Figure 5.

Localization of D1 receptor and Na⁺,K⁺-ATPase in renal tissue slices in response to nitecapone and forskolin: subcellular fractionation studies. Slices (250 μm) from outer cortex, rich in proximal tubules, were dissected from rat kidney. The tissues were treated in the absence (−) or presence (+) of either nitecapone (5 μM; 20 min) or forskolin (5 μM; 1 min), homogenized, and subjected to subcellular fractionation by using a 10–40% sucrose gradient. Each fraction (1–10) from vehicle-treated tissue was electrophoresed, blotted on a polyvinylene difluoride membrane, and probed for Na⁺,K⁺-ATPase. The amount of Na⁺,K⁺-ATPase was highest in fraction 2 and absent from fractions 4–10. After treatment with nitecapone or forskolin, the fractions were probed for the D1 receptor. The amount of the D1 receptor increased in the Na⁺,K⁺-ATPase-containing fractions, illustrated by fraction 2, and decreased in the fractions devoid of Na⁺,K⁺-ATPase, illustrated by fraction 10. Both nitecapone and forskolin caused an increase of D1 abundance in the plasma membrane fraction (3.13 ± 0.38 and 2.27 ± 0.37 times, respectively, compared with control, P < 0.01). Results are representative of three experiments.