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

Abstract

Homeostasis of the intracellular pH (pHi) in mammalian cells plays a pivotal role in maintaining cell function. Thus far, the housekeeping Na+-H+ exchanger (NHE) and the Na+-HCO3 − 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 [Ca2+]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 pHi and active acid extruders in cultured HRASMCs. The mechanism of pHi recovery from intracellular acidosis (induced by NH4Cl-prepulse) is determined using BCECF-fluorescence in cultured HRASMCs. It is seen that (a) the resting pHi is 7.19±0.03 and 7.10±0.02 for HEPES- and CO2/HCO3 −- buffered solution, respectively; (b) apart from the housekeeping NHE1, another Na+-coupled HCO3 − 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) pHi 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 pHi acid-extruding regulators: NHE1, NBCn1, NBCe1 and NBCe2, co-exist in cultured HRASMCs. LPS also increases cellular growth, pHi and NHE in a dose- and time-dependent manner.

Expressional and Functional Characterization of Intracellular pH Regulators and Effects of Ethanol in Human Oral Epidermoid Carcinoma Cells

“…However, in our previous study, mRNA/protein levels of three different isoforms of NBC were detected, i.e. NBCn1, NBCe1 (SLC4A4; electrogenic) and NBCe2 (SLC4A5) in human renal artery smooth muscle cells [25]. However, no related knowledge of OEC-M1 cells has been reported.…”

“…The active membrane transporters can be separated into two main groups: acid extruders and acid loaders. Acid extrusion regulators, such as NHE and NBC, can be triggered when cells are in acidic condition [15,25,26]. NHE is an electroneutral housekeeping transporter that mediates the exchange of [ [15,25,26].…”

“…Acid extrusion regulators, such as NHE and NBC, can be triggered when cells are in acidic condition [15,25,26]. NHE is an electroneutral housekeeping transporter that mediates the exchange of [ [15,25,26]. Under HEPES-buffered media (HCO 3 --free), the pH i recovery from acidification through NHE can be inhibited by removal of [Na + ] o or by the addition of a specific and potent NHE1 inhibitor, such as Hoe 694 [26,27].…”

“…Under HEPES-buffered media (HCO 3 --free), the pH i recovery from acidification through NHE can be inhibited by removal of [Na + ] o or by the addition of a specific and potent NHE1 inhibitor, such as Hoe 694 [26,27]. However, in CO 2 /HCO 3 -buffered Tyrode solution, NBC coexists with NHE to achieve acid-equivalent extrusion in many mammalian cells [24,25,27,28]. This cotransporter of NBC is largely (56% to 91%) 4, 4-diisothiocyanatostilbene-2, 2-disulphonic acid (DIDS) sensitive, while HOE 694-resistant [23,25,[27][28][29].…”

“…However, in CO 2 /HCO 3 -buffered Tyrode solution, NBC coexists with NHE to achieve acid-equivalent extrusion in many mammalian cells [24,25,27,28]. This cotransporter of NBC is largely (56% to 91%) 4, 4-diisothiocyanatostilbene-2, 2-disulphonic acid (DIDS) sensitive, while HOE 694-resistant [23,25,[27][28][29]. NBC is also inhibited by removal of external Na + [29,30].…”