Physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cells
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...
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| Format: | Article |
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Wolters Kluwer Medknow Publications
2015-01-01
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| Series: | Journal of Medical Sciences |
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| Online Access: | http://jms.ndmctsgh.edu.tw/article.asp?issn=1011-4564;year=2015;volume=35;issue=5;spage=208;epage=217;aulast=Chen |
| _version_ | 1811314957429506048 |
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| author | Gunng-Shinng Chen Ching-Hsia Wu Chi-Chiuan Liau Chih-Chin Hsu Jah-Yao Liu Gwo-Jang Wu Chi-Chung Chou Shih-Hurng Loh |
| author_facet | Gunng-Shinng Chen Ching-Hsia Wu Chi-Chiuan Liau Chih-Chin Hsu Jah-Yao Liu Gwo-Jang Wu Chi-Chung Chou Shih-Hurng Loh |
| author_sort | Gunng-Shinng Chen |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T11:21:43Z |
| format | Article |
| id | doaj.art-0dd787d7960f42ffbd0529e4528ccdbd |
| institution | Directory Open Access Journal |
| issn | 1011-4564 |
| language | English |
| last_indexed | 2024-04-13T11:21:43Z |
| publishDate | 2015-01-01 |
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| spelling | doaj.art-0dd787d7960f42ffbd0529e4528ccdbd2022-12-22T02:48:48ZengWolters Kluwer Medknow PublicationsJournal of Medical Sciences1011-45642015-01-0135520821710.4103/1011-4564.167775Physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cellsGunng-Shinng ChenChing-Hsia WuChi-Chiuan LiauChih-Chin HsuJah-Yao LiuGwo-Jang WuChi-Chung ChouShih-Hurng LohBackground: 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.http://jms.ndmctsgh.edu.tw/article.asp?issn=1011-4564;year=2015;volume=35;issue=5;spage=208;epage=217;aulast=ChenHuman umbilical artery smooth muscle cellsintracellular pHNa + -H + exchangerNa +-HCO3- cotransporterfluorescence dye-2′7′-bis(2-carboxethyl)-5(6)-carboxy-fluorescein-acetoxymethyl |
| spellingShingle | Gunng-Shinng Chen Ching-Hsia Wu Chi-Chiuan Liau Chih-Chin Hsu Jah-Yao Liu Gwo-Jang Wu Chi-Chung Chou Shih-Hurng Loh Physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cells Journal of Medical Sciences Human umbilical artery smooth muscle cells intracellular pH Na + -H + exchanger Na +-HCO3- cotransporter fluorescence dye-2′ 7′-bis(2-carboxethyl)-5(6)-carboxy-fluorescein-acetoxymethyl |
| title | Physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cells |
| title_full | Physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cells |
| title_fullStr | Physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cells |
| title_full_unstemmed | Physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cells |
| title_short | Physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cells |
| title_sort | physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cells |
| topic | Human umbilical artery smooth muscle cells intracellular pH Na + -H + exchanger Na +-HCO3- cotransporter fluorescence dye-2′ 7′-bis(2-carboxethyl)-5(6)-carboxy-fluorescein-acetoxymethyl |
| url | http://jms.ndmctsgh.edu.tw/article.asp?issn=1011-4564;year=2015;volume=35;issue=5;spage=208;epage=217;aulast=Chen |
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