Abstract 1028: Pivotal Role of Transglutaminase in Non-genomic Vasoconstrictor Effect of Aldosterone: Dimerization of Angiotenin II Type 1 Receptor in Smooth Muscle
Aldosterone (Aldo) exerts a vasoconstrictor effect by non-genomic mechanisms, but its precise mechanism is still unclear. Recent evidence shows that Aldo amplifies angiotensin II type 1 (AT1) signaling in vascular smooth muscle cells (VSMC), whereas transglutaminase (TG) enhances AT1 signaling by crosslinking AT1 receptor homodimers in monocytes. We investigated the role of TG and AT1 receptor dimerization in Aldo-mediated, non-genomic vasoconstrictor response of the mesenteric arterioles. The mesenteric arterioles (60 –160 μm) were isolated from C57BL/6J mice (WT) and AT1a knockout mice (AT1KO) at the age of 15–25 weeks, cannulated, and pressurized at 60 cmH2O. The effect of Aldo (10–13 to 10−6 M) on the vessel diameter was examined in endothelium-intact or endothelium-disrupted arterioles. Then the effects of spironolactone (10−6M) and 2 kinds of TG inhibitors, cystamine (5 × 10−4M) and monodansyl cadaverine (10−4M), on Aldo-induced vasomotor response were examined. Aldo produced a dose-dependent vasoconstriction in WT and the maximum diameter change was −7.2 ± 2.0% from the baseline (n = 24, p<0.05). This vasoconstrictor effect of Aldo was unaffected by spironolactone and denudation of the endothelium, suggesting that it is non-genomic and endothelium-independent. In AT1KO, vasoconstrictor effect of Aldo was completely abolished. Both TG inhibitors abolished the vasoconstrictor effect of Aldo (n=4, p<0.05) without affecting the vasoconstrictor effect of angiotensin II in WT. To examine the effect of Aldo on the formation of the AT1 dimer, WT mesenteric arterioles were exposed to 10−7 M Aldo for 15 minutes, and immunoblotted with AT1-specific antibody. AT1 dimer protein levels were increased in the arterioles treated with Aldo, and TG inhibitors blocked the Aldo-induced formation of AT1 dimer. To measure the cytosolic TG activity, in situ TG activity assay was subjected to cultured VSMC from WT. Treatment with 10−7 M Aldo for 15 minutes induced the increase in TG activity by 2.3 ± 0.7 fold (n = 8, p<0.05). These suggest that Aldo causes non-genomic, endothelium-independent vasoconstriction by inducing TG activation and formation of AT1 dimer in the mice mesenteric arterioles.