Endothelial cells (ECs), which line all blood vessels, are uniquely positioned to detect neurohumoral and mechanical signals and transform them into intracellular molecular signals that induce relaxation of the surrounding smooth muscle. EC Ca2+ plays a pivotal role in initiating a vasodilatory signal. There is a growing appreciation that increases in Ca2+ that are important for this vasoregulatory function are those that occur locally, not globally. An important source of such local Ca2+ increases in ECs is Ca2+ influx through TRPV (transient receptor potential vanilloid) channels on EC membranes. I recently discovered elementary Ca2+ influx events through single TRPV4 channels-Ca2+ sparklets-in ECs from intact mesenteric arteries (Sonkusare et al., Science, 2012), and further demonstrated that cooperative opening of as few as three TRPV4 channels per EC causes maximal vasodilation through activation of intermediate- and small- conductance, Ca2+-sensitive potassium channels in ECs. Although there is evidence for TRPV1 and V3 channels in ECs, their elementary properties and physiological modulators have not been explored. The ability to monitor unitary Ca2+ influx through TRPV1/V3/V4 channels is a powerful tool for investigating the physiological and pathological roles of these channels. My preliminary data show for the first time that activation of TRPV1 and V3 channels with selective agonists produces sparklets with biophysical properties that are distinct for each channel type. Moreover, physiological modulators, such as Gq protein-coupled receptor (GqPCR) agonists and temperature, differentially activate TRPV1/3/4 channels. The proposed research tests the hypothesis that TRPV1/V3/V4 channels are differentially modulated by physiological signals and differentially engage effector pathways to regulate vascular function, and should reveal pathways of local vasodilatory communication in ECs. As such, these studies will lay a solid foundation for understanding pathological mechanisms responsible for endothelial dysfunction in vascular disorders such as hypertension, diabetes, and atherosclerosis. During the mentored phase, I will employ state-of-the-art electrophysiology and high-resolution confocal Ca2+ imaging, a novel optogenetic approach and in vivo imaging of EC Ca2+ and arterial diameter to determine the distinct biophysical signatures of TRPV1/V3 channels and investigate the modulation of TRPV4 channels by GqPCR signaling. During this phase, I will also continue my professional and scientific career development with continued guidance from my advisory committee. During the independent phase, I will use Ca2+ imaging, pressure myography and EC patch-clamp to study differential activation of TRPV1/V3 channels by flow, temperature, and GqPCR signaling, and pathways downstream of TRPV1/V3 that mediate vasodilation to these modulators. This project will facilitate my continued technical, intellectual, and professional training, and will asist me in establishing an independent research laboratory at an academic research institute.
The cell layer (endothelium) that lines small blood vessels (arteries) regulates vascular function, serving as both a modulator of blood flow, and a physical barrier to the surrounding tissue. The endothelial function is critically regulated by Ca2+ in the cells. This project identifies novel signaling pathways in endothelial cells that increase Ca2+ inside the cells, relax arteries and regulate blood flow.
