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The electrochemical behaviour of Sb(III) and Sb(V) in water–HF mixtures (2.5 to 50% HF) and in anhydrous HF is described. A Teflon capillary dropping mercury electrode is proposed for polarographie investigations in these fluorinated media. It is shown, by classical or puise polarography, that Sb(III) can be reduced to Sb(0), as in acidic non-fluorinated media. The electrochemical reaction becomes reversible by addition of Cl−, Br−or I−. Sb(V) is not electroactive in the various HF-media, except when it is introduced as the SbCl5. In this case, the two polarographie waves of Sb(V) disappear with time because of SbF6−formation. Analytical applications to the determination of Sb(III) in water–HF and in anhydrous HF are discussed.

The variation of the function R0 (H) in water-hydrogen fluoride mixtures was determined on the basis of measurements of the hydrogen electrode potential versus the ferrocene-ferricinium system.

A decrease in platelet 5-HT content linked to partial inhibition of 5-HT uptake has been described in essential hypertension. Transport of 5-HT through platelet membrane is dependent upon transmembranal Naand Kgradients. It is inhibited by Na,K-ATPase inhibitors such as ouabain and endogenous digitalis-like compounds isolated from hemodiafiltrate. The activity of such compounds in plasma extracts, measured by inhibition of Na,K-ATPase or ouabain binding to human erythrocytes, and platelet 5-HT content were determined in parallel in essential hypertensive patients. Significant negative correlations were observed between these parameters in men, suggesting that high levels of digitalis-like compounds can affect platelet 5-HT content.

Cytosolic free calcium concentration Ca2was studied in platelets of hypertensive patients with the use of the fluorescent indicator Quin 2AM. Cytosolic free Ca2was significantly higher in platelets of hypertensive patients than in those of normotensive subjects (241 ± 9 versus 192 7 nmoll, n 58 and 57, respectively P< 0.001). When all 115 subjects were included, there was a significant correlation between cytosolic free Ca2and systolic or diastolic blood pressure (r 0.262, P< 0.0025 and r 0.251, P< 0.0025, respectively). Intracellular Quin 2 concentration was measured to evaluate the formaldehyde production (a product of Quin 2/AM hydrolysis which has been described as reducing the adenosine triphosphate (ATP) production). The Quin 2 concentrations in platelets of the two groups of subjects were observed to be similar (0.41 ± 0.03 versus 0.38 ± 0.03 mmoll, n 8 and 7 for hypertensives and normotensives, respectively). The effects of prostaglandin E1(PGE1), an adenylate cyclase stimulator, on cytosolic free Ca2were studied. The presence of 10-7mol/l PGE1lowered the Ca2in platelets of hypertensive patients only, suppressing the difference between the two groups.

To study the relationship between high blood pressure and hyperlipidaemia and the cytosolic calcium concentration in unstimulated platelets, focusing on the effects of an alteration in membrane dynamics.

To study membrane viscosity in various rat strains with genetic forms of experimental hypertension.

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Evidence exists which demonstrates the relationship between a Natriuretic Factor or Na+K+-ATPase inhibitor and volemic expansion, both in man and animal. Patients having extracellular volume expansion have been studied for the effect of their plasma on erythrocytes 3H-ouabain binding. High levels of ouabain-like activity was found in plasma from acromegalic patients and patients with chronic renal failure. High levels were also observed in some hypertensive patients. A partial purification of such a compound was performed from urine of hypertensives. The partially purified compound inhibited to a greater extent the Na+K+-ATPase semipurified from dog kidney than that from sheep brain. The present data are consistent with the possible regulation of the activity or the secretion of plasma ouabain-like activity by extracellular volume.

