Ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by a clathrin-dependent mechanism

BACKGROUND: We have demonstrated that ouabain causes dose- and time-dependent decreases in (86)Rb uptake in porcine proximal tubular (LLC-PK1) cells. The present study addresses the molecular mechanisms involved in this process. METHODS: Studies were performed with cultured LLC-PK1 and Src family kinase deficient (SYF) cells. RESULTS: We found that 50 nmol/L ouabain applied to the basal, but not apical, aspect for 12 hours caused decreases in the plasmalemmal Na/K-ATPase. This loss of plasmalemmal Na/K-ATPase reverses completely within 12 to 24 hours after removal of ouabain. Ouabain also increased the Na/K-ATPase content in both early and late endosomes, activated phosphatidylinositol 3-kinase (PI(3)K), and also caused a translocation of some Na/K-ATPase to the nucleus. Immunofluorescence demonstrated that the Na/K-ATPase colocalized with clathrin both before and after exposure to ouabain, and immunoprecipitation experiments confirmed that ouabain stimulated interactions among the Na/K-ATPase, adaptor protein-2 (AP-2), and clathrin. Potassium (K) depletion, chlorpromazine, or PI(3)K inhibition all significantly attenuated this ouabain-induced endocytosis. Inhibition of the ouabain-activated signaling process through Src by 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) significantly attenuated ouabain-induced endocytosis. Moreover, experiments performed in SYF cells demonstrated that ouabain induced increases in the endocytosis of the Na/K-ATPase when Src was reconstituted (SYF+), but not in the Src-deficient (SYF-) cells. CONCLUSION: These data demonstrate that ouabain stimulates a clathrin-dependent endocytosis pathway that translocates the Na/K-ATPase to intracellular compartments, thus suggesting a potential role of endocytosis in ouabain-induced signal transduction as well as proximal tubule sodium handling.     

Salt loading induces redistribution of the plasmalemmal Na/K-ATPase in proximal tubule cells

BACKGROUND: We have reported that digitalis-like substances (cardiotonic steroids), including marinobufagenin (MBG), induce endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells. The current report addresses the potential relevance of plasmalemmal Na/K-ATPase redistribution to in vivo salt handling. METHODS: Male Sprague-Dawley rats were given 1 week of a high salt (4.0% NaCl) or normal salt (0.4% NaCl) diet. Urinary sodium excretion, as well as MBG excretion, was monitored, and proximal tubules were isolated using a Percoll gradient method. Tubular (86)Rb uptake, Na/K-ATPase enzymatic activity, and Na/K-ATPase alpha1 subunit density were determined. RESULTS: The high salt diet increased urinary sodium (17.8 +/- 1.8 vs. 2.5 +/- 0.3 mEq/day, P < 0.01) and MBG excretion (104 +/- 12 vs. 26 +/- 4 pmol/day), and decreased proximal tubular (86)Rb uptake (0.44 +/- 0.07 vs. 1.00 +/- 0.10, P < 0.01) and Na/K-ATPase enzymatic activity (5.1 +/- 1.1 vs. 9.9 +/- 1.6 micromol/mg pr/hr, P < 0.01) relative to the normal diet. Proximal tubular Na/K-ATPase alpha1 protein density was decreased in the plasmalemma fraction but increased in both early and late endosomes following the high salt diet. In rats fed a high salt diet, anti-MBG antibody caused a 60% reduction in urinary sodium excretion, substantial increases in proximal tubule (86)Rb uptake, and Na/K-ATPase enzymatic activity, as well as significant decreases in the early and late endosomal Na/K-ATPase alpha1 protein content. CONCLUSION: These data suggest that redistribution of the proximal tubule Na/K-ATPase in response to endogenous cardiotonic steroids plays an important role in renal adaptation to salt loading.     

