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RECEPTORS AND SIGNAL TRANSDUCTION
1Department of Cardiology, Royal North Shore Hospital, St. Leonards, Sydney and 2Department of Medicine and 3School of Chemistry, University of Sydney, Sydney, Australia
Submitted 24 May 2006 ; accepted in final form 2 October 2006
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ABSTRACT |
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sodium transport; insulin; angiotensin II; cardiomyopathy; hyperglycemia
2- and 
1-isoforms but an increase in the 1-isoform with a discrepancy between protein expression and activity (9). Another study reported that expression of the 2-isoform was reduced while the 1-isoform was unchanged in diabetes. There was a decrease in overall K+-pNPPase activity (24). The abundance of Na+-K+ pumps in the sarcolemmal membrane or their maximal enzymatic activity does not necessarily reflect in situ Na+-K+ pump function with physiologically relevant intracellular and extracellular ion concentrations. A diabetes-induced change in partial reactions that are rate limiting under physiological conditions might not be reflected in pump abundance or maximal activity. Conversely, a change in abundance or maximal activity might be fully compensated for by a change in rate-limiting partial reactions and be of limited physiological relevance.
There is an association, and probably a causal relationship, between diabetes and congestive heart failure (1). Since raised levels of cytosolic Na+ play pivotal roles in the pathophysiology of heart failure (28), an understanding of the effect of diabetes on the sarcolemmal Na+-K+ pump may guide treatment and prevention. We have examined the effect of diabetes on the sarcolemmal Na+-K+ pump. We used the whole cell patch-clamp technique to measure electrogenic Na+-K+ pump current (Ip), arising from the exchange ratio 3Na+:2K+, in ventricular myocytes. The technique allows accurate control of membrane voltage and of the concentration of the pump's ligands on both sides of the sarcolemmal membrane. We found that diabetes induces a decrease in Ip measured at a physiological, rate-limiting intracellular Na+ concentration. The decrease was dependent on the intracellular K+ concentration in a pattern reminiscent of the effect of angiotensin II (ANG II) on Ip. Since diabetes is associated with upregulation of ANG II receptors, we examined the effect of treatment of rabbits with the ANG II receptor blocker losartan. The treatment reversed the diabetes-induced Na+-K+ pump inhibition.
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MATERIALS AND METHODS |
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To avoid ketoacidosis, most diabetic rabbits required subcutaneous low-dose (1–2 U/day) bovine insulin (Ultralente MC, Novo Nordisk). If urinary ketones were detected (monitored with Multistix 10 SG reagent strips, Bayer) the dose of insulin was increased to eliminate ketones and maintain blood glucose levels <28 mmol/l. Some diabetic rabbits were treated with daily full replacement doses of insulin to maintain euglycemia (4.4–6.5 mmol/l). A group of diabetic rabbits and a group of control rabbits were given the ANG II receptor-blocking drug losartan by oral gavage in a dose of 25 mg/kg body wt (4). A separate group was given losartan and insulin to maintain euglycemia. Blood was collected for biochemical studies from a marginal ear vein on day 1 before injection of alloxan and again on day 9, just before death. Blood pressure was measured in an ear lobe artery (16) on day 1 and day 8.
Rabbits were anesthetized with an intramuscular injection of 50 mg/kg ketamine and 20 mg/kg xylazine hydrochloride. The heart was removed when deep anesthesia was ensured by the absence of corneal reflex and withdrawal of the hindlimb to clamping of the paw. Single ventricular myocytes were isolated (16) and used on the day of isolation only. The institutional review committee for animal research approved all protocols.