Calcium binding properties of plasma membrane enriched fractions from various tissues were studied in young spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). In all tissues tested (heart, liver, nerve endings, erythrocytes), high affinity calcium binding sites were characterized. Their KD values were in the range of the cytosolic free calcium concentration : (10-8-10-7). Whatever the tissue, plasma membrane enriched fraction from SHR exhibited a reduced calcium binding capacity but no significant change in affinity. This decrease which averaged 15-25% was also observed in the presence of the calcium ionophore A23187 and thus does not result from a modified accessiblity of calcium to its binding sites. It is suggested that this abnormality may be of genetic origin and is possibly implicated in the pathogenesis of hypertension.

Distinct differences in central and peripheral noradrenaline (NA) were observed in the hypertension prone (SBH) and resistant (SBN) strain, derived from the Hebrew University SABRA rats. In the medulla oblongata NA concentration was 90% higher and tyrosine hydroxylase activity 88% lower in SBN when compared to SBH, suggesting marked strain differences in NA turnover. In this area, NA-induced cAMP generation was higher in SBH than in SBN, while in the hypothalamus, the reverse situation was present. The relevance to hypertension of the reciprocal cAMP changes is still uncertain. The concentration of NA in heart tissue was significantly higher in SBN than in SBH. Doca-salt treatment caused hypertension and depletion of atrial NA in SBH, but had no effect on either blood pressure or atrial NA in SBN rats. The results suggest that resistance to hypertension in SBN rats is associated with decreased NA turnover in medulla oblongata and reduced activity of cardiac neuronal sympathetic endings.

Our previous observation of a greater increase in erythrocyte Na+ in SHR than in WKY after an acute Na+ load may result either from a genetic membrane property or from a specific plasma influence. In order to elucidate this question, membrane characteristics were compared with or without an acute Na+ load. Na+ transport was measured in Ringer and in plasma on Na+ enriched and K+ depleted red cells. Platelet microviscosity was measured as an index of membrane structural changes. After acute Na+ load a similar reduction of net Na+ extrusion and of K+ influx was observed in both strains. This indicates an inhibition of the Na+,K +-pump. Platelet microviscosity was similarly increased in SHR and WKY. Thus an acute Na+ load induced alterations of membrane properties in both SHR and WKY. The higher erythrocyte Na+ content in SHR stems rather from their intrinsic membrane properties than from a plasma factor.

The catecholamine concentration and dopamine-β-hydroxylase activity were determined in several nuclei of the brain of spontaneously hypertensive rats (SHR) compared with Wistar Kyoto (WKY) controls. Catecholamines were measured by using liquid chromato-graphy coupled with electrochemical detection. The threshold of detection was 5 × 10-14 mole. Dopamine-β-hydroxylase (DBH) was assayed by a sensitive radioenzymatic assay using tyramine as the substrate. The limit of detection was 5 pmoles of octopamine per sample.Significantly lower noradrenaline content was observed in 4 week-old SHR in some medullary and hypothalamic areas which are involved in cardiovascular regulation. This abnormality was no longer detectable in 12 week-old rats. The changes in catecholamine levels observed in young rats were not observed during the development of deoxycorticosterone-salt hypertension and therefore probably do not represent a compensatory mechanism tending to limit the progressive rise blood pressure. No significant difference in DBH activity was observed between the young SHR and WKY in any brain region measured. The altered relationship between noradrenaline content and DBH activity observed in young SHR when compared to WKY suggests a change in noradrenergic neu-rones activity and/or structure which could correspond to a genetically transmitted neurochemical abnormality associated with the intitiation of hypertension in the SHR

To define better the relationships between lipid metabolism disturbances and platelet aggregation we have examined these parameters in hereditary hypertriglyceridemic and control Lewis rats. Hereditary hypertriglyceridemic rats are hypertensive and have high plasma triglycerides but not elevated plasma total cholesterol. In the present study, we have demonstrated that platelets from hereditary hypertriglyceridemic rats have lowered initial rate and maximal aggregation after stimulation with thrombin or ADP in comparison with controls. These two strains did not differ significantly in the inhibition of platelet aggregation by the thromboxane A2receptor inhibitor, SQ 29 548. In hereditary hypertriglyceridemic rats, the thrombin response, as well as the contribution of the thromboxane A2-sensitive pathway, were positively associated with the plasma level of triglycerides. Similar trend was found in Lewis rats. However, the slopes of these relationships were reduced in hereditary hypertriglyceridemic rats. These alterations of the aggregatory responses in hereditary hypertriglyceridemic rats were independent of blood pressure and plasma cholesterol level. In conclusion, our results showed a clear-cut platelet hypoaggregability to both thrombin and ADP in hypertensive hypertriglyceridemic rats. This hypoaggregability was not due to an impaired function of the thromboxane A2pathway but could be connected with disturbances of lipid metabolism.

