Abstract 928: Calcium-Dependent Tyrosine Kinase PYK2 Plays Crucial Role in Angiotensin II- But Not Norepineprine-Mediated Activation of Rac/Superoxide Pathway That Differentially Regulates Blood Pressure and Vasoconstriction
Background: AngII has been shown to be a key mediator in cardiovascular disease. Most of the effect of AngII is mediated by angiotensin type-I receptor (AT1R). The stimulation of AT1R caused vaso-constriction, NO-inactivation, cellular proliferation, and inflammation, leading to cardiovascular insufficiency. It has been reported that intracellular tyrosine kinases play crucial roles to transmit these versatile effects of AngII. We previously reported that PYK2 is involved in AT1R-induced activation of Rac/JNK. And also PYK2 is concerned with reactive oxygen species production. So we expected that PYK2 plays impotant roles in AT1R signaling, and analysed the PYK2-knockout mice (KO) that were newly generated.
Methods and Results: Basal blood pressure (BP) was higher in KO than the wild-type mice (WT) (mean 80 ± 2 vs. 74 ± 2 mmHg, P<0.05, n=10, each). Subcutaneous pump infusion of AngII (1.1mg/kg/day) increased BP in WT, whereas this increase was abolished in KO (mean BP, Day 7: 115 ± 4 vs. 88 ± 5, p<0.05, Day 14: 128 ± 4 vs. 95 ± 8mmHg, p<0.05). Infusion of norepinephrine (NE, 5.6mg/kg/day) increased BP in KO to the level comparable to WT. The level of superoxide in the aorta was measured using L-012 chemiluminescence. Although the basal level was similar between WT and KO, AngII infusion elevated the level of superoxide 6.9-fold in WT (p<0.005), whereas the elevation in KO was only 2.1-fold (p<0.05 vs. AngII-infused WT). NE-mediated elevation of superoxide was much smaller than that in AngII and similar between KO and WT (1.7-fold vs. 1.6-fold elevation from the baseline). Tyrosine phosphorylation of PYK2 in WT aorta was increased after AngII infusion, whereas its phosphorylation was lower after NE infusion. Activities of Rho and Rac in Ang-II-infused aorta were evaluated by ‘pull-down assay’. They increased 3.2-fold and 3.4-fold in WT, respectively, whereas the increases in KO were only 1.4-fold and 1.3-fold, respectively. And also AngII infusion increased Nox(1,2,4) expression in WT aorta, whereas the increase in KO was significantly smaller (p<0.05, respectively)
Conclusion: PYK2 plays critical role in Ang-II-mediated, but not Norepineprine-mediated activation of Rac/superoxide pathway that differentially regulates blood pressure and vascular function.