Measurement of Na+-K+ pump current. Myocytes were suspended in a tissue bath perfused with modified Tyrode solution that contained (in mmol/l) 140 NaCl, 5.6 KCl and 2.16 CaCl2, 0.44 NaH2PO4, 10 glucose, 1 MgCl2, and 10 HEPES. It was titrated with 1 mol/l NaOH to a pH of 7.40 ± 0.01 at 35°C. When the whole cell configuration was established, we switched to a superfusate that was nominally Ca2+ free and included 0.2 mmol/l CdCl2 and 2 mmol/l BaCl2. For measurement of Ip at a fixed test potential of –40 mV, patch pipettes were filled with solution that included (in mmol/l) 70 potassium glutamate, 10 sodium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, and 80 tetramethylammonium chloride (TMA-Cl). The solution was titrated to a pH of 7.40 ± 0.01 with KOH. In some experiments we increased the sodium glutamate concentration to 80 mmol/l and reduced the TMA-Cl concentration to 10 mmol/l to maintain osmotic balance. In other experiments we maintained the Na+ concentration in pipette solutions at 10 mmol/l but used one of four different K+ concentrations from 0 to 140 mmol/l. The solutions contained (in mmol/l) 9 sodium glutamate, 1 NaH2PO4, 5 HEPES, 2 MgATP, 5 EGTA, and 0, 35, 70, or 140 KCl. Osmotic balance was maintained by adjusting the TMA-Cl concentration from 150 to 10 mmol/l. Solutions were designed to eliminate time-dependent currents for measurements of the voltage dependence of Ip. They contained (in mmol/l) 10 sodium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, 60 TMA-Cl, 20 tetraethylammonium chloride, 70 CsCl, and 50 aspartic acid. We used wide-tipped patch pipettes to optimize control of the Na+-K+ pump's intracellular ligands. Ip was measured as the shift in membrane current induced by 100 µmol/l ouabain at a test membrane potential (Vm) of –40 mV unless otherwise indicated. For measurement of the voltage dependence of Ip we applied voltage steps of 320-ms duration in 20-mV increments to test potentials from –100 to +60 mV. The rationale for experimental protocols, characteristics of patch pipettes, and details for measurement of Ip at fixed (40) and variable (12) Vm have been described previously. Many representative traces of membrane currents similar to those in the present study have been published (see, for example, Refs. 4, 11–13, 16, 40).
Chemicals and reagents. TMA-Cl was purum grade (Fluka Chemicals). All other chemicals were analytical grade (BDH). Alloxan monohydrate and ouabain were obtained from Sigma-Aldrich (St. Louis, MO)
Statistical analysis. Results are expressed as means ± SE. Student's t-test for unpaired data was used for statistical comparisons. We used Dunnett's test when the same control group was used for more than one comparison. The Hill equation was fitted to data with nonlinear regression, and the slopes of Ip-Vm relationships were compared with linear regression. P < 0.05 is regarded as significant. Simulations of the Na+-K+ pump's partial reactions were carried out with the commercially available program Berkeley Madonna 7.0 via the variable step size Rosenbrock integration method for stiff systems of differential equations. The simulations yield the time course of the concentration of each enzyme intermediate, as well as the concentration of inorganic phosphate, from which the turnover number of the enzyme can be calculated (19).
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RESULTS |
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-hydroxybutyrate, free thyroxine, and thyroid-stimulating hormone. We also measured serum osmolarity. Results are shown in Table 1. Baseline in Table 1 refers to blood samples from rabbits never given alloxan or to samples taken before alloxan was given. Diabetes was associated with a significant increase in serum osmolarity consistent with the increase in glucose levels. There was also a small increase in serum cholesterol levels associated with diabetes. Abnormalities in glucose, osmolarity, and cholesterol levels were reversed by treatment with insulin aimed to achieve euglycemia. There was no evidence of ketosis in any group, as indicated by acetoacetate and -hydroxybutyrate levels. There were no differences in systolic or diastolic blood pressure among control rabbits, diabetic rabbits, and diabetic rabbits treated with insulin. There was no difference in the heart weight-to-body weight ratio among the groups.
