Myocardial Na+,K(+)-ATPase in tachycardia induced cardiomyopathy.

Na+,K(+)-ATPase is a major determinant of myocyte homeostasis and excitation-contraction. Cardiac glycosides such as digitalis and ouabain increase the inotropic state of the heart through the inhibition of Na+,K(+)-ATPase. While cardiac glycosides are commonly used in the setting of congestive heart failure, optimal therapy would depend upon an intact Na+,K(+)-ATPase system. Changes in Na+,K(+)-ATPase activity and glycoside receptor density with the development of cardiomyopathy have not been well defined. Accordingly, left ventricular (LV) function and Na+,K(+)-ATPase activity and glycoside binding were examined in 7 pigs with dilated cardiomyopathy and in 7 controls. Dilated cardiomyopathy was produced by pacing induced supraventricular tachycardia (SVT) for 3 weeks at 240 bpm. Left ventricular function was examined by simultaneous echocardiography and catheterization. Left ventricular fractional shortening significantly decreased with SVT (34 +/- 2 vs. 10 +/- 2%, P less than 0.05) and LV diastolic dimension and pressure significantly increased (3.8 +/- 0.3 vs. 5.1 +/- 0.4 cm, and 8 +/- 2 vs. 27 +/- 2 mmHg, respectively, P less than 0.05) as compared to controls. Na+,K(+)-ATPase activity was assayed as potassium dependent p-nitrophenol-phosphatase activity. Glycoside receptor density (Bmax) and affinity (KD) was determined using [3H]-ouabain binding assays. Na+,K(+)-ATPase activity, Bmax, and KD all significantly fell from control values with SVT induced cardiomyopathy (0.64 +/- 0.06 vs. 0.45 +/- 0.12 micrograms pNP/mg/h, 5.5 +/- 0.4 vs. 1.9 +/- 0.4 pmol/mg, and 15 +/- 3 vs. 9 +/- 3 nM, respectively, P less than 0.05). The distribution of Na+,K(+)-ATPase in LV sections taken from control and SVT hearts were examined using immunohistochemical techniques. A patchy distribution of Na+,K(+)-ATPase along the sarcolemma in SVT sections was observed as opposed to a more uniform distribution in control myocytes. There was no observable change in the relative content and distribution of the Na+,K(+)-ATPase isoforms alpha 2 and alpha 3 in the SVT sections as compared to controls. In an additional set of experiments, changes in LV as well as isolated myocyte responsiveness to ouabain were examined. Left ventricular fractional shortening and peak dP/dt were measured following administration of 20-60 micrograms/Kg of ouabain in control (n = 3) and SVT (n = 3) pigs. In the control group, 40 micrograms/Kg caused a 25% in LV fractional shortening and a 60% increase in peak dP/dt from baseline. Cumulative doses of 60 micrograms/Kg in the control pigs resulted in over a 75% increase in peak dP/dt from baseline values.(ABSTRACT TRUNCATED AT 400 WORDS)     

C-peptide, Na+,K(+)-ATPase, and diabetes

Na+,K(+)-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na+,K(+)-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na+,K(+)-ATPase activity was strongly related to blood C-peptide levels in non-insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene. A polymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na+,K(+)-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na+,K(+)-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na+,K(+)-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na+,K(+)-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na+,K(+)-ATPase activity. This impairment in Na+,K(+)-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetes-induced decrease in Na+,K(+)-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na+,K(+)-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na+,K(+)-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na+,K(+)-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.     

High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit.

Recently, a beta subunit for the rat gastric H+,K(+)-ATPase (HK beta), which is structurally similar to the beta subunit of Na+, K(+)-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta subunit assembly with different isoforms of the alpha subunit of Na+, K(+)-ATPase. Coexpression in yeast cells of the HK beta with both the sheep alpha 1 subunit and the rat alpha 3 subunit isoforms of Na+, K(+)-ATPase (alpha 1 and alpha 3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha 1 plus HK beta, alpha 3 plus HK beta) have a high affinity for ouabain (Kd, 5-10 nM) and are expressed at levels similar to those formed with the rat beta 1 subunit of Na+, K(+)-ATPase (beta 1) (alpha 1 plus beta 1 or alpha 3 plus beta 1). Potassium acts as a specific antagonist of ouabain binding by alpha 1 plus HK beta and alpha 3 plus HK beta just like sodium pumps formed with beta 1. Sodium pumps formed with the HK beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta subunit may play a role in sodium pump function.     

