Mechanism of action of the diuretic effect of muzolimine

Muzolimine is a diuretic which has been proposed in the treatment of hypertension. Muzolimine shared both the high ceiling effect of loop diuretics and the long duration of action of thiazides but has a chemical structure different from those of other loop diuretics. It may act as a prodrug and an active metabolite present in the urine may inhibit NaCl reabsorption in the Henle's loop. We studied the effect of urines of piretanide and muzolimine treated rats on Na+K+Cl- cotransport in renal cells in culture (MDCK). In the presence of ouabain (0.5 mM), the Na+K+Cl- cotransport measured by 86Rb influx, represented 92 p.100 of the total 86Rb influx (6.16 +/- 1.12 nmol 86Rb/min/mg prot, n = 10). Both diuretics were administered i.v. to rats where they induced marked diuresis. Excreted urine (dilution to 1/100) was tested for cotransport inhibition. After piretanide (27 mumol/kg) the urine inhibited the Na+K+Cl- cotransport in MDCK cells (72% and 41% inhibition at the 15th and 45th minutes after diuretic injection). After muzolimine (50 mumol/kg), urines also inhibited Na+K+Cl- cotransport but the effect was slower in onset and more prolonged (42% and 49% inhibition at the 15th and 60th min). Diuretic effects in vivo and Na+K+Cl- cotransport inhibition in vitro by the urine developed parallel for both diuretics. Probenecid (100 mumol/kg) suppressed simultaneously the diuretic effect of muzolimine and the Na+K+Cl- cotransport inhibition by the urine of muzolimine treated rats. Our results suggest that muzolimine acts as a prodrug. Its active metabolite is secreted into the tubular lumen through a probenecid sensitive pathway and inhibits, like other loop diuretics, Na+K+Cl- cotransport.     

Kinetic analysis of erythrocyte Na+-K+ pump and cotransport in essential hypertension

Alterations in red blood cell (RBC) Na+-K+ pump and Na+-K+ cotransport have been described in essential hypertension. We evaluated Na+-K+ pump and cotransport in 30 hypertensive and 26 normotensive subjects subdivided by race and family history of hypertension using an improved method to examine the kinetics of Na and K effluxes. RBCs were Na-loaded by the nystatin method to five different levels of internal Na with pump determined as ouabain-sensitive Na efflux and cotransport as furosemide-sensitive Na and K efflux. Two kinetic parameters were determined for both transport systems: the apparent affinity for Na (K0.5) and the velocity of efflux at saturating internal Na concentration (Vmax). Mean intracellular Na content in fresh RBCs (mmol/L cells) was higher in black hypertensive (12.6 +/- 1.8 mmol/L cells) and normotensive subjects (10.9 +/- 1.2 mmol/L cells) than in white hypertensive (8.7 +/- 1.0 mmol/L cells) or normotensive subjects (8.5 +/- 0.8 mmol/L cells). The Vmax and K0.5 for pump were not significantly different between study groups. The Vmax for cotransport was elevated in white hypertensive compared with normotensive subjects, but the K0.5 values were similar. Black normotensive and hypertensive subjects displayed a lower Vmax and increased K0.5 for cotransport compared with the white groups. A family history of hypertension had no influence on cotransport kinetics in blacks but did predict white normotensive and hypertensive subjects with low cotransport. The reduction in intracellular Na affinity for cotransport in black subjects may explain their higher intracellular Na in fresh RBCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Red cell membrane Na+ transport systems in hereditary spherocytosis: relevance to understanding the increased Na+ permeability

