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.

No abstract

Inhibition of Na؉ ,K ؉ -ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na ؉ ,K ؉ -ATPase activity in response to dopamine is associated with endocytosis of its ␣-and ␤-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na ؉ ,K ؉ -ATPase and inhibition of its activity were accompanied by phosphorylation of the ␣-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the ␣-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent ␣-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the ␣-subunit with a concomitant decrease in Na ؉ ,K ؉ -ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent ␣-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser 11 and Ser 18 ). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the ␣-subunit is essential for Na ؉ ,K ؉ -ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.

Catecholamines promote lung edema clearance via β-adrenergic-mediated stimulation of active Na+ transport across the alveolar epithelium. Because alveolar epithelial type II cell Na+-K+-ATPase contributes to vectorial Na+ flux, the present study was designed to investigate whether Na+-K+-ATPase undergoes acute changes in its catalytic activity in response to β-adrenergic-receptor stimulation. Na+-K+-ATPase activity increased threefold in cells incubated with 1 μM isoproterenol for 15 min, which also resulted in a fourfold increase in the cellular levels of cAMP. Forskolin (10 μM) also stimulated Na+-K+-ATPase activity as well as ouabain binding. The increase in Na+-K+-ATPase activity was abolished when cells were coincubated with a cAMP-dependent protein kinase inhibitor. This stimulation, however, was not due to protein kinase-dependent phosphorylation of the Na+-K+-ATPase α-subunit; rather, it was the result of an increased number of α-subunits recruited from the late endosomes into the plasma membrane. The recruitment of α-subunits to the plasma membrane was prevented by stabilizing the cortical actin cytoskeleton with phallacidin or by blocking anterograde transport with brefeldin A but was unaffected by coincubation with amiloride. In conclusion, isoproterenol increases Na+-K+-ATPase activity in alveolar type II epithelial cells by recruiting α-subunits into the plasma membrane from an intracellular compartment in an Na+-independent manner.

Thyroid hormones affect both renal morphology and function. They are required for kidney growth and development, and thyroid deficiency results in decreased renal plasma flow and glomerular filtration rate and in impaired urinary concentration and dilution. Thyroid hormones also influence membrane transport and electrolyte metabolism, and alterations in mineral metabolism in hyperthyroidism frequently cause calcium nephropathy which affects renal function adversely. The kidney plays an important role in the peripheral metabolism of iodine and thyroid hormones, and thyroid function is altered in certain kidney diseases, particularly chronic renal failure. The pathogenesis of these alterations is currently under active investigation.

A B S T R A C T The renal extraction and excretion of bovine proinsulin, insulin, and C-peptide and the contribution of the kidney to their total metabolic clearance rate (MCR) were studied in the rat. Metabolic clearance rates were measured by the constant infusion technique and plasma and urine concentrations of each polypeptide were determined by radioimmunoassay.The MCR of insulin (16.4±0.4 ml/min) was significantly greater than that of either proinsulin (6.7 ±0.3 ml/min) or C-peptide (4.6±0.2 ml/min). Metabolic clearance rates were independent of plasma levels over a range of steady-state plasma concentrations varying from 1 to 15 ng/ml.In contrast to the differences in their metabolic clearance rates, the renal disposition of the three polypeptides was similar, being characterized by high extraction and very low urinary clearance. The renal arteriovenous difference of proinsulin, insulin, and C-peptide averaged 36, 40, and 44%, respectively, and was linearly related to their arterial concentration between 2 and 25 ng/ml. When glomerular filtration was markedly reduced or stopped by ureteral obstruction, the renal extraction of proinsulin, insulin, and C-peptide was invariably greater than the simultaneously measured extraction of inulin, indicating that these polypeptides are removed from the renal circulation by both glomerular filtration and direct uptake from peritubular capillary blood. The fractional urinary clearance of each polypeptide never exceeded 0.6%, indicating that more than 99% of the amount filtered was sequestered in the kidney.The renal removal of proinsulin and C-peptide from the circulation accounts for 55 and 69% of their metabolic clearance rates, while the renal contribution to the peripheral metabolism of insulin was smaller, averThis work was published in abstract form in 1972 J. Clin. Invest. 51: 49a.Received for publication 18 September 1972 and in revised form 13 December 1972. aging 33%. This difference is due to the fact that insulin, but not the other two polypeptides, is metabolized to a significant extent by the liver. These results define the renal handling of proinsulin, insulin, and Cpeptide in the rat and indicate that in this species the kidney represents a major site for insulin metabolism and is the main organ responsible for the degradation of proinsulin and C-peptide. INTRODUCTIONDifferential secretion rates of insulin, proinsulin, and C-peptide by pancreatic beta cells have generally been considered to be the major determinant of their plasma concentrations (1-3). However, in vivo and in vitro studies have indicated that the peripheral distribution, metabolism, and degradation of insulin and proinsulin differ, suggesting that these factors may play an important role in determining their blood levels. Thus, the half disappearance time of proinsulin in dogs (4), swine (5), rats (6), and baboons (5) is two-to threefold longer than that of insulin. Experiments with the isolated perfused liver (7,8) have shown a high clearance of insulin compared with the removal...

Endocytosis of Na + ,K + -ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I A phosphoinositide-3 kinase (PI3K-I A ) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I A , through its p85α subunit-SH3 domain, binds to a proline-rich region in the Na + ,K + -ATPase catalytic α subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na + ,K + -ATPase α subunit and results in increased PI3K-I A activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na + ,K + -ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I A and its activation during Na + ,K + -ATPase endocytosis in response to G protein-coupled receptor signals.

Na-K-ATPase activity along the rabbit, rat, and mouse nephron was determined with a micromethod that measures directly labeled phosphate released by the hydrolysis of [gamma-32P]ATP. Na-K-ATPase activity was highest in the rat, intermediate in the mouse, and lowest in the rabbit nephron. With the exception of rabbit cortical thick ascending limb, the enzyme profile was similar in the three species: Na-K-ATPase activity per millimeter tubule length was highest in the distal convoluted tubule and thick ascending limb of Henle's loop, intermediate in the proximal convoluted tubule, and lowest in the pars recta and collecting tubule. The enzyme was present in the thin limbs of Henle's loop, but its activity was very low and measurements were close to the sensitivity limit of the method. Both the absolute activity and the fraction of the total enzyme represented by Na-K-ATPase were severalfold higher than in kidney homogenates. Finally, the Na-K-ATPase activity measured in certain segments of the rat and rabbit nephron in this study seems sufficient to account in theory for the active component of the net sodium transport found in the corresponding region of the nephron with either in vivo or in vitro single tubule microperfusion techniques.