PKD proteins in endothelial cells

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Project Summary Endothelial cells line the wall of all blood vessels and regulate a wide variety of functions, including contractility which controls systemic blood pressure. Dysfunctional endothelial cells are a hallmark of several cardiovascular diseases, but pathological mechanisms involved are poorly understood. Endothelial cells express both PKD1 (polycystin-1), an eleven transmembrane domain protein, and PKD2 (polycystin-2), a transient receptor potential (TRP) channel. Regulatory mechanisms, physiological functions and pathological involvement during hypertension of PKD1 protein and PKD2 channels in endothelial cells are unclear. Using a wide variety of approaches and inducible, endothelial cell-specific knockout mice, we provide evidence that physical coupling of PKD1 to PKD2 in endothelial cells stimulates vasodilation. Preliminary data also suggest that PKD1/PKD2 channel signaling is dysfunctional during hypertension, which attenuates this vasodilatory signaling mechanism. In this proposal, we will investigate three specific aims. Aim 1 will test the hypothesis that endothelial-dependent physiological stimuli activate PKD1/PKD2 coupling in endothelial cells, leading to vasodilation. Aim 2 will investigate the mechanisms by which endothelial-dependent stimuli activate PKD1/PKD2 channels in endothelial cells to produce vasodilation. Aim 3 will study the hypothesis that hypertension is associated with pathological alterations in PKD1/PKD2 channel signaling in endothelial cells that inhibits vasodilation mediated by these proteins. Methods used will include RT-PCR, Western blotting, biotinylation, FRET, RNAi, co-IP, immunofluorescence, patch-clamp electrophysiology, membrane potential recording, intracellular Ca2+ imaging, arterial myography and blood pressure telemetry. This project will provide significant novel information concerning vasoregulation by endothelial cell PKD1 and PKD2 proteins.