Effects of cardiac glycosides on sodium pump expression and function in LLC-PK1 and MDCK cells

BACKGROUND: The decreases in proximal tubule sodium reabsorption seen with chronic renal failure and volume expansion have been ascribed to circulating digitalis-like substances (DLS). However, the circulating concentrations of DLS do not acutely inhibit the sodium pump to a degree consistent with the observed changes in proximal tubule sodium reabsorption. METHODS: We examined how cell lines that simulated proximal (LLC-PK1) and distal tubule (MDCK) cells responded to acute (30 min) and long-term (up to 12 hours) Na+,K+-ATPase inhibition with DLS. RESULTS: In LLC-PK1, but not MDCK cells, low concentrations of ouabain decreased 86Rb uptake profoundly in a time and dose dependent manner. In LLC-PK1 cells grown to confluence, transcellular 22Na flux was markedly reduced in concert with the decreases in 86Rb uptake. Similar findings were observed with marinobufagenin (MBG) and deproteinated extract of serum derived from patients with chronic renal failure. However, inhibition of the Na+,K+-ATPase with low extracellular potassium concentrations did not produce any of these effects. Western and Northern blots detected no change in alpha1 Na+,K+-ATPase protein and message RNA, respectively, in LLC-PK1 cells treated with ouabain for 12 hours. However, the decrease in enzymatic activity of Na+,K+-ATPase of these cells was comparable to observed decreases in 86Rb uptake. Differential centrifugation as well as biotinylation experiments demonstrated a shift of the Na+,K+-ATPase from the plasmalemma with prolonged ouabain treatment. CONCLUSIONS: The results show that binding of cardiac glycosides by proximal (but not distal) tubular cells results in internalization of Na+,K+-ATPase with the net effect to amplify inhibition of the Na+,K+-ATPase. As the circulating concentrations of DLS increase with chronic renal failure and volume expansion, we suggest that this phenomenon explains some of the decreased sodium reabsorption by the proximal tubule seen in these conditions.     

Ouabain inhibits tubuloglomerular feedback in mutant mice with ouabain-sensitive alpha1 Na,K-ATPase

Initiation of tubuloglomerular feedback (TGF) depends on Na-K-2Cl co-transport in the macula densa (MD), but it is less clear whether Na,K-ATPase is responsible for establishing the inward Na+ gradient. It has been proposed that apical colonic H,K-ATPase, perhaps in concert with the Na/H exchanger (NHE2), may account for MD Na+ exit in these cells. This study evaluated TGF responses by micropuncture in mutant mice with altered ouabain sensitivity of the alpha1 and alpha2 Na,K-ATPase isoforms. TGF responses in alpha1-sensitive/alpha2-resistant mice were inhibited by intravenous ouabain (control stop-flow pressure = 9.7 +/- 0.9 versus 1.6 +/- 0.5 mmHg with intravenous ouabain). Subsequent inclusion of cyclohexyladenosine (10 microM) in the tubule perfusate confirmed the ability of the afferent arteriole to contract in the presence of ouabain. In alpha1-resistant/alpha2-resistant mice, ouabain infusion had no effect on TGF responses. In separate experiments, loop of Henle perfusion with 50 microM ouabain decreased TGF responses (control stop-flow pressure) from 10.5 +/- 1.1 to 3.9 +/- 1.0 mmHg in alpha1-sensitive/alpha2-resistant mice but had no effect in alpha1-resistant/alpha2-resistant mice, and afferent arteriole responsiveness again was confirmed by cyclohexyladenosine. TGF responses in NHE2 and colonic H,K-ATPase knockout mice were not different from those of wild-type mice. These data indicate that TGF requires activity of the alpha1 Na,K-ATPase, presumably in the MD. Furthermore, the data show that neither NHE2 nor colonic H,K-ATPase is essential for initiation of TGF responses.     

