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This study was designed to investigate the effects of a hypertensive stimulus, high salt intake, in hypertension-prone (SBH) and -resistant (SBN) Sabra rats on erythrocyte Na+ content (Na+i), Ca2+ influx and cytosolic Ca2+ concentration ([Ca2+]i). The relationships of these parameters to plasma lipids, circulating digoxin-like immunoreactivity and membrane microviscosity, determined by the fluorescence anisotropy of trimethylamino-diphenylhexatriene (TMA-DPH) and diphenylhexatriene (DPH), were also evaluated. Erythrocytes of SBH rats were characterized by increased [Ca2+]i, unchanged Ca2+ influx and reduced Na+i. There were no significant differences in the plasma digoxin-like immunoreactivity between the two strains. High-salt intake decreased membrane microviscosity (DPH anisotropy) in SBH rats but did not alter the above parameters. Erythrocyte [Ca2+]i correlated positively with diastolic blood pressure and negatively with erythrocyte Na+i. Membrane dynamics evaluated by the two fluorescent probes did not correlate with [Ca2+]i, Ca2+ influx or Na+i whereas DPH anisotropy was inversely related to blood pressure. These relationships were independent of plasma cholesterol or triglycerides. It can be concluded that 1) similarly to earlier observations in essential hypertension and spontaneously hypertensive rats, erythrocyte [Ca2+]i correlates positively with blood pressure in salt-dependent hypertension, and 2) increased erythrocyte Na+ content need not be a hallmark of hypertension.

Our previous experiments in human and rat platelets demonstrated that the absence of extracellular Na+ increased the fluorescence anisotropy of TMA-DPH (trimethylamino-diphenylhexatriene, probe preferentially incorporated into the outer leaflet of the plasma membrane). Here we investigated further the in vitro effects of Na+ ions on membrane dynamic properties. Na(+)-dependent changes were reversible and they required about 10-20 min to be induced. They were specifically located in the TMA-DPH environment because they were not observed with diphenylhexatriene (probe non-selectively incorporated into all hydrophobic domains of the cell). To evaluate the possible influence of the intracellular Na+, the effects of sodium replenishment, monensin, ouabain and thrombin on TMA-DPH anisotropy were measured. A rise in intracellular Na+ above the physiological level was associated with unchanged or slightly decreased TMA-DPH anisotropy whereas its decrease was accompanied by a pronounced rise in TMA-DPH anisotropy. Our data indicate that the changes in intracellular Na+ concentration, rather than those in extracellular Na+ concentration, are responsible for the alterations in platelet membrane fluidity probed by TMA-DPH.

The influence of transmembrane Na+ and Ca2+ gradients on cytosolic pH (pHi) and free Ca2+ concentration ([Ca2+]i) have been examined in unstimulated human platelets with the aid of BCECF and Fura-2 fluorescent dyes. The removal of external Na+ (Na+o) acidified the cytosol in a pHo-dependent manner which was insensitive to EIPA and DIDS, the inhibitors of the Na+/H+ exchanger and bicarbonate transporters. Na+o removal also increased [Ca2+]i by 17 +/- 5%, but the amplitude of the concomitant acidification was independent on Ca2+ influx or cytosolic Ca2+ concentration. In contrast, in the presence of 145mM Na+o, a rise in external Ca2+ concentration from 1 to 2mM increased [Ca2+]i by 38 +/- 11% and acidified the cytosol by 0.16 +/- 0.04 pH units. These results indicated that, in resting human platelets, the transmembrane Na+ gradient is a major determinant of pHi. Two Na(+)-dependent processes have been found: one is triggered by an external acidification and the other activated by a rise in Ca2+ influx or cytosolic concentration.