A putative fourth Na+,K(+)-ATPase alpha-subunit gene is expressed in testis.

The Na+,K(+)-ATPase alpha subunit has three known isoforms, alpha 1, alpha 2 and alpha 3, each encoded by a separate gene. This study was undertaken to determine the functional status of a fourth human alpha-like gene, ATP1AL2. Partial genomic sequence analysis revealed regions exhibiting sequence similarity with exons 3-6 of the Na+,K(+)-ATPase alpha isoform genes. ATP1AL2 cDNAs spanning the coding sequence of a novel P-type ATPase alpha subunit were isolated from a rat testis library. The predicted polypeptide is 1028 amino acids long and exhibits 76-78% identity with the rat Na+,K(+)-ATPase alpha 1, alpha 2 and alpha 3 isoforms, indicating that ATP1AL2 may encode a fourth Na+,K(+)-ATPase alpha isoform. A 3.9-kb mRNA is expressed abundantly in human and rat testis.     

Developmental differences in the responsiveness of gill Na+,K(+)-ATPase to cortisol in salmonids.

The ability of cortisol to increase gill Na+,K(+)-ATPase activity was examined in several salmonid species during development. Coho salmon (Oncorhynchus kisutch) parr were unresponsive to cortisol in vitro (10 micrograms/ml for 2 days) in November. Responsiveness was significant from January to March, peaking in January just prior to seasonal increases in gill Na+,K(+)-ATPase activity. Gill tissue became unresponsive to in vitro cortisol in April when in vivo gill Na+,K(+)-ATPase activity peaked. The ability of cortisol to stimulate gill, Na+,K(+)-ATPase activity in postemergent fry (2-3 months after hatching) was examined in chum (O. keta), chinook (O. tschawytscha), coho, and Atlantic salmon (Salmo salar). Initial levels of gill Na+,K(+)-ATPase activity were elevated in chum salmon, which normally migrate as fry. Cortisol (10 micrograms/ml for 4 days in vitro) increased gill Na+,K(+)-ATPase activity in chum salmon fry (48% above initial levels), had a limited but significant effect in chinook salmon fry, and had no effect in coho and Atlantic salmon fry. In an in vivo experiment, Atlantic salmon previously exposed to simulated natural photoperiod (SNP) and continuous light (L24) received four cortisol injections of 2 micrograms.g-1 every third day. SNP fish responded with increased gill Na+,K(+)-ATPase activity (+66%), whereas L24 fish were not affected. Atlantic salmon presmolts with initially low levels of gill Na+,K(+)-ATPase activity responded to cortisol in vitro, whereas smolts with initially high levels of gill Na+,K(+)-ATPase activity were unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation of Na+, K(+)-ATPase to delayed motor nerve conduction velocity: effect of aldose reductase inhibitor, ADN-138, on Na+, K(+)-ATPase activity

The role of sorbitol, myo-inositol, and Na+, K(+)-adenosine triphosphatase (ATPase) activity on motor nerve conduction velocity (MNCV) in streptozotocin (STZ)-diabetic rats was studied. Reduction of MNCV and Na+, K(+)-ATPase in caudal nerves appeared after 3 weeks of diabetes, and at this time treatment with aldose reductase inhibitor (ARI), ADN-138 and 1% myo-inositol supplement was begun. One percent myo-inositol supplement for 3 weeks resulted in a significant increase in myo-inositol levels in diabetic nerves, but left MNCV and sorbitol levels unchanged. In contrast, treatment with ADN-138 for 3 weeks reduced sorbitol levels in diabetic nerves and resulted in significant increases in MNCV and Na+, K(+)-ATPase in the nerves. Since ADN-138 did not restore myo-inositol levels, the increase in Na+, K(+)-ATPase levels by ADN-138 treatment was independent of myo-inositol levels. Also, nerve Na+ levels in ADN-138-treated rats were reduced and the ratio of K+ to Na+ was raised, while 1% myo-inositol supplement did not affect them. These results suggest that treatment with ADN-138 elevates MNCV through a series of processes: ARI----reduction of sorbitol level----increase in Na+, K(+)-ATPase activity----correction of K+, Na+ imbalance----increase in MNCV.     

Calcineurin mediates alpha-adrenergic stimulation of Na+,K(+)-ATPase activity in renal tubule cells.

