Na+/Li+ and Na+/H+ countertransport activity in hypertensive non-insulin-dependent diabetic patients: role of insulin resistance and antihypertensive treatment.

We measured erythrocyte Na+/Li+ and Na+/H+ countertransport (CT) activity (millimoles per liter per cell per hour) in 10 healthy control subjects (age, 38 +/- 4 years; body mass index, 25 +/- 1 kg/m2) and in 25 hypertensive patients with non-insulin-dependent diabetes mellitus ([NIDDM] age, 49 +/- 3 years; body mass index, 29 +/- 1 kg/m2; fasting plasma glucose, 157 +/- 12 mg/dL) 4 weeks after discontinuation of previous antihypertensive treatment. Na+/Li+ CT was significantly increased in hypertensive NIDDM patients compared with controls (0.56 +/- 0.04 v 0.30 +/- 0.03, P < .01), whereas Na+/H+ CT was similar to control levels (21 +/- 1 v 20 +/- 2). A positive correlation was found between Na+/Li+ CT and the severity of insulin resistance (r = .69, P < .01), mean arterial pressure ([MAP] r = .64, P < .01), plasma triglyceride concentration (r = .46, P < .05), and plasma total cholesterol (r = .41, P < .05). An inverse correlation was found between Na+/Li+ CT activity and plasma insulin concentration (r = -.47, P < .05). No relationship was observed between Na+/Li+ CT activity and either creatinine clearance or proteinuria. Stepwise multiple regression analysis for all metabolic variables and blood pressure showed that only the severity of insulin resistance was positively correlated with increased Na+/Li+ CT activity. Na+/H+ and Na+/Li+ CT activity were not altered by 3 hours of euglycemic physiologic hyperinsulinemia (84 +/- 3 microU/mL). Hypertensive NIDDM subjects were treated for 3 months with captopril, nifedipine, or doxazosin. After captopril, a reduction of Na+/H+ CT was observed (22 +/- 4 v 13 +/- 2, P < .05); Na+/Li+ CT decreased after doxazosin (0.56 +/- 0.06 v 0.45 +/- 0.05, P < .05) and nifedipine (0.52 +/- 0.06 v 0.42 +/- 0.05, P < .05). In conclusion, in hypertensive NIDDM subjects, (1) Na+/Li+ CT is increased and is correlated with the level of insulin resistance and the MAP; (2) acute physiologic hyperinsulinemia does not affect Na+/Li+ or Na+/H+ CT activity; and (3) Na+/H+ CT activity is reduced by captopril, and Na+/Li+ CT is decreased by doxazosin and nifedipine.

Elevated lymphocyte cytosolic calcium in a subgroup of essential hypertensive subjects.

Abnormalities of intracellular calcium homeostasis and sodium-proton exchange have been implicated in the pathophysiology of essential hypertension. To further define the nature of cytosolic calcium abnormalities and whether they relate to increased sodium-proton exchange in hypertension, we have studied peripheral lymphocytes from normotensive and hypertensive subjects. Lymphocyte cytosolic calcium was significantly increased (P < .01) in hypertensive compared with normotensive subjects while consuming a high salt diet. Using maximum likelihood analysis, we found that cytosolic calcium levels in our study population were not normally distributed and observed three modes (P < .02). The means of the first mode and the two upper modes were separated (+/-2 SD) at a cytosolic calcium level of 120 nmol/L. We conducted further analysis in the subgroups with cytosolic calcium levels > 120 nmol/L or < 120 nmol/L. The majority of the normotensive subjects (86%) and half of the hypertensive subjects (52%) had levels < 120 nmol/L. Clinical characteristics of the two subgroups did not differ. Subjects with levels < 120 nmol/L had a rise in cytosolic calcium when changed to a low salt diet; those with levels > 120 nmol/L did not show a change in cytosolic calcium but their blood pressure fell significantly with salt restriction. Hypertensive subjects also had increased sodium-proton exchange activity compared with normotensive subjects when both groups were studied in a high salt balance. A positive correlation between sodium-proton exchange and cytosolic calcium was observed in subjects with levels < 120 nmol/L. There was insufficient power to draw conclusions on this relationship in subjects with levels > 120 nmol/L. Thus, many hypertensive subjects have increased cytosolic calcium, but this abnormality is not associated with sodium-proton exchange activity in all individuals. The salt-induced change in cytosolic calcium in subjects with levels < 120 nmol/L and its link to sodium-proton exchange suggest regulation by factors involved in salt-volume homeostasis. Individuals with cytosolic calcium > 120 nmol/L, most of whom were hypertensive, may have abnormalities in this regulation, contributing to hypertension.

