The Effect and Underlying Mechanism of Ethanol on Intracellular H+-Equivalent Membrane Transporters in Human Aorta Smooth Muscle Cells

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Abstract
Background: The presence of intracellular pH (pH(i)) regulators, including Na+-H+ exchanger (NHE), Na+-HCO3- co-transporter (NBC), Cl-/OH- exchanger (CHE), and Cl-/HCO3- exchanger (AE), have been confirmed in many mammalian cells. Alcohol consumption is associated with increased risk of cardiovascular disorder. The aims of the study were to identify the possible transmembrane pH(i) regulators and to explore the effects of ethanol (EtOH) (10 to 300 mM) on the resting pH(i) and pH(i) regulators in human aorta smooth muscle cells (HASMCs). Methods: HASMCs were obtained from patients undergoing heart transplant. The pH(i) was measured by microspectrofluorimetry with the pH-sensitive dye, BCECF-AM. Results: The following results are obtained. (i) In cultured HASMCs, the resting pH(i) was 7.19 +/- 0.04 and 7.13 +/- 0.02 for HEPES- and CO2/HCO3--buffered solution, respectively. (ii) Two different Na+-dependent acid-equivalent extruders, including NHE and Na+-coupled HCO3- transporter, functionally coexisted. (iii) Two different Cl--dependent acid loaders (CHE and AE) were functionally identified. (iv) EtOH induced a biphasic, concentration-dependent change in resting pH(i) (+0.25 pH unit at 100 mM but only +0.05 pH unit at 300 mM) in bicarbonate-buffered solution, while caused a concentration-dependent decrease in resting pH(i) (-0.06 pH unit at 300 mM) in HEPES-buffered solution. (v) The effect of EtOH on NHE activity was also biphasic: increase of 40% at lower concentration of 10 mM, followed by decrease of 30% at higher concentration of 300 mM. (vi) The increase in Na+-coupled HCO3- transporter activity by EtOH was concentration dependent. (vii) The effect of EtOH on CHE and AE activities was both biphasic: increase of similar to 25% at 30 mM, followed by decrease of 10 to 25% at 100 mM, and finally increase of 15 to 20% at 300 mM. Conclusions: This study demonstrated that 2 acid extruders and 2 acid loaders coexisted functionally in HASMCs and that EtOH induced a biphasic, concentration-dependent change in resting pH(i) by altering the activity of the 2 acid extruders, NHE and Na+-coupled HCO3- transporter, and the 2 acid loaders, CHE and AE.
Funding Information
  • Ministry of Science and Technology (MOST 104-2314-B-016-044, MOST 104-2320-B-016- 020)
  • National Defense Medical Bureau (DOD102-I-30, D103-M084)
  • Tri-Service General Hospital (TSGH-C103-029)
  • Deh-Tzer Study Group for Human Medical Research Foundation (A1021028-2)

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