Ouabain-Sensitive alpha1 Na,K-ATPase enhances natriuretic response to saline load

Na,K-ATPase is ubiquitously expressed and is essential for maintaining electrochemical and osmotic gradients. The alpha subunit of Na,K-ATPase is the receptor for cardiotonic steroids, which act through the ouabain-binding site and are important in cardiovascular regulation. Interestingly, the presence of endogenous Na,K-ATPase ligands has been implicated in the natriuretic response to perturbations such as hypertension and salt loading; therefore, it is important to characterize the role of the ouabain-binding sites in this context. Because the alpha1 isoform of mice and rats is relatively ouabain resistant, gene-targeting strategies were used to produce mice with reversed responses of the alpha1 and/or alpha2 isoforms to ouabain to assess for altered natriuretic responses to acute salt loading. Regardless of the sensitivity of the alpha2 isoform to ouabain, conferring ouabain sensitivity to alpha1 augmented the natriuretic response to an acute salt load. In addition, when endogenous Na,K-ATPase inhibitors were sequestered with an anti-digoxin antibody fragment, the sodium excretion rates in the ouabain-sensitive alpha1 isoform mice were equivalent to the ouabain-resistant alpha1 isoform mice. These data suggest that the ouabain-binding site of the alpha1 Na,K-ATPase can participate in the natriuretic response to a salt load by responding to endogenous Na,K-ATPase ligands.     

Ouabain binds with high affinity to the Na,K-ATPase in human polycystic kidney cells and induces extracellular signal-regulated kinase activation and cell proliferation

In autosomal dominant polycystic kidney disease (ADPKD), cyst formation and enlargement require proliferation of mural renal epithelial cells and the transepithelial secretion of fluid into the cyst cavity. Na,K-ATPase is essential for solute and water transport in ADPKD cells, and ouabain blocks fluid secretion in these cells. By binding to the Na,K-ATPase, ouabain also induces proliferation in some cell types. Surprisingly, it was found that nanomolar concentrations of ouabain, similar to those circulating in blood, induced ADPKD cell proliferation but had no statistically significant effect on normal human kidney (NHK) cells. Ouabain, acting from the basolateral side of the cells, also caused an increase in the level of phosphorylated extracellular signal-regulated kinases (ERK). Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocked ouabain-induced ERK activation and cell proliferation, suggesting that ouabain effect is mediated through the MEK-ERK pathway. In contrast to NHK cells, the dose-response curve for ouabain inhibition of Na,K-ATPase activity indicated that approximately 20% of the enzyme in ADPKD cells exhibits a higher affinity for ouabain. The increased ouabain affinity of ADPKD cells was not due to differences in Na,K-ATPase isoform expression because these cells, like NHK cells, possess only the alpha1 and beta1 subunits. The gamma variants of the Na,K-ATPase also are expressed in the cells but are elevated in ADPKD cells. Currently, the basis for the differences in ouabain sensitivity of NHK and ADPKD cells is unknown. It is concluded that ouabain stimulates proliferation in ADPKD cells by binding to the Na,K-ATPase with high affinity and via activation of the MEK-ERK pathway.     

Comparison of contractile effects of sodium vanadate and ouabain in vascular smooth muscles of guinea-pigs and rats

Ouabain and vanadate are known as potent inhibitors of Na, K-ATPase in various tissues including smooth muscles. Both agents showed contractile action on various smooth muscles in a similar fashion: stronger contractile action on the aortae of rats (WKY and stroke prone spontaneously hypertensive rats, SHRSP) and guinea-pigs, and weaker contractile actions on basilar and mesenteric arteries of the same animals. Time to peak tension, however, was far longer in ouabain-induced contraction. Phentolamine depressed ouabain-induced contractions, while vanadate-induced contractions were not affected. Elevation of K+ concentration to 20 or 30 mM potentiated vanadate-induced contraction markely, while it potentiated ouabain-induced contraction only slightly. DIDS blocked vanadate-induced contraction but showed no effect on ouabain-induced contraction. Removal of Ca abolished ouabain-induced contractions, while vanadate-induced contractions of reduced height could be observed in the absence of Ca. Verapamil depressed both ouabain- and vanadate-induced contractions of WKY and SHRSP aorte aut exhibited no effect on the guinea-pig aorta. Thus, although similarities of the action of ouabain and sodium vanadate were observed, the modes of the actions were revealed to be different in the two agents. Inhibition of Na, K-ATPase might be involved in the case of ouabain-induced contractions, and inhibition of Ca-ATPase of membranous systems might be involved in vanadate-induced contraction.     