Effect of alpha-adrenergic blockers, ACE inhibitors, and calcium channel antagonists on renal function in hypertensive non-insulin-dependent diabetic patients.

In the present study we investigated the effect of a selective alpha 1-adrenergic blocker (doxazosin), an angiotensin-converting enzyme (ACE) inhibitor (captopril), and a calcium channel antagonist (nifedipine) on renal function in hypertensive non-insulin-dependent diabetic patients. 30 NIDD hypertensive patients (age = 50 +/- 3 years; BMI = 30 +/- 1 kg/m2) (mean +/- SEM) were studied before and after a 12-week period of antihypertensive treatment. Ten patients were treated with doxazosin (Cardura) (2-8 mg once daily or 8 mg b.i.d.), 9 with captopril (Capoten) (25-50 mg b.i.d.), and 11 with nifedipine (Procardia-XL) (30-60 mg once daily). Blood pressure, creatinine clearance, 24-hour urinary protein excretion, fasting plasma glucose concentration and glycosylated hemoglobin were measured before and after drug treatment. Fasting plasma glucose and glycosylated hemoglobin (HbA1c) were similar in all three groups prior to the start of antihypertensive therapy and did not change significantly from baseline in any treatment groups. In the doxazosin group creatinine clearance rose from 99 +/- 8 to 122 +/- 8 ml/1.73 m2.min (p < 0.01), while 24-hour urinary protein excretion declined from 2.66 +/- 0.05 to 1.76 +/- 0.02 mg/day/ml/1.73 m2.min (p < 0.01). In diabetics treated with captopril creatinine clearance rose from 93 +/- 6 to 109 +/- 9 ml/1.73 m2.min (p < 0.05), while the 24-hour urinary protein excretion fell from 2.70 +/- 0.05 to 2.03 +/- 0.04 mg/day/ml/1.73 m2.min (p < 0.05). In patients treated with nifedipine creatinine clearance did not change (97 +/- 6 vs. 94 +/- 7 ml/1.73 m2.min), while 24-hour urinary protein excretion decreased from 2.84 +/- 0.04 to 1.95 +/- 0.03 mg/day/ml/1.73 m2.min. Systolic and diastolic blood pressure were similar in doxazosin (150 +/- 3/95 +/- 2 mm Hg), captopril (153 +/- 3/93 +/- 1), and nifedipine (155 +/- 4/93 +/- 1) groups prior to the start of antihypertensive therapy and declined to 143 +/- 3/84 +/- 3 (doxazosin), 139 +/- 3/82 +/- 3 (captopril), and 141 +/- 3/84 +/- 1 (nifedipine) mm Hg (all p < 0.01 vs. pretreatment). In summary, both doxazosin and captopril treatment were associated with significant rises in GFR, while all three antihypertensive agents caused a significant decline in proteinuria. These results indicate that alpha-adrenergic blockers, ACE inhibitors, and calcium channel antagonists can safely and effectively be used in the clinical management of non-insulin-dependent diabetic patients with hypertension.

Effects of angiotensin-converting enzyme inhibitors, Ca2+ channel antagonists, and alpha-adrenergic blockers on glucose and lipid metabolism in NIDDM patients with hypertension.

