Mitzy Canessa

Increased sodium-lithium countertransport in red cells of patients with essential hypertension.

This paper describes experiments showing that one of the pathways of sodium transport across the red-cell membrane, sodium-lithium countertransport, is faster in patients with essential hypertension than in control subjects. This transport system accepts only sodium or lithium and is not inhibited by ouabain. The maximum rate of transport shows inherited differences. The mean maximum rate of sodium-lithium countertransport was found to be 0.55 +/- 0.02 (mean +/- S.E.M.) mmol (liter of red cells X hour)(-1) in a group of 36 patients with essential hypertension and 0.24 +/- 0.02 in 26 control subjects (P less than 0.001). The first-degree relatives of eight patients with essential hypertension and 10 control subjects had mean maximum rates of sodium-lithium countertransport of 0.54 +/- 0.05 and 0.23 +/- 0.02, respectively. Five patients with secondary hypertension had normal mean maximum rates of sodium-lithium countertransport. The relation between heritability of red-cell sodium-lithium countertransport and essential hypertension should be investigated further.

Predisposition to Hypertension and Susceptibility to Renal Disease in Insulin-Dependent Diabetes Mellitus

Only one third of patients with juvenile-onset insulin-dependent diabetes seem to be susceptible to diabetic nephropathy. To test whether this susceptibility is related to a predisposition to hypertension, we investigated the association of nephropathy with markers of risk for hypertension. We randomly selected 89 patients with insulin-dependent diabetes from a roster of children and adolescents who were seen between 1968 and 1972 at about the time the diagnosis was made. These 89 patients were recalled for examination, as young adults, in 1986 and 1987. Patients with nephropathy (cases, n = 33) were compared with controls without nephropathy (n = 56). Having a parent with hypertension tripled the risk of nephropathy (odds ratio, 3.7; 95 percent confidence interval, 1.4 to 10.1). Moreover, cases had significantly higher values for maximal velocity of lithium-sodium countertransport in red cells than controls (mean maximal velocity +/- SE, 0.51 +/- 0.04 vs. 0.38 +/- 0.02 mmol per liter of cells per hour; P less than 0.05). The excess risk associated with both these indicators of a predisposition to hypertension was evident principally in patients with poor glycemic control during their first decade of diabetes; the odds ratios were 4.5 (95 percent confidence interval, 1.1 to 18.7) for patients with a parental history of hypertension and 7.7 (95 percent confidence interval, 1.8 to 33.8) for patients with a maximal velocity of lithium-sodium countertransport greater than or equal to 0.35 mmol per liter of cells per hour. We conclude that the risk of renal disease in patients with juvenile-onset insulin-dependent diabetes is associated with a genetic predisposition to hypertension. Predisposition to hypertension appears to increase susceptibility for renal disease principally in patients with poor glycemic control.

Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange.

The cellular mechanisms responsible for abnormalities in spontaneously hypertensive rat (SHR) vascular smooth muscle cell (VSMC) growth and vasoreactivity are not defined. Because Na+/H+ exchange, which we have previously demonstrated in cultured VSMC, plays an essential role in mediating growth factor responses, we hypothesized that abnormalities in SHR growth regulation might be reflected in the activity of this transporter. To test this hypothesis, we studied DNA synthesis and Na+/H+ exchange (measured as the rate of amiloride-sensitive intracellular alkalinization or Na+ influx) in early subcultures (less than 6) of aortic VSMC from 12-wk-old SHR and Wistar Kyoto (WKY) animals. Serum-deprived SHR VSMC grew more rapidly in response to 10% serum with an increase in [3H]thymidine incorporation of 439% compared with 191% in WKY controls. Basal intracellular pH (pHi) values determined by fluorescent pH measurements were 7.37 +/- 0.04 and 7.27 +/- 0.03 (P less than 0.05) in early passage SHR and WKY, respectively. Acid recovery (initial pHi = 6.8) by SHR VSMC was faster than by WKY VSMC as measured by alkalinization (1.8 +/- 0.6 vs. 0.8 +/- 0.2 mmol H+/liter.min, P less than 0.05) or by amiloride-sensitive 22Na+ influx (14.5 +/- 1.2 vs. 4.0 +/- 0.5 nmol Na+/mg protein.min, P less than 0.05). In comparison to WKY cells early passage SHR VSMC exhibited 2.5-fold greater alkalinization and amiloride-sensitive 22Na+ influx in response to 100 nM angiotensin II. During serial passage, WKY cells acquired enhanced Na+/H+ exchange and growth rates so that by passage 6, these differences were no longer present. These findings in early cultures of SHR VSMC, removed from the in vivo neurohumoral milieu, suggest that increased Na+/H+ exchange in SHR may reflect alterations in Na+ homeostasis that might contribute to altered SHR VSMC function such as enhanced growth and vasoreactivity.

Volume-stimulated, Cl(-)-dependent K+ efflux is highly expressed in young human red cells containing normal hemoglobin or HbS.

