Calcium measurement in isolated arterioles during myogenic and agonist stimulation

Abstract

Vascular smooth muscle calcium was measured during agonist treatment or pressure-induced stimulation of the myogenic response in isolated first-order skeletal muscle arterioles. Arterioles (40-180 microns) with spontaneous tone were isolated from rat cremaster muscle and cannulated. Arterioles were loaded with the calcium-sensitive dye fura-2 and excited at 340 and 380 nm. Images of vessel fluorescence were formed with a fluorescence microscope and digitized using an image processor coupled to a low light level camera. The fluorescent images allowed individual vascular smooth muscle cells to be seen within the arteriolar wall. Fluorescent intensity of the vessel wall, expressed as the ratio of fluorescence at 340 nm/380 nm, was used to estimate changes in vessel wall calcium. Topical application of norepinephrine (10 microM) to the arterioles caused a rapid and sustained constriction of the arterioles (64% of basal diam). The calcium response was biphasic consisting of a transient spike to 271% of basal followed by a decrease to a new steady state at 143% of basal. In comparison, steady-state indolactam (1 microM) produced a similar degree of constriction without an increase in calcium. Adenosine significantly dilated (35%) the arterioles and produced a decrease (24%) in vessel wall calcium. To investigate the myogenic response, intravascular pressure was step increased from 90 to 130 cmH2O. Increasing intravascular pressure caused an initial increase in vessel diameter of approximately 5% followed by active constriction that returned diameter to basal diameter. In association with this diameter change, estimated vessel wall calcium increased rapidly 8 +/- 2% and then continued to increase more slowly and remained elevated at 10-15% above basal levels. This study demonstrates the successful application of calcium-imaging technology in isolated arterioles for study of the role of calcium in arteriolar function. Results indicate that the calcium-contraction relationship differs for different agonists and are further consistent with a role for pressure-induced increases in vascular smooth muscle calcium during the myogenic response.