Chlorothiazide

Human carbonic anhydrase II (Sigma-Aldrich, St. Louis, MO), ammonium acetate (Sigma-Aldrich, St. Louis, MO), and HPLC grade water (Millipore, Burlington, MA) were purchased. Ethoxzolamide, chlorothiazide, and furosemide were purchased from Sigma-Aldrich (St. Louis, MO). Equimolar protein/ligand solutions were prepared at a concentration of 10 μM with 100 mM ammonium acetate. A supercharging agent, m-nitrobenzyl alcohol (m-NBA, Sigma-Aldrich, St. Louis, MO), was added at 0.5% to the protein solutions to improve sequence coverage upon UVPD and generate extensive holo-product ions without disrupting the protein–ligand interactions. Solutions were desalted using Micro Biospin P-6 gel columns (Bio-Rad Laboratories Inc., Hercules, CA) for MS analysis.

C-21 (60 ng/kg/min for chronic intrarenal studies, 300 ng/kg/min for chronic systemic studies, and 100, 200, and 300 ng/kg/min for acute systemic studies; Vicore Pharma), a highly selective, non-peptide AT₂R agonist (K_i=0.4 mol/L) was employed to activate intrarenal and systemic AT₂Rs. C-21 demonstrates 25,000-fold selectivity at AT₂Rs compared with AT₁Rs (14 (link)). C-21 was administered systemically at doses that are selective for AT₂Rs in the rat (14 (link)). The intrarenal dose of C-21 was chosen as 20% of the systemic dose based on renal blood flow. Ang II (200 ng/kg/min; Bachem) was used to induce Ang II–dependent hypertension. PD-123319 (PD; 10 μg/kg/min; Parke-Davis) a specific AT₂R antagonist (IC₅₀=2×10⁻⁸ mol/L and >1×10⁻⁴ mol/L for AT₂R and AT₁Rs, respectively), was used to block intrarenal AT₂Rs. Amiloride (0.8 μg/kg/min; Tocris) was used to inhibit intrarenal E_NaC activity. Chlorothiazide (0.1 μg/kg/min; Sigma) was used to inhibit intrarenal NCC activity. Amiloride and Chlorothiazide doses were administered on the basis of dose-ranging studies with target incremental natriuretic responses of 1–2 μmol/min.