Na transport in autosomal recessive polycystic kidney disease (ARPKD) cyst lining epithelial cells

Autosomal dominant (ADPKD) and recessive (ARPKD) polycystic kidney disease are characterized by the progressive growth and expansion of cysts or ectatic collecting ducts, respectively, that ultimately destroy the normal renal parenchyma. Evidence from experimental models of ADPKD suggests that transepithelial Na and fluid secretion contribute to cyst growth, yet little is known about solute transport in ARPKD. This purpose of this study was to begin to characterize the expression and polarity of transport proteins involved in vectorial Na movement in ARPKD epithelium. Immunodetectable alpha1 and beta2 subunits of the Na/K-ATPase localized to the apical membrane of collecting duct cysts in tissue sections of human fetal ARPKD nephrectomy specimens and conditionally immortalized cells derived from these cysts. Measurements of transepithelial (22)Na transport performed on monolayers of ARPKD and age-matched collecting tubule (HFCT) cells grown on permeable supports revealed net Na absorption in both models. However, ARPKD cells absorbed Na at a rate approximately 50% greater than that of HFCT. Furthermore, Na absorption in ARPKD cells was partially inhibited by 100 micro M apical amiloride or 1 mM basolateral but not apical ouabain. Northern blot analyses of ARPKD whole kidney and Western immunoblot of ARPKD cells showed approximately twofold greater expression of the alpha-subunit of the epithelial Na channel (ENaC) compared with age-matched controls. These results suggest that, despite the presence of apical Na/K-ATPase, ARPKD cyst-lining cells absorb Na by a pathway that is modestly amiloride-sensitive. Whether Na absorption is mediated by ENaC, perhaps of nonclassical subunit composition, or another amiloride-sensitive transporter remains to be determined.     

Inhibition of Na,K-ATPase by dopamine in proximal tubule epithelial cells

In the current report we review the results that lay grounds for the model of intracellular sodium-mediated dopamine-induced endocytosis of Na,K-ATPase. Under conditions of a high salt diet, dopamine activates PKCzeta, which phosphorylates NKA alpha1 Ser-18. The phosphorylation produces a conformational change of alpha1 NH2-terminus, which through interaction with other domains of alpha1 exposes PI3K- and AP-2-binding domains. PI3K bound to the NKA alpha1 induces the recruitment and activation of other proteins involved in endocytosis, and PI3K-generated 3-phosphoinositides affect the local cytoskeleton and modify the biophysical conditions of the membrane for development of clathrin-coated pits. Plasma membrane phosphorylated NKA is internalized to specialized intracellular compartments where the NKA will be dephosphorylated. The NKA internalization results in a reduced Na+ transport by proximal tubule epithelial cells.     

Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments

To obtain a highly specific reagent that could be utilized for ultrastructural localization of Na,K-ATPase, monoclonal antibodies were produced using microsomal preparations of outer renal medulla of dog and rat enriched for Na,K-ATPase. The monoclonal antibody (C62.4) raised against dog antigen, immunoprecipitated a 96,000 Dalton protein from membranes labeled either with 35S methionine or 3H NAB ouabain. Na,K-ATPase, Na-ATPase, and KpNPPase activity were 25, 60, and 100% maximal after reaction with C62.4. Na,K-ATPase activated with SDS was inhibited, but Na,K-ATPase in tight right-side-out membrane vesicles was not. C62.4 inhibited ouabain binding in the presence of Na,K, and Mg, but did not inhibit ouabain binding in the presence of Mg and Pi. Labeling of broken membranes was readily seen using C62.4 labeled with colloidal gold. Intact right-side-out vesicles showed no evidence of labeling, demonstrating that the antibody is directed to an epitope of the cytoplasmic domain of the enzyme. Differential localization of C62.4 along the nephron was identified. Glomeruli showed no significant antibody binding except by occasional cells in the mesangial regions. Only basal lateral membranes of cells from all tubule segments labeled with C62.4. There was no evidence of specific apical labeling. The thick ascending limb of Henle's loop demonstrated the greatest concentration of antibody binding. In the cortical and outer medullary collecting duct, only principal cells showed abundant antibody binding. Intercalated cells showed no detectable evidence of antibody binding on any surface. These studies demonstrate that Na,K-ATPase is localized exclusively to the basal lateral membrane of renal tubular epithelial cells and varies in density and distribution in different nephron segments.     

The role of Na,K-ATPase in human sperm motility

A fourth Na,K-ATPase alpha isoform, which was found to be abundant in testes, was proved to be a catalytical subunit of the enzyme. Recently, it has been shown that the alpha 4 isoform along with alpha 1 is expressed in the midpiece of the flagellum of mature rat sperm and the inhibition of alpha 4 with ouabain led to sperm immotility. In this study, sperm from 135 males with normal semen profile and 50 males with oligoasthenospermia were treated with 10-5 and 10-2 M ouabain solutions to inhibit alpha 4, and alpha 4 plus alpha 1 isoforms, respectively. In males with normal semen profile, sperm motility has been demonstrated to decrease with time to almost the same level with both ouabain solutions. In oligoasthenospermic males motility was also found almost completely lost. These observations showed us that the alpha 4 isoform may be held responsible for human sperm motility. When sperm plasma membrane Na,K-ATPase activity was assayed for both normal and oligoasthenospermic males, a significant decrease in enzyme activity of males with oligoasthenospermia was observed (p < 0.05). In our recent study, sperm motility was found decreased by treatment with peroxynitrite (ONOO-). To investigate the effect of ONOO- on sperm Na,K-ATPase activity, sperm plasma membranes were treated with 100 microM ONOO- and plasma membrane Na,K-ATPase activity was observed to be significantly decreased (p < 0.05). Although total sulfhydryl (SH) content of sperm plasma membrane was also found significantly lower, no correlation was found between Na,K-ATPase activity and SH content.     

Regulation of tubular cell MCP-1 production by intracellular ions: a role for sodium and calcium

Proximal tubule cells (PTC) in chronic renal disease produce chemokines which cause renal interstitial inflammation, and also transport more Na+ than normal. To investigate whether these two events might be related, monocyte chemoattractant protein-1 (MCP-1) production was examined in rat PTC in primary culture. Amiloride reduced, while ouabain increased levels of MCP-1 mRNA and protein. Amiloride reduced MCP-1 in cells stimulated with ouabain, lipopolysaccharide (LPS) or albumin. Intracellular Na+ rose with ouabain, but not LPS or albumin. Effects of amiloride, ouabain, LPS and albumin were abrogated by sodium-free but not chloride-free culture medium, and were not explained by changes in intracellular pH. Intracellular Ca2+ rose with ouabain, LPS or albumin and sodium-free medium. BAPTA-AM reduced intracellular Ca2+ and MCP-1 mRNA levels in unstimulated cells, and cells stimulated with ouabain, LPS or albumin. Thus, amiloride and ouabain may alter tubular cell MCP-1 by changing intracellular Na+, with secondary changes in intracellular Ca2+, whereas stimulation by LPS and albumin may involve Ca2+ directly.     