1. Despite the fact that numerous studies provide evidence for the existence of an endogenous inhibitor of the cell membrane Na(+)-K+ pump in plasma, little is known about the relationships between this factor and the main haemodynamic parameters. 2. In order to shed some light on this, we attempted to correlate haemodynamic parameters, measured during heart catheterization in a group of 22 cardiopathic subjects, with plasma digitalis-like activity levels, determined by two different procedures. 3. The ability of plasma to inhibit a human renal Na(+)-K(+)-ATPase showed an inverse correlation with cardiac index and a direct correlation with peripheral resistance. Plasma cross-reactivity with digoxin antibodies correlated directly with left atrial pressure. 4. These results furnish confirmation of a number of theoretical assumptions which attribute to the digitalis-like factor the ability to modify the contractility of the cardiovascular system.

Na+ ions, which can play a pathogenic role in the development of high blood pressure, have been reported to regulate membrane enzymatic activities, receptor-ligand interaction and coupling of G-protein receptors to their effectors. This study was designed to investigate the in vitro effects of Na+ ions on membrane dynamic properties. The fluorescence anisotropy values of TMA-DPH (trimethylamino-diphenylhexatriene, probe selectively incorporated into the outer leaflet of the plasma membrane) was evaluated in platelets and erythrocytes of sodium-dependent hypertension-prone and -resistant rats of the Sabra Strain. Whereas no difference was observed between the 2 strains, TMA-DPH anisotropy was found to be strongly influenced in platelets by external Na+ ions. In the absence of external Na+, TMA-DPH anisotropy increased in human and rat platelets. In contrast, Na+ ions did not affect the anisotropy when the probe was inserted into erythrocyte ghosts. This indicates that Na+ ions can acutely regulate order parameter and microviscosity of platelet plasma membrane in the regions explored by the probe.

The kinetic properties of the Na+/H+ exchanger in human platelets were investigated by direct measurements of pHi as detected with the fluorescent dye, BCECF. In acid-loaded cells, the antiporter displayed a hyperbolic dependence regarding external Na+ with an apparent Km of 38 +/- 4 mM (pHo 7.2 at 25 degrees C) whereas its pHi-dependent activation between 7.3 to 6.4 did not obey a Michaelian model. External acidification from 7.7 to 6.5 decreased significantly the initial rate of Na(+)-dependent H+ efflux. The amiloride derivative, ethylisopropylamiloride blocked this exchanger and exerted a non-competitive inhibition with respect to Na+o (Ki = 17 nM). The cation selectivity of the external site of the antiporter was Na+ greater than Li+ greater than K+ and choline. These results indicate that the BCECF technique allows to evaluate the main features of the Na+/H+ exchanger in human platelets, which possesses kinetic properties similar to those reported in other cell types.

The activity of Na+-Ca+-exchange and ATP-dependent Na+ and Ca2+ transport by heart sarcolemmal membranes from male 3-4-week-old spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) were compared. Differences in active Ca2+ and Na+ transport between the two substrains were suppressed by addition of exogenous calmodulin. Calmodulin was active only in the presence of Ca2+. The rate of the Na+-dependent Ca2+ efflux, reflecting the activity of the Na+-Ca2+ exchange, was significantly higher in SHR than in WKY vesicles. An alteration of the intracellular calmodulin activity or content might thus be responsible for the modifications in Ca2+ handling, and limit the activity of the Na+ pump in SHR membranes. The platelet cytosolic free Ca2+ concentration of young SHR and WKY was measured by using the fluorescent indicator Quin-2/AM. In the absence or presence of added external Ca2+, no difference in the intracellular concentration of Ca2+ was observed between platelets of either origin. The intraerythrocytic sodium content, measured by flame spectrophotometry, was similar in SHR and WKY. This study, performed before the onset of hypertension, shows that membrane mechanisms controlling the intracellular ion content were already modified, whereas the resulting intracellular concentrations remained within the normal range.