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Dependence of Ip on extracellular K+. Since alloxan-induced diabetes has been reported to reduce the affinity of the cardiac sarcolemmal Na+-K+ pump for extracellular K+ (25), we examined the effect of diabetes on the dependence of Ip on the K+ concentration in the superfusate. We exposed voltage-clamped myocytes to superfusates containing K+ in different concentrations ranging from 0 to 15 mmol/l. Ip recorded with 10 mmol/l Na+ in pipette solutions is low and difficult to measure accurately when the extracellular K+ concentration is low. To facilitate detection of Na+-K+ pump activation at low extracellular K+ concentrations we used a high Na+ concentration in pipette solutions (80 mmol/l). Each myocyte was exposed to K+ in different concentrations in random order, and each exposure to a K+-containing superfusate was bracketed by exposure to K+-free superfusate to ensure that the holding current returned to baseline. The K+-induced shifts in holding currents are free of contamination of nonpump membrane currents, and rundown of the pump current does not occur during the period of experimentation when this protocol is used (11, 12). However, to minimize the time needed to maintain the myocyte in the whole cell configuration, we nevertheless chose not to measure membrane capacitance in these experiments. We normalized the data to the shift in current induced by 7 mmol/l K+ rather than to the capacitance. This also eliminated variability arising from the measurements of capacitance and hence facilitated the detection of any diabetes-induced change in K+ sensitivity. Results obtained in myocytes from control rabbits and rabbits with diabetes are summarized in Fig. 2. The K+ concentration for half-maximal Ip of a Hill equation fitted to the data was 2.4 and 2.2 mmol/l for myocytes from control rabbits and for myocytes from diabetic rabbits, respectively. Hill coefficients of both fits were 1.2. There was no significant difference between myocytes from control rabbits and diabetic rabbits in the sensitivity of Ip to extracellular K+.
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E2(K+)2 reaction. With the technique we used we cannot experimentally study directly binding of Na+ to the E1 conformation or the backward E1 + 2K+ E2(K+)2 reaction. We therefore evaluated the effect of a change in Na+ affinity or a change in backward rate constant with a model that simulates overall forward pump rate from the kinetics of partial reactions (19). We adjusted the microscopic dissociation constants in the model for the site on the pump's E1 conformation that is highly selective for Na+ binding (KN2) or for the nonselective sites where Na+ competes with K+ for binding (KN1). We first adjusted the dissociation constants to reproduce the dependence of Ip on [K]pip at 0, 35, 70, and 140 mmol/l for control myocytes. We then increased the dissociation constants to approximate the diabetes-induced decrease in Ip measured for a [K]pip of 70 mmol/l and used the model to calculate the pump's turnover number for an intracellular K+ concentration of 0 mmol/l. Results are shown in Fig. 3B. When KN1 or KN2 was adjusted to allow an approximation to experimental observations for a [K]pip of 70 mmol/l, the model predicted an 
24–29% decrease in turnover at an intracellular K+ concentration of 0 mmol/l, contrary to the experimental observation of no significant difference. In a second series of simulations we increased the rate constant of the backward E1 + 2K+ E2(K+)2 reaction. This reproduced a K+-dependent decrease in turnover number consistent with the decrease in Ip observed experimentally. It is an inevitable consequence of the model that the turnover number is identical when intracellular solutions are K+ free, regardless of the value of the rate constant for the backward reaction. This is in good agreement with experimental observations. Since insulin stimulates the Na+-K+ pump in cardiac myocytes in a voltage-dependent manner (12), a diabetes-induced change in pump function might be voltage dependent. Previous studies on the effect of diabetes on the Na+-K+ pump have not controlled membrane voltage. To examine whether there is an effect of diabetes on the pump's voltage dependence, we measured Ip of myocytes from control rabbits and diabetic rabbits at test potentials from –120 to +60 mV. The patch pipette solution included 10 mmol/l Na+. We replaced K+ in the solution with Cs+. The poor permeability of Cs+ in K+ channels should eliminate time-dependent currents of these channels during the voltage-clamp protocol, while a K+-like effect on the Na+-K+ pump should be retained because Cs+ can act as a K+ congener at intracellular pump sites (14). The Ip-Vm relationships for myocytes from the two groups of rabbits are summarized in Fig. 4. The relationships have been normalized to the Ip recorded at 0 mV to facilitate comparison of their slopes (12). There was no effect of diabetes on the slope.