We compared the effects of captopril, nifedipine, and doxazosin on glucose and lipid metabolism in 30 hypertensive non-insulin-dependent diabetes mellitus (NIDDM) patients (age = 50 +/- 3 years; body mass index = 30 +/- 1 kg/m2). Of these patients, 9 were treated with captopril, 11 with nifedipine, and 10 with doxazosin for 12 weeks. Blood pressure, fasting plasma glucose (FPG) concentration, HbA1c, oral glucose tolerance test (OGTT), euglycemic insulin clamp, and plasma lipids were measured before and after a 3-month period. Mean arterial blood pressure (114 +/- 2 mmHg) was similar in all groups before initiating antihypertensive therapy and declined to 102 +/- 2 (captopril), 103 +/- 1 (nifedipine), and 103 +/- 2 (doxazosin) mmHg (P < 0.001). Baseline FPG (148 +/- 11 mg/dl) and HbA1c (6.3 +/- 1%) were similar in all groups and did not change significantly with treatment. Plasma glucose, insulin, and free fatty acid (FFA) concentrations during the OGTT were similar in all groups before antihypertensive treatment and did not change with captopril and nifedipine; after doxazosin, plasma glucose and FFA concentrations during the OGTT decreased (both P < 0.05) without change in plasma insulin response. Insulin-mediated glucose uptake (144 +/- 11 mg.m-2.min-1), glucose oxidation (76 +/- 4 mg.m-2.min-1), and nonoxidative glucose disposal (71 +/- 6 mg.m-2.min-1) were similar in all groups before the start of antihypertensive treatment and did not change in captopril and nifedipine groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Impact of diabetes on sodium-lithium countertransport in pregnancy-induced hypertension.

The purpose of this study was to assess the impact of type I diabetes on sodium-lithium countertransport (CTT) in normotensive third trimester women and those with pregnancy-induced hypertension (PIH). CTT was measured in the following groups: nonpregnant diabetic and nondiabetic women, pregnant nondiabetics with and without PIH, and pregnant diabetics with and without PIH. CTT was determined as external sodium-stimulated lithium efflux from lithium loaded red cells [mmoles/liter of cells x hours = units (U)]. In the nonpregnant state, nondiabetics and diabetics demonstrated similar CTT activity (0.20 +/- 0.02 U, n = 22, v 0.26 +/- 0.03 U, n = 18). In pregnancy, normotensive nondiabetics and diabetics had CTT activity similar to each other, the values of both being significantly higher than those in nonpregnant women (0.39 +/- 0.03 U, n = 26, v 0.46 +/- 0.05 U, n = 17). Nondiabetics with PIH demonstrated higher CTT (0.53 +/- 0.03 U, n = 30) than normotensive pregnant nondiabetics (P = .002). However, diabetics with PIH and CTT activity (0.48 +/- 0.04 U) indistinguishable from normotensive pregnant diabetics (0.46 +/- 0.05 U). There were no differences in gestational age, renal function, or glycohemoglobin that could explain these observations. Therefore, we conclude that PIH in type I diabetic women, in contrast to PIH in nondiabetic pregnant women, is not associated with a further elevation in CTT.

Early and late effects of angiotensin-II on Ca2+ fluxes in bovine adrenal zona glomerulosa cells.

Previous studies have yielded conflicting results concerning the effects of angiotensin-II (Ang II) on Ca2+ fluxes in adrenal zona glomerulosa (ZG) cells. The present study was designed to investigate the kinetics and dose-dependency of Ang II-mediated changes in Ca2+ influx and efflux in cultured bovine ZG cells. At a high (10 nM) Ang II concentration, cytosolic Ca2+ (Cai) shows a peak-plateau response for the first 15 min, with small Cai transients commonly observed with longer stimulations. At 50 pM Ang II, more sustained Cai changes were elicited, typically consisting of Cai oscillations. The underlying changes in Ca2+ influx and efflux were studied. The early modifications of Ca2+ influx after 2 min of agonist stimulation were biphasic, with uptake increased by 90% between 1-100 pM Ang II and inhibited by 30% at 10 nM Ang II. Furthermore, high (10 nM) Ang II doses inhibited extracellular K(+)-stimulated Ca2+ influx. After 30 min of Ang II stimulation, the later dose response of Ca2+ influx was of similar magnitude but shifted to the left, showing a maximal influx at 10 pM Ang II and a modest enhancement at 10 nM. Basal Ca2+ efflux followed a two-compartment exponential decay, reflecting rapid Ca2+ displacement from extracellular sites (k1) and active Ca2+ transport (k2). A high (10 nM) Ang II concentration induced a transient large increase (130%) in k2 during the initial phase of Ang II stimulation, which returned to basal values within 10 min. A low (50 pM) Ang II concentration induced a small sustained increase (30%) in k2. A 10-nM Ang II concentration markedly reduced the exchangeable Ca2+ pool, as Ca2+ mobilized from intracellular stores into the cytosol was rapidly extruded, while Ca2+ influx was inhibited. A more physiological (50 pM) concentration of Ang II did not significantly alter the total exchangeable Ca2+ pool due to modest stimulation of both Ca2+ efflux and influx. In summary, the initial transient Cai response to high Ang II results from a large Ca2+ mobilization combined with inhibition of Ca2+ influx, which does not allow for the refilling of Ca2+ stores. At later times, small increases in Ca2+ influx allow for the eventual recovery of exchangeable cell Ca2+ and an enhanced elevation in Cai. At low Ang II concentrations, stimulation of both Ca2+ influx and efflux are concurrent and maintained, allowing for a sustained increase in Cai with little change in exchangeable cell Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of Ca2+ transport by platelet-derived growth factor-BB in rat vascular smooth muscle cells.

