Role of myosin phosphorylation and [Ca2+]i in myogenic reactivity and arteriolar tone.

The aim of this study was to define the relationship between intraluminal pressure, intracellular calcium concentration ([Ca2+]i), and myosin light-chain (MLC) phosphorylation in isolated arterioles exhibiting myogenic tone. Cremaster muscles were removed from anesthetized rats, and arterioles (approximately 100-microns diam) were dissected from surrounding tissues and cannulated on glass pipettes. Vessels were warmed to 34 degrees C and initially pressurized to 70 mmHg in the absence of intraluminal flow. For [Ca2+]i measurements, vessels were loaded with 5 microM fura 2, and fluorescence emitted by excitation at 340 and 380 nm was measured. Data were considered in terms of changes in the fluorescence ratio (340/380 nm) and collected at steady-state intraluminal pressures between 30 and 170 mmHg. 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 were visualized by silver staining. MLC phosphorylation was quantitated photodensitometrically, and results are expressed as percent total 20-kDa MLC. Increasing intraluminal pressure resultedin significant constriction with increased [Ca2+]i and MLC phosphorylation. For example, the fluorescence ratio was 0.80 +/- 0.04 at 30 mmHg compared with 1.02 +/- 0.05 at 120 mmHg (n = 7 vessels); corresponding MLC-phosphorylation values were 27.7 +/- 1.6 and 39.6 +/- 3.0% (n = 6). MLC phosphorylation in arterioles superfused with 0 mM Ca(2+)-2 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was 8.5 +/- 0.7%.(ABSTRACT TRUNCATED AT 250 WORDS)