Red cell sodium-proton exchange is increased in Dahl salt-sensitive hypertensive rats.

To investigate the relationship between red blood cell Na+/H+ exchange (EXC) and genetic factors in hypertension, we studied the maximal rate of the antiporter (mmol/liter cell x hr; flux units = FU) in three strains of genetically hypertensive rats. Salt-resistant Dahl rats (DR) were normotensive under low (0.02%) and high (8%) NaCl diets, while salt-sensitive Dahl rats (DS) became markedly hypertensive after four weeks on the high-NaCl diet. Na+/H+ exchange did not differ between DR and DS rats when both were fed with the low-NaCl diet (mean +/- SE, 31 +/- 3, N = 15, vs. 29 +/- 3 FU, N = 14). On the high-NaCl diet, the DR strain did not exhibit significant changes in blood pressure and antiporter activity, but the DS rats significantly increased their blood pressure and Na+/H+ exchange (57 +/- 4 FU, N = 13) versus DR rats (38 +/- 3 FU, N = 15, P < 0.02). DS rats also significantly increased blood pressure and antiporter activity when fed with high-NaCl diet for one week. These data indicate that high NaCl intake per se does not increase Na+/H+ EXC because the control DR strain did not exhibit transport and blood pressure alterations as observed in the DS strain. Milan hypertensive and spontaneously hypertensive rats (Charles River substrain) had higher blood pressures than Milan and Wistar-Kyoto normotensive rats when they were maintained for four weeks on a 1.5% NaCl diet; however, no differences were seen among normotensive and hypertensive strains in Na+/H+ exchange activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic abnormalities of the red blood cell sodium-proton exchange in hypertensive patients.

The present study was designed to examine the kinetics of Na(+)-H+ exchange in red blood cells of normotensive and hypertensive subjects and its relation to the previously reported abnormalities in Na(+)-Li+ exchange. The Na(+)-H+ antiporter activation kinetics were studied by varying cell pH and measuring net Na+ influx (mmol/l cell x hr = units) driven by an outward H+ gradient. The Na(+)-Li+ exchange was determined at pH 7.4 as sodium-stimulated Li+ efflux. Untreated hypertensive patients (n = 30) had a higher maximal rate of Na(+)-Li+ exchange (0.43 +/- 0.05 versus 0.26 +/- 0.02 units, p less than 0.0003), a higher maximal rate of Na(+)-H+ exchange (62.3 +/- 6.2 versus 47 +/- 4 units; p less than 0.02), but a similar affinity for cell pH compared with normotensive subjects (n = 46). The cell pH activation of the Na(+)-H+ antiporter exhibited a lower Hill coefficient than that of normotensive subjects (1.61 +/- 0.12 versus 2.56 +/- 0.14; p less than 0.0001). This index of occupancy of internal H+ regulatory sites was found reduced in most of the hypertensive patients (73%) whether their hypertension was untreated or treated. Hypertensive patients with Na(+)-Li+ exchange above 0.35 units (0.68 +/- 0.057 units, n = 16) did not exhibit elevated maximal rates of Na(+)-H+ exchange (57.3 +/- 10 units, NS) in comparison with those with Na(+)-Li+ exchange below 0.35 units (66.4 +/- 7.6 units, n = 26), but both groups exhibited reduced Hill coefficients. Hypertensive patients with enhanced Na(+)-H+ exchange activity (more than 90 units) had normal maximal rates of Na(+)-Li+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and stoichiometry of the human red cell Na+/H+ exchanger.