The alpha-adrenergic agonist oxymetazoline increased Na+,K(+)-ATPase activity of single proximal convoluted tubules dissected from rat kidney. Activation of the enzyme by oxymetazoline was prevented by either the alpha 1-adrenergic antagonist prazosin or the alpha 2-adrenergic antagonist yohimbine and was mimicked by the calcium ionophore A23187. The effect of oxymetazoline on Na+,K(+)-ATPase activity was prevented by a specific peptide inhibitor of calcineurin, as well as by FK 506, an immunosuppressant agent known to inhibit calcineurin; these results indicate that the action of oxymetazoline is mediated via activation of calcineurin (a calcium/calmodulin-dependent protein phosphatase). Activation of the Na+,K(+)-ATPase by either oxymetazoline or A23187 was associated with a greater than 2-fold increase in its affinity for Na+. The results provide a biochemical mechanism by which norepinephrine, released from renal nerve terminals, stimulates Na+ retention.     

Dietary soy prevents brain Na+, K(+)-ATPase reduction in streptozotocin diabetic rats

The aim of this study was to investigate Na+, K(+)-ATPase activity in cerebral cortex, hippocampus and hypothalamus of diabetic rats. The action of dietary soy protein on the effect produced by diabetes on this activity was also tested. Forty-nine-day-old Wistar were divided into two groups: diabetes streptozotocin (50 mg/kg body weight) and control (citrate solution). Rats were sacrificed 56 days later. In other set of experiments, rats received a dietary with casein (control) from day 21 to the 49 of postnatal-age and were subjected to diabetes or received citrate (control). One week later, rats received a special dietary with soy protein with isoflavones or casein (control) from day 56 to the 105 of postnatal-age. Results showed that diabetic rats presented a reduction ( approximately 40%) of Na+, K(+)-ATPase activity in all structures studied. Pretreatment with soy protein prevented the inhibitory effects of diabetes on the enzyme activity. Assuming the possibility that these effects might also occur in the human condition, our findings may be relevant to explain, at least in part, the neurologic dysfunction associated with diabetes and might support a novel therapeutic strategy (soy protein) to slow the progression of neurodegeneration in this disorder.     

Phosphorylation of the catalytic subunit of Na+,K(+)-ATPase inhibits the activity of the enzyme.

We have examined two distinct protein kinases, cAMP-dependent protein kinase and protein kinase C, for their ability to phosphorylate and regulate the activity of three different types of Na+,K(+)-ATPase preparation. cAMP-dependent protein kinase phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 1 mol of phosphate per mol of alpha subunit. Protein kinase C phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 2 mol of phosphate per mol of alpha subunit. The phosphorylation by each of the kinases was associated with an inhibition of Na+,K(+)-ATPase activity of about 40-50%. These two protein kinases also inhibited the activity of a partially purified preparation of Na+,K(+)-ATPase from rat renal cortex and the activity of Na+,K(+)-ATPase present in preparations of basolateral membrane vesicles from rat renal cortex.     

Bidirectional regulation of Na+,K(+)-ATPase activity by dopamine and an alpha-adrenergic agonist.

Catecholamines have pronounced effects on the renal handling of sodium and water, dopamine-promoting sodium and water excretion, and norepinephrine-promoting sodium and water retention. In the present study, using isolated permeabilized renal tubule cells and intact rats, we have shown that these effects can be attributed to opposing actions of these transmitters on renal tubular Na+,K(+)-ATPase activity. The ability of each of these catecholamines to regulate Na+,K(+)-ATPase activity is affected by the concentration of Na+ as well as by the absence or presence of the opposing catecholamine.     

Ventricular function and Na+,K(+)-ATPase activity and distribution with chronic supraventricular tachycardia.