Platelet cytosolic free calcium concentration ([Ca2+]i) and intracellular pH (pHi) (including their responses to thrombin), as well as erythrocyte [Ca2+]i and 45Ca2+ influx, were studied in Lyon hypertensive (LH) and normotensive (LN) rats aged 3 months. Platelets of LH rats were characterized by substantially elevated basal [Ca2+]i values, higher [Ca2+]i levels after thrombin stimulation, and enhanced initial rate of thrombin-induced Mn2+ entry through receptor-operated Ca2+ channels. Basal platelet pHi values were not significantly different in LH and LN animals but thrombin elicited a significant alkalinization only in LH platelets. Erythrocytes of LH rats had an enhanced initial rate of 45Ca2+ and tended to elevated [Ca2+]i levels. Our data indicate profound alterations in cell Ca2+ handling in platelets and erythrocytes of LH rats, similar to those previously described in spontaneously hypertensive rats of the Okamoto-Aoki strain. The analysis of the relations between blood pressure, plasma lipids, and cell Ca2+ handling suggested that triglycerides, but not cholesterol, might be involved in altered platelet Ca2+ handling in LH rats.

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.

1. The metabolism of blood platelets, taken as an accessible model of excitable cells, has been reported to be altered in hypertension. Most of the identified alterations concern the functions of various plasma membrane constituents. 2. A possible modification of membrane microviscosity was investigated by 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene fluorescence depolarization. In order to determine whether or not the membrane structures probed by these indicators were related to platelet physiological functions, the cytosolic free Ca2+ concentration was determined in parallel. 3. At physiological temperature, the fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene was decreased in untreated hypertensive patients (0.276 ± 0.002 versus 0.288 ± 0.002, n = 23 and 22, P < 0.001), indicating a lowered microviscosity at the lipid-water interface of cell membrane. It correlated inversely with blood pressure (P < 0.001) and cytosolic free Ca2+ concentration (P < 0.030). On the contrary, 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy was observed to vary with sex but not with blood pressure. 4. These results suggest that structural membrane modifications may participate in the various functional abnormalities observed in platelets from hypertensive patients.

1. Blood cells and vascular endothelial cells are subjected to a wide range of haemodynamically generated shear stress forces. In vitro, membrane stretching or shear stress have been observed to activate ion channels and cell metabolism and to facilitate erythrocyte and platelet aggregation. 2. The present study was designed to evaluate the participation of shear stresses in the control of apparent platelet cytosolic free Ca2+ concentration in hypertensive patients. 3. Shear conditions and platelet cytosolic free Ca2+ concentration in vitro were studied after a dynamic perturbation induced by 3 months of double-blind treatment with one of two β-antagonists, carteolol and atenolol. Brachial artery wall shear rate and stress were estimated by means of a pulsed Doppler velocimeter, and blood viscosity was measured by a co-axial viscometer at a shear rate of 96 s−1. Platelet cytosolic free Ca2+ concentration was simultaneously measured by using the Quin-2 fluorescent chelator. The direct effect of atenolol and carteolol on platelet cytosolic free Ca2+ concentration in vitro was also measured after addition of the β-blockers to plateletrich plasma. 4. Atenolol and carteolol decreased blood pressure similarly but their effects on shear rate (P < 0.02), shear stress (P < 0.01) and platelet cytosolic free Ca2+ concentration (P < 0.05) differed after 3 months of therapy. In contrast, neither of the drugs significantly altered platelet cytosolic free Ca2+ concentration, in vitro per se. 5. In the overall population, strong positive correlations existed not only between changes in platelet cytosolic free Ca2+ concentration and those in shear rate (r = 0.81, P < 0.001) and shear stress (r = 0.83, P < 0.001), but also between their absolute values, suggesting a possible haemodynamic shear-dependent modulation of transmembrane Ca2+ transport.

