Dissociation of P2 purinoceptor-mediated increase in intracellular Ca2+ level from myosin light chain phosphorylation and contraction in rat aorta

Abstract

1 The effects of P2 agonists, adenosine-5′-triphosphate (ATP), α,β-methylene-adenosine-5′-triphosphate (α,β-me-ATP) and adenosine-5-O-(3-thiotriphosphate) (ATPγS), on the intracellular free Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction were examined in vascular smooth muscle of rat aorta. 2 ATP (0.1 μM-1 mM), α,β-me-ATP (0.1-100 μM) and ATPγS (1-100 μM) induced transient increases followed by sustained increase in [Ca2+]i. The effects of these agonists were concentration-dependent. Compared with the effects of a high concentration of KCl (17.5-72.4 mM), the contractions induced by these P2 purinoceptor agonists were smaller at a given [Ca2+]i. 3 In the absence of extracellular Ca2+ (with 0.5 mM EGTA), ATPγS (10 μM) induced large transient increase in [Ca2+]i with only small contraction in Ca2+-free solution. In contrast, α,β-me-ATP (10 μM) induced only a very small increase in [Ca2+]i and contraction. 4 ATP (1 mM), α,β-me-ATP (10 μM) and ATPγS (10 μM), added during stimulation with 0.1 μM noradrenaline, induced additional and transient increases in [Ca2+]i which were also not associated with contraction. 5 High K+ (72.4 mM) increased MLC phosphorylation with a similar time course to that of the increase in [Ca2+]i (peak phosphorylation was 56% when [Ca2+]i increased to 100%). In contrast, the time course of the increase in MLC phosphorylation due to ATP (1 mM) did not coincide with that of the large increases in [Ca2+]i; MLC phosphorylation increased to only 31% when [Ca2+]i increased to 163%. The MLC phosphorylation due to α,β-me-ATP (10 μM) and ATPγS (10 μM), measured at peak [Ca2+]i, were only 19% and 14%, respectively, irrespective of a large increase in [Ca2+]i (138% and 188%, respectively). 6 The absence of a clear relationship between P2-purinoceptor-mediated increase in [Ca2+]i (either by Ca2+ influx or Ca2+ release) and MLC phosphorylation or force generation appears to imply that elevation in [Ca2+], does not contribute to these responses.