The Mechanism of Increasing Ca2+ Responsiveness by .ALPHA.1-Adrenoceptor Stimulation in Rat Ventricular Myocytes.

Abstract

It has been reported that ␣ 1 -adrenoceptor stimulation increases contraction with or without a significant change in the peak of Ca 2ϩ transient, and an increase in the Ca 2ϩ responsiveness of the contractile element is considered to be one of the possible mechanisms [1,2]. The increase in the Ca 2ϩ responsiveness of the contractile element by ␣ 1 -adrenoceptor stimulation is explained by intracellular alkalinization or phosphorylation of the contractile elements or both, but the relative contribution of the two mechanisms is still unclear owing to controversy in experimental results [3][4][5][6].For the assessment of Ca 2ϩ responsiveness, a phase-plane loop (trajectory), obtained by measuring the relation between the intracellular Ca 2ϩ transient and cell shortening in a single isolated myocyte loaded with a fluorescent dye, has been widely used [7]. The ␣ 1 -adrenoceptor stimulation shifts the trajectory, in particular the re-lengthening phase, to the left, which suggests an increase in Ca 2ϩ responsiveness. However, the re-lengthening phase of the trajectory, particularly in unloaded shortening, is significantly influenced by repulsion (restoring) force of the elastic components including cytoskeleton, connectin, and other factors rather than the active cross-bridges [8]. Therefore, the re-lengthening phase of the trajectory, in particular the later phase, does not directly reflect the Ca 2ϩ responsiveness measured using the pCa-ten- Key words: Ca 2ϩ sensitivity, phenylephrine, Na/H exchange, intracellular pH, protein kinase C (PKC). Abstract:We investigated the mechanism of ␣ 1 -adrenoceptor stimulation on the myofibrillar Ca 2ϩ responsiveness at steady-state in intact rat ventricular myocytes. We produced tetanus, and an instantaneous plot of [Ca 2ϩ ] i vs. cell length (Ca-L trajectory) was constructed to estimate the Ca 2ϩ responsiveness. An ␣ 1 -agonist, phenylephrine, dose-dependently shifted the Ca-L trajectory to the left, corresponding to sensitization of the myofilaments. An ␣ 1 -antagonist, prazosin, and inhibition of the Na/H exchange by ethylisopropylamiloride (EIPA) completely reversed the phenylephrine-induced shift. Phenylephrine increased pH i (⌬pH i ϭϩ0.1), which was reversed by prazosin and EIPA. Chelerythrine, an inhibitor of protein kinase C (PKC), completely blocked the effects of phenylephrine on Ca 2ϩ responsiveness and pH i . When pH i was increased (⌬pH i ϭ ϩ0.1) without phenylephrine by changing pH o , the Ca-L trajectory was shifted to the same extent as that observed with phenylephrine. We conclude that ␣ 1 -adrenoceptor stimulation activates Na/H exchange through a PKC-mediated pathway and that an increase in pH i is mainly responsible for the increase in Ca 2ϩ responsiveness.

α 1 -Adrenoceptor stimulation potentiates L-type Ca 2+ current through Ca 2+ /calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes

“…1 shows a representative result of the effect of 10 M phenylephrine on I Ca,L . The effect of phenylephrine on the Ca 2ϩ responsiveness of the contractile element in rat ventricular myocytes is almost saturated at this concentration (5). As reported (8, 9), we observed a transient decrease followed by a sustained increase of I Ca,L amplitude.…”

Role of Ca2+/calmodulin-dependent protein kinase II in the regulation of the cardiac L-type Ca2+current during endothelin-1 stimulation

“…Single ventricular myocytes were freshly prepared from adult male Wistar rats (300 -400 g), as described previously (19). Briefly, the rats were anesthetized with inhalation of ether, followed by intraperitoneal injection of pentobarbital.…”

Use of the Ca-Shortening Curve to Estimate the Myofilament Responsiveness to Ca2+ in Tetanized Rat Ventricular Myocytes

“…We used the K d value of 0.24 µM, the R min value of 0.292, and the R max value of 7.43 estimated in previous studies [7,11]. The third parameter, β, was estimated to be 8.95 [7,12].…”

“…Equilibrium between cell length and [Ca 2+ ] i , however, may not always be achieved during twitch relaxation, particularly with interventions that increase the decay rate of the [Ca 2+ ] i transient (e.g., β-adrenoceptor stimulation). Previously, we reported that an instantaneous relation between [Ca 2+ ] i and cell length (Ca-L trajectory) during tetanus could estimate the relative changes in myofibrillar Ca 2+ responsiveness in isolated rat ventricular myocytes [6,7]. For quantitative analysis, we measured the [Ca 2+ ] i , which caused 5% shortening from the resting cell length during the relaxation phase (Ca 5% ) and calculated the change in Ca 5% from the control level.…”