Red blood cells (RBCs) in hereditary spherocytosis (HS) show high sodium (Na+) and potassium (K+) movement across the membrane, resulting in dehydration. In general, these abnormal cation fluxes have been interpreted as "increased leaks" due to passive or electrodiffusional permeability of the RBC membrane. A study to elucidate the contribution of concomitant ouabain-resistant pathways (Na-K-2Cl cotransport and Na-Li countertransport) to abnormal Na+ permeability present in RBCs of subjects with HS has been undertaken. Accordingly, erythrocyte Na+ and K+ content and transmembrane cation movements via the Na-K pump, Na-K-2Cl cotransport, Na-Li countertransport, and Na+ passive diffusion, were measured in 25 non-splenectomized patients with HS and compared with the results obtained from the study of 11 patients with congenital non-spherocytic haemolytic anaemia (CNSHA) due to hereditary elliptocytosis (7 cases) and RBC enzyme defects (4 cases) and of 30 normal controls. Compared to the controls, patients with HS exhibited a highly significant (P<0.001) increase in all the Na+ transmembrane movements via passive diffusion (411+/-243 vs 105+/-40), Na-K pump (2615+/-970 vs 1874+/-359), Na-K-2Cl cotransport (males: 371+/-138 vs 190+/-42; females: 401+/-134 vs 104+/-44) and Na-Li countertransport (207+/-131 vs 98+/-41). This was associated with increased Na+ and decreased K+ content, resulting in a reduction of total cation (Na+ + K+) RBC concentration. Furthermore, significant correlations were also found between the patients' RBC cationic content and the mean corpuscular haemoglobin concentration (MCHC) (r=0.51, P<0.05) and between the Na+ passive leak and the haematocrit value (r=-0.44, P<0.05). In the patients with CNSHA, a less significant (P<0.01) increase of active (Na-K pump) and passive (leak) transmembrane permeability to Na+ was associated with normal transmembrane movements via Na-K-2Cl cotransport and Na-Li countertransport. The present study demonstrates that in HS, RBCs are characterized by a variable, but always significant increase of all the membrane transport systems leading to the extrusion of Na+, and that these abnormalities, regardless of their relation to membrane structural defects, may probably be valuable for the differential diagnosis between HS and other congenital defects of RBCs.     

Sodium pump activity and calcium relaxation in vascular smooth muscle of deoxycorticosterone acetate-salt rats

The Na+-K+ pump activity was determined in femoral arterial smooth muscle from deoxycorticosterone acetate (DOCA)-salt hypertensive rats using potassium relaxation and ouabain-sensitive 86Rb uptake as indices. The membrane-stabilizing effect of calcium and its relation to Na+-K+ pump activity also were examined. Femoral arteries from DOCA-salt rats exhibited a greater relaxation in response to potassium addition after contraction with norepinephrine in a low potassium (0.6 mM) Krebs solution. The concentration of potassium required to produce a 50% relaxation was significantly less in DOCA-salt rats. Ouabain-sensitive 86Rb uptake was significantly greater at 3, 10, and 20 minutes of 86Rb incubation in femoral arteries from DOCA-salt rats. Linear regression analysis revealed a significant correlation between the uptake of 86Rb and time of incubation in both control and DOCA-salt rats. A significant difference in the slopes of the regression lines showed that the rate of uptake was greater in DOCA-salt rats. No difference was observed in ouabain-insensitive 86Rb uptake. A dose-dependent relaxation in response to increasing concentrations of calcium following contraction to norepinephrine was observed in femoral arteries from control and DOCA-salt rats. The relaxation was directly dependent on the level of extracellular potassium and was blocked by ouabain. Femoral arteries from DOCA-salt rats relaxed to a significantly greater extent in response to calcium at each level of potassium when compared with controls. These results provide further evidence for an increase in Na+-K+ pump activity in vascular smooth muscle from DOCA-salt hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered cations and muscle membrane ATPase activity in deoxycorticosterone acetate-salt spontaneously hypertensive rats.

