This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zerbini, G. Articles by Semplicini, A. Search for Related Content PubMed PubMed Citation Articles by Zerbini, G. Articles by Semplicini, A.

(Hypertension. 1995;25:986-993.)
© 1995 American Heart Association, Inc.

Articles

Sodium-Lithium Countertransport Has Low Affinity for Sodium in Hyperinsulinemic Hypertensive Subjects

Gianpaolo Zerbini; Giulio Ceolotto; Cynthia Gaboury; Lucio Mos; Achille C. Pessina; Mitzy Canessa; Andrea Semplicini

From the Endocrine Hypertension Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Mass (G.Z., C.G., M.C.); and the Department of Clinical Medicine, University of Padova (Italy) Medical School (G.C., L.M., A.C.P., A.S.).

Correspondence to Mitzy Canessa, PhD, Endocrine Hypertension Division, Brigham and Women's Hospital, 221 Longwood Ave, Boston, MA 02115.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract We recently reported that incubation of red blood cells with insulin markedly decreases the affinity for external Na⁺ and increases the maximal transport rate (V_max) of Na⁺-Li⁺ countertransport. The association of hypertension with insulin resistance and its compensatory hyperinsulinemia led us to investigate the relationship between insulin levels in vivo and the Na⁺ activation kinetics of this antiporter. We studied normotensive (n=28) and hypertensive (n=25) subjects after they had fasted overnight and determined their plasma glucose and insulin concentrations. Insulin levels were higher in the hypertensive subjects (11.7±1.5 µU/mL, mean±SEM) than in the normotensive subjects (8.2±1.2 µU/mL), but glucose levels were similar and within normal limits. Antiporter activity was measured as sodium-stimulated Li⁺ efflux by a new procedure that uses isosmotic conditions to raise external Na⁺ to 280 mmol/L. In normotensive subjects, V_max was reached between 50 and 100 mmol/L Na⁺, whereas in most hypertensive subjects, Na⁺ concentrations higher than 150 mmol/L were needed. This different kinetic behavior was because the Na⁺ concentration for half-maximal activation (K_m) was twofold higher in hypertensive subjects (58.9±5.3 mmol/L) than in normotensive subjects (29.8±2.6 mmol/L, P<.001). Hypertensive subjects with fasting insulin levels greater than 10 µU/mL (n=12) had a higher K_m for Na⁺ than subjects with insulin levels less than 10 µU/mL (n=13) (73.4±8.7 versus 45.6±3.9 mmol/L, respectively, P<.01) and similar V_max (0.57±0.05 versus 0.41±0.05 mmol · L^-1 · h^-1). In contrast, normotensive subjects with insulin levels greater than 10 µU/mL (n=6) had V_max and K_m values similar to those with insulin levels less than 10 µU/mL (n=22). Simple regression analysis showed that body mass index, insulin, and blood pressure correlated with both kinetic parameters. However, stepwise multiple regression analysis showed that the main determinant of V_max was blood pressure and for K_m, blood pressure and insulin levels. The association of a low affinity for Na⁺ with in vivo hyperinsulinemia and its concomitant insulin resistance observed in hypertensive subjects agrees with the in vitro effects of insulin on this antiporter.

Key Words: hyperinsulinism • hypertension, essential • sodium affinity • sodium exchanger • erythrocytes • lithium

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Numerous studies have confirmed the observation that red blood cell (RBC) Na⁺-Li⁺ countertransport activity is elevated in hypertensive compared with normotensive individuals¹ (for review see Reference 2). Several epidemiological surveys have consistently found an association between elevated RBC Na⁺-Li⁺ countertransport and blood pressure (BP).^{3 4} Family studies have established Na⁺-Li⁺ countertransport as the best-characterized intermediate phenotype with a very high heritability component.^{5 6} Within the hypertensive population, elevated countertransport activity is associated with non-modulating hypertension⁷ and alterations of body mass index (BMI), serum triglycerides, uric acid, and cholesterol.^{8 9 10} These latter metabolic abnormalities are part of the syndrome characterized by resistance to insulin-stimulated glucose disposal and compensatory hyperinsulinemia.^{11 12} Ferrannini et al¹³ have established the coexistence of hypertension and insulin resistance using the euglycemic insulin-clamp technique, and this has been confirmed in numerous studies. Hyperinsulinemia has been proposed as a potential link between these metabolic and BP abnormalities.^{11 12 13}

Insulin has been shown to stimulate the Na⁺-H⁺ antiporter (an Na⁺ exchange mode activated by low intracellular pH) in skeletal muscle cells¹⁴ and renal proximal tubular cells.¹⁵ Although insulin receptors have been very well characterized in human erythrocytes,¹⁶ the targets of insulin action in RBCs are unknown. We have recently demonstrated that physiological concentrations of insulin augmented the maximal transport rate (V_max) of Na⁺-H⁺ and Na⁺-Li⁺ exchanges in RBCs of normotensive subjects studied after an overnight fast. In addition, insulin action in vitro induced a marked increase in the Na⁺ concentration necessary for half-maximal activation (K_m) of Na⁺-H⁺ and Na⁺-Li⁺ exchanges.^{17 18 19} These findings led us to investigate whether the enhanced activity of RBC Na⁺-Li⁺ countertransport observed in hypertensive subjects reflects in vivo responsiveness of this antiporter to hyperinsulinemia. In fact, it has been shown that hypertensive subjects with elevated Na⁺-Li⁺ countertransport activity have greater insulin resistance than hypertensive subjects with normal countertransport activity.²⁰ Recent studies in young normotensive and hypertensive blacks have also reported the association between insulin resistance and elevated V_max of RBC Na⁺-H⁺ and Na⁺-Li⁺ exchanges.²¹

