Dopamine inhibits Na؉ ,K ؉ -ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the ␣ subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat ␣ 1 subunit isoform. To identify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat ␣ 1 subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na ؉ ,K ؉ -ATPase activity and increased ␣ subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type ␣ 1 subunit or the S11A ␣ 1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A ␣ 1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affect per se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na ؉ ,K ؉ -ATPase activity, although it did not alter the increased ␣ subunit phosphorylation induced by this agonist.We conclude that dopamine-induced inhibition of Na ؉ ,K ؉ -ATPase activity in rat renal tubule cells requires endocytosis of the ␣ subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process.

Site-directed polyclonal antibodies were generated against four oligopeptides derived from the rat alpha-isoforms of Na(+)-K(+)-adenosinetriphosphatase (i.e., Na(+)-K+ pump) and were used to assess the phylogenetic conservation of the target regions. The first antibody was directed against a region near the phosphorylation site that is conserved among alpha-subunits from numerous species. The remaining antibodies were specific for the isoforms of alpha and recognized a region near the fluorescein isothiocyanate-reactive lysine of the adenine binding site. Immunoblots of protein from a number of species probed with these antibodies revealed dramatic differences in the conservation of the target sequences. The region derived from alpha 1 was found in four of the six mammals screened. In contrast, the analogous region derived from alpha 2 was found in all the screened mammals. Most conserved was the region derived from alpha 3, which was found in all the screened mammals as well as in chickens, catfish, dogfish, and sea slugs. Conservation of the region from alpha 3 suggests functional significance and implies that strong selective pressures have discouraged mutational change. Results from the immunoblots further suggest that isoforms are present in all vertebrates.

The normal composition of the effluent samples at Abbott Laboratories is within the maximum tolerated impurity levels as listed in the Illinois State Standards (1968). These tolerances are partially summarized in Table I and do not interfere in the assay. Wiersma (1970) employed 2,3-diaminonaphthalene as a fluorometric reagent for nitrite ion and in addition performed Table I. Illinois Effluent Standards Constituent" Standard Ammonia nitrogen 2.5 ppm Arsenic 1.0 ppm BOD 40 mg/1.

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.

Gastrointestinal stasis during sepsis may be associated with gastrointestinal smooth muscle dysfunction. Endotoxin [lipopolysaccharide (LPS)] impairs smooth muscle contraction, in part through inducible nitric oxide synthase (NOS II) and enhanced nitric oxide production. We studied the roles of tumor necrosis factor-α (TNF) and interleukin-1 (IL-1) in this process by using TNF binding protein (TNFbp) and IL-1 receptor antagonist (IL-1ra). Rats were treated with TNFbp and IL-1ra, or their vehicles, 1 h before receiving LPS or saline. At 5 h after LPS, contractility was measured in strips of ileal longitudinal smooth muscle, and NOS II activity was measured in full-thickness segments of ileum. LPS decreased maximum stress (mean ± SE) from 508 ± 55 (control) to 355 ± 33 g/cm2( P < 0.05). Pretreatment with TNFbp plus IL-1ra prevented the LPS-induced decrease. Separate studies of TNFbp alone or IL-1ra alone indicated that, at the doses and timing used, TNFbp was more effective. LPS also increased NOS II activity by >10-fold ( P < 0.01) over control. This increase was prevented by TNFbp plus IL-1ra ( P = not significant vs. control). We conclude that the LPS-induced increase in NOS II activity and the decrease in ileal muscle contractility are mediated by TNF and IL-1.

Na+ reabsorption is regulated in proximal tubules by hormones that stimulate protein kinase C (PKC). To determine whether stimulation of PKC causes a reduction in intracellular Na+ concentration ([Na+]i) that might link Na+ pump activation to increased Na+ reabsorption, [Na+]i was measured in kidney cells loaded with the Na+-sensitive fluorescent indicator SBFI. Rapid digital imaging fluorescence microscopy determinations were performed in epithelial kidney cells transfected with the rodent Na+ pump alpha1 cDNA. In 42 determinations, the basal [Na+]i was 19.7 +/- 2.4 mM. Stimulation of PKC reduced the [Na+]i to 5.6 +/- 0.6 mM in approximately 10 sec. This drastic change in [Na+]i requires a transient 74-120-fold increase in Na+ pump activity. After the new steady state [Na+]i is reached, the Na+ pump is 58% activated. The entry of Na+ into the cells is not affected by stimulation of PKC; therefore, the reduction in [Na+]i is exclusively dependent on activation of the Na+ pump. Accordingly, PKC stimulation does not affect the [Na+]i of cells expressing a mutant Na+ pump that is not stimulated by PKC. The decrease in [Na+]i observed in cells transfected with the rodent Na+ pump alpha1 cDNA is large and sufficiently fast that it is expected to stimulate rapidly passive Na+-influx into the cells, thereby accounting for the observed PKC-induced stimulation of Na+ reabsorption.

Cardiac glycosides exert a positive inotropic effect by inhibiting sodium pump (Na,K-ATPase) activity, decreasing the driving force for Na ϩ -Ca ϩϩ exchange, and increasing cellular content and release of Ca ϩϩ during depolarization. Since the inotropic response will be a function of the level of expression of sodium pumps, which are ␣␤ heterodimers, and of Na ϩ -Ca ϩϩ exchangers, this study aimed to determine the regional pattern of expression of these transporters in the heart. Immunoblot assays of homogenate from atria, ventricles, and septa of 14 nonfailing human hearts established expression of Na,K-ATPase ␣ 1, ␣ 2, ␣ 3, ␤ 1, and Na ϩ -Ca ϩϩ exchangers in all regions. Na,K-ATPase ␤ 2 expression is negligible, indicating that the human cardiac glycoside receptors are ␣ 1 ␤ 1,, ␣ 2 ␤ 1, and ␣ 3 ␤ 1. ␣ 3, ␤ 1, sodium pump activity, and Na ϩ -Ca ϩϩ exchanger levels were 30-50% lower in atria compared to ventricles and/or septum; differences between ventricles and septum were insignificant. Functionally, the EC 50 of the sodium channel activator BDF 9148 to increase force of contraction was lower in atria than ventricle muscle strips (0.36 vs. 1.54 M). These results define the distribution of the cardiac glycoside receptor isoforms in the human heart and they demonstrate that atria have fewer sodium pumps, fewer Na ϩ -Ca ϩϩ exchangers, and enhanced sensitivity to inotropic stimulation compared to ventricles. ( J. Clin. Invest. 1996. 98:1650-1658.)

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