Arterial diameter is regulated by three distinct intracellular calcium (Ca) signaling modalities that occur in smooth muscle cells. These modalities, termed """"""""Ca2+ sparks"""""""", """"""""Ca2+ waves"""""""" and """"""""global Ca2+ concentration"""""""" ([Ca2+]i), differ with respect to their frequency, amplitude, spatial-temporal properties and physiological functions. Mechanisms that regulate different Ca2+ signaling events in smooth muscle cells are poorly understood. This proposal is based on preliminary data suggesting mitochondria regulate Ca2+ sparks, Ca2+ waves, and global [Ca2+]i in cerebral artery smooth muscle cells, thereby regulating arterial diameter. The unifying hypothesis of this proposal is that mitochondria regulate local and global intracellular Ca2+ signals and Ca2+-activated K+ (Kca) channel activity in cerebral artery smooth muscle cells and arterial diameter by generation of reactive oxygen species (ROS) and permeability transition pore opening. This proposal combines information obtained at molecular, cellular and intact tissue levels.
Four specific aims will be addressed.
Aim 1 will examine the hypothesis that KATP channel openers depolarize mitochondria generating ROS that stimulate Ca2+ sparks and transient Kca currents in cerebral artery smooth muscle cells.
Aim 2 will explore the hypothesis that mitochondria-derived reactive oxygen species activate transient Kca currents.
Aim 3 will determine the functional significance of mitochondria-derived ROS-mediated activation of Ca2+ sparks and transient Kca currents.
Aim 4 will investigate mechanisms by which small and large mitochondrial depolarizations lead to differential regulation of Ca2+ sparks and transient Kca currents. Sparks, waves and global [Ca2+]i will be imaged in arterial smooth muscle cells using laser-scanning confocal Ca2+ imaging. Mitochondrial potential and ROS will be measured by imaging fluorescent indicators. Patch clamp electrophysiology will assess ion channel activity. [Ca2+]i and diameter will be measured in pressurized arteries using simultaneous imaging and edge detection techniques. Since mitochondria modulate both physiological and pathological processes, investigating the regulation of intracellular Ca2+ signaling by these organelles should not only provide a better understanding of mechanisms that regulate arterial diameter, but may provide insights into cardiovascular disorders, including hypertension and stroke. ? ?
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