In the present study, we investigated the sodium activation kinetics of the exchange of Li⁺ for Na⁺ in normotensive and hypertensive subjects. Both study groups were characterized by fasting levels of insulin and glucose, V_max, and the affinity for Na⁺ determined from measurements of K_m. We have developed a novel procedure that accurately determines V_max and K_m for Na⁺ because saturation kinetics can be shown for external Na⁺ in both normotensive and hypertensive subjects. Our results indicate that Na⁺-Li⁺ countertransport has two abnormalities in hypertensive subjects: (1) a low affinity for Na⁺ that correlates with increased insulin levels in vivo and (2) a high V_max associated with high BP but not with hyperinsulinemia. Our study therefore provides information about the in vivo responsiveness of the antiporter in the presence of insulin levels.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Subject Selection
Participants in this study were untreated hypertensive (n=25) and normotensive (n=28) volunteers of Caucasian origin. The hypertensive subjects had been consecutively admitted to the outpatient clinic for hypertension assessment; the normotensive subjects were recruited from a population study aiming to define normal limits for ambulatory BP. Twenty-five subjects were enrolled in Boston and 28 in Padova. All subjects provided written informed consent before enrollment. The protocol was approved by the Committee of Human Studies of both institutions. At enrollment, none of the subjects was taking any antihypertensive medications. Subjects known to have type I or type II diabetes mellitus were excluded.

Study Protocol
Subject and control groups followed a diet containing 200 mEq/d NaCl. On the morning of the study, the subjects reported to the outpatient clinic after an overnight fast, and height and weight were measured while the subjects were wearing light clothing and no shoes. After a 5-minute rest, mean systolic and diastolic (fifth Korotkoff sound) BP values were obtained by averaging two supine BP measurements. Mean BP was calculated by adding one third of pulse pressure to diastolic pressure. Hypertension was defined as a diastolic BP higher than 90 mm Hg or systolic higher than 140 mm Hg in the absence of treatment. Venous blood was drawn for blood glucose and insulin measurements and for measurement of RBC Na⁺-Li⁺ countertransport (herein called countertransport); blood was transported to the laboratory for processing within 2 hours of venipuncture.

Plasma glucose concentration was measured by the glucose oxidase method and insulin by radioimmunoassay in samples frozen at -20°C.

Measurements of RBC Na⁺-Li⁺ Countertransport
The Na⁺-Li⁺ countertransport assay measures the transport of Li⁺ coupled to the Na⁺ gradient as external Na⁺-stimulated Li⁺ efflux.²² Saturation of internal sites is obtained by loading RBCs with 8 mmol/L cell Li⁺ with the use of the nystatin procedure.²² For determination of the affinity for external Na⁺, it is necessary to increase the Na⁺ concentration in the efflux media to more than twice the apparent affinity constant (K_m). According to previous studies in a few healthy subjects,^{23 24} K_m for Na⁺ varied between 20 and 25 mmol/L, and saturation was reached between 70 and 150 mmol/L. Preliminary results in our laboratory provided similar values in RBCs from normotensive subjects, as shown in Fig 1 in a representative example. However, the same experiments performed in some hypertensive subjects indicated that Li⁺ efflux was a linear function of external Na⁺ between 0 and 150 mmol/L and did not reach saturation even at 150 mmol/L Na⁺ (Fig 1). Since this may invalidate the use of any kinetic analysis based on the MichaelisMenten equation, a different method was developed to increase external Na⁺ to 280 mmol/L and to avoid cell shrinkage by setting isosmotic conditions. In this procedure, intracellular Li⁺ is increased to 15 mmol/L cell (22 mmol/L) to give precision to the measurements of Li⁺ efflux at low external Na⁺. This new 600 mOsm/L procedure is fully described below.

View larger version (14K): [in this window] [in a new window]   Figure 1. Line graph shows external Na+ activation of Na+-Li+ countertransport in red blood cells of one normotensive (NT) subject () and one essential hypertensive (HT) subject () using the 300 mOsm/L procedure. Both subjects were fasted for 12 hours. Kinetic constants were calculated by fitting the experimental data by a nonlinear regression program as indicated in "Methods." In the normotensive subject, Vmax was 0.37±0.10 mmol/L cellxh and Km for Na+ was 23±9 mmol/L (mean±SEM). Li+ efflux reached maximal and constant values over 60 mmol/L Na+. In the hypertensive subject, calculated Vmax was 1.02±0.31 mmol/L cellxh and Km for Na+ was 155±41 mmol/L Na+. Li+ efflux increased linearly with external Na+ and did not reach constant values even over 100 mmol/L Na+. Bars indicate SEM of the flux, calculated from the regression line of Li+ content of the media per liter cell vs time for six samples.

Li⁺ Loading of RBCs
Four milliliters of packed RBCs was suspended at 4°C for 20 minutes in 20 mL nystatin Li⁺-loading solution containing (mmol/L) LiCl 20, KCl 265, sucrose 50, and nystatin 40 mg/L; the final osmolarity was 600±10 mOsm/L. Subsequently, the cell suspension was centrifuged, the supernatant discarded, and the loading solution (without nystatin) renewed and incubated for an additional 10 minutes at 4°C. Nystatin was removed by adding 5 mL of warm (37°C) nystatin washing solution and by incubating the cell suspension for 10 minutes at 37°C. Nystatin washing solution contained (mmol/L) KCl 265, sucrose 50, glucose 10, potassium-phosphate buffer 1.0 (pH 7.4 at 37°C, 600±10 mOsm/L), and albumin 0.1%. Nystatin removal was ensured by four more washes with nystatin washing solution. The mean cation content of Li⁺-loaded RBCs was Li⁺ 15±2, K⁺ 238±10, and Na⁺ 1.4±0.2 mmol/L cell (mean±SEM, n=50).