STUDY OBJECTIVE--The molecular and cellular mechanisms responsible for the dilated cardiomyopathy associated with chronic supraventricular tachycardia are not well understood. The purpose of this study was to examine Na+,K(+)-ATPase activity and distribution in a pacing induced model of dilated cardiomyopathy. DESIGN--Left ventricular function and Na+,K(+)-ATPase activity and distribution were examined in two groups of pigs: (1) atrially paced for 3 weeks (supraventricular tachycardia, 240 beats.min-1); (2) sham operated controls. SUBJECTS--10 Yorkshire male swine (23-25 kg) were randomly assigned to the control group or the supraventricular tachycardia group. MEASUREMENTS AND MAIN RESULTS--Left ventricular function was examined using simultaneous pressure echocardiography. Na+,K(+)-ATPase activity was determined in tissue homogenates by measuring the rate of p-nitrophenol-phosphate (pNPP) hydrolysis. Changes in content and distribution of Na+,K(+)-ATPase were examined immuno-histochemically in tissue sections. Left ventricular fractional shortening decreased significantly with supraventricular tachycardia as compared to controls, at 15 (SEM 3)% v 31(3)%, respectively p less than 0.05. Supraventricular tachycardia resulted in a significant increase in end diastolic dimension [5.0(0.3) cm v 3.5(0.2) cm, respectively p less than 0.05] and pressure [22(4)mm Hg v 6(2)mm Hg, respectively p less than 0.05]. Maximal Na+,K(+)-ATPase activity (microgram pNPP.mg-1 protein.h-1) was significantly lower with supraventricular tachycardia than in controls, at 0.45(0.12) v 0.64(0.06), respectively p less than 0.05. In the presence of 7 microM digitalis, Na+,K(+)-ATPase activity was inhibited by 68% in control and by 45% in supraventricular tachycardia homogenates (p less than 0.05). In control sections all left ventricular myocytes showed a uniform immunostaining pattern along the sarcolemma for Na+,K(+)-ATPase, whereas a focal loss of staining was observed in myocytes from the supraventricular tachycardia group. CONCLUSIONS--The congestive cardiomyopathy produced by supraventricular tachycardia was associated with a reduction in sarcolemmal Na+,K(+)-ATPase activity and changes in enzyme distribution. The findings also suggest a reduction in digitalis sensitivity with chronic supraventricular tachycardia. These alterations in Na+,K(+)-ATPase activity may be one potential mechanism responsible for the depressed left ventricular function associated with chronic supraventricular tachycardia.     

Role of Na+,K(+)-ATPase in the centrally mediated hypotensive effects of potassium in anaesthetized rats.

Several investigators have demonstrated the antihypertensive properties of potassium in various models of hypertension. The present studies were conducted to determine whether central mechanisms contribute to these salutary effects of potassium. In Inactin-anaesthetized rats, intracerebroventricular administration of KCl solutions (0.375, 0.75 and 1.25 mumol/5 microliters) produced concentration-dependent reductions in arterial pressure and heart rate. These effects were significantly attenuated by prior central administration of ouabain, a selective inhibitor of the sodium pump. In a separate series of experiments, prior central administration of alpha 1- and alpha 2-antagonist phentolamine, or the dopamine receptor (DA1 and DA2) antagonist RS-sulpiride, was also effective in inhibiting the hypotensive and bradycardiac effects of intracerebroventricular administration of potassium. Thus, these data suggest that activation of Na+,K(+)-ATPase and central noradrenergic and dopaminergic mechanisms are involved in the central actions of potassium and these central mechanisms may contribute to the salutary effects of a potassium-rich diet in hypertensive subjects. The present studies demonstrate a potentially important relationship between Na+,K(+)-ATPase activity in the central nervous system and neural regulation of arterial blood pressure.     

Differential expression and enzymatic properties of the Na+,K(+)-ATPase alpha 3 isoenzyme in rat pineal glands.

We have used immunoblotting and biochemical techniques to analyze expression of Na+,K(+)-ATPase alpha and beta subunits in rat pineal glands. Western blot analysis of pineal microsomal membrane fractions with antisera specific for each of the three rat alpha and two rat beta subunits revealed similar levels of expression of alpha 1 and alpha 3 subunits in pineal glands of 5-day-old rats. High levels of alpha 3 and beta 2 subunits and low levels of alpha 1 subunits were detected in adult glands. No alpha 2 or beta 1 subunits were detectable at either developmental stage. Examination of the enzymatic properties of the pineal gland alpha 3 isoform suggests that this enzyme is a ouabain-sensitive ATPase whose activity is dependent upon Na+ and K+. This ATPase exhibited a lower apparent Km for Na+ than the kidney alpha 1 isoenzyme and did not show positive cooperative Na+ activation. Our results suggest that the activity of the Na+,K(+)-ATPase alpha 3 isoenzyme may be adapted to function under conditions of hyperpolarizing transmembrane potentials.     

Local translational diffusion rates of membranous Na+,K(+)-ATPase measured by saturation transfer ESR spectroscopy.

