Hille Carsten, Walz Bernd
University of Potsdam, Institute of Biochemistry and Biology, Department of Animal Physiology, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany.
J Exp Biol. 2007 Apr;210(Pt 8):1463-71. doi: 10.1242/jeb.001529.
Cells of the dopaminergically innervated salivary ducts in the cockroach Periplaneta americana have a vacuolar-type H(+)-ATPase (V-ATPase) of unknown function in their apical membrane. We have studied whether dopamine affects intracellular pH (pH(i)) in duct cells and whether and to what extent the apical V-ATPase contributes to pH(i) regulation. pH(i) measurements with double-barrelled pH-sensitive microelectrodes and the fluorescent dye BCECF have revealed: (1) the steady-state pH(i) is 7.3+/-0.1; (2) dopamine induces a dose-dependent acidification up to pH 6.9+/-0.1 at 1 micromol l(-1) dopamine, EC(50) at 30 nmol l(-1) dopamine; (3) V-ATPase inhibition with concanamycin A or Na(+)-free physiological saline (PS) does not affect the steady-state pH(i); (4) concanamycin A, Na(+) -free PS and Na(+)/H(+) exchange inhibition with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) each reduce the rate of pH(i) recovery from a dopamine-induced acidification or an acidification induced by an NH(4)Cl pulse; (5) pH(i) recovery after NH(4)Cl-induced acidification is almost completely blocked by concanamycin A in Na(+)-free PS or by concanamycin A applied together with EIPA; (6) pH(i) recovery after dopamine-induced acidification is also completely blocked by concanamycin A in Na(+)-free PS but only partially blocked by concanamycin A applied together with EIPA. We therefore conclude that the apical V-ATPase and a basolateral Na(+)/H(+) exchange play a minor role in steady-state pH(i) regulation but contribute both to H(+) extrusion after an acute dopamine- or NH(4)Cl-induced acid load.
在美洲大蠊中，多巴胺能神经支配的唾液腺导管细胞的顶端膜上有一种功能未知的液泡型H(+)-ATP酶（V-ATP酶）。我们研究了多巴胺是否影响导管细胞内的pH值（pH(i)），以及顶端V-ATP酶是否以及在何种程度上参与pH(i)的调节。使用双管pH敏感微电极和荧光染料BCECF进行的pH(i)测量结果显示：（1）稳态pH(i)为7.3±0.1；（2）多巴胺在1 μmol l(-1)多巴胺时诱导剂量依赖性酸化，直至pH值达到6.9±0.1，30 nmol l(-1)多巴胺时的半数有效浓度（EC(50)）；（3）用 concanamycin A或无钠生理盐水（PS）抑制V-ATP酶不影响稳态pH(i)；（4）concanamycin A、无钠PS以及用5-(N-乙基-N-异丙基)-amiloride（EIPA）抑制Na(+)/H(+)交换，均会降低pH(i)从多巴胺诱导的酸化或NH(4)Cl脉冲诱导的酸化中恢复的速率；（5）在无钠PS中，concanamycin A或与EIPA一起应用的concanamycin A几乎完全阻断了NH(4)Cl诱导酸化后的pH(i)恢复；（6）在无钠PS中，concanamycin A也完全阻断了多巴胺诱导酸化后的pH(i)恢复，但与EIPA一起应用的concanamycin A仅部分阻断。因此，我们得出结论，顶端V-ATP酶和基底外侧Na(+)/H(+)交换在稳态pH(i)调节中起次要作用，但在急性多巴胺或NH(4)Cl诱导的酸负荷后，两者都有助于H(+)的排出。
