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Studies on the role of cyclic AMP in the regulation of vascular smooth muscle tension

University of British Columbia

The precise role of cyclic AMP (cAMP) in the regulation of smooth muscle contraction has been a subject of controversy for the last two decades. It has been hypothesized that drug-induced elevation of cAMP levels is responsible for vascular smooth muscle relaxation. In the present studies this hypothesis was examined in vascular smooth muscle (1) by comparing the effects of prostacyclin (PGI₂) on cAMP levels and tension in two different vascular smooth muscles (bovine coronary arteries and rabbit aortic rings) and (2) by studying the effects of prostaglandin E1(PGE₁), isoproterenol and forskolin on cAMP levels, cyclic AMP-dependent protein kinase activity and tension in rabbit aortic rings. In bovine coronary arteries, PGI₂ elevated cAMP levels and relaxed the potassium-depolarized muscles. The PGI₂-induced cAMP elevation preceded the relaxation and both parameters were altered in a dose-dependent manner by increasing concentrations of PGI₂ (0.3, 3, and 30 μM). These results are consistent with a role for cAMP as a mediator of vascular smooth muscle relaxation. Cyclic AMP levels were also elevated by PGI₂ in a concentration- and time-dependent manner in rabbit aortic rings. However, in direct contrast to the-results in the bovine coronary arteries, PGI₂-induced elevation of cAMP in the aortic rings was accompanied by contraction rather than relaxation. Isoproterenol, a drug which is generally believed to relax smooth muscles by virtue of its ability to increase tissue cAMP levels, relaxed PGI₂-contractedaortic rings with no further elevation of cAMP beyond that caused by the PGI₂ alone. These results indicate that drug-induced elevation of cAMP in vascular smooth muscle is not always accompanied by relaxation. Forskolin, a direct stimulant of adenylate cyclase, has been suggested to be a valuable tool for elucidating the role of cAMP in various physiological processess. We studied the effects of forskolin, PGE₁ and isoproterenol on cAMP levels and tension in rabbit aortic rings to further examine the relationship between drug-induced elevation of cAMP levels and tension in vascular smooth muscle. PGE₁, isoproterenol and forskolin all increased cAMP levels in rabbit aortic rings. Isoproterenol and forskolin relaxed phenylephrine-contracted aortic rings, but PGE₁ contracted the rings in the presence or absence of phenylephrine. Isoproterenol relaxed these PGE₁ contracted aortic rings without a further change in the total cAMP levels, which were already elevated by PGE₁ alone. Pretreatment with forskolin potentiated the effects of PGE₁ on cAMP levels. PGE₁ contracted muscles partially relaxed by forskolin even though very large increases in cAMP levels (30 fold) were produced by PGE₁ in the presence of forskolin. Isoproterenol was able to relax these forskolin-stimulated, PGE₁-contracted muscles with no further increase in cAMP levels. Thus, based on estimations of total tissue levels of cAMP, there does not appear to be a good correlation between changes in cAMP levels and tension in rabbit aortic rings under various conditions. 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). As prevously noted in cardiacmuscle, a differential activation of cA kinase in specific compartments (soluble vs particulate) by different drugs might possibly explain the differences in pharmacological responses observed in our experiments on rabbit aorta. In order to investigate this possibility, we studied the effects of isoproterenol, PGE₁ and forskolin on soluble and particulate cA kinase activity in rabbit aortic rings. A concentration of isoproterenol which produced a moderate increase in cAMP levels did not change the protein kinase activity in the soluble fraction. This could be partly due to the technical limitations of the assay. Both forskolin and PGE₁ significantly increased the kinase activity although they exerted opposite effects on the tension of the preparations. Isoproterenol relaxed the PGE₁-contracted muscles without any further activation of the kinase. Forskolin potentiated the effects of PGE₁ on protein kinase activity but PGE₁ still contracted the forskolin-relaxed aortic rings. Once again, isoproterenol was able to relax these preparations without further activating the kinase. Thus, as was the case with the cAMP data described above, activation of the kinase in the soluble fraction of aortic rings occurred whether the muscles were relaxed or contracted. It was anticipated that a difference in activation of cA kinase in the particulate fraction might possibly explain our results. However, the increase in cA kinase activity in the pellet appears to be the same with isoproterenol and PGE₁, alone or in combination. Significant increases in cA kinase activity were observed with forskolin and also with the combination of forskolin, PGE₁ and isoproterenol in the particulate fraction. Our results thus demonstrate a clear dissociation between tension, elevation of cAMP and activation of cA kinase under various conditions in rabbit aortic rings. It can be suggested that elevation of cAMP and activation of cA kinase may not be directly responsible for the regulation of vascular smooth muscle tension or, alternatively, that some form of functional compartmentation of cA kinase might exist in this tissue. Finally, an attempt was made to study the role of calcium in phenylephrine- and PGE₁-induced contractions and the effect of isoproterenol under these conditions. Phenylephrine produced a phasic contraction and PGE₁ produced a sustained contraction in the absence of extracellular calcium, suggesting that intracellular calcium is partially responsible for these contractions. Pretreatment with isoproterenol resulted in complete inhibition of the phenylephrine-induced phasic contraction and also relaxed the PGE₁-induced sustained contraction under similar conditions. These results suggest that at least part of isoproterenol's relaxant effect is mediated by an action at an intracellular site in rabbit aorta.

Text

2010-08-09

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http://hdl.handle.net/2429/27219

Pharmaceutical Sciences

University of British Columbia

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