Lithium Efflux
To study the external Na⁺ activation kinetics of Li⁺ efflux, we varied the Na⁺ concentration of the efflux solutions while balancing the osmolarity at 600 mOsm/L with choline chloride. Nine Na⁺ media were prepared containing 10, 20, 50, 80, 120, 150, 200, 250, and 280 mmol Na⁺-Li⁺. The Na⁺ concentration of each solution was validated by measurements with atomic absorption spectrophotometry. All Na⁺ media also contained (mmol/L) 3-[N-morpholino]propanesulfonic acid (MOPS)–Tris 10 (pH 7.4 at 37°C), MgCl₂ 1.0, and ouabain 0.1. The choline medium contained (mmol/L) choline chloride 300, MgCl₂ 1.0, MOPS-Tris 10 (pH 7.4 at 37°C), and ouabain 0.1.

Efflux was started by addition of 0.7 mL of 50% hematocrit cell suspension to 7 mL of media preincubated at 37°C (final hematocrit, 3% to 4%). Duplicate samples were taken after incubation times of 10, 25, and 40 minutes and the media separated by centrifugation at 4°C for 10 minutes at 6000g. The Li⁺ concentration of the efflux medium samples was determined by atomic absorption spectrophotometry using standards with the same composition as the flux medium. At every time point, the Li⁺ content of the media (Li_m) in millimoles per liter of cells (or micromoles per milliliter of cells) was calculated as follows:

where Li_c is Li⁺ concentration of the efflux media (micromoles per liter), Hto is the hematocrit of the cell suspension, and CS is the cell suspension.

Li⁺ efflux into every Na⁺ media was calculated from the slope±SEM of the linear regression analysis of Li⁺ content of the media (millimoles per liter of cells) versus time (n=6 time samples). Na⁺-stimulated Li⁺ efflux was calculated as the difference between Li⁺ efflux into the Na⁺ and choline media. The standard error (SE) of this difference was calculated from

Kinetic Analysis
Kinetic parameters for the sodium activation of Na⁺-Li⁺ countertransport were estimated by using the ENZFITTER software computer program (Elsevier Science Publishers). The Na⁺ activation of Li⁺ efflux was fitted by a nonlinear regression program to determine the Na⁺ concentration for K_m and V_max. Nonlinear regression fitting has the advantage that it does not change the error distribution as it occurs with transformations such as Lineweaver-Burk and Eadie-Hofstee plots.²⁵

Statistical Analyses
All statistical analyses were performed using the SPSS/PC statistical software program (version 4.0, SPSS Inc). Paired and unpaired Student's t test and one-way and two-way ANOVAs were used when needed. Linear regression and stepwise multiple regression analyses were performed to dissociate the effects of several variables on the kinetic parameters of countertransport. Data are reported as mean±SEM; a value of P<.05 was regarded as statistically significant.

Chemicals
NaCl, MgCl₂, and glucose were obtained from Fisher Scientific Co. Ouabain, Tris, MOPS, nystatin, and albumin (bovine fraction V) were purchased from Sigma Chemical Co. Choline chloride was obtained from Calbiochem, Behring Diagnostic.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Characteristics of the Study Population
The data from the subjects enrolled in Boston and Padova were examined separately for age, sex, BMI, BP, and fasting insulin levels and were not found to be different (data not shown). Therefore, subsequent analyses used the pooled data of these clinical characteristics from both study sites (Table 1). As shown in Table 1, besides BP, the hypertensive subjects did not differ from the normotensive subjects in BMI, age, or glucose and insulin levels.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Normotensive and Hypertensive Subjects

Sodium Activation Kinetics of Na⁺-Li⁺ Countertransport in Normotensive and Hypertensive Subjects
Fig 2 shows experiments performed with the 600 mOsm/L procedure in the same normotensive and hypertensive subjects reported in Fig 1. In the normotensive subject, Li⁺ efflux increased sharply between Na⁺ concentrations of 0 to 75 mmol/L and reached maximal and constant values between 80 and 280 mmol/L. The curve fitted by nonlinear regression yielded a V_max of 0.37 mmol · L^-1 · h^-1 and a K_m for Na⁺ of 21 mmol/L. These values were similar to those obtained in the same subject with the activation curve performed at 300 mOsm/L, which yielded a V_max of 0.37 mmol · L^-1 · h^-1 and K_m for Na⁺ of 23 mmol/L (Fig 1).

View larger version (13K): [in this window] [in a new window]   Figure 2. Line graph shows external Na+ activation of Na+-Li+ countertransport in red blood cells of subjects using the 600 mOsm/L procedure. Red blood cells of the same normotensive (NT) () and hypertensive (HT) () subjects studied in Fig 1 were used. Both subjects were fasted for 12 hours. Kinetic constants were calculated by fitting the experimental data by a nonlinear regression program as indicated in "Methods." In the normotensive subject, Vmax was 0.37±0.07 mmol · L-1 · h-1 and Km was 21±5 mmol/L. Li+ efflux reached maximal values over 60 mmol/L Na+; constant values were observed between 90 and 280 mmol/L Na+. In the hypertensive subject, Vmax was 0.55±0.16 mmol/L cellxh and Km for Na+ was 50±15 mmol/L. Li+ efflux increased linearly with external Na+ up to 100 mmol/L and reached constant values between 150 and 280 mmol/L Na+. Notice that SEM values of both kinetic parameters are lower than those of measurements done with the 300 mOsm/L procedure (Fig 1). Bars indicate SEM of the flux calculated from the regression line of Li+ content of the media per liter cell vs time for six samples. Duplicate samples were taken at 10, 25, and 40 minutes with and without Na+.

Fig 2 also shows the external Na⁺ dependence of Li⁺ efflux in RBCs from a hypertensive subject. Li⁺ efflux increased up to 150 mmol Na⁺/L and reached constant values between 125 and 280 mmol/L Na⁺; V_max was 0.55 mmol/L cellxh, and K_m for Na⁺ was 50 mmol/L. The V_max and K_m for Na⁺ were higher in the hypertensive than in the normotensive subjects. Fig 2 also demonstrates that countertransport follows Michaelis-Menten saturation kinetics in RBCs of a hypertensive subject when high Na⁺ concentrations are used. Note that the kinetic parameters yielded by the 300 mOsm/L procedure in this hypertensive subject (Fig 1) are higher than those obtained in the same subject using the 600 mOsm/L procedure (Fig 2). These experiments demonstrate the importance of using sufficiently high Na⁺ concentrations so that the rate of Li⁺ efflux can approach constant maximal values. As shown in Fig 1, the lack of saturation with external Na⁺ can lead to erroneous estimations of V_max and K_m.

