MODULATION OF INTRACELLULAR pH BY Na+/H+ EXCHANGER AND HCO3 TRANSPORTER IN SALIVARY ACINAR CELLS

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Journal of the Korean Academy of Pediatric Dentistry 1998;25(2):352-367.
Published online May 31, 1998.

MODULATION OF INTRACELLULAR pH BY Na⁺/H⁺ EXCHANGER AND HCO₃ TRANSPORTER IN SALIVARY ACINAR CELLS

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Na⁺/H⁺ exchanger와 HCO₃ transporter에 의한 흰쥐 타액선 선세포내 pH 조절

박동범¹, 서정택², 손흥규¹, 이종갑¹

¹연세대학교 치과대학 소아치과학교실
²연세대학교 구강과학연구소

Abstract

Intracellular pH (pHi) plays an important role in the regulation of cellular processes by influencing the acitivity of various enzymes in cells. Therefore, almost every type of mammalian cell possesses an ability to regulate its pHi. One of the most prominent mechanisms in the regulation of pHi is Na⁺/H⁺ exchanger. This exchanger has been known to be activated when cells are stimulated by the binding of agonist to the muscarinic receptors. Therefore, the aims of this study were to compare the rates of H⁺ extrusion through Na⁺/H⁺ exchanger before and during muscarinic stimulation and to investigate the possible existence of HCO₃ transporter which is responsible for the continuous supply of HCO₃ ion to saliva. Acinar cells were isolated from the rat mandibular salivary glands and loaded with pH-sensitive fluoroprobe, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), for 30min at room temperature. Cells were attached onto the coverglass in the perfusion chamber and the changes in pHi were measured on the iverted microscope using spectrofluorometer. 1. By switching the perfusate from HCO₃-free to HCO₃-buffered solution, pHi decreased by 0.39{\pm}0.02 pH units followed by a slow increase at an initial rate of 0.04±0.007 pH units/min. The rate of pHi increase was reduced to 0.01±0.002 pH units/min by the simultaneous addition of 1 mM amiloride and 100μM DIDS. 2. An addition and removal of NH₄⁺ caused a decrease in pHi which was followed by an increase in pHi. The increase of pHi was almost completely blocked by 1mM amiloride in HCO₃-free perfusate which implied that the pHi increase was entired dependent on the activation of Na⁺/H⁺ exchanger in HCO₃-free condition. 3. An addition of 10μM carbachol increased the initial rate of pHi recovery from 0.16±0.01 pH units/min to 0.28±0.03pH units/min. 4. The initial rate of pHi decrease induced by 1mM amiloride was also increased by the exposure of the acinar cells to 10μM carbachol (0.06±0.008pH unit/min) compared with that obtained before carbachol stimulation (0.03±0.004pH unit/min). 5. The intracellular buffering capacity β1 was 14.31±1.82 at pHi 7.2-7.4 and β1 increased as pHi decreased. 6. The rate of H⁺ extrusion through Na⁺/H⁺ exchanger was greatly enhanced by the stimulation of the cells with 10μM carbachol and there was an alkaline shift in the activity of the exchanger. 7. An intrusion mechanism of HCO₃ was identified in rat mandibular salivary acinar cells. Taken all together, I observed 3-fold increase in Na⁺/H⁺ exchanger by the stimulation of the acinar cells with 10μM carbachol at pH 7.25. In addition, I have found an additional mechanism for the regulation of pHi which transported HCO₃ into the cells.

Key Words: amiloride, cotransporter, DIDS, intracellular pH, HCO₃ BCECF, Na⁺/H⁺ exchanger, spectrofluorometer