Alicia A. McDonough

The sodium pump needs its beta subunit.

The sodium pump Na,K‐ATPase, located in the plasma membrane of all animal cells, is a member of a family of ion‐translocating ATPases that share highly homologous catalytic subunits. In this family, only Na,K‐ATPase has been established to be a heterodimer of catalytic (α) and glycoprotein (β) subunits. The β subunit has not been associated with the pumps transport or enzymatic activity, and its role in Na,K‐ATPase function has been, until recently, a puzzle. In this review we describe what is known about the structure of β and summarize evidence that expression of both a and β subunits is required for Na,K‐ATPase activity, that inhibition of glycosylation causes a decrease in accumulation of both α and β subunits, and we provide evidence that pretranslational up‐regulation of β alone can lead to increased abundance of sodium pumps. These findings are all consistent with the hypothesis that the β subunit regulates, through assembly of αβ heterodimers, the number of sodium pumps transported to the plasma membrane.— McDonough, A. A.; Geering, K.; Farley, R. A. The sodium pump needs its β subunit. FASEB J. 4: 1598‐1605; 1990.

Reduced Sodium Pump α1, α3, and β1-Isoform Protein Levels and Na+,K+-ATPase Activity but Unchanged Na+-Ca2+ Exchanger Protein Levels in Human Heart Failure

Background−Cardiac glycosides initiate an increase in force of contraction by inhibiting the sarcolemmal sodium pump (Na+,K+-ATPase), thereby decreasing Ca2+ extrusion by the Na+-Ca2+ exchanger, which increases the cellular content of Ca2+. In patients with heart failure the sensitivity toward cardiac glycosides is enhanced. Methods and Results−Because the inotropic effect of cardiac glycosides may be a function of the sodium pump and Na+-Ca2+ exchanger (NCE) expression levels, the present study aimed to investigate protein expression of both transporters (immunoblot with specific antibodies against the sodium pump catalytic α1-, α2-, α3-, and glycoprotein β1-isoforms and against NCE) in left ventricle from failing (heart transplantations, New York Heart Association class IV, n=21) compared with nonfailing (donor hearts, NF, n=22) human myocardium. The density of 3H-ouabain–binding sites (Bmax) and the Na+,K+-ATPase activity were also measured. In NYHA class IV, protein levels of Na+,K+-ATPase α1- (0.62±0...

AT1A Angiotensin Receptors in the Renal Proximal Tubule Regulate Blood Pressure

Hypertension affects more than 1.5 billion people worldwide but the precise cause of elevated blood pressure (BP) cannot be determined in most affected individuals. Nonetheless, blockade of the renin-angiotensin system (RAS) lowers BP in the majority of patients with hypertension. Despite its apparent role in hypertension pathogenesis, the key cellular targets of the RAS that control BP have not been clearly identified. Here we demonstrate that RAS actions in the epithelium of the proximal tubule have a critical and nonredundant role in determining the level of BP. Abrogation of AT(1) angiotensin receptor signaling in the proximal tubule alone is sufficient to lower BP, despite intact vascular responses. Elimination of this pathway reduces proximal fluid reabsorption and alters expression of key sodium transporters, modifying pressure-natriuresis and providing substantial protection against hypertension. Thus, effectively targeting epithelial functions of the proximal tubule of the kidney should be a useful therapeutic strategy in hypertension.

Mechanisms of proximal tubule sodium transport regulation that link extracellular fluid volume and blood pressure

