Increase in endothelial cell Ca 2+ in response to mouse cremaster muscle contraction

We addressed the role of endothelial cells (ECs) in metabolic dilatation of skeletal muscle arterioles in anaesthetized mice in situ . Electrical field stimulation was used to contract the cremaster muscle for 15 s at 30 Hz. Diameter was observed using bright field microscopy. In controls, muscle contraction produced a 15.7 ± 1.5 μm dilatation from a baseline of 17.4 ± 1.6 μm. Endothelial denudation (−EC) via intraluminal perfusion of air abolished this response (1.6 ± 1.2 μm in −EC, P < 0.05), identifying endothelium as the primary vascular cell type initiating the dilatation. To investigate the role of EC Ca 2+ in metabolic dilatation, arteriolar ECs were loaded with Fluo‐4 AM or BAPTA AM by intraluminal perfusion, after which blood flow was re‐established. Ca 2+ activity of individual ECs was monitored as a function of change from baseline fluorescence using confocal microscopy. In ECs, whole cell Ca 2+ increased (>10%, P < 0.05) during muscle contraction, and localized Ca 2+ transients were increased (>20%, P < 0.05) during the first minute after contraction. Chelation of EC Ca 2+ abolished the dilatations in response to muscle contraction (1.1 ± 0.7 μm, P < 0.05). Inhibition of P 1 purinergic receptors (with xanthine amine congener) did not alter the rate of onset of the dilatation ( P > 0.05) but decreased its magnitude immediately post stimulation (7.1 ± 0.9 μm, P < 0.05) and during recovery. These findings demonstrate obligatory roles for endothelium and EC Ca 2+ during metabolic dilatation in intact arterioles. Furthermore, they suggest that at least two separate pathways mediate the local response, one of which involves stimulation of endothelial P 1 purinergic receptors via endogenous adenosine produced during muscle activity.