Increased Na+-H+ Exchange in Red Blood Cells of Patients With Primary Aldosteronism
We measured Na+-H+ exchange as the amiloride-inhibited fraction of H+ efflux from red blood cells into a sodium-containing medium (pHo 7.95 to 8.05) at pHi values of 6.05 to 6.15, 6.35 to 6.45, 6.95 to 7.05, and 7.35 to 7.45 in 12 drug-free patients with primary aldosteronism before and after excision of histologically proven aldosterone-producing adrenal adenoma, 12 drug-free essential hypertensive patients, and 12 healthy control subjects. Red blood cell Na+-H+ exchange was increased in patients with primary aldosteronism similarly to the mean exchanger velocity in essential hypertensive patients compared with values in healthy subjects (334±25 and 310±29 versus 139±21 μmol H+/L cells per minute, respectively; P<.001 and .01). The kinetic parameters of Na+-H+ exchange returned to normal on day 2 after removal of the aldosterone-producing mass. Km for [Na+]o was not affected by aldosterone, whereas Km for [H+]i was decreased in patients with primary aldosteronism. The kinetic characteristics did not differ in essential hypertensive patients and control subjects. Protein kinase C inhibition in vitro by calphostin C (60 nmol/L) increased Km for [H+]i and caused up to a 65% suppression of Na+-H+ exchange (pHi 6.05 to 6.15), while diminishing Km for [Na+]o in red blood cells of patients with primary aldosteronism. The calmodulin antagonist W-13 (60 mmol/L) decreased exchanger velocity and increased Km for both H+ and Na+. We conclude that aldosterone stimulates red blood cell Na+-H+ exchange by a nongenomic mechanism that augments the exchanger affinity to Na+ and H+. In primary aldosteronism, protein kinase C and calmodulin seem to have synergistic stimulatory effects on red blood cell Na+-H+ exchange, and both increase the affinity of the exchanger to H+, while their effect on Na+ binding is opposite.
Enhancement of NHE (a ubiquitous membrane exchanger of intracellular H+ for extracellular Na+) has been proposed as a marker of primary hypertension.1 2 Preliminary data have showed that similar enhancement of NHE is observed in secondary hypertension due to PA.3 To confirm these findings and assess the reversibility of aldosterone-induced NHE changes, in this study we evaluated red blood cell NHE in PA patients before and 6 months after excision of an aldosterone-producing adrenal mass. We compared results with those from EH patients and healthy control subjects.
Three groups of individuals were studied (Table 1⇓). Group 1 included 12 patients with PA (6 men and 6 women), 30 to 60 years of age (mean, 47±4). All patients had clinical and laboratory evidence of hyperaldosteronism, adrenal mass shown by computed tomographic scan, and no family history of arterial hypertension. In 6 patients, the histological evidence of adrenal adenoma was confirmed postoperatively. Group 2 included 12 patients with EH (6 men and 6 women), 30 to 60 years of age (mean, 46±4). Hypertension was established according to World Health Organization stage II,4 and EH was diagnosed after the absence of secondary hypertension was established on the basis of clinical examination and normal laboratory values for serum electrolytes and creatinine, urinalysis, kidney ultrasonographic study, renal isotopic radiograms, and/or intravenous pyelogram where indicated. Group 3 was a control group of 12 healthy volunteers (6 men and 6 women), 30 to 60 years of age (mean, 45±2). Subjects had no history of arterial hypertension, normal results of physical and routine laboratory examinations, and no first- or second-degree hypertensive relatives.
After informed consent was obtained, all medications were stopped in the patients in groups 1 and 2, under a physician's supervision, for at least 7 days before the investigation. The investigation protocols were as follows: For group 1, in 6 patients who underwent surgery for the adrenal adenoma, blood samples were taken for NHE determination before excision of an aldosterone-producing mass and on days 2, 7, and 30 thereafter. In 5 of these patients, NHE was also determined 6 months after surgery. Six patients who did not undergo surgery had single measurements of NHE. For groups 2 and 3, NHE levels were determined once and served as baseline measurements against which the experimental group was compared.
Blood samples were taken between 8 and 10 am after subjects had fasted 8 to 10 hours; samples were stored with heparin (50 IU/mL) and kept ice-cold for no more than 8 hours. After sedimentation (2000g for 10 minutes at −2°C), plasma and white blood cells were removed and red blood cells were washed twice with a medium containing 150 mmol/L NaCl and 5 mmol/L sodium phosphate buffer (pH 7.4).
NHE was defined as the amiloride-inhibited fraction of H+ efflux from red blood cells into an Na+-containing medium, as described by Escobales and Canessa5 and Orlov et al.6 Packed red blood cells (100 μL) were placed into 1.9 mL of medium consisting of (mmol/L) NaCl 150, KCl 1, MgCl2 1, and glucose 10 and were incubated for 5 minutes at 37°C. pH of the cell suspension was adjusted by slow addition of 0.2N HCl solution in 150 mmol/L choline chloride. The anion exchanger was inhibited by 200 μmol/L 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and the medium pH was adjusted rapidly to 7.95 to 8.05 by addition of 0.05N NaOH solution in 150 mmol/L choline chloride. Each experiment was redone with 500 μmol/L amiloride (half-inhibitory effect seen at 50 μmol/L) added before DIDS. The kinetics of the proton efflux were registered by means of a 91-15 electrode (Orion) connected to a pH meter (PHM-64, Radiometer).