Measurements of the Na⁺ activation of countertransport using the 600 mOsm/L procedure were made in 28 normotensive and 25 hypertensive subjects (Table 2). We observed that V_max and the assay of countertransport activity at 150 mmol/L Na⁺ (v₁₅₀) were significantly higher in RBCs of hypertensive subjects than of normotensive subjects. In both normotensive and hypertensive subjects, V_max was higher than the v₁₅₀ assay (Table 2); however, the difference between V_max and the standard assay was twofold larger in the hypertensive subjects.

View this table: [in this window] [in a new window]   Table 2. Kinetic Parameters of Na+-Li+ Countertransport in Normotensive and Hypertensive Subjects

We also examined the relation between V_max and the v₁₅₀ assay (Fig 3). V_max was strongly correlated with the countertransport assay at 150 mmol/L Na⁺ both in the entire study group and separately in the two groups of normotensive and hypertensive subjects. However, when the v₁₅₀ assay had values higher than 0.4 mmol · L^-1 · h^-1 (mainly in hypertensive subjects), it underestimated V_max, as shown by its significant deviation from the identity line. This behavior is due to the incomplete saturation of the exchanger with 150 mmol/L Na⁺ observed in hypertensive subjects (Fig 1). The mean value for K_m for Na⁺ to stimulate Li⁺ efflux was in fact twofold higher in the hypertensive than in the normotensive subjects (58.9±5.3 mmol/L, n=25, versus 29.8±2.6 mmol/L, n=28; P<.001). These results clearly indicate that the affinity for Na⁺ of Na⁺-Li⁺ countertransport is markedly and significantly reduced in RBCs of hypertensive patients.

View larger version (18K): [in this window] [in a new window]   Figure 3. Scatterplot shows relation between Na+-Li+ countertransport activity measured at 150 mmol/L Na+ (v150) and Vmax determined from the sodium activation curve (n=53, r=.90, P<.001). In the entire population, the independent variable Vmax is described by the equation Vmax=0.06+1.31x(v150). The slope was significantly higher than 1 (P<.001). In the normotensive group (n=28), the regression between both measurements is described by the equation Vmax=0.03+1.11x(v150) (r=.80, P<.001). In the hypertensive subjects (n=25), the regression between both measurements is described by the equation Vmax=0.04+1.26x(v150) (r=.91, P<.001). This analysis underlines the fact that for countertransport values lower than 0.4 mmol/L cellxh (mostly in normotensive subjects), Vmax is close to v150. However, when the antiporter activity has values higher than 0.40 mmol/L cellxh (mostly in hypertensive subjects), Vmax has higher values and is dissociated from the identity line to a significant extent. indicates normotensive subjects; , hypertensive subjects.

Kinetic Parameters of Na⁺-Li⁺ Countertransport and Fasting Insulin Levels
In this study, fasting insulin was higher than 10 µU/mL in 6 of the 28 normotensive subjects (21%, 3 men) and in 12 of 25 hypertensive subjects (48%, 7 men). Insulin levels higher than 10 µU/mL have been shown to be highly correlated with reduced insulin-stimulated glucose disposal.^{11 13} Table 3 shows the clinical characteristics of normotensive and hypertensive subjects classified according to their fasting insulin levels. According to one-way ANOVA with Tukey's conservative correction, only BMI was modestly but significantly (P<.05) higher in hyperinsulinemic hypertensive subjects than in normoinsulinemic normotensive subjects.

View this table: [in this window] [in a new window]   Table 3. Clinical Characteristics of Normoinsulinemic and Hyperinsulinemic Subjects

Fig 4 reports the V_max of countertransport in the study population subdivided according to BP and insulin category. Two-way ANOVA revealed that both BP (F=13.3, P<.01) and fasting insulin levels (F=4.26, P<.05) affected V_max, with no interaction. One-way ANOVA with Tukey's conservative correction also showed that V_max was higher in hyperinsulinemic hypertensive subjects than in normotensive subjects, independent of their insulin levels. Within the normotensive and hypertensive group, insulin levels did not affect V_max. These findings suggest that insulin levels are not the main determinants of the elevated V_max observed in the hypertensive subjects. It should be mentioned that in both normotensive and hypertensive subject groups, the assay of Na⁺-Li⁺ countertransport at 150 mmol/L Na⁺ did not show significant differences between the group with normal or high insulin levels (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 4. Plots show maximal rate of Na+-Li+ countertransport in red blood cells of subjects classified according to their blood pressure and fasting insulin levels. To convert insulin values to picomoles per liter, multiply by 7.175. Vmax was estimated in every subject from the sodium activation curve of Li+ efflux using the 600 mOsm/L procedure. A, Vmax values determined in normotensive subjects with fasting insulin levels less than 10 µU/mL (n=22) and greater than 10 µU/mL (n=6). B, Vmax values in hypertensive subjects with fasting insulin levels less than 10 µU/mL (n=13) and greater than 10 µU/mL (n=12). According to one-way ANOVA with Tukey's conservative correction, Vmax was significantly (P<.05) higher in hyperinsulinemic hypertensive subjects than in both groups of normotensive subjects.

Fig 5 shows the K_m for Na⁺ in the four study groups. Two-way ANOVA indicated that both BP (F=20, P<.001) and fasting insulin levels (F=11.9, P<.001) affected K_m, with no interaction. According to one-way ANOVA with Tukey's conservative correction, K_m was higher in hyperinsulinemic hypertensive subjects (73.4±8.7 mmol/L Na⁺) than in normoinsulinemic hypertensive subjects (45.6±3.9 mmol/L) and in normotensive subjects with normal (28.0±2.9 mmol/L) and high (36.4±4.5 mmol/L) insulin levels. The K_m for Na⁺ of normoinsulinemic hypertensive subjects was also higher than that of normotensive subjects. In contrast, the K_m for Na⁺ was not significantly different in the normotensive group with normal or high insulin levels.