The role of ions and cell membrane function in the pathogenesis of benign and malignant hypertension was investigated in spontaneously hypertensive rats (SHR). Ten-week-old male SHR (n = 50) and SHR treated with deoxycorticosterone acetate (DOCA; n = 70) and 1% NaCl drinking water were studied weekly for 14 weeks. Malignant hypertension developed only in DOCA-salt SHR and was characterised by severe hypertension, failure to thrive and renal fibrinoid necrosis. Fourteen DOCA-salt SHR and one SHR died. Extracellular (serum) and intracellular (erythrocyte and muscle) Na+, K+, Mg2+, Ca2+ and muscle membrane Na+,K(+)-adenosine triphosphatase (ATPase), Ca(2+)-ATPase and Mg(2+)-ATPase were measured at various stages in the development of malignant hypertension. Three developmental phases were defined: benign, premalignant and malignant. DOCA-salt SHR showed persistent hypokalaemia. In the benign phase, there were no differences in Na+, Mg2+ and Ca2+ between SHR and DOCA-salt SHR. In the premalignant phase, serum and erythrocyte Mg2+ and ATPase activity were significantly lower in DOCA-salt SHR compared with SHR. During the late premalignant and malignant phases, intracellular Ca2+ and Na+ were significantly higher in the DOCA-salt SHR compared with SHR. In view of these findings, the abnormalities in DOCA-salt SHR during the early phases of blood pressure elevation could be contributory factors to the development of malignant hypertension.     

A genetic approach to the geography of hypertension : examination of Na+ - K+ cotransport in Ivory Coast Africans

Outward Na+ - K+ cotransport in erythrocytes from essential hypertensive Caucasian subjects was found to be excessively low (Co -) compared to normotensives (Co +) carefully selected for their negative family history of hypertension. Since the frequency of essential hypertension varies widely among different populations and is particularly high in certain coloured peoples, we compared erythrocyte Na+ - K+ cotransport in normotensive and hypertensive subjects in Paris (France) and in Abidjan (Ivory Coast) to seen whether defective cotransport was related to high blood pressure in the African group as well. Of the 66 French unselected normotensives investigated, 26 (39%) were Co - whereas 14 of the 18 Ivory Coast unselected normotensives (79%) were Co -. 64 (80%) of the 80 essential hypertensives examined in France were Co -, but the proportion of Co - subjects among the Ivory Coast hypertensives was even higher. In addition, both hypertensives and normotensives in the African groups often had undetectable outward Na+ effluxes, a rare finding in the French subjects. We suggest that the high incidence of abnormal Na+ - K+ cotransport in the Ivory Coast series may reflect a genetic propensity to hypertension in this population, and that consequently, Na+ - K+ erythrocyte cotransport measurements might prove useful in defining geographic variations in hypertension.     

Natural variation in passive sodium permeability in human erythrocytes

The rate of influx of 22Na+ into human erythrocytes (RBC) varies greatly depending upon the donor. A high rate of influx may be related to a congenital predisposition to essential hypertension. In Northern Europeans, we find a threefold difference in the rate of 22Na+ influx between those with the least (LP) and most highly permeable (HP) RBC (from less than 0.15 to greater than 0.60 mmol Na+/liter RBC/hr). In order to further define determinants of these apparently hereditary differences in passive membrane Na+ transport, we identified two groups of normal laboratory and hospital personnel differing markedly (greater than twofold) in RBC 22Na+ influx rate. We find that the loop diuretics furosemide and bumetanide decrease by about 50% the influx of 22Na+ into HP RBC, but have a lesser influence on LP RBC. Impermeant polyanions such as citrate and pyrophosphate also specifically diminish 22Na+ influx into HP, but not LP, RBC. Therefore, the exaggerated 22Na+ influx into HP RBC probably occurs through a discrete pathway (perhaps via "Na/K/Cl cotransport"), which appears to be almost absent in LP RBC. The differences between HP and LP RBC most likely do not involve polymorphisms of RBC anion transport per se. The rate of RBC anion (35SO4(2-)) transport is the same in HP and LP RBC and is equally inhibited by furosemide and (to a lesser extent) bumetanide. Furthermore, the potent inhibitor of RBC anion transport, DIDS (diisothiocyanostilbene disulfonate) does not affect RBC Na+ permeability in either group. Nonetheless, the preferential reduction of Na+ permeation of HP RBC by loop diuretics may be of help in experimentally distinguishing HP from LP phenotypes. This information may be crucial in unraveling the structural basis of intrinsic differences in cell membrane Na+ permeability and their possible relationship to essential hypertension.     