We have investigated the kinetic properties of the human red blood cell Na+/H+ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H+ (Hi) and external Na+ (Nao) in red blood cells of normal subjects. Red blood cells with different cell Na+ (Nai) and pH (pHi) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na+ influx were measured by varying pHi (from 5.7 to 7.4), external pH (pHo), Nai and Nao and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na+ influx (Nai less than 2.0 mmol/liter cell, Nao = 150 mM) increased sigmoidally (Hill coefficient 2.5) when pHi fell below 7.0 and the external pHo was 8.0, but increased linearly at pHo 6.0. The net Na+ influx driven by an outward H+ gradient was estimated from the difference of Na+ influx at the two pHo levels (pHo 8 and pHo 6). The H+-driven Na+ influx reached saturation between pHi 5.9 and 6.1. The Vmax had a wide interindividual variation (6 to 63 mmol/liter cell.hr, 31.0 +/- 3, mean +/- SEM, n = 20). The Km for Hi to activate H+-driven Na+ influx was 347 +/- 30 nM (n = 7). Amiloride (1 mM) or DMA (20 microM) partially (59 +/- 10%) inhibited red cell Na+/H+ exchange. The stoichiometric ratio between H+-driven Na+ influx and Na+-driven H+ efflux was 1:1. The dependence of Na+ influx from Nao was studied at pHi 6.0, and Nai lower than 2 mmol/liter cell at pHo 6.0 and 8.0. The mean Km for Nao of the H+-gradient-driven Na+ influx was 55 +/- 7 mM. An increase in Nai from 2 to 20 mmol/liter cell did not change significantly H+-driven net Na+ influx as estimated from the difference between unidirectional 22Na influx and efflux. Na+/Na+ exchange was negligible in acid-loaded, DIDS-treated cells. Na+ and H+ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na+ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na+/H+ exchanger. It is concluded that human red cell Na+/H+ exchange performs 1:1 exchange of external Na+ for internal protons, which is partially amiloride sensitive.(ABSTRACT TRUNCATED AT 400 WORDS)

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: individual differences in the number of channels.

We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K+ (Ca-K) channel of human red cells (RBC). Cytosolic Ca2+ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na+ media containing CaCl2. The Ca-K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8 +/- 2.4 mmol/liter cell.hr, to 3.7 +/- 1.0 mmol/liter cell.hr by 6 nM CTX (n = 3). The kinetic of Ca-K efflux was studied by increasing cell ionized Ca2+ using A23187 (60 mumol/liter cell), and buffering with EGTA or citrate; initial rates of net K+ efflux (90 mmol/liter cell K+) into Na+ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca-K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca2+ was raised, with a Km of 0.37 microM and saturating between 2 and 10 microM Ca2+. Ca-K efflux was partially blocked (71 +/- 7.8%, mean +/- SD, n = 17) by CTX with high affinity (IC50 0.8 nM), a finding suggesting that is a high affinity ligand of Ca-K channels. CTX also blocked 72% of the Ca-activated K+ efflux into 75 mM K+ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K+ channel in Na+ media. CTX did not block Valinomycin-activated K+ efflux into Na+ or K+ medium and therefore it does not inhibit K+ movement coupled to anion conductive permeability. The Vmax, but not the Km-Ca of Ca-K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell.min (FU). In red cells with Hb A, Vmax was 9.36 +/- 3.0 FU (mean +/- SD, n = 17). The Vmax of the CTX-sensitive, Ca-K efflux was 6.27 +/- 2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75 +/- 3.2 FU, n = 8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of the Vmax of Ca-K+ efflux. Estimation of the number of CTX-sensitive Ca-activated K+ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K+ efflux (2.7 +/- 0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.

Na-K-C1 cotransport and Na pump in red cells of young blacks and blood pressure response to salt loading.

The functional operation of Na and K fluxes, mediated by the furosemide-sensitive (FS) Na-K-C1 cotransport and the ouabain-sensitive Na pump, was studied in a representative young (18-22 years) black population. Cation fluxes (mumol/L cell X hour = FU) were studied prior to salt loading; results were compared with the blood pressure response to chronic oral salt loading. All subjects (n = 26) took 10 g/day NaC1 plus their usual diets for 14 days. A mean arterial blood pressure increase higher than 5 mmHg was considered a sodium-sensitive (SS) response; a response lower than 5 mmHg was considered sodium-insensitive (SI). The Km for Nac to stimulate FS Na efflux was significantly greater in SS (SS = 13.8 +/- 1.39 mmol/L cells, n = 15; SI = 8.5 +/- 0.8 mmol/L cells, n = 11; p less than 0.001). Inward cotransport of Na and K was also studied at constant Nac (13 mmol/L cells) and incubation of 140 mM Na and 4 mM K. No significant differences were found in the Vmax of outward FS Na and K efflux, FS86 Rb, Na influx, net Na movement (efflux minus influx), and the Na/K ratio of inward cotransport. Several parameters of the ouabain-sensitive Na pump (Vmax of the Na efflux, Rb influx, Na/K coupling ratio (2.2), and fresh Nac) were found to be similar in both groups. The results indicate that in young blacks, the Na sensitivity of the blood pressure may be associated with lower affinity for internal Na of the outward Na-K-C1 cotransport system.