Diffusion-controlled Heisenberg spin exchange between spin-labeled Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), EC 3.6.1.37] proteins has been studied by saturation transfer ESR spectroscopy in reconstituted membranes. Na+,K(+)-ATPase from the salt gland of Squalus acanthias was solubilized in a polyoxyethylene ether detergent, octa(ethylene glycol) dodecyl monoether. Part of the solubilized enzyme was covalently spin-labeled with a nitroxide derivative of indanedione and recombined with various proportions of the unlabeled enzyme while the native lipid/protein ratio was maintained. Purified membranes were then reconstituted from the various samples by precipitation with divalent ions. The reciprocal integrated intensities of the saturation transfer ESR spectra were found to increase linearly with the fraction of protein that was spin-labeled, and the gradient of the concentration dependence increased with increasing temperature over the range 4 degrees-25 degrees C. Comparison with theoretical analyses of the effects of weak Heisenberg spin exchange [Marsh, D. & Horváth, L. I. (1992) J. Magn. Reson. 97, 13-26] suggests that the effects on the saturation transfer ESR intensity are attributable to short-range diffusional collisions between the spin-labeled protein molecules. The effective value of the local translational diffusion coefficient is 1.8-2.9 microns2.s-1 at 15 degrees C, depending on the diffusion model used, which is much larger than the values obtained for the long-range diffusion coefficient in cells by photobleaching techniques. The temperature dependence of the translational diffusion is larger than expected but correlates with the anomalous temperature dependence of the rotational diffusion observed in the same system.     

The presence of H+,K(+)-ATPase in the crypt of rabbit distal colon demonstrated with monoclonal antibodies against gastric H+,K(+)-ATPase

Indirect evidence indicates the presence of an active H+/K+ antiporter for the secretion of acid in the distal colon. It was examined whether the H+/K+ antiporter in the rabbit distal colon was hydrogen-potassium-stimulated adenosine triphosphatase (H+,K(+)-ATPase), which acts as a proton pump in the gastric mucosa. For this purpose, four monoclonal antibodies against hog gastric H+,K(+)-ATPase were raised. Three monoclonal antibodies dose-dependently inhibited the ouabain-insensitive gastric ATP-ase activity. Antibody HK4001 completely inhibited the ATPase activity. In indirect immunofluorescence studies, all four monoclonal antibodies stained H+,K(+)-ATPase in gastric mucosae of various animal species. Two monoclonal antibodies including antibody HK4001 cross-reacted with H+,K(+)-ATPase located in crypts of the transverse and descending colon and rectum of rabbits. Because the other two antibodies did not cross-react with the H+,K(+)-ATPase in the colon, this colonic enzyme is similar but not identical to gastric H+,K(+)-ATPase. On the other hand, HK4001 and SCH 28080 did not inhibit ouabain-sensitive K(+)-dependent ATPase activity in the guinea pig distal colon, and the antibodies did not stain the enzyme in the tissue. Therefore, ouabain-sensitive H+/K+ antiporter in the guinea pig is not similar to ouabain-insensitive rabbit colonic H+,K(+)-ATPase.     

Immunocytochemical and enzyme histochemical localization of Na+,K(+)-ATPase in normal and ischemic porcine myocardium

Na+,K(+)-ATPase is involved in generating transmembrane ion gradients and the associated potential difference necessary for contraction of cardiac myocytes. It is possible that changes in the activity or membrane content of this enzyme may occur under ischemic conditions. To investigate this question, right ventricular (RV) ischemia was produced in closed chest pigs and the RV ejection fraction was measured using a fast response thermistor in the pulmonary artery. Sections of RV collected at 15, 30, 45, and 60 min of ischemia were assayed for changes in sarcolemmal Na+,K(+)-ATPase activity using an enzyme coupled histochemical reaction as well as a biochemical assay. Similar sections were examined for changes in the distribution and content of Na+,K(+)-ATPase using an immunocytochemical procedure. The RV ejection fraction fell significantly from baseline after 15 min of ischemia (62 +/- 3% vs 39 +/- 3% respectively, P less than 0.05, n = 10). A decrease in sarcolemmal Na+,K(+)-ATPase activity was first detected after 30 min of occlusion and a significant reduction in enzyme activity was present at 45 min of ischemia. In contrast no changes were detected in the distribution or content of immunoreactive Na+,K(+)-ATPase in the sarcolemma at any time point. In addition, the amount of Na+,K(+)-ATPase in tissue homogenates showed no significant changes after 45 min of ischemia. These findings show that acute ischemia results in the disruption of sarcolemmal Na+,K(+)-ATPase activity and suggests that the decrease in enzyme activity is not due to the loss or redistribution of sarcolemmal Na+,K(+)-ATPase.     