The present study investigates the effects of platelet-derived growth factor (PDGF) isoform BB (PDGF-BB) on cytosolic Ca2+ concentration ([Ca2+]i), Ca2+ transport, and Ca2+ pools in rat vascular smooth muscle (VSM) cells. VSM cells from thoracic aorta of Milan normotensive rats were enzymatically dispersed, cultured in 10% serum medium, and made quiescent by 72 hours in 0.3% serum medium. [Ca2+]i, Ca2+ influx, Ca2+ efflux, and exchangeable cell Ca2+ pool were evaluated by ratiometric fluorescent and radioisotope techniques. Ca2+ transport showed time-dependent changes during stimulation with PDGF-BB. The initial early responses to this peptide were transient rise in [Ca2+]i, a 30% decrease in Ca2+ influx, and a 3.6-fold increase in the rate constant for active Ca2+ efflux. Stimulation of Ca2+ efflux and inhibition of Ca2+ influx were associated with a substantial 30% reduction in the cell Ca2+ pool. This initial stimulation of Ca2+ efflux is concomitant with Ca2+ mobilization into the cytosol and is due to activation of Na(+)-independent Ca2+ efflux via the Ca2+ pump. After a 10-minute stimulation, Ca2+ influx returned to the basal value, whereas Ca2+ efflux remained 2.2-fold above control values, leading to a decline in [Ca2+]i below basal levels and a further decrease in the cell Ca2+ pool. Nearly half of this late Ca2+ efflux appears to be driven by Na(+)-Ca2+ exchange, as evidenced by its external Na+ dependence. After a 120-minute stimulation with PDGF-BB, nifedipine-sensitive Ca2+ influx is increased 37% above basal levels, and Ca2+ efflux remains elevated. During prolonged stimulation by PDGF-BB, both Ca2+ influx and efflux are stimulated, resulting in a new intracellular Ca2+ homeostasis marked by the recovery of the cell Ca2+ pool but a lowered [Ca2+]i. These final events coincide with the initiation of cell proliferation in VSM cells by PDGF-BB.

Calcium-mediated intracellular acidification and activation of Na(+)-H+ exchange in adrenal glomerulosa cells stimulated with potassium.

Intracellular pH (pHi) regulation by Na(+)-H+ exchange is important in cellular responses to hormones and growth factors, particularly those which raise cytosolic Ca2+ (Cai). Since elevation of Cai occurs when adrenal glomerulosa cells are stimulated by angiotensin II (Ang II) and high external K+ (Ko), we evaluated the relationship of Na(+)-H+ exchange to calcium movement particularly during high Ko stimulation of bovine glomerulosa cells. Inhibition of Na(+)-H+ exchange by dimethylamiloride markedly reduced aldosterone secretion in response to 8 mM Ko. This high Ko stimulation was accompanied by a rise in dimethylamiloride-sensitive Na+ influx and pHi acidification which was extracellular Ca2+ (Cao) dependent. High Ko also produced a rise in Ca2+ influx, Cai levels and Ca2+ efflux at 37 C. However, at 4 C, Ca2+ influx remained intact, but Ca2+ efflux and cellular acidification were inhibited. In contrast, Ang II produced protein kinase C (PKC) activation accompanied by a Na(+)-H+ exchange-dependent rise in pHi which was independent of Cao. After PKC depletion by phorbol ester pretreatment, Ang II also produced a Cao-dependent cell acidification as with high Ko. Thus, Na(+)-H+ exchange is activated by both Ang II and high Ko but by different mechanisms. High Ko stimulation induces an enhanced cellular acidification, whereas Ang II induces alkalinization driven by PKC activation of the antiporter. These findings suggest that a physiological role of Na(+)-H+ exchange may be, in part, to counteract the acidification produced by enhanced active Ca2+ efflux (via Ca2+ pumping) during both high Ko and Ang II stimulation of aldosterone secretion.