The digitalis-like activities of plasma extracts from 108 patients and normal subjects were measured by their ability to compete with ouabain for binding to the digitalis sites of the Na+-pump. High levels were found in 18 of 54 untreated patients with moderate hypertension, 10 of 14 patients with end-stage renal failure and six patients with active acromegaly. These levels returned to control values after dialysis in the patients with renal insufficiency and high levels of the inhibitor, and after successful surgery and cobalt therapy in seven acromegalic patients. An increase in circulating Na+, K+-ATPase inhibitor was also found in rats after chronic sodium loading. These results indicate that levels of the circulating compound with digitalis-like properties do not result from high blood pressure but, rather, are related to blood volume and Na+ balance.

The effects of a high salt diet (8% NaCl) on blood pressure and intra-erythrocytic Na+ content were studied in Wistar rats. The ability of the plasma to inhibit the renal Na+,K+-ATPase activity and to cross-react with digoxin antibodies was also investigated. After 1 week, neither systolic blood pressure nor intra-erythrocytic Na+ content were modified, but plasma extracts slightly inhibited renal Na+,K+-ATPase (70.9 +/- 1.7 versus 76.3 +/- 2.1 mumol Pi/mg per h, P = 0.05). After 2 weeks, the plasma inhibitory activity, systolic blood pressure and intra-erythrocytic Na+ content were higher than corresponding values in control animals (65.5 +/- 1.6 versus 79.1 +/- 2.8 mol Pi/mg per h, P less than 0.001; 132 +/- 2 versus 114 +/- 4 mmHg, P less than 0.001, and 4.95 +/- 0.32 versus 3.81 +/- 0.36 mmol/l cells, P less than 0.05, respectively). After 3 months, the plasma digoxin-like immunoreactivity and its ability to inhibit the Na+ pump were elevated (68.7 +/- 7.9 versus 48.2 +/- 5.4 pg/ml, P less than 0.02; 57.8 +/- 1.8 versus 72.9 +/- 1.8 mumol Pi/mg per h, P less than 0.001, respectively) whereas intra-erythrocytic Na+ content had returned to control levels. The results demonstrated that this high salt intake led to simultaneous increases in systolic blood pressure and in the activity of a digitalis-like compound present in plasma. The inhibition of Na+,K+-ATPase was correlated with systolic blood pressure and digoxin-like immunoreactivity (r = 0.569, n = 76, P less than 0.001 and r = 0.414, n = 34, P less than 0.02, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating digitalislike compounds have been proposed to be involved in some Na+-dependent types of experimental hypertension and in human essential hypertension. The level of circulating Na+-K+ pump inhibitor(s) was investigated in the spontaneously hypertensive rat of the Okamoto strain (SHR), its normotensive control, Wistar-Kyoto rat (WKY), and the regular Wistar rat using the following criteria: the ability of whole plasma to inhibit the total active Na+ efflux from Wistar rat erythrocytes and to cross-react with digoxin antibodies and the ability of plasma extracts to inhibit Na+,K+-adenosine triphosphatase (ATPase) activity of membranes from rat kidney. SHR plasma inhibited the net Na+ efflux from Wistar erythrocytes by up to 27% compared with WKY or Wistar plasma. For a given number of cells, the inhibition increased with the amount of available plasma. Cross-reactivity with digoxin antibodies was twice as high in SHR as in WKY or Wistar plasma. It was already enhanced in 3- to 4-week-old rats. Plasma extracts from SHR significantly inhibited Na+,K+-ATPase activity when compared with WKY extracts (75.6 +/- 2.6 vs 89.3 +/- 2.4 mumol Pi/mg/hr; p less than 0.01) but did not differ from Wistar plasma extracts. These results strongly suggest that circulating digitalislike compound(s) are present in elevated amounts in SHR as early as 3 to 4 weeks of age, but their exact participation in blood pressure elevation or maintenance remains to be clarified.