One-hundred years ago, Starling articulated the interdependence of renal control of circulating blood volume and effective cardiac performance. During the past 25 years, the molecular mechanisms responsible for the interdependence of blood pressure (BP), extracellular fluid volume (ECFV), the renin-angiotensin system (RAS), and sympathetic nervous system (SNS) have begun to be revealed. These variables all converge on regulation of renal proximal tubule (PT) sodium transport. The PT reabsorbs two-thirds of the filtered Na(+) and volume at baseline. This fraction is decreased when BP or perfusion pressure is increased, during a high-salt diet (elevated ECFV), and during inhibition of the production of ANG II; conversely, this fraction is increased by ANG II, SNS activation, and a low-salt diet. These variables all regulate the distribution of the Na(+)/H(+) exchanger isoform 3 (NHE3) and the Na(+)-phosphate cotransporter (NaPi2), along the apical microvilli of the PT. Natriuretic stimuli provoke the dynamic redistribution of these transporters along with associated regulators, molecular motors, and cytoskeleton-associated proteins to the base of the microvilli. The lipid raft-associated NHE3 remains at the base, and the nonraft-associated NaPi2 is endocytosed, culminating in decreased Na(+) transport and increased PT flow rate. Antinatriuretic stimuli return the same transporters and regulators to the body of the microvilli associated with an increase in transport activity and decrease in PT flow rate. In summary, ECFV and BP homeostasis are, at least in part, maintained by continuous and acute redistribution of transporter complexes up and down the PT microvilli, which affect regulation of PT sodium reabsorption in response to fluctuations in ECFV, BP, SNS, and RAS.

The absence of intrarenal ACE protects against hypertension.

Activation of the intrarenal renin-angiotensin system (RAS) can elicit hypertension independently from the systemic RAS. However, the precise mechanisms by which intrarenal Ang II increases blood pressure have never been identified. To this end, we studied the responses of mice specifically lacking kidney angiotensin-converting enzyme (ACE) to experimental hypertension. Here, we show that the absence of kidney ACE substantially blunts the hypertension induced by Ang II infusion (a model of high serum Ang II) or by nitric oxide synthesis inhibition (a model of low serum Ang II). Moreover, the renal responses to high serum Ang II observed in wild-type mice, including intrarenal Ang II accumulation, sodium and water retention, and activation of ion transporters in the loop of Henle (NKCC2) and distal nephron (NCC, ENaC, and pendrin) as well as the transporter activating kinases SPAK and OSR1, were effectively prevented in mice that lack kidney ACE. These findings demonstrate that ACE metabolism plays a fundamental role in the responses of the kidney to hypertensive stimuli. In particular, renal ACE activity is required to increase local Ang II, to stimulate sodium transport in loop of Henle and the distal nephron, and to induce hypertension.

Regional expression of sodium pump subunits isoforms and Na+-Ca++ exchanger in the human heart.

Cardiac glycosides exert a positive inotropic effect by inhibiting sodium pump (Na,K-ATPase) activity, decreasing the driving force for Na+-Ca++ exchange, and increasing cellular content and release of Ca++ during depolarization. Since the inotropic response will be a function of the level of expression of sodium pumps, which are alpha(beta) heterodimers, and of Na+-Ca++ 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 alpha1, alpha2, alpha3, beta1, and Na+-Ca++ exchangers in all regions. Na,K-ATPase beta2 expression is negligible, indicating that the human cardiac glycoside receptors are alpha1beta1, alpha2beta1, and alpha3beta1. alpha3, beta1, sodium pump activity, and Na+-Ca++ exchanger levels were 30-50% lower in atria compared to ventricles and/or septum; differences between ventricles and septum were insignificant. Functionally, the EC50 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 microM). 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+-Ca++ exchangers, and enhanced sensitivity to inotropic stimulation compared to ventricles.

Recent Advances in Understanding Integrative Control of Potassium Homeostasis

The potassium homeostatic system is very tightly regulated. Recent studies have shed light on the sensing and molecular mechanisms responsible for this tight control. In addition to classic feedback regulation mediated by a rise in extracellular fluid (ECF) [K(+)], there is evidence for a feedforward mechanism: Dietary K(+) intake is sensed in the gut, and an unidentified gut factor is activated to stimulate renal K(+) excretion. This pathway may explain renal and extrarenal responses to altered K(+) intake that occur independently of changes in ECF [K(+)]. Mechanisms for conserving ECF K(+) during fasting or K(+) deprivation have been described: Kidney NADPH oxidase activation initiates a cascade that provokes the retraction of K(+) channels from the cell membrane, and muscle becomes resistant to insulin stimulation of cellular K(+) uptake. How these mechanisms are triggered by K(+) deprivation remains unclear. Cellular AMP kinase-dependent protein kinase activity provokes the acute transfer of K(+) from the ECF to the ICF, which may be important in exercise or ischemia. These recent advances may shed light on the beneficial effects of a high-K(+) diet for the cardiovascular system.