Angiotensin II-induced activation of Na(+)-H+ exchange in adrenal glomerulosa cells is mediated by protein kinase C.

Modulation of Na(+)-H+ exchange (Na/H EXCH) by hormones and growth factors may be important in cell growth. We have previously shown that Na/H EXCH in adrenal glomerulosa cells is activated by angiotensin II (ANG II), a potent stimulator of aldosterone secretion. In the present paper, we have investigated the role of protein kinase C (PKC) in the activation of Na/H EXCH by ANG II. To accomplish this, we monitored cytosolic pH (pHi) in cells loaded with 2,7-biscarboxyethyl-5 (6)-carboxyflourescein and measured initial rates off 22Na uptake into glomerulosa cells in the presence or absence of dimethylamiloride. Both phorbol 12-myristate 13-acetate (PMA) and ANG II increased the activity of Na/H EXCH similarly when studied under basal conditions (pH 7.1). This was accompanied by alkalinization of pH and an increase in dimethylamiloride-sensitive Na+ influx. Study of kinetics of the antiporter activation showed that both ANG II and PMA led to an increase in the maximal rate (Vmax) and a decrease in the Michaelis-Menten constant (Km) for external Na+. The pHi dependence of Na+ influx was half-minimal (pK) at pHi 7.09 and remained unchanged in the presence of ANG II (pK 7.03) and PMA (pK 7.14). Depleting the cells of PKC by exposing them to PMA (1 microM) for 3 h caused a marked reduction in control and ANG II-stimulated Na+ influx and control pK (7.09 to 6.85, P less than 0.05). However, with PKC depletion, the kinetics of Na/H EXCH (Vmax, Km for external Na+, and pK) were unaffected by ANG II. Thus, Na/H EXCH in adrenal glomerulosa cells functions under basal conditions, and its relatively alkaline pK (7.09) is dependent upon PKC activity. In addition, the ANG II-induced activation of Na/H EXCH is modulated by PKC. These effects suggest an important role for PKC in pHi regulation of adrenal glomerulosa cells, particularly during ANG II stimulation of aldosterone secretion.

Na(+)-H+ exchanger kinetics in adrenal glomerulosa cells and its activation by angiotensin II.

We have studied the kinetic properties of basal and angiotensin II (ANG II) stimulated Na(+)-H+ exchange in adrenal glomerulosa cells by measuring changes in cytosolic pH (pHi) and initial rates of 22Na uptake in the presence or absence of dimethylamiloride (DMA). The cells were studied 1) under basal conditions, 2) at constant pHi (6.8) with varied external sodium (Na+o), and 3) at varied pHi with constant Na+o (50 mM). In 2,7-biscarboxyethyl-5(6)-carboxyfluorescein loaded cells under basal conditions, pHi rose from 7.09 +/- 0.02 to 7.19 +/- 0.02 (P less than 0.05) with addition of ANG II (100 nM). Similarly, DMA-sensitive Na influx was enhanced from 9.2 +/- 1.3 to 14.8 +/- 2.1 nmol Na+/mg protein x min (P less than 0.01) by ANG II. In cells acid-loaded by preincubation in Na(+)-free media (pHi 6.8), addition of varying Na+o resulted in a rapid H+ efflux that was markedly inhibited by DMA. DMA-sensitive Na+ influx into these acidified cells with varied Na+o exhibited a Michaelis-Menten constant (Km) of 23 mM and a maximum velocity (Vmax) of 43 nmol Na+/mg protein x min. By varying pHi (from pHi 7.1 to 6.2), DMA-sensitive Na+ influx likewise showed activation with cellular acidification with a pK at pHi 7.09. At pHi 6.8, ANG II decreased the Km for Na+o from 23 to 17 mM and increased the Vmax from 43 to 53 nmol Na+/mg protein x min. The pHi dependence of DMA-sensitive Na+ influx was not affected by ANG II (pK at pHi 7.03). DMA also inhibited AII-stimulated aldosterone secretion and Na+ influx similarly. These results indicate that Na(+)-H+ exchange in adrenal glomerulosa cells is 1) functioning under basal conditions, and 2) is modulated by ANG II with enhanced Na+o affinity and Vmax but without a shift in pHi dependence (similar to ANG II effects on vascular smooth muscle cells). These effects suggest an important role for Na(+)-H+ exchange during ANG II stimulation of aldosterone production by glomerulosa cells.

