Abstract 1429: The Proline-Rich Tyrosine Kinase PYK2 Regulates NO Production by eNOS under Physiologic and Pathophysiologic Conditions.
In endothelial cells, the proline-rich tyrosine kinase (PYK2) is activated by fluid shear stress and phosphorylates the endothelial NO synthase (eNOS) on tyrosine residue 657. We have previously shown that eNOS phosphorylation on Tyr657 inhibits NO production in vitro and therefore addressed the consequences of PYK2 activation in a more physiological situation i.e., in perfused isolated carotid arteries as well as in response to other stimuli. Pressurised perfused murine carotid arteries subjected to step-wise increases in luminal flow responded with a graded endothelium-dependent vasodilatation that was partially sensitive to NOS inhibition. Carotid arteries in which the endothelium was infected with an adenovirus expressing a dominant-negative PYK2 mutant displayed normal flow-dependent vasodilatation (maximal response: Rmax 49 ± 6 μm) that was sensitive to the NOS inhibitor L-NAME (Rmax 28 ± 2 μm). However, flow-induced vasodilatation was markedly reduced in arteries overexpressing wild-type PYK2 and was insensitive to L-NAME (Rmax 27 ± 4 μm and 28 ± 5 μm, respectively) indicating loss of the NO-dependent component of vasodilatation. Given that angiotensin II is known to stimulate PYK2 phosphorylation and activation in smooth muscle cells we assessed the effects of angiotensin II on endothelial PYK2 activation and NO-dependent vasodilatation. We observed a time-dependent phosphorylation of PYK2 immuno-precipitated from porcine aortic endothelial cells stimulated with angiotensin II for 30 min up to 24 hrs. This correlated with an angiotensin II-induced decrease in NO-dependent vasodilatation. These data indicate that under physiological conditions, the tyrosine phosphorylation of eNOS by PYK2 in response to fluid shear stress attenuates the activity of the enzyme and limits NO-mediated flow-dependent vasodilatation. This PYK2-dependent inhibition of NO production may at least partially limit the detrimental consequences of maintained high NO output e.g. the generation of peroxynitrite. Conversely, under pathophysiological conditions in which angiotensin II levels are elevated, the increased activity of PYK2 can contribute to endothelial dysfunction by reducing eNOS activity.