Low doses of ouabain protect from serum deprivation-triggered apoptosis and stimulate kidney cell proliferation via activation of NF-kappaB

It now generally is agreed that Na,K-ATPase, in addition to its role in the maintenance of Na+ and K+ gradients across the cell membrane, plays a role in communicating information from the extracellular environment to intracellular signaling pathways. It was reported recently that interaction between ouabain-bound Na,K-ATPase and the 1,4,5-trisphosphate receptor (IP3R) triggers slow calcium oscillations and activation of NF-kappaB. Here it is demonstrated that this signaling pathway can serve to prevent cell death and promote cell growth. Rat renal proximal tubular cells in primary culture first were grown in the presence of 10% serum and then exposed to 0.2% serum for 24 h to induce apoptosis. Serum starvation increased the apoptotic index from 1.21 +/- 0.26 to 14.01 +/- 1.17%. Ouabain in concentrations that did not inhibit Na,K-ATPase activity (1 to 10 nM) completely abolished the apoptotic effect of serum starvation. Ouabain protection from apoptosis was not observed when release of calcium from intracellular stores via the IP3R was prevented. It was shown that the NH2 terminal tail of the Na,K-ATPase alpha subunit plays a key role in ouabain-triggered calcium oscillations. It was found that ouabain did not protect from apoptosis in serum-deprived cells that expressed a mutant Na,K-ATPase alpha subunit with deletion of the NH2 terminal tail. Ouabain exposure (10 nM for 24 h) significantly increased translocation of NF-kappaB from cytoplasm to nucleus. Helenalin, an inhibitor of NF-kappaB, abolished the antiapoptotic effect of ouabain. Ouabain (0.1 to 10 nM) also was found to stimulate proliferation and increase the viability of kidney cells. These effects were abolished when release of calcium via the IP3R was prevented.     

Developmental change in Na,K-ATPase alpha1 and beta1 expression in normal and hypothyroid rat renal cortex

Renal Na,K-ATPase drives active reabsorption of sodium and cotransported solutes along the nephron. There is a large increase in net Na(+) reabsorption in the postnatal rat kidney. It has previously been established that the postnatal increase in expression of sodium pump isoforms in the brain, but not the heart, is blunted in the hypothyroid neonate. The aims of this study were to establish whether the developmental increase in renal sodium transport is associated with coordinate increases in the abundance of the sodium pump alpha(1) catalytic and beta(1) glycoprotein subunits and Na,K-ATPase activity, and to determine whether thyroid status influences the postnatal increase in renal Na,K-ATPase expression. Pregnant rats were made hypothyroid with low iodine diet, propylthiouracil and perchlorate. Offspring were hypothyroid assessed by triiodothyronine/thyroxine RIA. Renal cortical membranes were prepared from euthyroid and hypothyroid rats from 6 to 24 days of age. There was no change in Na,K-ATPase activity or expression between 6 and 15 days. Between 15 and 24 days, Na,K-ATPase activity increased 1.35-fold while sodium pump alpha(1) and beta(1) subunit abundance increased coordinately to 1.7- and 2-fold over the previous period, respectively. In hypothyroid neonates, kidney weight was less than in euthyroids, and Na,K-ATPase activity, alpha(1) and beta(1) subunit pool sizes did not significantly increase as a function of age between 6 and 24 days. We conclude that the postnatal increase in sodium pump activity can be accounted for by coordinate increases in the pool sizes of alpha(1) and beta(1) subunits and that, like in brain, this increased Na,K-ATPase expression is dependent on normal thyroid status.     

