Intracellular Acid-Extruding Regulators and the Effect of Lipopolysaccharide in Cultured Human Renal Artery Smooth Muscle Cells

Homeostasis of the intracellular pH (pH i ) in mammalian cells plays a pivotal role in maintaining cell function. Thus far, the housekeeping Na + -H + exchanger (NHE) and the Na + -HCO 3 − co-transporter (NBC) have been confirmed in many mammalian cells as major acid extruders. However, the role of acid-extruding regulators in human renal artery smooth muscle cells (HRASMCs) remains unclear. It has been demonstrated that lipopolysaccharide (LPS)-induced vascular occlusion is associated with the apoptosis, activating calpain and increased [Ca 2+ ] i that are related to NHE1 activity in endothelia cells. This study determines the acid-extruding mechanisms and the effect of LPS on the resting pH i and active acid extruders in cultured HRASMCs. The mechanism of pH i recovery from intracellular acidosis (induced by NH 4 Cl-prepulse) is determined using BCECF-fluorescence in cultured HRASMCs. It is seen that (a) the resting pH i is 7.19±0.03 and 7.10±0.02 for HEPES- and CO 2 /HCO 3 − - buffered solution, respectively; (b) apart from the housekeeping NHE1, another Na + -coupled HCO 3 − transporter i.e. NBC, functionally co-exists to achieve acid-equivalent extrusion; (c) three different isoforms of NBC: NBCn1 (SLC4A7; electroneutral), NBCe1 (SLC4A4; electrogenic) and NBCe2 (SLC4A5), are detected in protein/mRNA level; and (d) pH i and NHE protein expression/activity are significantly increased by LPS, in both a dose- and time- dependent manner, but NBCs protein expression is not. In conclusion, it is demonstrated, for the first time, that four pH i acid-extruding regulators: NHE1, NBCn1, NBCe1 and NBCe2, co-exist in cultured HRASMCs. LPS also increases cellular growth, pH i and NHE in a dose- and time-dependent manner.