High Na+ intake has been proposed to induce a rise in the activity of a circulating inhibitor of the Na+, K+-pump. The effects on male Wistar rats of a high sodium diet (8 per cent NaCl) on the activity of such a plasma Na+, K+-ATPase inhibitor were investigated. Systolic blood pressure, body weight, urinary Na+ excretion, haematocrit, intraerythrocytic Na+ content and the activity of a Na+ dependent transport system, i.e. the uptake of 5-HT by blood platelets were measured in parallel. After one week, neither systolic blood pressure nor intraerythrocytic Na+ content were modified, but the ability of the plasma extracts to inhibit renal Na+, K+-ATPase increased (70.9 +/- 1.7 vs 76.3 +/- 2.1 mumol Pi/mg/h; p = 0.05). After two weeks, the plasma inhibitory activity, the systolic blood pressure and the intraerythrocytic Na+ content were higher than that of control animals (65.5 +/- 1.6 vs 79.1 +/- 2.8 mumol Pi/mg/h, p less than 0.001; 132 +/- 2 vs 114 +/- 4 mmHg, p. +/- 0.001 and 4.95 +/- 0.32 vs 3.81 +/- 0.36 mmol/l.cells, p less than 0.05). After three months, the ability of plasma extracts to inhibit the Na+ pump and the systolic blood pressure were still elevated (57.8 +/- 1.8 vs 72.9 +/- 1.8 mumol Pi/mg/h, p less than 0.001; 145 +/- 4 vs 118 +/- 2 mmHg, p less than 0.001) whereas intraerythrocytic Na+ content had returned to control levels and 5-HT uptake was not modified.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the activity of semipurified renal Na+, K+-ATPase and of 3H-Ouabain binding to erythrocytes was used to titrate the level of a plasmatic endogenous inhibitor of the Na+, K+-pump. The inhibition effect of plasma extracts was specific and unrelated to vanadate, calcium and products of proteolysis. Similar results were obtained with the two procedures. An endogenous pump inhibitor could be detected in 28 plasmas of the 42 normotensives investigated so far. In 18 out of 21 normotensives born of hypertensive parent(s) the level was found to be higher than that observed in normotensive controls devoid of hypertensive heredity. 13 untreated essential hypertensives out of 21 also exhibited higher levels of the sodium-potassium pump inhibitor. Plasma levels of the inhibitor appear stable during several months in male subjects. No clear relationship could be established either in normotensives or hypertensives between the pump inhibitor level, the urinary output of Na+, and blood pressure. Neither could any relationship be established between the inhibitor and the intraerythrocytic Na+ concentration. The accuracy of the methodology allows a large scale clinical investigation. It can also be used in the purification of the substance, and preliminary results precised that its molecular weight was inferior to 1 000 daltons and that it was anionic. Purified fractions were obtained after gel filtration, ion exchange chromatography, and high pressure liquid chromatography. Preliminary results suggest that the purified fraction may interfere with the mechanisms of hypertension. They inhibited the Na+-dependent serotonin uptake by human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Active transport of Na+,K+ and Ca2+ was compared in heart plasma membranes from 3-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). ATP-dependent Ca2+ accumulation, which reflects Ca2+, Mg2+-ATPase activity, was higher in SHR than in WKY membranes. At a free calcium concentration of 4 X 10(-7)M, the addition of 2 X 10(-7)M calmodulin enhanced the active Ca2+-transport more in WKY than in SHR vesicles. Na+- and K+-dependent ATPase activity was two fold higher in SHR than in WKY. From ouabain binding studies this seemed to be due to an increased density of enzyme units. Physiological concentrations of calmodulin and calcium ions reduced Na+,K+-ATPase activity in the two strains but more in SHR than in WKY. This study demonstrates that active Na+ and Ca2+ transport is enhanced in young SHR; the Ca2+-calmodulin complex may regulate Na+,K+-ATPase activity and sensitivity of Na+,K+-ATPase and Ca2+,Mg2+-ATPase activities to Ca2+-calmodulin differs between SHR and WKY.