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-cells eliminates production of insulin and the C-peptide derived from proinsulin. The peptide is reported to stimulate the Na+-K+ pump in a variety of tissues via mechanisms independent of the mechanisms that mediate insulin-induced stimulation (37). Since the insulin preparation used to reverse the diabetes-induced inhibition of Ip shown in Fig. 1A does not contain C-peptide, it seems unlikely that C-peptide deficiency accounts for the decrease in Ip associated with diabetes. To further examine whether C-peptide plays a role, we examined the effect of exposing myocytes to C-peptide in vitro. Myocytes from control rabbits were exposed to 100 nmol/l C-peptide included in the superfusate for 10 min before Ip was measured. We chose this protocol because C-peptide-induced Na+-K+ pump stimulation in renal cells was maximal when these concentrations and exposure times were used (36). Mean Ip of seven myocytes from four rabbits was 0.42 ± 0.04 pA/pF. This was not significantly different from the mean Ip shown in Fig. 1A of myocytes exposed to solution not containing C-peptide. Effect of ANG II receptor blockade on Ip. ANG II receptors in the heart are upregulated in diabetes (31), and angiotensin-converting enzyme inhibitors and ANG II receptor-blocking drugs are used to prevent noncardiac complications of diabetes. It may therefore be of therapeutic importance to determine whether ANG II receptor blockade can reverse diabetes-induced Na+-K+ pump inhibition. We measured Ip of myocytes isolated from normal rabbits treated with the ANG II type 1 (AT1) receptor antagonist losartan, from diabetic rabbits that were treated with losartan, and from diabetic rabbits that were treated with insulin to restore euglycemia and that were given losartan. The Na+ and K+ concentrations in pipette solutions were 10 and 70 mmol/l, respectively.
Treatment with losartan induced a decrease in the blood pressure that per se has no effect on Ip of isolated myocytes (16). There was no effect of losartan on blood glucose levels. Mean levels of Ip recorded in myocytes from the three groups of rabbits treated with losartan are shown in Fig. 5. Mean levels for myocytes from control rabbits and from diabetic rabbits not treated with losartan are also shown. In agreement with previous studies (4, 15), treatment of nondiabetic rabbits with losartan induced a statistically significant increase in mean Ip to 148% of control levels. Losartan induced a statistically significant 188% increase in mean Ip of myocytes isolated from diabetic rabbits and abolished the diabetes-induced decrease in mean Ip relative to Ip of control myocytes. Combined treatment of diabetic rabbits with losartan and insulin induced a statistically significant 251% increase in mean Ip to a level similar to that induced when rabbits without diabetes were treated with losartan.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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A comparison of Fig. 1, A and B, indicates that diabetes induced an apparent decrease in the sensitivity of Ip to intracellular Na+. However, Fig. 3A shows that there was no effect of diabetes when pipette solutions were K+ free and contained 10 mmol/l Na+. Since a Na+ concentration of 10 mmol/l is nonsaturating when pipette solutions are K+ free (13, 40) a diabetes-induced decrease in the apparent affinity of the pump for Na+ should have been reflected by a decrease in Ip. The simulation of the effect of a diabetes-induced increase in the microscopic dissociation constants for binding of Na+ to the Na+-K+ pump's E1 conformation shown in Fig. 3B indicates that the decrease in Ip expected from a change in Na+ binding measured with K+-free pipette solutions should be easy to detect experimentally.
