Upregulation of apical NHE3 in renal OK cells overexpressing the rodent alpha(1)-subunit of the Na(+) pump.
Vectorial Na(+) reabsorption across the proximal tubule is mediated by apical entry of Na(+), primarily via Na(+)/H(+) exchanger isoform 3 (NHE3), and basolateral extrusion via the Na(+) pump (Na(+)-K(+)-ATPase). We hypothesized that regulation of Na(+) reabsorption should involve not only the activity of the basolateral Na(+)-K(+)-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na(+)-K(+)-ATPase, opossum kidney (OK) cells were transfected with the rodent Na(+)-K(+)-ATPase alpha(1)-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na(+)-K(+)-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na(+)-K(+)-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC(50) values for ouabain: the first value was similar to the IC(50) of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum alpha(1)-isozymes. It is shown that transfection of OK cells with Na(+)-K(+)-ATPase increased Na(+)-K(+)-ATPase and NHE3 activities. This was associated with overexpression of the Na(+)-K(+)-ATPase alpha(1)-subunit and NHE3 in transfected OK cells. The abundance of the Na(+)-K(+)-ATPase beta(1)-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na(+)-K(+)-ATPase in cells transfected with the rodent Na(+) pump alpha(1)-subunit cDNA is expected to stimulate apical Na(+) influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.
Gomes
Pedro
P
Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200-319 Porto, Portugal.
Soares-da-Silva
Patrício
P
D000818:Animals; D002470:Cell Survival / Q000187:drug effects*; D017097:Electric Impedance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D009893:Opossums; D010042:Ouabain / Q000494:pharmacology*; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation
D004791:Enzyme Inhibitors; D017923:Sodium-Hydrogen Exchangers; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase