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ORIGINAL ARTICLE
Year : 2015  |  Volume : 35  |  Issue : 5  |  Page : 208-217

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


1 Division of Orthodontics and Dentofacial, Orthopedics and Pedodontics, Department of Dentistry, Tri-Service General Hospital; School of Dentistry, National Defense Medical Center; Gaduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC
2 Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan, ROC
3 Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC
4 Department of Pharmacology, National Defense Medical Center, Taipei; Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan, ROC
5 Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan, ROC

Correspondence Address:
Shih-Hurng Loh
Department of Pharmacology, National Defense Medical Center, No. 161, Section 6, Minquan E Rd., Neihu District, Taipei 11490, Taiwan
ROC
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1011-4564.167775

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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 + /HCO3 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 4 Cl-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 /HCO3 -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% CO2/HCO3 -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 HCO3 -transporter, that is, NBC, functionally coexists to responsible for acid-extruding in HUASMCs.


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