Transmembrane electrical potential difference regulates Na+/HCO3- cotransport and intracellular pH in hepatocytes.

We have examined the hypothesis that a regulatory interplay between pH-regulated plasma membrane K+ conductance (gK+) and electrogenic Na+/HCO3- cotransport contributes importantly to regulation of intracellular pH (pHi) in hepatocytes. In individual cells, membrane depolarization produced by transient exposure to 50 mM K+ caused a reversible increase in pHi in the presence, but not absence, of HCO3-, consistent with voltage-dependent HCO3- influx. In the absence of HCO3-, intracellular alkalinization and acidification produced by NH4Cl exposure and withdrawal produced membrane hyperpolarization and depolarization, respectively, as expected for pHi-induced changes in gK+. By contrast, in the presence of HCO3-, NH4Cl exposure and withdrawal produced a decrease in apparent buffering capacity and changes in membrane potential difference consistent with compensatory regulation of electrogenic Na+/HCO3- cotransport. Moreover, the rate of pHi and potential difference recovery was several-fold greater in the presence as compared with the absence of HCO3-. Finally, continuous exposure to 10% CO2 in the presence of HCO3- produced intracellular acidification, and the rate of pHi recovery from intracellular acidosis was inhibited by Ba2+, which blocks pHi-induced changes in gK+, and by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, which inhibits Na+/HCO3- cotransport. These findings suggest that in hepatocytes, changes in transmembrane electrical potential difference, mediated by pH-sensitive gK+, play a central role in regulation of pHi through effects on electrogenic Na+/HCO3- cotransport.     

Inhibition of Na+,K+ pump affects nucleic acid synthesis and smooth muscle cell proliferation via elevation of the [Na+]i/[K+]i ratio: possible implication in vascular remodelling

OBJECTIVES : Na+,K+ pump inhibition is known to delay the development of apoptosis in vascular smooth muscle cells (VSMC). This study examines Na+,K+ pump involvement in the regulation of VSMC macromolecular synthesis and proliferation. METHODS : DNA, RNA and protein synthesis in VSMC from the rat aorta was studied by the incorporation of [3H]-labelled thymidine, uridine and leucine. Cell cycle progression was estimated by flow cytometry. Intracellular Na+ and K+ content and Na+,K+ pump activity were quantified as the steady-state distribution of 22Na and 86Rb and the rate of ouabain-sensitive 86Rb uptake in Na+-loaded cells, respectively. RESULTS : Ouabain inhibited the Na+,K+ pump with a Ki of 0.1 mmol/l. At concentrations less than 0.1 mmol/l, neither [Na+]i nor [K+]i was affected by ouabain; elevation of ouabain concentration sharply increased the [Na+]i/[K+]i ratio with a K0.5 of approximately 0.3 mmol/l. At concentrations higher than 0.1 mmol/l, ouabain time- and dose-dependently activated RNA and DNA syntheses in serum-deprived VSMC and inhibited cell cycle progression triggered by serum. In quiescent VSMC, ouabain did not affect protein synthesis, total cell number, but slightly increased the percentage of cells in the S-phase (4.25 versus 1.46%) and attenuated cell death assessed by staining with trypan blue and lactate dehydrogenase release. CONCLUSIONS : Elevation of the [Na+]i/[K+]i ratio caused by Na+,K+ pump inhibition markedly enhances nucleic acid synthesis in quiescent VSMC and blocks cell cycle progression in serum-supplied VSMC. The relative contribution of this phenomenon as well as the anti-apoptotic action of increased [Na+]i/[K+]i ratio to vascular remodelling under augmented content of endogenous Na+,K+ pump inhibitors, seen in volume-expanded hypertension, should be investigated by in-vivo studies.     

Sodium cotransport in vascular smooth muscle cells.