View larger version (17K): [in this window] [in a new window]   Figure 5. Plots show external Na+ concentration for Km of Na+-Li+ countertransport in red blood cells of subjects classified by blood pressure and fasting insulin levels. To convert insulin values to picomoles per liter, multiply by 7.175. Km was determined in every subject from the sodium activation curve of Li+ efflux using the 600 mOsm/L procedure. A, Km for Na+ obtained in normotensive subjects with fasting insulin levels less than 10 µU/mL (n=22) and greater than 10 µU/mL (n=6). B, Km values in hypertensive subjects with fasting insulin levels less than 10 µU/mL (n=13) and greater than 10 µU/mL (n=12). According to one-way ANOVA with Tukey's conservative correction, Km was significantly (P<.05) higher in hyperinsulinemic hypertensive subjects than in the other three groups of subjects and in the normoinsulinemic hypertensive subjects than in the normoinsulinemic normotensive subjects.

Regression Analysis of the Kinetic Parameters of Na⁺-Li⁺ Countertransport
We examined the entire sample for the relationship between the kinetic parameters of countertransport and fasting insulin levels, mean BP, and BMI by simple linear correlation (Table 4) and stepwise multiple regression analysis (Table 5).

View this table: [in this window] [in a new window]   Table 4. Correlation Matrix of Insulin, Body Mass Index, Mean Blood Pressure, Vmax, and Km of Na+-Li+ Countertransport

View this table: [in this window] [in a new window]   Table 5. Stepwise Multiple Regression Analysis of Vmax and Km of Na+-Li+ Countertransport on Insulin, Body Mass Index, and Mean Blood Pressure

Simple linear correlation analysis revealed that V_max correlated with mean BP, insulin, and BMI (Table 4). Fig 6A plots V_max versus insulin and shows that V_max correlated with insulin levels in the entire population and in hypertensive subjects but not in normotensive subjects. A stepwise multiple regression analysis was performed in the entire sample with V_max as dependent variable and mean BP, insulin, and BMI as independent variables (Table 5). The analysis showed that only BP was significantly and independently correlated to V_max. The same analysis applied to the hypertensive group showed that V_max was independently correlated to insulin levels (ß=0.46, P<.05).

View larger version (16K): [in this window] [in a new window]   Figure 6. Scatterplots show correlation between Vmax and Km of Na+-Li+ countertransport and fasting insulin levels. A, Dependence of Vmax from fasting insulin levels in normotensive () and hypertensive () subjects. For the entire population, r=.44, P<.01, n=53. Vmax was significantly correlated with insulin levels in the hypertensive subjects (r=.46, P<.05, n=25) but not in the normotensive subjects (r=.22, n=28). B, Dependence of Km from fasting insulin levels. For the entire population, r=.51, P<.001, n=53. Km for Na+ was significantly correlated with insulin levels in the hypertensive subjects (r=.54, P<.01, n=25) and in the normotensive subjects (r=.36, P<.05, n=28). To convert insulin values to picomoles per liter, multiply by 7.175.

On simple regression, K_m correlated to mean BP, BMI, and insulin (Table 4 and Fig 6B). Stepwise multiple regression analysis was applied to the entire group, taking K_m as dependent variable and mean BP, BMI, and insulin as independent variables. Both mean BP and insulin were significantly and independently correlated to K_m (Table 5). The same analysis applied to the hypertensive group revealed that K_m was independently correlated to insulin (ß=0.54, P<.01).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study provides evidence that the Na⁺ activation kinetics of Na⁺-Li⁺ countertransport in hypertensive subjects exhibit elevated V_max and low apparent affinity for external Na⁺ (ie, increased K_m). This study also shows that the elevated K_m for Na⁺ observed in hypertensive subjects is largely accounted for by the presence of fasting hyperinsulinemia. Essential hypertension has been recognized as an insulin-resistant state, a defect that results in a rise in fasting and postprandial plasma levels of insulin.^{11 12 13} The correlation of increased K_m with fasting hyperinsulinemia suggests that this abnormality is linked to insulin-resistant glucose disposal.

To accurately assess the Na⁺ activation of this antiporter in RBCs of fasting individuals, we used a new procedure that measures the effect of high external Na⁺ concentrations (up to 280 mmol/L) on Li⁺ efflux. This experimental approach has been previously used to study the chloride activation of the Cl^--[HCO₃]^- exchanger.²⁶ With the use of this procedure, the Na⁺ activation curve of Li⁺ efflux reached constant and maximal values at higher external Na⁺ concentrations in hypertensive subjects compared with normotensive subjects. Our results also indicate that measurements of Li⁺ efflux by varying external Na⁺ concentrations between 0 and 280 mmol/L give V_max values higher than those estimated from the standard assay (at 150 mmol/L Na⁺), and this was evident mainly in the hypertensive subjects. This was due to the fact that K_m for external Na⁺ was increased twofold in the hypertensive group as a whole.

The Na⁺ activation of Li⁺ efflux from RBCs has been studied by several investigators. Initial studies by Sarkadi et al²³ and Duhm et al²⁴ using six different Na⁺ concentrations reported K_m values for Na⁺ between 20 and 30 mmol/L. Hannaert and Garay,²⁷ using two different Na⁺ concentrations in three subjects, reported a K_m for Na⁺ that was 10-fold higher than the values reported by Sarkadi et al²³ and Duhm et al.²⁴ More recently, Rutherford et al,^{28 29 30} using five different Na⁺ concentrations ranging from 37 to 150 mmol/L NaCl, studied the activation kinetics of countertransport in control subjects, hypertensive subjects, and diabetics. In control subjects, they found the K_m for Na⁺ to be 148 mmol/L (range, 100 to 263 mmol/L),²⁸ similar to the finding of Hannaert and Garay²⁷ but fivefold to sixfold higher than the values reported by Sarkadi et al,²³ Duhm et al,²⁴ and the present study.