If the Na+-K+ pump cycle is described by the formalism of the Post-Albers scheme (10, 17) a shift in the E1 
E2 equilibrium toward E2, rather than a change in intrinsic binding affinity, can cause an apparent decrease in the sensitivity of the pump to intracellular Na+ (30). In principle, a change in any partial reaction may shift the E1 E2 equilibrium toward E2. However, an effect of diabetes on a rate-limiting reaction is most likely to affect overall forward pump rate. Since the main rate-limiting forward E2 E1 conversion (22) is sensitive to cytosolic K+ (30), we determined the [K]pip-Ip relationship. The [K]pip-Ip relationships shown in Fig. 3A were consistent with a diabetes-induced increase in the rate of the backward E1 + 2K+ E2(K+)2 reaction and hence an increase in the apparent affinity constant for binding of K+ to the Na+-K+ pump's Na+-sensitive E1 conformation. While the effect of K+ on Na+ activation of the pump is not purely competitive (7), our findings are in good qualitative agreement with the suggestion that modulation of K+/Na+ antagonism regulates the Na+-K+ pump in the heart (34).
The absence of a significant effect of diabetes on the Ip-Vm relationship in this study does not necessarily indicate that diabetes has no effects on voltage-dependent steps of the pump cycle. The major charge-translocating step occurs with release of Na+ extracellularly. Since the step is very fast (30), only a large diabetes-induced change would have an effect easily detected in the overall forward pump rate reflected in the Ip-Vm relationships. Binding of extracellular K+ is also voltage dependent. This voltage dependence is only detected experimentally when the extracellular K+ concentration is nonsaturating (26), below the physiological level we used. A diabetes-induced change would have to be large to be detected. However, it is unlikely that diabetes has any effect on extracellular K+ binding because we did not detect a change in Ip measured at fixed Vm when the K+ concentration was nonsaturating (Fig. 2).
The absence of an effect of diabetes on the sensitivity of the pump to extracellular K+ is at odds with a reported approximately threefold decrease in the affinity of Na+-K+ pump for K+ in cardiac myocytes isolated from dogs with alloxan-induced diabetes. However, the pump in that study was assessed with the K+-pNPPase assay (25). The assay only reflects the partial reaction of E2(K2) P-E2K2 in a "low-energy" phosphorylation without subsequent conformational change essential for ion transport (23). The reaction, sometimes referred to as "backdoor phosphorylation," is not physiological and cannot be assumed to reflect changes in the entire forward pump cycle reflected by Ip in this study.
A direct toxic effect of alloxan seems unlikely to have caused sarcolemmal Na+-K+ pump inhibition in our study because alloxan is highly specific for pancreatic -cells (20), it did not cause pump inhibition unless diabetes developed, and the alloxan-induced inhibition was reversible with restoration of euglycemia. Insulin deficiency also seems unlikely. The effect of insulin on the pump is voltage dependent at the rate-limiting intracellular Na+ levels we used (12), and there was no significant effect of diabetes on the voltage dependence (Fig. 4). Diabetes was associated with an increase in serum cholesterol (Table 1). However, while marked hypercholesterolemia in rabbits is associated with Na+-K+ pump inhibition, a modest increase in serum cholesterol such as that seen in this study causes pump stimulation (11). The consistent association between hyperglycemia and pump inhibition implicates hyperglycemia in the mechanism mediating the inhibition. The reversal of pump inhibition by treatment with losartan is consistent with this conclusion since in vivo (31) and in vitro (29) studies indicate that raised extracellular glucose levels directly upregulate AT1 receptors in cardiac myocytes.
Treatment of rabbits with the angiotensin-converting enzyme inhibitor captopril or with losartan induces a [K]pip-dependent increase in Ip, while exposure of myocytes isolated from captopril-treated rabbits to ANG II reduces Ip to control levels in a [K]pip-dependent manner (4) consistent with the decrease induced by diabetes in this study. The decrease is mediated by protein kinase C (PKC) (3, 4). The [K]pip dependence is consistent with an effect of ANG II to accelerate the backward E1 + 2K+ E2(K+)2 reaction (5). ANG II increases Na+-K+-ATPase activity and enhances phosphorylation of peptide fragments cleaved from the intact enzyme in rat proximal renal tubule (42). However, the effects of PKC-mediated phosphorylation of the Na+-K+ pump are varied and tissue specific. Many studies have reported stimulation, while others have reported inhibition. Furthermore, there may not be a causal relationship between phosphorylation of the Na+-K+ pump and its activity since there is considerable evidence for pump regulation that depends on PKC but does not involve phosphorylation of the pump itself (35).