SummaryWe report here that a Cl−-dependent K− (K∶Cl) efflux, which is stimulated by N-ethylmaleimide, (NEM) and by increased red cell volume, exists in young red cells of individuals with normal hemoglobin A (AA) and in those homozygous for hemoglobin S (SS). We have investigated this K∶Cl efflux in several density-defined red cell fractions obtained from Percoll-Stractan continuous density gradients. We found high activity of the NEM-stimulated K∶Cl transport in reticulocytes and young red cells from nine sickle cell (SS) patients (43±27 mean±sd mmol K+/liter of cells/hr=flux units (FU)) and in the young cell fraction of three AA individuals with high reticulocytosis recuperating from nutritional anemias (41.7±10 FU). In addition, we observed significant interindividual variation of this K∶Cl efflux in the discocyte fraction of SS blood. Cell swelling markedly stimulated the K∶Cl efflux, in SS whole blood (9.8±7.4 FU, in SS young cells (13±13 FU), and in AA young cells (21.4±11 FU). The activity of the Na−K−Cl cotransport, as estimated by the bumetanide sensitive K+ efflux was not found to be cell-age dependent in either AA or SS cells.Measurements of red cell density by isopycnic gradients indicated that 27% of the young cells reduce their volume by a Cl−-dependent process in hypotonic or low pH-induced swelling.The large volume-stimulated K∶Cl efflux in AA young cells raises the possibility that these fluxes may be involved in the maturation of erythropoietic precursors. The high activity in the red cells of sickle cell anemia patients and its interindividual variation may have pathophysiological consequences since it reverses the decrease in the intracellular concentration of hemoglobin which occurs in response to low pH or osmolarity, an unwelcome pro-sickling event.

Volume‐dependent and NEM‐stimulated K+Cl−1 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 ) 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< 0.001, n = 22) and the acid‐pH‐induced swelling (r = 0.969,p< 0.001, n = 22), indicating that it is driven by the K+Cl− transporter.

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 (Li1-0) countertransport and outward sodium-potassium (Na-K) cotransport in red cells from normotensive and hypertensive subjects. Li1-Nao countertransport was assayed by measuring the Nao- Li efflux from cells loaded to contain 10 mmoles Li per liter of cells by incubation in isotonic LiCI. 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- 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 (/cells × hour): Na countertransport = 0.29 ± 0.02 vs 0.51 ± 0.03 (p < 0.001); Na cotransport = 0.30 ± 0.03 vs 0.51 ± 0.05 (p < 0.005); and K cotransport = 0.34 ± 0.03 vs 0.60 ± 0.04 (p < 0.005). Li1-Na0 countertransport correlated significantly with Na cotransport (r = 0.50, n = 38, p < 0.005) and K cotransport (r = 0.57, p < 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 < 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. (Hypertension 4: 795–804, 1982)

Amiloride-sensitive Na+ transport in human red cells: evidence for a Na/H exchange system

SummaryThe role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na+ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 μm) and methazolamide (400 μm). Red cells with different internal pH (pHi=6.4, 7.0 and 7.8) were prepared and Na+ influx was measured at different external pH (pHo=6.0, 7.0, 8.0). Na+ influx into acid-loaded cells (pHi=6.4) markedly increased when pHo was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na+/H+ exchange systems blocked about 60% of the H+-induced Na+ entry, while showing small inhibitory effects in the absence of pH gradients. When pH0 was kept at 8.0, the amiloride-sensitive Na+ entry was abolished as pHi was increased from 6.4 to 7.8. Moreover, measurements of H+ efflux into lightly buffered media indicated that the imposition of an inward Na+ gradient stimulated a net H+ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na+ (Nai+=Na0+=15mm,Em=+6.7 mV), an outward H+ gradient (pHi=6.4, pH0=8.0) promoted a net amiloride-sensitive Na+ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na+/H+ exchange system in human red cells.

Outward sodium and potassium cotransport in human red cells

SummaryThis paper reports some kinetic properties of Na−K cotransport in human red cells. All fluxes were measured in the presence of 10−4 M ouabain. We measured Na and K efflux from cells loaded by the PCMBS method to contain different concentrations of these ions into a medium that contained neither Na nor K (MgCl2-sucrose substitution) in the absence and presence of furosemide. Furosemide inhibited 30–60% of the total efflux depending on the internal ion concentration and the individual subject. We took the furosemide-sensitive fluxes to be a measure of Na−K cotransport. The ratio of Na to K cotransport was 1 over the entire range of internal Na and K concentrations studied. When Na was substituted for K as the only internal cation, cotransport was maximally activated when the Na and K concentrations were between 20 and 90 mmol/liter cells. The concentration of internal Na required to produce half-maximal cotransport was about 13±4 mmol/liter cells (n=4), while the comparable concentration of K was somewhat lower. The activation curve was definitely sigmoid in character, suggesting that at least two Na ions are involved in the transport process. The maximum of Na−K cotransport was about 0.5±0.15 mmol/liter cells × hr (n=5); it had a flat maximum in the medium at about pH 7.0, decreasing in both the acid and alkaline sides. furosemide-resistant effluxes were found to be linear functions of internal Na and K concentrations and to yield rate coefficients of 0.019±0.002 hr−1 and 0.014±0.002 hr−1 (n=7), respectively. These values are of the same order of magnitude expected of ions moving across phospholipid bilayers.

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/1 cellxhr=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<0.0003), a higher maximal rate of Na+-H+ exchange (62J±62 versus 47±4 units; p<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<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 035 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+-LJ+ 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. We propose that the elevated maximal rate of Na+-Li+ exchange and the low Hill coefficient for Na+-H+ exchange activation seen in many hypertensive patients might reflect a higher number of antiporter sites or abnormal antiporter regulation by phosphorylation.

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)