Reduced glomerular sodium/potassium adenosine triphosphatase activity in acute streptozocin diabetes and its prevention by oral sorbinil

To explore metabolic changes associated with the sorbitol accumulation and myo-inositol depletion observed in glomeruli of rats with experimental diabetes, we examined total and ouabain-inhibited adenosine triphosphatase (ATPase) activity in glomeruli isolated from control and streptozocin (STZ)-diabetic rats. Glomerular Na/K-ATPase activity (ouabain-inhibited) was significantly reduced in diabetic animals, while total (composite) ATPase activity remained unchanged. Treatment with insulin partially restored the Na/K-ATPase activity. Administration of the aldose reductase inhibitor, sorbinil, which normalizes glomerular contents of both sorbitol and myo-inositol in diabetes, completely prevented the diminution of Na/K-ATPase activity. These results establish that glomerular Na/K-ATPase activity is reduced in acute experimental diabetes. The ability of sorbinil to prevent this decrease suggests that it is related to polyol accumulation and/or myoinositol depletion, although an effect of the drug unrelated to its aldose reductase inhibiting property has not been excluded. Since increased polyol pathway flux, decreased myo-inositol, and reduced Na/K-ATPase activity have also been described in peripheral nerve, another tissue in which typical diabetic complications characteristically occur, the consequences of these metabolic changes may be implicated in the pathogenesis of diabetic nephropathy.     

Contrary to rat-type, human-type Na,K-ATPase is phosphorylated at the same amino acid by hormones that produce opposite effects on enzyme activity

Renal sodium homeostasis is a major determinant of BP and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular secondary messengers, either activate or inhibit proximal tubule Na,K-ATPase. It was shown previously that phorbol esters and angiotensin II and serotonin induce the phosphorylation of both Ser-11 and Ser-18 of the Na,K-ATPase alpha-subunit. This results in the recruitment of Na,K-ATPase molecules to the plasma membrane and an increased capacity to transport sodium ions. Treatment of the same cells with dopamine leads to phosphorylation of the Na,K-ATPase alpha-subunit Ser-18. The subsequent internalization of Na,K-ATPase molecules results in a reduced capacity to transport sodium ions. These effects are observed in cells that express the rat-type Na,K-ATPase. However, the Na,K-ATPase alpha1-subunit of several species, such as human, pig, and mouse, does not have a Ser-18 in their N-terminal region. Therefore, the possibility exists that, in those species, the Na,K-ATPase is not regulated by the hormones that regulate natriuresis. This study presents evidence that in cells that express the human-type Na,K-ATPase, dopamine inhibits and phorbol esters activate the Na,K-ATPase-mediated transport. These opposite effects are mediated by the phosphorylation of the same amino acid residue, Ser-11 of Na,K-ATPase alpha1, and the presence of alpha1 Ser-18 is not essential for the hormonal regulation of Na,K-ATPase activity in LLCPK1 cells. It was observed that, whereas the regulatory stimulation of Na,K-ATPase is mediated by protein kinase Cbeta, the regulatory inhibition is mediated by protein kinase Czeta. This is similar to what was demonstrated previously in cells that express the rat-type Na,K-ATPase.     

Plasma conjugated androgens in a dialysis-dependent male as immunoreactive digitalis-like factors

Endogenous digitalis-like factors (DLF) including those which were immunoreactive with digoxin antibody and those which displaced ouabain from Na,K-ATPase, were isolated from the plasma of a dialysis-dependent male patient not taking digoxin. Plasma was passed through a C18 disposable column, the DLF eluted with methanol and separated by HPLC on a C8 column. Immunoreactive DLF were measured in each 1 ml HPLC fraction using radioimmunoassay (RIA) for digoxin. The immunoreactive peaks were determined and aliquots from each peak analyzed by fast atom bombardment mass spectroscopy (FAB-ms). The compounds in the five major HPLC immunoreactive peaks were identified as: 1) dehydroepiandrosterone glucuronide and tetrahydrocortisone glucuronide; 2) epiandrosterone glucuronide; 3) dehydroepiandrosterone sulfate and androsterone glucuronide; 4) epiandrosterone sulfate and 5) androsterone sulfate and androstanediol glucuronide. These immunoreactive DLF represent 70% of the total plasma immunoreactive DLF of 0.124 micrograms digoxin equivalents/l. Aliquots of the HPLC fractions were also assayed for ability to displace ouabain from Na,K-ATPase. The ouabain displacing DLF gave a very different elution pattern from that obtained by RIA with the major Na,K-ATPase ouabain displacing DLF eluting in the more polar fractions. They remain unidentified.     