The reasons for discrepancies in the K_m for Na⁺ have been recently discussed in detail and may arise from many sources,¹⁸ such as the lack of saturation kinetics, which invalidates the use of any transformation of the Michaelis-Menten equation, especially when the measured values are all lower than the estimated K_m. Second, different study conditions (prandial status, insulin levels, and insulin sensitivity) may contribute to discrepancies and may affect the saturation kinetics of external Na⁺, as reported in previous publications^{17 18 19} and in the present article. Third, the Na⁺ affinity may actually vary in different populations.

The present article shows that the K_m for Na⁺ of Na⁺-Li⁺ countertransport in RBCs of hyperinsulinemic hypertensive patients is higher than in normotensive subjects and in normoinsulinemic hypertensive subjects. These findings are in agreement with the observed effects of insulin in vitro.^{18 19} In previous studies, we reported that the incubation of RBCs with physiological doses of insulin increased the K_m for Na⁺ from 37 to 84 mmol/L in normotensive subjects.^{17 18} However, we studied the in vitro effects of insulin by incubating RBCs in K⁺ media to maximize conditions for insulin binding.^{17 18} The results of the present investigation indicate that the elevated K_m observed in the hyperinsulinemic hypertensive subjects is related not only to BP but to the long-term exposure of subjects to high plasma insulin levels. However, other interpretations should also be considered because many hypertensive subjects with insulin levels lower than 10 µU/mL had K_m values higher than those observed in RBCs incubated with insulin in vitro from normotensive subjects.

Previous studies showed that insulin in vitro did not change Na⁺-Li⁺ exchange activity at 150 mmol/L Na⁺ but significantly increased V_max from 0.32 to 0.42 mmol/L cellxh.¹⁹ In the present study, the highest V_max was found in hyperinsulinemic hypertensive subjects and correlated with BP, insulin, and BMI. However, the stepwise multiple regression analysis showed that the only determinant of V_max was mean BP. In a study of Na⁺-H⁺ exchange activity in RBCs of blacks with mild hypertension, in whom insulin sensitivity had been characterized by the euglycemic insulin clamp, the V_max of Na⁺-H⁺ exchange was found to be elevated in hypertensive subjects with insulin resistance.²¹ V_max also was inversely correlated with the degree of insulin resistance as measured by insulin-stimulated glucose disposal but not with insulin levels per se. These results suggested that fasting insulin levels might not be a sensitive monitor of insulin resistance as it is in insulin-stimulated body glucose disposal. The association of elevated countertransport with low high-density lipoprotein cholesterol,^{31 32} obesity,⁹ hyperuricemia,¹⁰ and high triglyceride levels has been well documented.^{8 33} These observations further support the conclusion that abnormalities of both V_max and K_m of Na⁺-Li⁺ countertransport are linked to the metabolic defects associated with the insulin resistance in white or black hypertensive subjects. Nevertheless, further studies on countertransport kinetics are required in other populations because the association with hyperinsulinemia may be influenced by ethnic factors, obesity, and body fat distribution.

This new methodology developed to study the kinetic properties of Na⁺-Li⁺ countertransport in hypertensive subjects also made it possible to differentiate abnormalities present in insulin-dependent diabetics with and without nephropathy.¹⁸ It was shown that patients with nephropathy exhibited an elevation of V_max and K_m for Na⁺ similar to that of insulin-resistant hypertensive subjects.¹⁸ A recent study compared type I diabetics with high and normal Na⁺-Li⁺ countertransport matched for age, sex, BMI, metabolic control, and duration of diabetes. It was found that insulin-stimulated glucose disposal rates, a measure of insulin sensitivity, were lower in those subjects with elevated antiporter activity.³⁴

Our findings therefore document that the antiporter abnormalities are not only a marker of hypertension but also a measure of insulin action on Na⁺ influx into RBCs. A similar action of insulin in the kidney may induce antinatriuresis,¹⁵ a mechanism that has been suggested to confer salt sensitivity to BP.³⁵ Nosadini et al³² have shown that hypertensive patients with elevated countertransport have enhanced renal Na⁺ reabsorption (lower lithium clearance) and increased total body exchangeable Na⁺. Ferrannini and Natali³⁶ have recently suggested that the link between hyperinsulinemia and Na⁺ retention may be explained by a shift along the pressure-natriuresis curve to higher BP levels to achieve a similar natriuretic effect. Our findings agree with the hypothesis that increased fasting insulin levels are associated with abnormal Na⁺ homeostasis as reflected by abnormal Na⁺-Li⁺ countertransport. Measurements of the sodium activation kinetics of this antiporter may help to unmask a unique subgroup of subjects prone to develop hypertension.

In summary, we conclude that Na⁺-Li⁺ countertransport in RBCs exhibits two different abnormalities in subjects with essential hypertension: a low affinity for Na⁺, related to increased BP and hyperinsulinemia, and increased V_max, mainly related to increased BP.

	Acknowledgments

 
This work was supported by grants HL-42120 and DK-4610701 from the National Institutes of Health, Bethesda, Md. Dr G. Zerbini was a recipient of a grant from the Ministero Italiano della Publica Istruzione. Drs A.C. Pessina and A. Semplicini were recipients of a grant from the Italian National Research Council Project "Prevention and control of disease factors" (FATMA, Subproject 8, "Cardiovascular Diseases" 91.00.218 PF 41. 115.06.654).