Hemodialysis and red cell cation transport in uremia: role of membrane free fatty acids.

Active and facilitated cation transport in erythrocytes of uremic patients may be improved acutely by hemodialysis, although the mechanisms remain unknown. As nonesterified fatty acids (NEFA) can affect Na+ pump activity in vitro, changes in plasma and red cell membrane NEFA content following a single hemodialysis procedure were examined and compared with acute changes in erythrocyte cation flux rates in 34 hemodialysis patients. In nonsodium-loaded cells, small changes in Na+ pump flux with dialysis did correlate with changes in intracellular Na+ content (r = 0.59; N = 17; P less than 0.01). On average, neither maximal Na+ pump activity nor Na+/Li+ counter-transport flux improved with dialysis, but Na+/K+/Cl- cotransport rates rose 25% post-dialysis (P less than 0.02). Plasma NEFA levels rose 87% following hemodialysis but erythrocyte membrane NEFA content declined by 23% (P less than 0.001). Importantly, 24 of the 34 subjects studied had a decrease in erythrocyte membrane NEFA content of greater than 10%, and in these patients, the fall in membrane NEFA correlated with an increase in ouabain-sensitive Na+ efflux (r = 0.564; P less than 0.01). The effects of hemodialysis on both erythrocyte NEFA content and Na+ pump flux could be reproduced by incubating pre-dialysis cells in fatty acid-free albumin. We conclude that acute changes in membrane NEFA may modulate active cation transport in uremic erythrocytes.

The Na-K-Cl cotransport in essential hypertension: cellular functions and genetic environment interactions.

The present paper examines factors involved in the expression of the Na-K-Cl cotransport system present in erythrocytes and in vascular cells. This transport system is modulated by vasoactive peptides such as atrial natriuretic factor and bradykinin in vascular smooth muscle and endothelial cells. The Vmax of the Na-K-Cl in human red cells displays large interindividual differences which can be mainly accounted for by genetic factors. Elevation of the Km for Na of the outward Na cotransport is found in red cells of some Caucasian hypertensives and in Black normotensives born of hypertensive parents whose blood pressure increases with salt loading. Reduction of Na intake from 200 to 10 mEq/day does not influence the activity of the cotransport in normotensive individuals but decreases the Km for Na of hypertensive subjects to values similar to those of the normotensives. These findings indicate that the Na-K-Cl cotransport is an important probe of genetic and environmental factors in the hypertensive process.

Vascular smooth muscle Na+-H+ exchanger kinetics and its activation by angiotensin II.

We have studied the kinetic properties of basal and angiotensin II (ANG II)-stimulated Na+-H+ exchange in cultured rat aortic smooth muscle cells. Initial rates of 22Na+ influx were measured in the presence of ouabain (1 mM) and bumetanide (0.1 mM) with and without amiloride after intracellular acidification by preincubation in Na-free media. The kinetics of amiloride (100 microM)-sensitive Na+ influx were studied under the following conditions: 1) constant intracellular pH (pHi; 6.8) and varying external Na+ (Na+o), which gave a Km of 23.6 +/- 2.0 (SD, n = 3) mM and a maximum velocity (Vmax) of 25 nmol.mg protein-1.min-1 (varying the amiloride concentration gave a Ki of 22 microM for inhibition under these conditions); and 2) constant Na+o (100 mM) and varying pHi (from 7.4 to 6.2), which indicated that amiloride-sensitive Na+ influx was stimulated by cell acidification when an outward H+ gradient was imposed. ANG II-stimulated amiloride-sensitive Na+ influx for up to 30 min with a half-maximal activation 10(-8) M. The pHi dependence from cell pH (pHi 7.2-6.2) of amiloride-sensitive Na+ influx stimulated by ANG II was similar to that of the basal values, a finding indicating that ANG II did not change the affinity of Na+-H+ exchange for intracellular H+. However, at pHi 6.8, ANG II increased the Vmax of amiloride-sensitive Na+ influx from 25 to 33 nmol.mg protein-1.min-1 and markedly decreased the Km for Na+o from 23.6 +/- 7.4 to 3.7 (SD, n = 4; P less than 0.005) mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic differences in lithium-sodium exchange and regulation of the sodium-hydrogen exchanger in essential hypertension.