In vivo PTH provokes apical NHE3 and NaPi2 redistribution and Na-K-ATPase inhibition

The aim of this study was to test the hypothesis that in vivo administration of parathyroid hormone (PTH) provokes diuresis/natriuresis through redistribution of proximal tubule apical sodium cotransporters (NHE3 and NaPi2) to internal stores and inhibition of basolateral Na-K-ATPase activity and to determine whether the same cellular signals drive the changes in apical and basolateral transporters. PTH-(1-34) (20 U), which couples to adenylate cyclase (AC), phospholipase C (PLC), and phospholipase A2 (PLA2), or [Nle8,18,Tyr34]PTH-(3-34) (10 U), which couples to PLC and PLA2 but not AC, were given to anesthetized rats as an intravenous bolus followed by low-dose infusion (1 U. kg-1. min-1 for 1 h). Renal cortex membranes were fractionated on sorbitol density gradients. PTH-(1-34) increased urinary cAMP excretion 3-fold, urine output (V) 2.0 +/- 0.1-fold, and lithium clearance (CLi) 2.8 +/- 0.3-fold. With this diuresis/natriuresis, 25% of NHE3 and 18% of NaPi2 immunoreactivity redistributed from apical membranes to higher density fractions containing intracellular membrane markers, and basolateral Na-K-ATPase activity decreased 25%. [Nle8,18,Tyr34]PTH-(3-34) failed to increase V or CLi or to provoke redistribution of NHE3 or NaPi2, but it did inhibit Na-K-ATPase activity 25%. We conclude that in vivo PTH stimulates natriuresis/diuresis associated with internalization of apical NHE3 and NaPi2 and inhibition of Na-K-ATPase activity, that cAMP-protein kinase A stimulation is necessary for the natriuresis/diuresis and NHE3 and NaPi2 internalization, and that Na-K-ATPase inhibition is not secondary to depressed apical Na+ transport.The aim of this study was to test the hypothesis that in vivo administration of parathyroid hormone (PTH) provokes diuresis/natriuresis through redistribution of proximal tubule apical sodium cotransporters (NHE3 and NaPi2) to internal stores and inhibition of basolateral Na-K-ATPase activity and to determine whether the same cellular signals drive the changes in apical and basolateral transporters. PTH-(1-34) (20 U), which couples to adenylate cyclase (AC), phospholipase C (PLC), and phospholipase A2(PLA2), or [Nle8,18,Tyr34]PTH-(3-34) (10 U), which couples to PLC and PLA2 but not AC, were given to anesthetized rats as an intravenous bolus followed by low-dose infusion (1 U ⋅ kg-1 ⋅ min-1for 1 h). Renal cortex membranes were fractionated on sorbitol density gradients. PTH-(1-34) increased urinary cAMP excretion 3-fold, urine output (V) 2.0 ± 0.1-fold, and lithium clearance (CLi) 2.8 ± 0.3-fold. With this diuresis/natriuresis, 25% of NHE3 and 18% of NaPi2 immunoreactivity redistributed from apical membranes to higher density fractions containing intracellular membrane markers, and basolateral Na-K-ATPase activity decreased 25%. [Nle8,18,Tyr34]PTH-(3-34) failed to increase V or CLi or to provoke redistribution of NHE3 or NaPi2, but it did inhibit Na-K-ATPase activity 25%. We conclude that in vivo PTH stimulates natriuresis/diuresis associated with internalization of apical NHE3 and NaPi2 and inhibition of Na-K-ATPase activity, that cAMP-protein kinase A stimulation is necessary for the natriuresis/diuresis and NHE3 and NaPi2 internalization, and that Na-K-ATPase inhibition is not secondary to depressed apical Na+ transport.