Phosphorylated Mr 32,000 dopamine- and cAMP-regulated phosphoprotein inhibits Na+,K(+)-ATPase activity in renal tubule cells.

Dopamine inhibits Na+,K(+)-ATPase activity in several renal tubule segments and thereby regulates urinary Na+ excretion. We now show that a phosphopeptide of 31 amino acids, corresponding to residues 8-38 of the protein phosphatase inhibitor DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32,000), mimics the inhibitory action of dopamine on Na+,K(+)-ATPase activity in renal tubule cells from the ascending limb of the loop of Henle. The dephosphorylated form of the peptide is ineffective. The results indicate that dopamine acts through a protein phosphorylation pathway to regulate the activity of an ion pump. In addition, the data suggest that inhibition of protein phosphatase 1 by phophorylated DARPP-32 is a component of the mechanism by which dopamine regulates urinary Na+ excretion.     

Erythrocyte sodium fluxes, ouabain binding sites, and Na+,K(+)-ATPase activity in hyperthyroidism

Erythrocyte sodium pump activity, in contrast to other tissues, is decreased in hyperthyroidism. In order to examine whether the effect of thyroid hormones on erythrocytes is part of a generalized effect on other transport pathways, we measured sodium pump activity, Na+,K(+)-adenosine triphosphatase (ATPase) activity, ouabain binding sites, bumetanide-sensitive sodium potassium cotransport (SPC), sodium lithium countertransport (SLC), and ouabain- and bumetanide-insensitive passive efflux of sodium (sodium "leak") in erythrocytes from 20 healthy subjects and 18 untreated hyperthyroid subjects. Sodium pump activity (ouabain-sensitive sodium efflux rate constant), Na+,K(+)-ATPase activity, and the number of ouabain binding sites were lower and the erythrocyte sodium content was higher in hyperthyroid subjects. The rate constants of erythrocyte SPC (P less than .05), SLC (P less than .001), and sodium "leak" (P less than .05) were also significantly lower in hyperthyroidism. In 11 of the hyperthyroid subjects, sodium flux measurements were repeated after 20 weeks of treatment. Sodium pump activity, the number of ouabain binding sites, and the rate constant for SLC increased. These results suggest that the effect of thyroid hormones on the erythrocyte sodium pump is part of a generalized effect on membrane proteins, rather than a specific effect.     

Enhanced Angiotensin II-Induced Activation of Na+, K+-ATPase in the Proximal Tubules of Obese Zucker Rats

Renal angiotensin II (AII) is suggested to play a role in the enhanced sodium reabsorption that causes a shift in pressure natriuresis in obesity related hypertension; however, the mechanism is not known. Therefore, to assess the influence of AII on tubular sodium transport, we determined the effect of AII on the Na⁺, K⁺-ATPase activity (NKA), an active transporter regulated by the AT₁ receptor activity, in the isolated proximal tubules of lean and obese Zucker rats. Also, we determined the levels of the tubular AT₁ receptor and associated signal transducing G proteins, as the initial signaling components that mediate the effects of AII on Na⁺, K⁺-ATPase activity. In the isolated proximal tubules, AII produced greater stimulation of the NKA activity in obese compared with lean rats. Determination of the AT₁ receptors by Scatchard analysis of the [¹²⁵I] Sar-Ang II binding and Western blot analysis in the basolateral (BLM) and brush border membrane (BBM) revealed a modest but significant increase (23%) in the AT₁ receptor number mainly in the BLM of obese compared with lean rats. The AII affinity for AT₁ receptors, as determined by IC₅₀ values of AII to displace [¹²⁵I] Sar-Ang II binding in BLM and BBM were similar in lean and obese rats. Western blot analysis revealed significant increases in Giα₁, Giα₂, Giα₃, and Gq/₁₁α in BLM and Giα₁, Giα₃, and Gq/₁₁α in BBM of obese as compared with lean rats. The increase in the levels of the AT₁ receptor and G proteins, mainly in the BLM, may be contributing to the enhanced AII-induced activation of NKA in the proximal tubules of obese rats. This phenomenon, in part, may be responsible for the increased sodium reabsorption and the development of hypertension in obese Zucker rats.