Maximal initial velocity (Vmax) of the exchanger was determined as (ΔpH1−ΔpH2)×b/mt, where ΔpH1 and ΔpH2 are the initial rates of the medium acidification in the absence and presence of amiloride, respectively; b is the buffer capacity of the incubation medium (determined by titration of 1.9 mL medium with NaOH and HCl from pH 6.0 to 8.0); m is the red blood cell volume; and t is the incubation time. The Hill equation was used for determination of Km as substrate concentration yielding half of Vmax.
In a separate series of experiments, calphostin C and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13) were used as antagonists of PKC and calmodulin, respectively. Their half-maximal inhibitory concentrations were estimated as 20 nmol/L and 25 mmol/L (curves not shown), respectively, and for NHE study, the supramaximal concentrations (60 nmol/L and 60 mmol/L, respectively) were used.
DIDS, amiloride, calphostin C, and W-13 solutions were prepared in dimethyl sulfoxide. NaCl, KCl, MgCl2, Na2HPO4, NaH2PO4, HCl, and NaOH were obtained from BDH; amiloride, staurosporine, dimethyl sulfoxide, calphostin C, and W-13 were from Sigma Chemical Co; and DIDS was from Calbiochem.
Paired two-tailed Student's t test was used for assessment of the differences between every two groups. The Pearson product moment method was used for calculation of the correlation coefficient. A value of P<.05 was considered statistically significant. Results are expressed as mean±SE.
The exchanger was characterized in PA by increased Vmax. The increase of NHE Vmax appeared when pHi achieved values of 7.35 to 7.45, was maximal at pHi 6.35 to 6.45, and persisted until pHi reached nearly 6.0. Km for [Na+]o did not differ between PA patients and healthy subjects. On the other hand, Km for [H+]i was decreased in PA (Table 2⇑). Thus, the activated state of NHE in PA appears to be associated with increased exchanger affinity to intracellular H+, whereas affinity to Na+ in PA remains unchanged.
EH patients also had an increased initial velocity of NHE, but the affinity of the exchanger to Na+ and H+ was similar to that found in healthy control subjects (Table 2⇑). In both, the increase in Vmax appeared at pHi 7.0 and was maximal at pHi 6.1.
On day 2 after removal of the aldosterone-producing mass, the NHE values in PA patients returned to normal (Fig 2⇓) and were stable during the 6 months of follow-up. Thus, the exchanger appears to be activated in PA by a reversible, nongenomic mechanism.
There was no correlation between red blood cell NHE and age, sex, and disease duration in any of the groups. Mean arterial pressure correlated positively with the exchanger velocity (r=.41, P=.03) in all groups.
Treatment of red blood cells in vitro with calphostin C (a selective inhibitor of PKC) resulted in a reduction of NHE in both PA and EH patients (Table 3⇓). The reduction in Vmax reached 65% in PA and 84% in EH patients (pHi 6.05 to 6.15). Inhibition of PKC increased Km for [H+]i and for [Na+]o in PA patients, but calphostin C did not affect these parameters in EH patients. The calmodulin antagonist W-13 decreased the Vmax of NHE and increased Km for both H+ and Na+ in PA but not EH patients (Table 3⇓). Therefore, it appears that hyperaldosteronism augments the exchanger affinity to extracellular Na+ and intracellular H+. In PA, PKC and calmodulin seem to have a synergistic effect on red blood cell NHE activity, and both increased the affinity of the exchangers to [H+]i, whereas their effects on NHE affinity to Na+ were opposite and neutralized each other.
NHE is a plasma membrane transport protein expressed in essentially all types of human cells,7 including red blood cells, lymphocytes, smooth muscle cells, and the basolateral membrane of polarized epithelia, where it represents an amiloride-sensitive isoform referred to as NHE-1.8 Entry of Na+ into the cells in exchange for an intracellular proton is the main effect of NHE, which therefore is involved in cellular pH, volume balance, and initiation of cell growth and proliferation.9
Enhanced NHE has been described in red blood cells,6 platelets,10 and lymphocytes11 of EH patients as one of the membrane alterations that include increased membrane permeability for monovalent cations, depressed activity of intracellular calcium binders (eg, calmodulin), rapid turnover of phosphoinositol, and intracellular redistribution of isoenzymes of PKC and that result in “cytosol calcium overload.”12
Arterial hypertension is a common clinical sign of PA. The precise mechanism of blood pressure elevation in mineralocorticoid excess remains undetermined, although sodium retention and “baroreceptor failure” with volume expansion have been suggested. There is some evidence of membrane alterations in experimental mineralocorticoid hypertension. Jones13 has shown increased membrane cation transport in smooth muscle cells from deoxycorticosterone acetate–treated rats. Kornel et al14 correlate this effect with the hormone binding to the cell receptors. The present study confirms our preliminary observation3 that red blood cell NHE is enhanced in PA to the level characterizing at least some patients with EH (Table 2⇑). The activity of red blood cell NHE in PA in vivo correlates positively with plasma aldosterone levels (Fig 1⇑).