Vascular smooth muscle cells possess a number of Na cotransport systems. Three of these cotransport systems, Na/Ca exchange, Na/H exchange and Na-K-Cl cotransport, have been the subject of an increasing number of investigations to determine the respective roles of these transporters in vascular smooth muscle cell function. Evidence has been obtained that the Na/Ca exchange system participates in regulation of intracellular Ca in vascular smooth muscle cells. The Na/H exchange system appears to function in concert with a Cl/HCO3 exchange system to regulate intracellular pH. The Na-K-Cl cotransport system is a major contributor to K flux across the plasma membrane of vascular smooth muscle cells and is regulated by a number of vasoactive agents, suggesting that this Na cotransport system is also an important component of vascular smooth muscle cell function. Cultured vascular smooth muscle cells derived from spontaneously hypertensive rats have been found to exhibit reduced Na-K-Cl cotransport activity compared to smooth muscle cells from normotensive controls. Thus, alteration of vascular smooth muscle Na-K-Cl cotransport activity may be related to changes in vascular tone. However, the precise function of Na-K-Cl cotransport in vascular smooth muscle cells remains to be clarified. Recent studies of Na-K-Cl cotransport in vascular endothelial cells provide evidence that the co-transporter is important for regulation of endothelial cell volume and suggest that this Na cotransport system may be vitally important for normal function of the vasculature.     

Na(+)-K+ pump and Na(+)-K+ co-transport in cultured vascular smooth muscle cells from spontaneously hypertensive and normotensive rats: baseline activity and regulation.

OBJECTIVE: This paper examines the hypothesis that aberrations in vascular smooth muscle univalent ion transport systems play an important role in the pathogenesis of hypertension. DESIGN: Baseline Na(+)-K+ pump and Na(+)-K(+)-2Cl- co-transport activities and the regulation of these ion transport systems by angiotensin II and second messenger molecules have been studied in cultured aortic smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: Ion transport was studied using isotopic univalent cations (86Rb and 22Na). RESULTS: Baseline Na(+)-K+ pump activity was comparable between SHR- and WKY-derived VSMC. Baseline Na(+)-K(+)-2Cl- and K(+)-Cl- co-transport activity as well as K+ leakage were significantly greater in SHR VSMC. Baseline Na(+)-K(+)-2Cl- co-transport was sensitive to inhibition by forskolin and ethyleneglycol-bis-(beta-amino ethylester)-N,N,N',N'-tetraacetic acid, whereas cyclic guanosine monophosphate and phorbol 12-myristate, 13-acetate had no effect. Angiotensin II-stimulated Na(+)-K(+)-2Cl- co-transport activity did not differ between WKY and SHR VSMC. Angiotensin II increased Na(+)-K(+)-pump activity to a significantly greater extent in SHR VSMC. The stimulatory effect of angiotensin II upon Na(+)-K+ pump activity was reduced under Na(+)-free buffer conditions and in the presence of the Na(+)-H+ exchange inhibitor, ethylisopropyl amiloride. Na(+)-K+ pump activity was also stimulated by the protein kinase C activator, phorbol 12-myristate, 13-acetate, and this was completely inhibited under Na(+)-free buffer conditions. CONCLUSIONS: SHR VSMC exhibit anomalous Na(+)-K(+)-pump and Na(+)-K(+)-2Cl- co-transport activities. The influence of these univalent ion transport systems upon cellular Na+ and Ca2+ homeostasis invoke their participation in the pathogenesis of hypertension.     

Potassium-induced relaxation as an indicator of Na+-K+ ATPase activity in vascular smooth muscle.

Helical strips of rat tail artery were observed to relax in response to potassium after contraction induced by 10(-7) g/ml norepinephrine in potassium-free solution. After several minutes of relaxation, the strips showed an abrupt redevelopment of tension. The amplitude of the potassium-induced relaxation was employed as an index of the activity of the electrogenic sodium-potassium pump and hence of the Na+-K+ ATPase. This assumption seemed justified because the observed amplitude of potassium-induced relaxation paralleled known effects of the following variables on Na+-K+ ATPase: (1) intracellular sodium concentration; (2) ouabain administration; (3) magnesium; (4) temperature, and (5) potassium concentration. The relaxation that occurred in response to potassium is suggested to be due to an enhanced Na+-K+ ATPase resulting in increased electrogenic transport of sodium and potassium and, consequently, hyperpolarization. We propose that potassium-induced relaxation of rat tail artery may be used as a functional indicator of Na+-K+ ATPase activity in vascular smooth muscle.     