Received June 6, 1994; first decision August 3, 1994; accepted November 29, 1994.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Canessa M, Adragna N, Solomon HS, Connolly TM, Tosteson DC. Increased sodium-lithium countertransport in red cells of patients with essential hypertension. N Engl J Med. 1980;302:772-776. [Abstract]

2. Semplicini A. The Na⁺/Li⁺ countertransport in hypertension. In: Coca A, Gary R, eds. Ionic Transport in Hypertension: New Perspectives. Boca Raton, Fla: CRC Press; 1993:90-117.

3. Turner S, Johnson M, Boerwinkle E, Richelson E, Taswell HF, Sing CF. Sodium-lithium countertransport and blood pressure in healthy blood donors. Hypertension. 1985;7:955-962. [Abstract/Free Full Text]

4. Laurenzi M, Trevisan M. Sodium-lithium countertransport and blood pressure: the Gubbio population study. Hypertension. 1989;13:408-415. [Abstract/Free Full Text]

5. Williams RR, Hunt SC, Kuida H, Smith JB, Ash KO. Sodium-lithium countertransport in erythrocytes of hypertension prone families in Utah. Am J Epidemiol. 1983;118:338-344. [Abstract/Free Full Text]

6. Hasstedt SJ, Wu LL, Ash KO, Kuida H, Williams RR. Hypertension and sodium-lithium countertransport in Utah pedigrees: evidence for major locus inheritance. Am J Hum Genet. 1988;43:14-22. [Medline] [Order article via Infotrieve]

7. Redgrave J, Canessa M, Gleason R, Hollenberg NK, Williams GH. Red blood cell lithium-sodium countertransport in non-modulating essential hypertension. Hypertension. 1989;13:721-726. [Abstract/Free Full Text]

8. Corrocher R, Steinmayr M, Ruzzenente O, Brugnara C, Bertinato L, Mazzi M, Furri C, Bonfanti F, DeSandre G. Elevation of red cell sodium-lithium countertransport in hyperlipidemias. Life Sci. 1985;36:649-655. [Medline] [Order article via Infotrieve]

9. Trevisan M, Ostrow D, Cooper R, Liu K, Sparks S, Okonek A, Stevens E, Marquard J, Stamler J. Abnormal red blood cell ion transport and hypertension: the People's Gas Company study. Hypertension. 1983;5:363-367. [Abstract/Free Full Text]

10. Strazzullo P, Cappuccio FP, Trevisan M, Iacovello L, Iacone R, Barba G, De Leo A, Farinaro E, Mancini M. Red blood cell sodium-lithium countertransport, blood pressure and uric acid metabolism in untreated healthy men. Am J Hypertens. 1989;2:634-636. [Medline] [Order article via Infotrieve]

11. DeFronzo RA. Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care. 1991;14:173-194. [Abstract]

12. Reaven GM. Hypertension as a disease of carbohydrate and lipoprotein metabolism. Am J Med. 1989;87:2S-5S. [Medline] [Order article via Infotrieve]

13. Ferrannini E, Buzzigoli G, Bonadonna R, Giorico MA, Oleggini M, Graziadei L, Pedrinelli R, Brandi L, Bevilacqua S. Insulin resistance in essential hypertension. N Engl J Med. 1987;317:350-357. [Abstract]

14. Moore RD. Stimulation of Na:H exchange by insulin. Biophys J. 1981;33:203-210. [Medline] [Order article via Infotrieve]

15. Gesek FA, Schoolwerth AC. Insulin increases Na⁺/H⁺ exchange activity in proximal tubules from normotensive and hypertensive rats. Am J Physiol. 1991;260:F695-F703. [Abstract/Free Full Text]

16. Grigorescu F, White MF, Kahn RC. Insulin binding and insulin-dependent phosphorylation of the insulin receptor solubilized from human erythrocytes. J Biol Chem. 1983;258:13708-13716. [Abstract/Free Full Text]

17. Pontremoli R, Zerbini G, Rivera A, Canessa M. Insulin activation of red blood cell Na⁺/H⁺ exchange decreases the affinity of sodium sites. Kidney Int. 1994;46:365-375. [Medline] [Order article via Infotrieve]

18. Canessa M, Zerbini G, Laffel LMB. Sodium activation kinetics of red blood cell Na⁺/Li⁺ countertransport in diabetes: methodology and controversy. J Am Soc Nephrol. 1992;3:S41-S49.

19. Canessa M, Zerbini G. Insulin modulation of Na⁺/Li⁺ and Na⁺/H⁺ countertransport: impact on hypertension and diabetes. Acta Diabetol. 1992;29:186-190.

20. Doria A, Fioretto P, Avogaro A, Carraro A, Morocutti A, Trevisan R, Frigato F, Crepaldi G, Viberti G, Nosadini R. Insulin resistance is associated with high sodium-lithium countertransport in essential hypertension. Am J Physiol. 1991;261:E684-E691. [Abstract/Free Full Text]

21. Canessa M, Falkner B, Hulman S. Red blood cell sodium-proton exchange in hypertensive blacks with insulin-resistant glucose disposal. Hypertension. 1993;22:204-213. [Abstract/Free Full Text]

22. Canessa M. Kinetic properties of the human red cell Na⁺/H⁺ and Li⁺/Na⁺ exchanges. Methods Enzymol. 1989;173:176-191. [Medline] [Order article via Infotrieve]

23. Sarkadi B, Alifimoff JK, Gunn RB, Tosteson DC. Kinetics and stoichiometry of Na⁺-dependent Li⁺ transport in human red blood cells. J Gen Physiol. 1978;72:250-265.

24. Duhm J, Becker BF. Studies on the lithium transport across the red cell membrane, IV: interindividual variations in the Na⁺-dependent Li⁺ countertransport system of human erythrocytes. Pflugers Arch. 1977;370:211-219. [Medline] [Order article via Infotrieve]

25. Leatherbarrow RJ. Using linear and non-linear regression to fit biochemical data. Trends Biochem Sci. 1990;15:455-458. [Medline] [Order article via Infotrieve]

26. Gunn R. Transport of anions across red cell membranes. In: Tosteson DC, ed. Membrane Transport in Biology. Berlin, Germany: Springer-Verlag; 1979;12:59-80.

