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NBCn1 (slc4a7) Mediates the Na⁺-Dependent Bicarbonate Transport Important for Regulation of Intracellular pH in Mouse Vascular Smooth Muscle Cells

From The Water and Salt Research Center (E.B., J.P., C.A.); Department of Physiology (E.B., C.A.), Institute of Physiology and Biophysics; and Institute of Anatomy (J.P.), University of Aarhus, Denmark.

Correspondence to Christian Aalkjaer, Department of Physiology, University of Aarhus, Ole Worms Allé, Bldg 1160, DK-8000 Aarhus C, Denmark. E-mail ca{at}fi.au.dk

The contribution of sodium-dependent bicarbonate transport to intracellular pH (pH_i) regulation in vascular smooth muscle cells is controversial, partly because the molecular identity of the transporter(s) responsible has not been identified. Here, using the pH-sensitive fluorophore 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), we show that smooth muscle cells of intact mouse mesenteric, coronary, and cerebral small arteries all display a sodium- and bicarbonate-dependent pH_i recovery after an NH₄⁺-prepulse. The sodium-dependent bicarbonate flux was largely 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) sensitive (56% to 91%) and of a magnitude similar to the amiloride-sensitive flux. Additionally, steady-state pH_i was lower (0.2 to 0.4 pH units magnitude) in all 3 vascular beds when CO₂/bicarbonate was omitted. RT-PCR analyses showed that NBCn1 (slc4a7) is the only Na⁺-dependent bicarbonate transporter of the slc4 family detectable at the mRNA level in all 3 vascular beds investigated. Whole-mount immunolabeling and immunogold electron microscopy confirmed the presence of NBCn1 protein in the sarcolemma of mouse mesenteric small arterial smooth muscle cells. Intact mouse mesenteric small arteries were electropermeated to facilitate transfection with small interfering RNA targeting NBCn1, which resulted in an approximate 43% decrease in the ratio of NBCn1 to glyceraldehyde-3-phosphate dehydrogenase mRNA. After knock-down, we found a decreased steady-state pH_i (0.21±0.08 pH units) as well as a 68±10% decrease in the net Na⁺-dependent, amiloride-insensitive base influx after acid load. Finally, omission of CO₂/bicarbonate resulted in a decreased contractile response to norepinephrine after sustained exposure to the agonist, underlining the importance of CO₂/bicarbonate for vascular contractility. We conclude that NBCn1 mediates the Na⁺-dependent bicarbonate transport important for pH_i regulation in smooth muscle cells of mouse mesenteric, coronary, and cerebral small arteries.

Key Words: vascular smooth muscle cells • bicarbonate • intracellular pH • siRNA • NBC

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