A61603

Hearts were excised into ice-cold modified Krebs-Henseleit perfusion buffer. The aorta was perfused at 80 mmHg with perfusion buffer equilibrated with 95% O₂ and 5% CO₂ at 37°C and pH 7.4. A fluid-filled balloon was inserted via the mitral valve, and inflated to a diastolic pressure of ∼5 mmHg. Hearts were maintained at 37±0.1°C in a water-jacketed bath. Experiments, performed in separate groups, were: 1) perfusion with successively increasing concentrations of A61603 (0.1 nM−1.0 µM; Sigma, Australia), a selective α_1A-AR agonist; 2) before and after perfusion with angiotensin II (AngII, 100 nM, 10 min, MP Biomedicals, Australia) or with one of two selective α_1A-AR antagonists, RS100329 (50 nM, 10 min, Sigma, Australia) or KMD3213 dihydrobromide (100 nM, 10 min, Kissei Pharmaceutical Co. Matsumoto, Japan); 3) for contractile protein measurements, perfusion with saline or RS100329 (50 nM, 8 min), then snap-frozen (liquid nitrogen); 4) for RhoA/ROCK pathway, perfusion with saline or Y-27632, a selective ROCK inhibitor (1 µM, 5 min, Merck Millipore, MA), then snap-frozen; 5) for RhoA/ROCK signaling in agonist-induced responses, A61603 (0.1 nM−1.0 µM) in absence or presence of Y-27632.

TAMRA‒AngII was purchased from AnaSpec. BODIPY FL‒prazosin, biotinylated EGF conjugated to Alexa Fluor 488 streptavidin, and Alexa Fluor 488–conjugated human transferrin were bought from Thermo Scientific. BODIPY FL‒SKF83566 and BODIPY FL‒(S)-propranolol were from HelloBio. Candesartan and SCH 23390 were purchased from Tocris. [Sar¹,Ile⁴,Ile⁸]-angiotensin II was bought from Bachem. The compound library consisting of randomly selected 180 molecules was from BioAscent. Native coelenterazine and coelenterazine h were from Regis Technologies. Furimazine and the Luciferase Assay System were from Promega; DeepBlueC (coelenterazine 400a) was from Perkin-Elmer. Fura-2/AM was from Molecular Probes. ICI118,552 EGF, transferrin, angiotensin II, A61603, prazosin, and otherwise not stated materials were from Sigma-Aldrich.