Dissociation of P2 purinoceptor-mediated increase in intracellular Ca2+ level from myosin light chain phosphorylation and contraction in rat aorta.
1. The effects of P2 agonists, adenosine-5'-triphosphate (ATP), alpha, beta-methylene-adenosine-5'-triphosphate (alpha, beta-me-ATP) and adenosine 5-O-(3-thiotriphosphate) (ATP gamma S), 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 microM-1 mM), alpha, beta-me-ATP (0.1-100 microM) and ATP gamma S (1-100 microM) 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), ATP gamma S (10 microM) induced large transient increase in [Ca2+]i with only small contraction in Ca(2+)-free solution. In contrast, alpha, beta-me-ATP (10 microM) induced only a very small increase in [Ca2+]i and contraction. 4. ATP (1 mM), alpha, beta-me-ATP (10 microM) and ATP gamma S (10 microM), added during stimulation with 0.1 microM 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 alpha, beta-me-ATP (10 microM) and ATP gamma S (10 microM), 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+]i does not contribute to these responses.
Kitajima
S
S
Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Japan.
Harada
K
K
Hori
M
M
Ozaki
H
H
Karaki
H
H
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D008297:Male; D009119:Muscle Contraction / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D011983:Receptors, Purinergic / Q000187:drug effects*; D013997:Time Factors
D018994:Myosin Light Chains; D011983:Receptors, Purinergic; D000255:Adenosine Triphosphate; D002118:Calcium