The presence in plasma extracts of a sodium pump inhibitor with digitalis-like properties was investigated by two complementary tests: decrease in the affinity of ouabain binding to human red blood cells and inhibition of Na+,K+-ATPase. The results of the two methods were correlated (r = 0.76, n = 44, p less than 0.01), suggesting that the same factor may be responsible for both effects. All subjects with elevated values were hypertensive or normotensive and had a family history of hypertension. Forty percent of the subjects in these two groups had high inhibition values. The elevation was significant (p less than 0.01) when compared with values in normotensive subjects with no hypertensive heredity. Increased inhibition was observed in patients taking beta-blocking agents; conversely, diuretics normalized the values. No correlation was found between pump inhibition and age, sex, blood pressure, levels of plasma K+ or Na+, or plasma renin activity. These data show the existence of a sodium pump inhibitor in the plasma of some subjects and point to a possible association with hypertension. They also underline the importance of genetic background and the heterogeneity of essential hypertension.

Active Na+ and Ca2+ transports by sarcolemmal vesicles from young spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) were compared. The effects of the calmodulin and the calcium antagonist nifedipine on Ca2+ binding ATP-dependent accumulation of Ca2+ were studied at free Ca2+ concentrations of 2.10(-8)M and 4.10(-7)M. 2.10(-7)M calmodulin stimulated Ca2+ binding to SHR membranes up to a level equivalent to that in WKY, whereas it enhanced active Ca2+ transport more in WKY than in SHR, thus suppressing the difference between the two substrains. At a 2.10(-8)M free Ca2+ concentration low concentrations of nifedipine (10(-7) to 10(-6)M) induced an increases in ATP-dependent Ca2+ transport by SHR vesicles. Inhibition of NA+, K+-adenosine triphosphatase activity by ouabain was also studied. Na+, K+ATPase activity in SHR membranes was double that in membranes from WKY (22.1 +/- 2.8 v.s. 11.3 +/- 1.1. mumole Pi/h/mg protein). These differences, observed on 3 week-old rats, before a significant rise blood pressure, may reflect genetic characteristics of these hypertensive-prone rats.

The effects of chronic sodium load on the activity of a circulating inhibitor of the Na+, K+-pump were measured in male Wistar rats. The inhibition by deproteinized plasma of renal Na+, K+-ATPase was higher in rats with high Na+ intake than in control animals (53.4 +/- 2.0 mumoles Pi.h-1 X mg-1 vs 66.6 +/- 2.6 mumoles Pi.h-1 X mg-1, p less than 0.01). The simultaneous rise in systolic blood pressure and the decrease in the hematocrit suggest that blood pressure, volemic expansion and the activity of this inhibitor may be somehow linked.

The presence in human plasma of compounds able to interact with the sodium pump has been investigated. The effects of whole and deproteinized plasma on the parameters of ouabain binding to the digitalis sites of the sodium pump were compared. Changes in the apparent affinity were potentialized by deproteinization. High levels of ouabain-like activity were observed to be present not only in some hypertensive patients but also in normotensive patients with chronic renal failure and active acromegaly, indicating a probable relationship with positive sodium balance and volemic expansion.

1. The clinical applications of a simple laboratory test for the diagnosis of essential hypertension were further investigated on a large number of patients (124 individuals). 2. The reduced erythrocyte ratio of Na+/K+ net fluxes observed in essential hypertensives might be secondary to a functional defect in a Na+-K+ cotransport system. 3. A similar reduction in the ratio of Na+/K+ net fluxes was also observed in erythrocytes of some normotensive subjects born of hypertensive parents and of genetically hypertensive rats. This suggests that the erythrocyte membrane defect follows the genetic transmission of hypertension.

Much experimental and clinical evidence points to the fact that increased intracellular Na+ concentration could play an important pathogenic role in hypertension, especially where cells of excitable tissues are concerned. Active extrusion of Na+ (from the cells to the extracellular space) depends mainly on the activity of the system of membrane transport known as the Na+ pump. The extrusion of Na+ occurs in exchange for K+ and the activity of the pump depends on the enzyme Na+-K+-ATPase. The Na+ pump is inhibited by cardiotonic glycosides such as digitalis and ouabain . The results obtained in our laboratory suggest that the activity of the Na+ pump is controlled by an endogenic system acting in a similar manner to ouabain and digitalis. Experimental and clinical studies show: -- that the administration of Na+ increases the inhibitor effect both in urine and plasma; -- that the inhibitor is increased in about 50 p. 100 of patients with essential hypertension; -- that this increase seems to depend on familial or genetic factors. The biochemical identification of this endogenic inhibitor is now under way; it has a small molecular weight (less than 3000), is thermostable and anionic. This factor could have both a regulating role on Na+ turnover and a pathological role in hypertension. Its activity comes into competition cations also capable of inhibiting the Na+ pump, including K+.

