Role of myosin phosphorylation and [ca2+]i in myogenic reactivity and arteriolar tone. Zou, Hui, Paul H. Ratz, and Michael A. Hill. Departments of Physiology and Pharmacology, Eastern Virginia Medical School, Virginia 23501
APStracts 2:0225H, 1995.
The aim of this study was to define the relationship between intraluminal pressure, intracellular calcium ([Ca2+]i), and myosin light chain (MLC) phosphorylation in isolated arterioles exhibiting myogenic tone. Cremaster muscles were removed from anesthetized rats, arterioles (diam. approx. 100[mu]m) dissected from surrounding tissues and cannulated on glass pipettes. Vessels were warmed to 34 degrees C and initially pressurized to 70mmHg in the absence of intraluminal flow. For [Ca2+]i measurements, vessels were loaded with 5 [mu]M Fura-2 and fluorescence emitted by excitation at 340nm and 380nm measured. Data were considered in terms of changes in fluorescence ratio (340/380) and collected at intraluminal pressures (steady state) between 30 and 170mmHg. For measurement of MLC phosphorylation, vessels were frozen in acetone/dry ice followed by sonication in homogenizing buffer. Homogenates were separated by two dimensional gel electrophoresis and proteins visualized by silver staining. MLC phosphorylation was quantitated photo-densitometrically and results expressed as % total 20 kDa MLC. Increasing intraluminal pressure resulted in significant constriction with increased [Ca2+]i and MLC phosphorylation. For example, at 30 mmHg fluorescence ratio was 0.80 0.04 compared to 1.02 0.05 at 120mmHg (n=7); corresponding MLC phosphorylation values were 27.7 1.6% and 39.6 3.0% (n=6). MLC phosphorylation in arterioles superfused with 0 mM Ca2+/2mM EGTA was 8.5 0.7%. To provide further evidence for participation of MLC phosphorylation in steady-state myogenic tone, studies were performed in the absence or presence of the myosin light chain kinase (MLCK) inhibitor ML-7 (n=5). ML-7 caused dose-dependent inhibition of tone but did not prevent pressure-induced increases in [Ca2+]i . Collectively, the results demonstrate a major role for MLC phosphorylation in arteriolar myogenic phenomena and setting of basal tone. 

Received 12 January 1995; accepted in final form 11 May 1995.
APS Manuscript Number H29-5.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on  8 June 1995.