Mechanisms of Endothelial Cell Hyperpolarization

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DESCRIPTION (provided by applicant): Adenosine triphosphate (ATP) is a potent regulator of vascular tone in the cerebral circulation. This physiological agonist is released from a variety of sources in the cardiovascular system under normal and pathological conditions. When presented to the endothelium, ATP stimulates artery dilation through multiple pathways including endothelium-derived hyperpolarizing factor (EDHF) mediated mechanisms. Endothelial cell (EC) hyperpolarization plays a crucial role in EDHF mediated dilation and is a fundamental determinant of arterial tone in multiple vascular beds. While the mechanism by which ECs become hyperpolarized by agonists is still poorly defined, several lines of evidence indicate a significant role for activation of endothelial intermediate-conductance KCa (IKCa) channels. IKCa channels are primarily regulated by cytosolic Ca2+ concentration, but the source of the activating Ca2+ is not known. We propose that ATP stimulates Ca2+ influx through endothelial transient receptor potential (TRP) channels which promotes IKCa channel activation with subsequent EC hyperpolarization and artery dilation. Specifically, we propose to: Aim 1: Define the role of TRP channels in ATP signaling in cerebrovascular endothelial cells. ATP promotes endothelial Ca2+ influx and subsequent dilation via NO and EDHF dependent mechanisms. In this specific aim we will determine if ATP activates TRP channels to promote Ca2+ influx in cerebrovascular endothelial cells. We will identify the TRP channels expressed in ECs (RT-PCR and immunohistochemistry), measure [Ca2+]i and Ca2+ influx in response to ATP in freshly isolated middle cerebral artery (MCA) ECs and in the ECs of pressurized MCA (Fura 2 dye), and demonstrate the role of the identified TRP channels by pharmacological and RNA silencing techniques (organ culture with siRNA). Aim 2: Elucidate the role of TRP channels in IKCa channel activation, EC hyperpolarization, and EDHF-mediated dilation in cerebral arteries. EDHF-mediated dilation of cerebral arteries requires EC Ca2+ influx, IKCa channel activation, and EC hyperpolarization. We will determine the role of Ca2+ influx through TRP channels on IKCa channel activation and EC hyperpolarization. Specifically, we will demonstrate that Ca2+ influx via TRP channels is critical for activation of IKCa channels and subsequent EC hyperpolarization using techniques to measure membrane potential and IKCa channel activation (whole cell patch clamp) in ECs from organ cultured MCA. We will also demonstrate that Ca2+ influx via TRP channels is critical for EDHF-mediated dilation by measuring ATP stimulated dilation in intact organ cultured MCA. These studies should lead to novel therapeutic strategies for controlling blood flow in the brain by defining the role of specific TRP channels in the regulation of EC Ca2+ concentration, EC hyperpolarization, and EDHF-mediated dilation. Adenosine triphosphate (ATP) is released into the cerebral circulation and acts on endothelial cells within cerebral arteries to control blood flow in the brain. However, we do not presently understand the mechanism by which ATP controls this endothelial cell function. These studies will define this mechanism of blood flow control and thus provide novel therapeutic strategies for regulating cerebral blood flow in health and disease states.