Physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cells

Abstract

Background: Intracellular pH (pH_i) is a pivotal factor for cellular functions and homeostasis. Apart from passive intracellular buffering capacity, active transmembrane transporters responsible for kinetic changes of pH_i impacts. Acid extrusion transporters such as Na^+/H^+ exchanger (NHE) and Na^+/HCO_3^− cotransporter (NBC) have been found to be activated when cells are in an acidic condition in different cell types. However, such far, the pH_i regulators have not been characterized in human umbilical artery smooth muscle cells (HUASMCs).Materials and Methods: We, therefore, investigated the mechanism of pH_i recovery from intracellular acidosis, induced by NH_4Cl-prepulse, using pH-sensitive fluorescence dye: 2',7'-bis(2-carboxethyl)-5(6)-carboxy-fluorescein in HUASMCs. Cultured HUASMCs were derived from the segments of the human umbilical artery that were obtained from women undergoing children delivery. Results: The resting pH_i is 7.23 ± 0.03 when cells in HEPES (nominally HCO_3^−-free) buffered solution. The resting pH_i is higher as 7.27 ± 0.03 when cells in CO_2/HCO_3^−-buffered solution. In HEPES-buffered solution, a pH_i recovery following induced intracellular acidosis could be inhibited completely by 30 μM HOE 694 (a specific NHE inhibitor) or by removing [Na^+]_o. In 5% CO_2/HCO_3^−-buffered solution, 30 μM HOE 694 slowed the pH_i recovery from the induced intracellular acidosis only. On the contrary, HOE 694 adding together with 0.2 mM 4, 4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (a specific NBC inhibitor) or removal of [Na^+]_o entirely blocked the acid extrusion. By using Western blot technique, we demonstrated that four different isoforms of NBC, that is, SLC4A8 (NBCBE), SLC4A7 (NBCn1), SLC4A5 (NBCe2) and SLC4A4 (NBCe1), co-exist in the HUASMCs. Conclusions: We demonstrate, for the 1^(st) time, that apart from the housekeeping NHE1, another Na^+ couple HCO_3^−-transporter, that is, NBC, functionally coexists to responsible for acid-extruding in HUASMCs.