Disturbances in Na+ Transport Systems Induced by Ethanol in Human Red Blood Cells

The effects of ethanol on fluxes catalyzed by four Na+ transport systems (ouabain-sensitive Na+, K+ pump, bumetanide-sensitive Na+, K+ cotransport system, Na+:Li+- countertransport and anion carrier) and on Na+ and K+ leaks were investigated in human red blood cells. Ethanol concentrations higher than 32 mM were required in order to significantly modify erythrocyte Na+ transport function. The observed changes can be summarized as follows: (a) stimulation of Na+ efflux through the Na+, K+ pump (by 21-32% at 160-400 mM) and Na+:Li+ countertransport (by 34-59% at 160-400 mM); (b) inhibition of outward Na+, K+ cotransport (by 23-34% at 160-400 mM) and LiCO3- influx through the anion carrier (by 17-21% at 64-400 mM); and (c) increase in Na+ and K+ leaks (by 13-16% at 64-400 mM). The effects of ethanol on the Na+,K+ pump and Na+,K+ cotransport system resulted from changes in maximal rates of Na+ efflux (increased and decreased, respectively) without any significant effect on the apparent affinities for internal Na+. Erythrocytes preincubated for 1 hr with 160 mM ethanol, washed and further incubated in flux media, recovered a normal Na+ transport function. In conclusion, high concentrations of ethanol induced reversible perturbations of fluxes catalyzed by erythrocyte Na+ transport systems. The observed effects may reflect disturbances in Na+ transport function associated with severe intoxication.     

Erythrocyte Na+,K+ cotransport and Na+,K+ pump in black and caucasian hypertensive patients

Alterations in red blood cell (RBC) Na+,K+ pump and in Na+,K+ cotransport (CoT) have been described in essential hypertension (EH). We examined pump and CoT in 50 normotensive (NT) subjects and 58 EH subjects subdivided by race and family history of hypertension (+ FH). RBCs were preloaded with Na+ to obtain intracellular levels of 25 mM/liter cells by using the p-chloromercuribenzene sulfonic acid (pCMBS) method. Na+ and K+ efflux rates into a magnesium-sucrose medium were quantitated in the presence of ouabain and ouabain plus furosemide to define pump and CoT activity respectively. Mean intracellular Na+ content was higher (p less than 0.05) in black NT and HT subjects compared to Caucasians. Mean RBC CoT was lower in black EH compared to NT and compared to Caucasian NT and HT subjects. Conversely, Caucasian HT patients had higher mean CoT than NT subjects. Subdivision into + FH revealed very little effect of + FH on CoT in black NT and HT subjects. In Caucasian NT and HT subjects with + FH, mean CoT was significantly reduced (less than 0.3 mM/liter cells/hr) compared to those without + FH. A subgroup of Caucasian EH subjects displayed high CoT (greater than 0.6 mM/liter cells/hr); a + FH had little impact on the high CoT group. There was no correlation between RBC CoT activity and age, sex, severity of hypertension, urinary sodium excretion, and plasma aldosterone. There was a positive correlation (r = + 0.47; p less than 0.01) between CoT and upright plasma renin activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic factor stimulates Na/K/Cl cotransport in vascular smooth muscle cells.