Reversible effects of acute hypertension on proximal tubule sodium transporters

Acute hypertension provokes a rapid decrease in proximal tubule sodium reabsorption with a decrease in basolateral membrane sodium-potassium-ATPase activity and an increase in the density of membranes containing apical membrane sodium/hydrogen exchangers (NHE3) [Y. Zhang, A. K. Mircheff, C. B. Hensley, C. E. Magyar, D. G. Warnock, R. Chambrey, K.-P. Yip, D. J. Marsh, N.-H. Holstein-Rathlou, and A. A. McDonough. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F1004-F1014, 1996]. To determine the reversibility and specificity of these responses, rats were subjected to 1) elevation of blood pressure (BP) of 50 mmHg for 5 min, 2) restoration of normotension after the first protocol, or 3) sham operation. Systolic hypertension increased urine output and endogenous lithium clearance three- to fivefold within 5 min, but these returned to basal levels only 15 min after BP was restored. Renal cortex lysate was fractionated on sorbitol gradients. Basolateral membrane sodium-potassium-ATPase activity (but not subunit immunoreactivity) decreased one-third to one-half after BP was elevated and recovered after BP was normalized. After BP was elevated, 55% of the apical NHE3 immunoreactivity, smaller fractions of sodium-phosphate cotransporter immunoreactivity, and apical alkaline phosphatase and dipeptidyl-peptidase redistributed to membranes of higher density enriched in markers of the intermicrovillar cleft (megalin) and endosomes (Rab 4 and Rab 5), whereas density distributions of the apical cytoskeleton protein villin were unaltered. After 20 min of normalized BP, all the NHE3 and smaller fractions of the other apical membrane proteins returned to their original distributions. These findings suggest that the dynamic regulation of proximal tubule sodium transport by acute changes in BP may be mediated by rapid reversible regulation of sodium pump activity and relocation of apical sodium transporters.

SKELETAL MUSCLE NA,K-ATPASE ALPHA AND BETA SUBUNIT PROTEIN LEVELS RESPOND TO HYPOKALEMIC CHALLENGE WITH ISOFORM AND MUSCLE TYPE SPECIFICITY

During potassium deprivation, skeletal muscle loses K+ to buffer the fall in extracellular K+. Decreased active K+ uptake via the sodium pump, Na,K-ATPase, contributes to the adjustment. Skeletal muscle expresses α1, α2, β1, and β2 isoforms of the Na,K-ATPase αβ heterodimer. This study was directed at testing the hypothesis that K+ loss from muscle during K+ deprivation is a function of decreased expression of specific isoforms expressed in a muscle type-specific pattern. Isoform abundance was measured in soleus, red and white gastrocnemius, extensor digitorum longus, and diaphragm by immunoblot. α2 expression was uniform across control muscles, whereas α1 and β1 were twice as high in oxidative (soleus and diaphragm) as in fast glycolytic (white gastrocnemius) muscles, and β2 expression was reciprocal: highest in white gastrocnemius and barely detectable in soleus and diaphragm. Following 10 days of potassium deprivation plasma K+ fell from 4.0 to 2.3 mM, and there were distinct responses in glycolytic versus oxidative muscles. In glycolytic white gastrocnemius α2 and β2 fell 94 and 70%, respectively; in mixed red gastrocnemius and extensor digitorum longus both fell 60%, and β1 fell 25%. In oxidative soleus and diaphragm α2 fell 55 and 30%, respectively, with only minor changes in β1. Although decreases in α2 and β2 expression are much greater in glycolytic than oxidative muscles during K+ deprivation, both types of muscle lose tissue K+ to the same extent, a 20% decrease, suggesting that multiple mechanisms are in place to regulate the release of skeletal muscle cell K+.