Disruption of Na ⁺ ,HCO ₃ ⁻ Cotransporter NBCn1 (slc4a7) Inhibits NO-Mediated Vasorelaxation, Smooth Muscle Ca ²⁺ Sensitivity, and Hypertension Development in Mice

Abstract

BACKGROUND: Disturbances in pH affect artery function, but the mechanistic background remains controversial. We investigated whether Na(+), HCO₃- transporter NBCn1, by regulating intracellular pH(pH₁), influences artery function and blood pressure regulation. METHODS AND RESULTS: Knockout of NBCn1 in mice eliminated Na+, HCO₃⁻ cotransport and caused a lower steady-state pH(i) in mesenteric artery smooth muscle and endothelial cells in situ evaluated by fluorescence microscopy. Using myography, arteries from NBCn1 knockout mice showed reduced acetylcholine-induced NO-mediated relaxations and lower rho-kinase-dependent norepinephrine-stimulated smooth muscle Ca²⁺ sensitivity. Acetylcholine-stimulated NO levels (electrode measurements) and N-nitro-l-arginine methyl ester-sensitive l-arginine conversion (radioisotope measurements) were reduced in arteries from NBCn1 knockout mice, whereas relaxation to NO-donor S-nitroso-N-acetylpenicillamine, acetylcholine-induced endothelial Ca²⁺ responses (fluorescence microscopy), and total and Ser-1177 phosphorylated endothelial NO-synthase expression (Western blot analyses) were unaffected. Reduced NO-mediated relaxations in arteries from NBCn1 knockout mice were not rescued by superoxide scavenging. Phosphorylation of myosin phosphatase targeting subunit at Thr-850 was reduced in arteries from NBCn1 knockout mice. Evaluated by an in vitro assay, rho-kinase activity was reduced at low pH. Without CO₂/HCO₃⁻, no differences in pH(i), contraction or relaxation were observed between arteries from NBCn1 knockout and wild-type mice. Based on radiotelemetry and tail-cuff measurements, NBCn1 knockout mice were mildly hypertensive at rest, displayed attenuated blood pressure responses to NO-synthase and rho-kinase inhibition and were resistant to developing hypertension during angiotensin-II infusion. CONCLUSIONS: Intracellular acidification of smooth muscle and endothelial cells after knockout of NBCn1 inhibits NO-mediated and rho-kinase-dependent signaling in isolated arteries and perturbs blood pressure regulation.