The effect of canrenone, an antialdosterone and partial ouabain-agonist drug, was studied in rats that developed volume expansion and hypertension after renal mass reduction and excess Naintake (RRM-salt). The RRM-salt was characterized by (1) increased endogenous “digitalis-like” compounds in plasma cross reactivity with digoxin-antibodies (57.5 ± 5.0 vs. 42.1 ± 3.8 pg/ml, p < 0.02); inhibition of kidney Na,K-ATPase activity (135 ± 5 vs. 154 ± 5 μmol/mg/h, p < 0.01); and inhibition of Naextrusion from normal erythrocytes (5.96 ± 0.40 vs. 7.68 ± 0.34 mmol/L cells/h, p < 0.01); (2) reduced Na,K-pump activity (7.34 ± 0.29 vs. 10.88 ± 0.41 mmol/L cells/h, p < 0.001) and increased Nacontent (4.66 ± .08 vs. 4.16 ± 0.11 mmol/L cells, p < 0.01) in erythrocytes; and (3) low plasma renin activity (2.1 ± 0.9 vs. 12.6 ± 1.6 ng/ml/h). Ninety minutes after the administration to RRM-salt of a single oral dose of 60 mg/kg of canrenone, the systolic blood pressure decreased by 36 ± 4 mm Hg (mean ± SEM). Chronic canrenone administration (60 mg/kg/day) resulted in a marked antihypertensive effect associated to a correction of volume expansion, a decrease in endogenous “digitalis- like” compounds, and a partial recovery of Na,K-pump activity and Nacontent in erythrocytes. Our results suggest that the antihypertensive effect in RRM-salt rats results, at least in part, from antagonism with endogenous “digitalis-like” compounds.

The reduction by nitric oxide donors of Ca2+mobilization in stimulated platelets lead us to investigate the direct effect of authentic NO on ATP-dependent Ca2+uptake into platelet membrane vesicles. The effects of NO were compared to those of thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone, two specific inhibitors of the sarco/endoplasmic reticulum Ca2+-ATPases. All three compounds modulated the initial rate of ATP-dependent Ca2+uptake. NO effects on the initial rate of active Ca2+uptake were biphasic, with an inhibition above 10 nmol/L and a stimulation below this concentration. These effects could not be attributed to cGMP, its usual effector molecule, or to nitrite ions, its metabolic product. NO inhibitory effects were decreased after a five min incubation, indicating that they were due to a short-lived compound and reversible. These results suggest that NO is functionally coupled to SERCA pumps of the dense tubular system through a cGMP-independent mechanism.

Catalytic performance of Na₂CO₃-K₂CO₃ and Li₂CO₃-K2CO₃ immobilized on lithium aluminate towards the oxidative dimerization of methane was investigated at 750, 800 and 850°C. CH₄ conversion of 15-16%, C₂ yield of 9-10%, and C₂ selectivity if 58-62% was obtained at 800°C. Catalytic activity increased with temperature and melt decarbonation, which can be correlated with a stabilization of peroxide species. Comparison between these melts and with Li₂CO₃-Na₂CO₃ shows that a higher amount of C₂ hydrocarbons, formed per second and per gram of carbonate, was obtained in Li₂CO₃-K₂CO₃.Copyright 1995, 1999 Academic Press

The kinetic properties of the Na +/H +exchanger in human platelets were investigated by direct measurements of pH ias detected with the fluorescent dye, BCECF. In acid-loaded cells, the antiporter displayed a hyperbolic dependence regarding external Na +with an apparent Kmof 38 ± 4 mM (pH o7.2 at 25°C) whereas its pH i-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 +> Li +> 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.

