Intracellular pH activates membrane-bound Na(+)/H(+) exchanger and vacuolar H(+)-ATPase in human embryonic kidney (HEK) cells.
Mammalian cells regulate their cytosolic pH through a variety of proton extruding and bicarbonate loading mechanisms. The human embryonic kidney cell line HEK 293 is thought to show some characteristics of proximal tubule cells. The present study was performed to investigate the activity of proton extruding mechanisms (i.e. Na(+)/H(+) exchange and/or V-H(+)-ATPases) and the influence of intracellular pH (pH(i)) on the activation of these transport processes. At resting pH(i) (7.4) and in the absence of bicarbonate, removal of extracellular Na(+) did not alter pH(i) Intracellular acidification (pH(i) approximately 6.2) after a NH(4)Cl prepulse (20 mM) followed by exposure to a Na(+) free bath solution led to a slow pH(i) recovery (with a delay of 5 min), which was inhibited by the specific vacuolar H(+)-ATPase inhibitor bafilomyocin. There was no Na(+)-dependent pH(i) recovery upon exposure to Na(+) after a short intracellular acidification (less than 5 min). However, when the intracellular acidification phase was extended, the activation of a Na(+)-dependent pH(i) recovery was seen. This Na(+)-dependent pH(i) recovery was inhibited by 30 microM EIPA but not by 2 microM EIPA or less, suggesting the involvement of NHE3. Western blot analysis confirmed the presence of NHE3 protein in HEK 293 cells. Disruption of the microtubular network by colchicine (20 microM) did not significantly inhibit the rate of Na(+)-independent or dependent pH(i) recovery indicating that activation of both H(+)-ATPase, and the NHE were not due to stimulated trafficking of the transport proteins or some activators to the membrane. We conclude that a) the plasma membrane of HEK293 cells contains both, NHE and H(+)-ATPase and b) both H(+) extrusion systems are inactive at neutral pH(i), c) a decrease of cytosolic pH to 6.5 activates both transport proteins in a slow, time-dependent manner.
Department of Cellular and Molecular Physiology, Yale University, New Haven 06510, USA.
Karl
K
Lang
Carsten
C
Wagner
Gabriel
G
Haddad
Olga
O
Burnekova
John
J
Geibel
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
D020033:Protein Isoforms; D017923:Sodium-Hydrogen Exchangers; D025262:Vacuolar Proton-Translocating ATPases