Atrial natriuretic factor (ANF) is a collective term used to describe a group of peptides isolated from mammalian atria which have vasorelaxant activity as well as diuretic and natriuretic activity. Recently, ANF peptides have been shown to bind to specific receptors on vascular smooth muscle cells (VSMC) and to cause an elevation in cGMP levels. We have previously demonstrated that VSMC possess a prominent, cyclic-nucleotide-sensitive Na/K/Cl cotransport system. In the present study, the effects of the ANF peptide rat atriopeptin III (rAP III) were measured on Na/K/Cl cotransport of VSMC by using primary cultures derived from rat thoracic aorta. It was found that rAP III caused a marked elevation of Na/K/Cl cotransport. Maximal stimulation occurred at 100 nM, and the dose of rAP III required for half-maximal potassium influx (K1/2) was 9 nM. We also investigated the effect of rAP III on cGMP levels in VSMC. It was found that rAP III increased cGMP in a dose-dependent manner, with a K1/2 value of 10 nM. Finally, we measured the effect of the permeable cGMP analog 8-bromo-cGMP on Na/K/Cl cotransport. It was found that 8-bromo-cGMP stimulated cotransport to the same extent as did a saturating dose of rAP III (K1/2 = 0.2 microM). Saturating doses of rAP III and 8-Br-cGMP in combination did not stimulate cotransport in an additive manner, suggesting that rAP III probably does not elevate cGMP via inhibition of phosphodiesterase. These findings suggest that activation of Na/K/Cl cotransport via elevations in cGMP may be associated with ANF-mediated vasorelaxation and/or ANF-mediated diuresis and natriuresis.     

Outward Na+-K+-Cl- cotransport function in erythrocytes from essential hypertensives

We have performed a kinetic analysis of the interaction of the outward Na+-K+-Cl- cotransport system with intra-cellular Na+ in erythrocytes from 30 normotensive controls and 72 patients with essential hypertension. Neither maximal rate of ouabain-resistant, bumetanide-sensitive sodium efflux (Vmax) nor intracellular Na+ content required for half-maximal stimulation (K50%) were significantly different between normotensives and hypertensives. Nevertheless, using 95% confidence limits of the K50% in the normotensive group as a cut-off point, 21 (29.17%) essential hypertensive patients exhibited values above the upper normal limit of 20.11 mmol/l cells, revealing a decreased apparent affinity of outward Na+-K+-Cl- cotransport for internal Na+ ('Co-' hypertensives). The Vmax of Na+-K+-Cl- cotransport exhibited a great variability among hypertensives but 'Co-' patients tended to have increased values of this parameter when compared with the remaining essential hypertensives (959 +/- 84 vs 652 +/- 39 mumol/l cells/h, P = 0.0024). Mean BP values were significantly lower in the 'Co-' subset (121.4 +/- 1.6 mmHg), compared with the remaining 51 hypertensive patients (126.4 +/- 1.3 mmHg, P = 0.0297). We conclude that an abnormal function of outward Na+-K+-Cl- cotransport is present in 19% to 40% of Spanish patients with essential hypertension and this abnormality may be implicated in the mechanisms of hypertension.     

Blood pressure and erythrocyte Na+ transport systems in a French urban male population

This paper reports an investigation of blood pressure (taken as a continuous variable) as a function of: erythrocyte Na+ content; Na+,K+ pump; Na+,K+ cotransport and Na+,Li+ countertransport fluxes, and passive cation permeabilities in fresh erythrocytes from 129 French males who were living in an urban area and were not under treatment for any medical condition (after allowing for the effects of age, body mass index, alcohol and tobacco consumption). In contrast with previous findings in a North American population, we were unable to confirm that blood pressure was correlated with erythrocyte Na+ content and Na+,K+-AT-Pase activity. Conversely, the only transport parameter correlated (negatively) with blood pressure was outward Na+,K+ cotransport [r = -0.20, P less than 0.05 and r = -0.19, P less than 0.05, for systolic (SAP) and diastolic arterial pressure (DAP), respectively; n = 114]. When allowing for age, body mass index and alcohol consumption, the correlation coefficient between the Na+,K+ cotransport system and blood pressure increased from -0.20 to -0.28 (P less than 0.01) for SAP and from -0.19 to -0.28 (P less than 0.01) for DAP (n = 105). We conclude that the correlations between blood pressure and erythrocyte Na+ transport function could differ between North American and French (or Mediterranean) populations. In any case, a decreased pump or outward Na+,K+ cotransport activity may lead hypertensive subjects to a similar increase in cell Na+ (and Ca2+) content in the vascular wall.     