Various abnormalities in platelet metabolism, including increased sensitivity to several aggregating agents, have been described in essential hypertension. Platelet response is controlled by Ca2and cyclic AMP-dependent mechanisms (stimulatory and inhibitory, respectively) which oppose one another. In the present study, the cyclic AMP contents of unstimulated platelets were measured by radio-immunoassay and observed to be lower in hypertensive than in normotensive subjects, either in the basal state or after prostaglandin E1 (PGE1) stimulation. In the presence of 7-bromo-1,5,dihydro-3,6-dimethylimidazo 2,1-b quinazolin-2(3H)-one (Ro 15-2041), a specific inhibitor of phosphodiesterase, the increases in cyclic AMP content were similar in platelets from both groups, indicating that this enzyme was not responsible for the alterations in cyclic AMP metabolism observed in hypertension. Low external Ca2reduced basal and PGE1-stimulated cyclic AMP content in both normotensive and hypertensive groups but cyclic AMP levels remained lower in hypertensive patients than in normotensive subjects, indicating that Ca2influx is not responsible for this altered metabolism of cyclic AMP in hypertension. These data suggest that the reduced platelet cAMP content may participate in the hyperreactivity to various aggregating agents previously reported to accompany essential hypertension.

Alterations in the metabolism of intracellular messengers, such as calcium and cyclic adenosine 5'-phosphate (cAMP), have been reported in essential hypertension. Since intracellular pH (pHi) participates in the control of fundamental cell functions, we looked for changes in platelet cytosolic Hconcentration (H i) in hypertension and investigated whether or not its impaired metabolism is linked to the calcium handling abnormalities. The fluorescent pH indicator BCECF has been used to evaluate intracellular Hconcentration in platelets, unstimulated ex vivo, from normotensive (n20) and hypertensive patients (n20). Cytosolic Hwas 20 lower in hypertensive than in normotensive subjects (49.5 ± 3.4 and 61.8 ± 2.2nmol/l cells, respectively, P<0.005; mean pH, values were 7.21 and 7.33, respectively). Platelet cytosolic Hand free Ca2concentrations (Ca2i) were determined in parallel in 15 normotensive and 15 hypertensive patients. Ca2iwas found to be 19 higher (P< 0.01), and H i22 lower (P< 0.02), in the hypertensive patients compared with the normotensive subjects. Platelet pHiand Ca2 iwere increased simultaneously in some hypertensive patients. These results are compatible with the hypothesis of an in vivo activation of platelets in hypertension. If a similar alkalinization exists in smooth muscle cells, it may participate in cell proliferation and in an enhanced sensitivity to agonists, two parameters thought to be involved in blood pressure elevation.

The effects.of a high salt diet (8 NaCI) on blood pressure and intra-erythrocytic Nacontent 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.

Cytosolic free Ca2and serotonin (5-HT) content were measured in platelets from 22 untreated hypertensive patients and 16 normotensive subjects. In hypertensive patients, cytosolic free Ca2was significantly higher (239 ± 13 nmol/l versus 186 7 nmol/l, n22 and 16, P<0.01) and 5-HT content was significantly lower than those measured in cells from control subjects (3.22 ± 0.26 versus 4.99 ± 0.38 x 10-7mol/1011cells, n22 and 16, P<0.001). These two parameters were closely correlated (r-0.565, n38, P<0.001). These two concomitant changes in platelet characteristics might result from a common cause, such as cell membrane alterations. As ritanserine, a specific 5-HT2 receptor antagonist, did not modify the cytosolic free Ca2, a higher stimulation of 5-HT2receptors is not likely to be responsible for the enhanced free Ca2levels.