Some investigators15 16 propose that NHE acts as a modifier of calcium influx into the cells. In this case, aldosterone via increased NHE may augment the intracellular calcium content—an assumption confirmed partly by the elevated ionized calcium concentrations found in the cytosol of aldosterone-treated white blood cells by Wehling et al.17 “Cytosol calcium overload” in hyperaldosteronism might predispose to disturbed motility of arterial vessels. Indeed, Pan and Young18 showed that a marked elevation of peripheral resistance and the secondary rise in cardiac output contribute to blood pressure elevation in deoxycorticosterone acetate–treated dogs rather than sodium retention and volume expansion due to the direct renal action of aldosterone.19
Our results show that aldosterone influences red blood cell NHE through a nongenomic mechanism, since a decrease of the exchanger activity in mature red blood cells (that do not possess their own gene material) was demonstrated on day 2 after removal of the aldosterone-producing adenoma. This rapid effect can be explained by aldosterone-induced carboxymethylation20 of the membrane protein that forms the NHE or by phosphorylation of the exchanger by one or more intracellular phosphorylation mechanisms. In favor of the latter assumption is the effect of staurosporine (a nonspecific kinase antagonist) to prevent the NHE enhancement in red blood cells of PA patients in vitro (100 nmol/L, incubation for 1 hour, data not shown). This staurosporine effect in PA patients was similar to that observed in EH patients, in whom more extensive phosphorylation of the NHE had been found in platelets.10
Aldosterone in vitro augments NHE in red blood cells of healthy subjects by a consequent very rapid (seconds to minutes) mechanism that is independent of phosphorylation and a slower mechanism (half an hour to hours) that depends on PKC (W.K., unpublished data). The present study proposes also that PKC is involved in NHE regulation by hyperaldosteronism. Indeed, PKC inhibition by calphostin C in red blood cells of PA patients in vitro caused a 65% decline in initial velocity of NHE (Table 3⇑). An additional 20% of exchanger velocity seemed to be calmodulin dependent.
In EH, up to 84% of NHE represents the PKC-dependent fraction, and the contribution of the calmodulin-dependent fraction in the exchanger stimulation is minimal. The difference between the PKC- and calmodulin-dependent fractions of NHE in PA and EH, respectively, are statistically significant (Table 3⇑). Nevertheless, the fractional analysis of NHE, which shows a wide zone of overlap, can hardly be used as a diagnostic measure to differentiate the NHE enhancement of PA from that of EH.
Enhanced NHE in EH is not associated with elevated levels of aldosterone or its analogues. To explain the similarly high NHE rates in hyperaldosteronism and EH without aldosteronism, we can assume that at least two models of NHE activation exist in hypertension: (1) the “intrinsic model” of NHE overstimulation due to enhanced NHE gene transcription,21 activated PKC, and minimal involvement of calmodulin-dependent processes, which is represented by EH and is associated with normal or low plasma levels of aldosterone; and (2) the “acquired” activation of NHE by plasma aldosterone in PA, involving PKC- and calmodulin-dependent pathways stimulated by high plasma concentrations of aldosterone.
Aldosterone modulates NHE in red blood cells by increasing the affinity of the exchanger to intracellular H+ and causing a shift of the NHE curve toward the higher pHi. This mechanism differs from the reported ones for angiotensin II in vascular smooth muscle cells,22 which increases the exchanger affinity for Na+ but not for H+; for epidermal growth factor,23 which increases the exchanger affinity for H+; and for insulin,24 which decreases the affinity of the red blood cell exchanger to extracellular Na+ without a change in intracellular H+ binding.
We have also shown that PKC increases the exchanger affinity to [H+]i and decreases its binding with [Na+]o. Calmodulin increases the exchanger affinity to both Na+ and H+. To date, we have no sufficient explanation of the fact that these kinetic effects of PKC are seen in PA but not in EH as a classic state of PKC overactivity.25
Interestingly, no elevation of red blood cell Na+-Li+ countertransport (the system that is authentic or structurally close to NHE) was revealed in PA.26 The above-discussed effect of aldosterone that increases the NHE affinity to H+ without a change in Na+ binding may explain the discrepancy between the NHE and Na+-Li+ countertransport measurements in PA. Indeed, at pH 7.4 and Na+ concentration of 150 mmol/L in the preincubation medium (the usual conditions of Na+-Li+ countertransport study), the saturation of the exchanger may be incomplete and the velocity of the countertransporter is therefore submaximal.
Selected Abbreviations and Acronyms
|NHE||=||Na+-H+ exchange, exchanger|
|PKC||=||protein kinase C|
- Received August 20, 1996.
- Revision received August 21, 1996.
- Accepted August 21, 1996.
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