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WATER AND ELECTROLYTE HOMEOSTASIS

Upregulation of apical NHE3 in renal OK cells overexpressing the rodent ₁-subunit of the Na⁺ pump

Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal

Submitted 14 February 2005 ; accepted in final form 3 November 2005

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 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 IC₅₀ values for ouabain: the first value was similar to the IC₅₀ of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum 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 ₁-subunit and NHE3 in transfected OK cells. The abundance of the Na⁺-K⁺-ATPase 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 ₁-subunit cDNA is expected to stimulate apical Na⁺ influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.

sodium-potassium-ATPase ₁-subunit; sodium-hydrogen exchanger; transfection

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