Volume-dependent and NEM-stimulated K+,Cl- transport is elevated in oxygenated SS, SC and CC human red cells.

Mechanisms involved in cell volume regulation are important in SS, SC cells as they might be involved in determining the extent of sickling and the generation of dense cells and irreversibly sickled cells. We have studied in these cells the response to cell swelling of the K+,Cl- transporter. We found that Hb SS, SC and CC red cells have higher values of a ouabain-resistant, chloride-dependent and NEM-stimulated K+ efflux than AA red cells. In contrast, the Na+,K+,Cl- cotransport estimated from the bumetanide-sensitive component of K+ efflux was not significantly different in SS, SC and CC red cells. The (ouabain + bumetanide)-resistant K+ efflux from SS, SC and CC red cells was stimulated by cell swelling induced by reduction of the osmotic pressure (300 to 220 mosmol/l) and pH (8 to 7) of the flux media (140 mM NaCl). The Cl--dependent K+ efflux stimulated by osmotic swelling highly correlated with the NEM-stimulated component (r = 0.8, p less than 0.001, n = 22) and the acid-pH-induced swelling (r = 0.969, p less than 0.001, n = 22), indicating that it is driven by the K+,Cl- transporter.

Red cell sodium countertransport and cotransport in normotensive and hypertensive blacks.

We have previously described elevated Lii -Nao countertransport (CT) and Na-K cotransport (CO) in red cells of Caucasian patients from Boston. In this study, we report both transport systems in black patients from Philadelphia. The maximal rate (Vmax) of CT was assayed by measuring the Nao-stimulated Li efflux from cells containing +/- 6 mmol Li/liter. The Vmax of outward cotransport was assayed by measuring the furosemide-sensitive component of Na and K efflux into Mg medium from cells containing 50 mmol/liter of both ions. The mean value of CT for 18 normotensive (NT) subjects with no family history of hypertension, (-) FHH , was 0.18 +/- 0.05 (mmol/liter cells X hour); and in 14 hypertensive (HT) patients, 0.18 +/- 0.07. The mean values of Na and K cotransport were, respectively (mmol/liter cells X hour), in 18 NT subjects with (-) FHH , 0.38 +/- 0.24 and 0.50 +/- 0.28 in 18 HT subjects, 0.25 +/- 0.17 and 0.24 +/- 0.14. We conclude that there is no difference in the Vmax for CT between the two groups of black subjects, but that the Vmax for Na-K CO was significantly reduced in the HT group. Notably, the offspring of HT patients (age 14 years, n = 17) also had a marked reduction in the Vmax of Na (0.15 +/- 0.17) K cotransport (0.19 +/- 14) in comparison with the mean value of Na (0.40 +/- 0.2) and K (0.60 +/- 0.3) cotransport measured in offspring (n = 10) of NT subjects (age 14 years).(ABSTRACT TRUNCATED AT 250 WORDS)

Family history of hypertension and red cell cation transport in high school students.

High school students who had at least one parent with hypertension (n = 22) were compared to schoolmates of the same age with a negative family history of hypertension in the parents (n = 21). We investigated in both groups the maximal rate of the ouabain-sensitive Na pump and the Na-K cotransport in nystatin-loaded cells and the Lii-Nao countertransport in lithium-loaded cells. The two groups were significantly different only in the sum of net Na transport mediated by the Na-K pump and Na-K cotransport. The mean diastolic blood pressure in the positive family history group was significantly higher. With control for blood pressure the difference in the maximal rate of Na transport was no longer significant; it remains uncertain if control for blood pressure represents "over-adjustment'. The finding of a higher maximal rate of Na transport in these adolescents who are at increased risk of future hypertension, yet currently well within the normal range, suggests that abnormal sodium metabolism may be a useful marker and appears early in the pathogenesis of this disease.