Na/K-ATPase in intercalated cells along the rat nephron revealed by antigen retrieval

The Na/K-ATPase plays a fundamental role in the physiology of various mammalian cells. In the kidney, previous immunocytochemical studies have localized this protein to the basolateral membrane in different tubule segments. However, intercalated cells (IC) of the collecting duct (CD) in rat and mouse were unlabeled with anti-Na/K-ATPase antibodies. An antigen retrieval technique has been recently described in which tissue sections are pretreated with sodium dodecyl sulfate before immunostaining. This procedure was used to reexamine the presence of Na/K-ATPase in IC along the rat nephron using monoclonal antibodies against the Na/K-ATPase alpha-subunit. Subtypes of IC along the nephron were identified by their distinctive staining with polyclonal and monoclonal antibodies to the 31-kD vacuolar H+ -ATPase subunit, whereas principal cells (PC) were labeled with a polyclonal antibody to the water channel aquaporin-4 (AQP-4). In PC, the Na/K-ATPase and AQP-4 staining colocalized basolaterally. In contrast to previous reports, we found that IC of all types showed basolateral labeling with the anti-Na/K-ATPase antibody. The staining was quantified by fluorescence image analysis. It was weak to moderate in IC of cortical and outer medullary collecting ducts and most intense in IC of the initial inner medullary collecting duct. IC in the initial inner medulla showed a staining intensity that was equivalent or stronger to that in adjacent principal cells. Models of ion transport at the cellular and epithelial level in rat kidney, therefore, must take into account the potential role of a basolateral Na/K-ATPase in intercalated cell function.     

Effects of uremic serum on isolated cardiac myocyte calcium cycling and contractile function.

BACKGROUND: Diastolic dysfunction occurs in patients with chronic renal failure. Moreover, serum from uremic patients contains one or more inhibitors of the plasmalemmal Na,K-ATPase (sodium pump). We hypothesized that a circulating substance present in uremic sera contributes to both sodium pump inhibition and diastolic dysfunction. METHODS: Serum samples were obtained from six patients with chronic renal failure and diastolic dysfunction. RESULTS: Their serum samples caused marked inhibition of Na,K-ATPase purified from dog kidney at all concentrations studied (all P < 0.01) and also impaired ouabain-sensitive rubidium uptake by myocytes isolated from Sprague-Dawley rats (P < 0.01). These cardiac myocytes were studied for their contractile function with video-edge detection and calcium metabolism with indo-1 fluorescence spectroscopy after exposure to these uremic sera. These uremic sera caused increases in myocyte fractional shortening (P < 0.01) as well as an increase in the time constant of relengthening (P < 0.01). Examining the calcium transient, the time constant for calcium recovery was also increased (P < 0.01). Exposure of these cells to sera from age- and sex-matched healthy subjects did not result in significant changes in contraction or calcium cycling. Extracts of uremic serum samples inhibited isolated Na,K-ATPase whereas extracts of normal serum samples did not. The effect of uremic serum extracts on contractile function and calcium cycling were quite similar to that of intact serum or the addition of ouabain. Co-incubation of uremic serum extract with an antibody fragment directed against digoxin markedly attenuated the inhibition of Na,K-ATPase activity and completely prevented any effects on calcium cycling or contractile function. CONCLUSION: These data show that one or more substances are present in uremic sera that acutely cause increased force of contraction and impaired recovery of cardiac myocyte calcium concentration as well as impaired relaxation. As these effects are similar to that seen with ouabain and can be prevented by co-incubation with an antibody fragment to digitalis, which also attenuates the sodium pump inhibitory effect, we suggest that this (these) substance(s) circulating in uremic sera and inhibiting the sodium pump also causes the acute diastolic dysfunction seen in our system.