A heat-stable, low molecular weight, anionic substance(s) capable of inhibiting 3H-ouabain binding and Na+-K+-ATPase activity could be extracted from human urine and plasma. The level of the inhibitor was elevated in 40%-50% of essential hypertensives, compared to controls, and also in some of the offspring of hypertensive parents. Higher levels of the inhibitor were measured in patients treated with beta-blocking agents than in those treated with diuretics. The inhibitor extracted from plasma also appeared capable of (1) inhibiting the uptake of serotonin in human platelets, an Na+-dependent mechanism, and (2) inducing an increase in blood pressure when injected intracerebroventricularly. From these various data, we propose that the increase in the endogenous inhibitor may play a role in essential hypertension and may modulate, at least partially, some of the various cell functions that depend on a transmembrane Na+ gradient, including cellular excitability.

Angiotensin II receptors from rat adrenal cortex and myometrium were studied with the use of tritiated angiotensin under conditions where the sensitivity of the target organs for angiotensin II is modified. Sodium status was found to modulate the number of angiotensin receptors both in adrenal gland and uterus. In both target tissues low Na+ diet increases the number of receptors, while a high Na+ diet results in an increase in uterine receptors without modifying adrenal cortical receptors. However, a more markedly positive sodium balance, such as that observed in deoxycorticosterone acetate (DOCA) hypertension and in one-kidney Goldblatt hypertension, resulted in a reduction of the adrenocortical angiotensin II binding capacity. The endogenous angiotensin II level may also regulate the number of receptor sites as demonstrated by an increased number of receptors after suppression of circulating angiotensin II. It is proposed that the number of angiotensin II receptors is determined by the combined influences of sodium status and angiotensin II concentration. Some changes in the sensitivity of the target organ can be secondary to variations in the number of angiotensin receptors. However, others cannot be so explained and stem, therefore, from events occurring beyond the hormone-receptor interaction.

Recent studies in essential hypertensive patients and rats with genetic hypertension strongly suggested that the development of primary hypertension results from a transient and chronic "cascade" of events; I) excess Na+ intake, II) secretion of natriuretic factors, III) abnormal cell Na+ homeostasis in the vascular wall, due to the presence of inherited and induced abnormalities in different Na+ transport system and IV) increase in cytosolic free Ca2+ content and sympathetic drive. In vitro studies have previously shown that canrenone, an antihypertensive antialdosterone drug, behaves like a partial agonist at the digitalis-receptor site of the Na+, K+-pump. In particular, it has been shown that canrenone counterbalances the increases in internal Na+ and cytosolic free Ca2+ contents induced by ouabain in cultured smooth muscle cells. We thus investigated the effect of canrenone administration in a model of experimental hypertension with increased endogenous "ouabain-like" factors (rats with reduced renal mass under excess Na+ intake: RRM-salt rats). Results presented here confirm that RRM-salt rats exhibit: volume expansion, strongly decreased plasma renin activity, increased endogenous "ouabain-like" factors and (IV) decreased Na+, K+-pump activity and increased Na+ content in erythrocytes. In addition, we found that canrenone is antihypertensive in this model and this is associated with a tendency to normalize volume expansion, plasma levels of endogenous "ouabain-like" factors, Na+, K+-pump activity and Na+ content in erythrocytes. In conclusion, our results suggest that administration of canrenone to RRM-salt rats may induce a lowering of blood pressure by antagonism with endogenous "ouabain-like" factors at the vascular wall.