27. Hannaert PA, Garay RP. A kinetic analysis of Na⁺/Li⁺ countertransport in human red blood cells. J Gen Physiol. 1986;87:353-368. [Abstract/Free Full Text]

28. Rutherford PA, Thomas TH, Wilkinson R. Increased erythrocyte sodium-lithium countertransport activity in essential hypertension is due to an increased affinity for extracellular sodium. Clin Sci. 1990;79:365-369. [Medline] [Order article via Infotrieve]

29. Rutherford PA, Thomas TH, Carr SJ, Taylor R, Wilkinson R. Kinetics of sodium-lithium countertransport activity in patients with uncomplicated type I diabetes. Clin Sci. 1992;82:291-299. [Medline] [Order article via Infotrieve]

30. Rutherford PA, Thomas TH, Carr SJ, Taylor R, Wilkinson R. Changes in erythrocyte sodium-lithium countertransport kinetics in diabetic nephropathy. Clin Sci. 1992;82:301-307. [Medline] [Order article via Infotrieve]

31. Hunt SC, Williams RR, Smith JG, Ash KO. Associations of three erythrocyte cation transport systems with plasma lipids in Utah subjects. Hypertension. 1986;3:30-36.

32. Nosadini R, Semplicini A, Fioretto P, Lusiani L, Trevisan R, Donadon V, Zanette G, Nicolosi GL, Dall'Aglio V, Zanuttini D. Sodium-lithium countertransport and cardiorenal abnormalities in essential hypertension. Hypertension. 1991;18:191-198. [Abstract/Free Full Text]

33. Strazzullo P, Cappuccio FP, Trevisan M, Siani A, Barba G, Ragone E, Pagano E, Mancini M. The relationship of erythrocyte sodium-lithium countertransport to blood pressure and metabolic abnormalities in a sample of untreated middle-age male workers. J Hypertens. 1993;11:815-822. [Medline] [Order article via Infotrieve]

34. Catalano C, Winocour Ph, Thomas TH, Walker M, Sum CF, Wilkinson R, Alberti KGM. Erythrocyte sodium-lithium countertransport activity and total body insulin-mediated glucose disposal in normoalbuminuric normotensive type I (insulin-dependent) diabetic patients. Diabetologia. 1993;36:52-56. [Medline] [Order article via Infotrieve]

35. Rocchini AP, Katch V, Kveseli D, Moorhead C, Martin M, Lampman R, Gregory M. Insulin and renal sodium retention in obese adolescents. Hypertension. 1989;14:367-374. [Abstract/Free Full Text]

36. Ferrannini E, Natali A. Insulin resistance and hypertension: connections with sodium metabolism. Am J Kidney Dis. 1993;21(suppl 2):37-42.

This article has been cited by other articles:

				G. Zerbini, D. Gabellini, D. Ruggieri, and A. Maestroni Increased Sodium-Lithium Countertransport Activity: A Cellular Dysfunction Common to Essential Hypertension and Diabetic Nephropathy J. Am. Soc. Nephrol., January 1, 2004; 15(90010): S81 - 84. [Abstract] [Full Text]

				G. Zerbini, A. Maestroni, D. Breviario, R. Mangili, and G. Casari Alternative Splicing of NHE-1 Mediates Na-Li Countertransport and Associates With Activity Rate Diabetes, June 1, 2003; 52(6): 1511 - 1518. [Abstract] [Full Text] [PDF]

				F. D. Grant, J. R. Romero, X. Jeunemaitre, S. C. Hunt, P. N. Hopkins, N. H. Hollenberg, and G. H. Williams Low-Renin Hypertension, Altered Sodium Homeostasis, and an {alpha}-Adducin Polymorphism Hypertension, February 1, 2002; 39(2): 191 - 196. [Abstract] [Full Text] [PDF]

				K. Tsuda, Y. Kinoshita, K. Kimura, I. Nishio, and Y. Masuyama Electron Paramagnetic Resonance Investigation on Modulatory Effect of 17{beta}-Estradiol on Membrane Fluidity of Erythrocytes in Postmenopausal Women Arterioscler Thromb Vasc Biol, August 1, 2001; 21(8): 1306 - 1312. [Abstract] [Full Text] [PDF]

				P. Mead, R. Wilkinson, and T. H. Thomas Thiol Protein Defect in Sodium-Lithium Countertransport in Subset of Essential Hypertension Hypertension, December 1, 1999; 34(6): 1275 - 1280. [Abstract] [Full Text] [PDF]

				P. Strazzullo, A. Siani, F. P. Cappuccio, M. Trevisan, E. Ragone, L. Russo, R. Iacone, and E. Farinaro Red Blood Cell Sodium-Lithium Countertransport and Risk of Future Hypertension : The Olivetti Prospective Heart Study Hypertension, June 1, 1998; 31(6): 1284 - 1289. [Abstract] [Full Text] [PDF]

				K. van Norren, J. M. P. M. Borggreven, A. Hovingh, H. L. Willems, T. d. Boo, L. D. Elving, J. H. M. Berden, and J. J. H. H. M. De Pont Comparison of Methods for Measurement of Na+/Li+ Countertransport Across the Erythrocyte Membrane Clin. Chem., June 1, 1997; 43(6): 1090 - 1092. [Full Text] [PDF]

				Y. Chi, D. M. de Freitas, M. Sikora, and V. K. Bansal Correlations of Na+-Li+ Exchange Activity With Na+ and Li+ Binding and Phospholipid Composition in Erythrocyte Membranes of White Hypertensive and Normotensive Individuals : A Nuclear Magnetic Resonance Investigation Hypertension, March 1, 1996; 27(3): 456 - 464. [Abstract] [Full Text]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zerbini, G. Articles by Semplicini, A. Search for Related Content PubMed PubMed Citation Articles by Zerbini, G. Articles by Semplicini, A.