Effects of angiotensin II on intracellular Ca2+ and pH in isolated beating rabbit hearts and myocytes loaded with the indicator indo-1.

1. Angiotensin II increases myocardial contractility in several species, including the rabbit and man. However, it is controversial whether the predominant mechanism is an increase in free cytosolic [Ca2+]i or a change in myofilament Ca2+ sensitivity. To address this question, we infused angiotensin II in isolated perfused rabbit hearts loaded with the Ca2+ indicator indo-1 AM and measured changes in beat-to-beat surface transients of the Ca2+i-sensitive 400:500 nm ratio and left ventricular contractility. The effects of angiotensin II were compared with the response to a Ca(2+)-dependent increase in the inotropic state produced by a change in the perfusate [Ca2+] from 0.9 to 3.6 nM. 2. In the isolated beating heart, an increase in perfusate [Ca2+] caused an increase in left ventricular pressure +dP/dt in association with an increase in peak systolic [Ca2+]i. Angiotensin II perfusion caused a similar increase in left ventricular +dP/dt in the absence of any increase in peak systolic [Ca2+]i. 3. To exclude any contribution of non-myocyte sources of Ca(2+)-sensitive fluorescence which may be present in the intact heart, we also compared the effects of angiotensin II and a change in superfusate [Ca2+] in collagenase-dissociated paced adult rabbit ventricular myocytes loaded with indo-1 AM. In the isolated rabbit myocytes a change in perfusate [Ca2+] from 0.9 to 3.6 mM caused an increase in peak systolic cell shortening coincident with an increase in peak systolic [Ca2+]i. In contrast, angiotensin II caused a similar increase in peak systolic cell shortening whereas there was no increase in peak systolic [Ca2+]i. There was also no change in inward Ca2+ current (ICa) in response to angiotensin II. 4. To investigate further the mechanism of the positive inotropic action of angiotensin II, its effects on intracellular pH were studied in isolated rabbit myocytes loaded with the fluorescent H+ probe SNARF 1. These experiments demonstrated that angiotensin II induced a 0.2 pH unit increase coincident with the development of a positive inotropic effect in isolated rabbit myocytes. 5. In summary, angiotensin II has a direct positive inotropic effect in beating rabbit hearts and in isolated paced rabbit myocytes. These experiments provide support for the hypothesis that the predominant mechanism is not an increase in free cytosolic Ca2+ but is due in part to an increase in myofilament Ca2+ sensitivity due to intracellular alkalosis.