The elevation of Vmax of red cell Li/Na exchange in some hypertensives and in their normotensive offspring is mainly accounted for by genetic factors. In order to understand the pathophysiological meaning of this finding, we have studied if it is an operational mode of H/Na exchange, a transport system involved in the regulation of cell pH in vascular and kidney cells. To define the relationship of the Li/Na exchange to the H/Na exchange pathway, we have investigated the kinetic effects of internal and external H+ on H/Na, H/Li, and Li/Na exchange. These three exchange modes have highly asymmetric affinities for H+ much greater than Li+ greater than Na+, are activated by internal H+ (Hi) with a high "Hill coefficient" (n = 2.5), and competitively inhibited by external H+ (Ho). Moreover, cell Li activates H/Na exchange while being transported by Li/Na exchange at a lower rate than Hi. These observations support the conclusion that Li/Na exchange is a mode of operation of H/Na exchange. Li+ discriminates better than cell H+ between regulatory and transport sites in red cells of normotensive subjects. Kinetic studies of H/Na exchange in red cells of hypertensive subjects with high Li/Na exchange indicate that the Vmax of both pathways are not linearly related. Thus, Li/Na exchange does not measure the number of Na/H sites but assesses the occupancy of an internal site. We found that H/Na exchange in hypertensive subjects has a reduced Hill coefficient for the cell pH dependence of Na influx, which is interpreted as a defective Hi regulatory site.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of digitalis-like factors in human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiac glycoside-specific antibodies.

Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin-specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma.

Red cell lithium-sodium countertransport and sodium-potassium cotransport in patients with essential hypertension.

Alterations in sodium countertransport and cotransport have been reported in red cells of patients with essential hypertension. We have investigated the relationship between these two systems by performing simultaneous measurements of the maximal rates of lithium-sodium (Li1-Na0) countertransport and outward sodium-potassium (Na-K) cotransport in red cells from normotensive and hypertensive subjects. Li1-Na0 countertransport was assayed by measuring the Na0-stimulated Li efflux from cells loaded to contain 10 mmoles Li per liter of cells by incubation in isotonic LiCl. Na-K cotransport was assayed by measuring the furosemide-sensitive component of Na and K efflux into magnesium-sucrose medium from cells loaded by the p-chloromercuribenzene sulfonic acid (PCMBS) procedure to obtain 50 mmoles of both ions per liter of cells. The mean values (+/- SE) for 16 normotensives and 22 hypertensives were (mmole/liter cells x hour): Na countertransport = 0.29 +/- 0.02 vs 0.51 +/- 0.03 (p less than 0.001); Na cotransport = 0.30 +/- 0.03 vs 0.51 +/- 0.05 (p less than 0.005); and K cotransport = 0.34 +/- 0.03 vs 0.60 +/- 0.04 (p less than 0.005). Li1-Na0 countertransport correlated significantly with Na cotransport (r = 0.50, n = 38, p less than 0.005) and K cotransport (r = 0.57, p less than 0.005). This observation suggests that both transport systems are somehow regulated to be more active in this group of hypertensive patients. The increased cotransport in hypertensive patients is also apparent from two other measurements of Na and K fluxes in red cells suspended in Na medium. First, the furosemide-sensitive net Na efflux into Na medium was (mean +/- SE) 0.25 +/- 0.05 in 10 normotensive subjects and 0.50 +/- 0.09 in 12 hypertensive patients. Second, the furosemide-sensitive net K efflux into Na medium was (mean +/- SE) 0.25 +/- 0.04 in 13 normotensive subjects and 0.43 +/- 0.04 in 16 hypertensive patients (p less than 0.005). We conclude that mean values for both Na countertransport and Na-K cotransport are significantly higher in the group of hypertensives than in the group of normal control subjects.