Erythrocyte cation transport systems and plasma lipids in a general male population

The relationships between five erythrocyte cation transport systems (Na(+)-K+ pump, Na(+)-K+ cotransport, Na(+)-Li+ countertransport and Na+ and K+ passive permeabilities) and plasma lipids (total plasma cholesterol, high-density lipoprotein cholesterol and triglycerides) were investigated in 129 male adult subjects with no known history of hypertension. Na+ and K+ erythrocyte contents were also considered for their possible relationships with plasma lipids. Na(+)-K+ cotransport and passive Na+ permeability were both significantly correlated with plasma triglycerides. Conversely, no significant correlation was found between erythrocyte cation transport systems or erythrocyte cation contents and total cholesterol. These findings suggest that plasma lipids can modulate erythrocyte ion transport activity in the general population.     

Angiotensin increases Na+ entry and Na+/K+ pump activity in cultures of smooth muscle from rat aorta.

Angiotensin markedly altered the Na+ permeability of smooth muscle cells cultured from explants of rat aorta. The rate of net Na+ uptake was followed in the presence of ouabain in order to block Na+ efflux via the Na+/K+ pump. Angiotensin II (AII) or angiotensin III (AIII) increased net Na+ uptake by about 3-fold. Maximal stimulation of Na+ uptake was produced by about 10 nM AII. Bradykinin and the angiotensin antagonist [Sar1, Ileu5, Ala8]AII had no significant effect on net Na+ uptake. Angiotensin also enhanced the activity of the Na+/K+ pump, which was assayed by following the rate of ouabain-sensitive 86Rb+ uptake by the cells. AII and AIII nearly doubled ouabain-sensitive 86Rb+ uptake, but bradykinin, norepinephrine, and [Sar1, Ileu5, Ala8]AII had no effect. In the presence of ouabain, 86Rb+ uptake was not significantly affected by AII or AIII, indicating that angiotensin did not alter passive permeability to Rb+. Loading the cells with Na+, either by incubation in K+-free medium or exposure to the Na+-selective ionophore monensin, markedly increased ouabain-sensitive 86RB+ uptake. This result indicates that the activity of the Na+/K+ pump is limited by the low level of Na+ that is normally in the cells. AII had no effect on the activity of the Na+/K+ pump in Na+-loaded cells. These results suggest that AII or AIII stimulates the Na+/K+ pump in cultured aortic muscle cells by increasing its Na+ supply.     

Hypertensive dog plasma inhibits the Na+-K+ pump of cultured vascular smooth muscle

We investigated the effects of plasma from dogs with perinephritic hypertension on the Na+-K+ pump of cultured dog vascular smooth muscle cells. We also measured [3H]ouabain binding by myocardium and vascular tissue. Fresh, unprocessed plasma from healthy dogs during the first 6 weeks of benign one-kidney, one wrapped hypertension and from paired normotensive control dogs was layered over confluent primary cultured puppy aortic smooth muscle cells that had been sodium-loaded with monensin. In 26 paired assays of plasma from four pairs of dogs, cells incubated in the presence of plasma from hypertensive dogs had significantly reduced total (p less than 0.01) and ouabain-sensitive (p less than 0.001) 86Rb+ uptakes, but their intracellular sodium content did not differ from cells incubated in paired normotensive plasma. We no longer detected these uptake differences when passaged cells or cells cocultured with bovine endothelial cells were used for assay or when plasma was treated with protease inhibitors or boiled. However, boiled plasma increased the sodium content of the assay cells, suggesting an ionophorelike effect. Levels of pump inhibitory activity in plasma appeared to remain constant during Weeks 1 to 6 of hypertension. We found no evidence for altered numbers of pump sites in cardiovascular tissues from these hypertensive dogs. These findings support the hypothesis that plasma factors inhibit the membrane Na+-K+ pump in vascular smooth muscle cells in this form of hypertension. These plasma inhibitory factors apparently do not induce pump molecules.