forskolin as a tool in elucidating a role of cyclic nucleotides in vascular smooth muscle function. Our results suggest that cAMP may not be the only mechanism responsible for relaxation of coronary arteries by these drugs or alternatively, that some form of functional compartmentalization of cAMP and cA kinase exists in this tissue.

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