This study examined whether elevated intravascular pressure stimulates asynchronous Ca2+ waves in cerebral arterial smooth muscle cells and if their generation contributes to myogenic tone development. The endothelium was removed from rat cerebral arteries, which were then mounted in an arteriograph, pressurized (20-100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (VM) were monitored using conventional techniques; Ca2+ wave generation and myosin light chain (MLC20)/MYPT1 (myosin phosphatase targeting subunit) phosphorylation were assessed by confocal microscopy and Western blot analysis, respectively. Elevating intravascular pressure increased the proportion of smooth muscle cells firing asynchronous Ca2+ waves as well as event frequency. Ca2+ wave augmentation occurred primarily at lower intravascular pressures (2+, eliminated these events. Ca2+ wave generation was voltage insensitive as Ca2+ channel blockade and perturbations in extracellular [K+] had little effect on measured parameters. Ryanodine-induced inhibition of Ca2+ waves attenuated myogenic tone and MLC20 phosphorylation without altering arterial VM. Thapsigargin, an SR Ca2+-ATPase inhibitor also attenuated Ca2+ waves, pressure-induced constriction and MLC20 phosphorylation. The SR-driven component of the myogenic response was proportionally greater at lower intravascular pressures and subsequent MYPT1 phosphorylation measures revealed that SR Ca2+ waves facilitated pressure-induced MLC20 phosphorylation through mechanisms that include myosin light chain phosphatase inhibition. Cumulatively, our findings show that mechanical stimuli augment Ca2+ wave generation in arterial smooth muscle and that these transient events facilitate tone development particularly at lower intravascular pressures by providing a proportion of the Ca2+ required to directly control MLC20 phosphorylation. Tissue blood flow is controlled by a network of resistance arteries. Under normal conditions, the diameter of an artery is regulated by: (1) agents released from nerves and surrounding tissue; and (2) mechanical forces including blood pressure. Bayliss first described the ability of arteries to constrict to blood pressure and since his pioneering work in 1902, studies have been interested in defining the signalling events underlying this important biological response. Using a range of physiological and biochemical techniques, this study showed that Ca2+ waves play an important role in enabling arteries to respond to elevated blood pressure. Ca2+ waves are discrete events that spread in an asynchronous manner from one end of a smooth muscle cell to the other. These events depend on the release of Ca2+ from an internal store called the sarcoplasmic reticulum.
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