Since reactive oxygen species directly inhibit isolated Na+-K+-ATPase (8) and since ANG II promotes their synthesis via a PKC-dependent mechanism, we have speculated that reactive oxygen species may mediate ANG II-induced Na+-K+ pump inhibition in cardiac myocytes. Preliminary studies support this under in vitro experimental conditions (39). It cannot necessarily be assumed that similar inhibition occurs in vivo. However, since hyperglycemia increases synthesis of superoxide and other reactive oxygen species (32), it is reasonable to speculate that reactive oxygen species play a role in the diabetes-induced inhibition of Ip in this study, perhaps by oxidizing sulfhydryl groups on regulatory amino acids of the Na+-K+ pump itself or by inducing lipid peroxidation of membrane phospholipids associated with the pump. Since effects of oxidation on the Na+-K+ pump are poorly reversible (23), such a scheme might also account for the resilience of the diabetes-induced change in Ip to myocyte isolation and subsequent whole cell patch-clamp studies.
Hyperglycemia with diabetes is an independent risk factor for development of heart failure in humans (1, 41). In its early stages, heart failure associated with diabetes is characterized by diastolic dysfunction. Sarcolemmal Na+-K+ pump inhibition and raised cytosolic levels of Na+ may contribute to this because the transmembrane electrochemical potential gradient for Na+ contributes to removal of cytosolic Ca2+ via Na+/Ca2+ exchange and hence to diastolic relaxation (38). The Na+ gradient also mediates cellular uptake of glucose via Na+-glucose cotransporter 1 (SGLT1). Reduced SGLT1-mediated glucose uptake may contribute to a defective energy metabolism in heart failure (33), and, conversely, overexpression of SGLT1 provides protection from experimentally induced heart failure (21). Raised cytosolic levels of Na+ with Na+-K+ pump inhibition in diabetes are expected to reduce SGLT1-mediated glucose uptake and hence perhaps promote heart failure. Abundantly expressed SGLT1 in human cardiac myocytes (43) suggests that this is relevant to human diabetes. Losartan abolished the diabetes-induced Na+-K+ pump inhibition, suggesting that ANG II receptor antagonists or angiotensin-converting enzyme inhibitors should be considered early to treat or prevent heart failure in diabetes.
While diabetes is a risk factor for heart failure, the converse also applies: heart failure of any cause is associated with increased incidence and prevalence of diabetes (1). Since raised levels of cytosolic Na+ are believed to be of pivotal pathogenetic importance in heart failure (28), interventions that restore cellular Na+ homeostasis are desirable. Our study indicates that Na+-K+ pump inhibition associated with hyperglycemia is reversed when euglycemia is restored. Aggressive management of hyperglycemia may be useful in the treatment of heart failure.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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  A. S. Barth and G. F. Tomaselli Cardiac Metabolism and Arrhythmias Circ Arrhythm Electrophysiol, June 1, 2009; 2(3): 327 - 335. [Full Text] [PDF]
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 C. N. White, G. A. Figtree, C.-C. Liu, A. Garcia, E. J. Hamilton, K. K. M. Chia, and H. H. Rasmussen Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase Am J Physiol Cell Physiol, April 1, 2009; 296(4): C693 - C700. [Abstract] [Full Text] [PDF]
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) and for 14 myocytes isolated from 5 diabetic rabbits (
). There was no significant difference between the relationships.
): KN1 was increased to 27.5 mmol/l. All other parameters were as for control simulations. Simulations of an effect of diabetes on the backward rate constant for the reaction E1 + 2K+ 