In stimulated platelets, endothelin-3 (ET-3) has been previously shown to attenuate Ca2+mobilization. Using the calcium indicator chlortetracycline, the present study demonstrates that 0.5 μM ET-3 produced a 24% reduction in the Ca2+pool mobilized by A23187. ET-3 up to 1 μM dose-dependently decreased the initial velocity and steady state value of 45Ca2+-uptake into platelet membrane vesicules (p<0.001). In addition, ET-3 partially reversed the inhibitory effects of half maximally effective concentrations of thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone, two specific inhibitors of the sarco/endoplasmic reticulum Ca2+-ATPases. These results suggest that ET-3 is functionally coupled to Ca2+-pumps of the dense tubular system. Based on these findings, we propose that ET-3 decreases the activity of Ca2+-pumps in the dense tubular system which accumulates less Ca2+, leading to lowered Ca2+release in response to agonists.

1. Calmodulin and calcium effects on cardiac ouabain-sensitive adenosine triphosphatase (ATPase) activity were studied in young spontaneously hypertensive rats (SHR) and in their normotensive control Wistar—Kyoto rats (WKY). 2. Cardiac sarcolemmal membranes from SHR showed significantly higher ouabain-sensitive ATPase activity than membranes from WKY rats. This activity was unaffected by calmodulin or calcium alone. 3. However, when both calmodulin and calcium were added, ouabain-sensitive activity was significantly reduced without changes in the total ATPase activity. 4. The calcium-dependent calmodulin effect was dose-dependent and greater in SHR than in WKY membranes. 5. An altered interaction between the calcium-calmodulin system and sodium handling by the plasma membrane in SHR may play a role in the pathogenesis of hypertension.

1. Fluorescence Dolarization of dbhenvlhexa-triene embedded in membranes was used as an index of ‘microviscosity’ in platelets and ervthro—cyte ghosts of spontaneously hypertensive rats of the Okamoto-Aoki strain (SHR), Wistar-Kyoto strain (WKY) and of the hypertension-prone and -resistant Sabra strains (SBH and SBN), and the original Sabra strain (SB). 2. Microviscosity was increased both in erythrocyte ghosts and platelet membranes of male but not female SHR rats compared with WKY rats and in hypertension-prone Sabra rats compared with the original Sabra rats. 3. Acute and chronic salt loading increased the microviscosity of platelet membranes in all strains of rats but had no effect on the erythrocyte membranes. 4. Microviscosities of vesicles made of lipids extracted from SHR and WKY erythrocyte ghosts were similar. This supports the hypothesis that membrane proteins play a major role in the differences in microviscosity observed in SHR rats.

The oxidative dimerization of methane was investigated at 750–850°C in Li2CO3-Na2CO3-K2CO3 immobilized within LiAlO2 or Li2TiO3 supports. Catalytic performance was enhanced with moderate melt decarbonation (i.e. with molten phase/LiAlO2 at 850°C: CH4 conversion of 25% and C2 yield of 12.5%), then dramatically fell with the precipitation of sodium and lithium oxide. The effect of the partial pressure of CO2 was analyzed. As in the case of binary carbonate eutectics, catalytic activity of the ternary melt was correlated with the presence of peroxide species. This activity was more important when using LiA1O2 support.

Inhibitors of the Na+ pump have been proposed as participating in sodium excretion, xtracellular fluid regulation, and in the rise of blood pressure.The presence of digitalis-like compounds in human plasma has been investigated by comparing the effects of plasma extracts to those of ouabain in 4 tests.-competition with ouabain for binding to the Na+ pump-inhibition of Na+ and K+ dependent hydrolysis-inhibition of serotonin uptake by human platelets-central hypertensive effectPlasma fractions exhibited digitalis-like properties in the 4 tests. The effects of plasma extracts of 42 normotensive subjects (21 with family history of hypertension) and 38 patients with essential hypertension (15 with antihypertensive treatment) and 9 patients with chronic renal failure were compared.Plasma from Forty per cent of untreated hypertensive patients and normotensives with hypertensive heredity had a high inhibition level. Inhibition was enhanced in beta-blocker treated patients and decreased in those on diuretics.No digitalis-like activity was observed in uremic plasma.These observations strongly suggest the presence of digitalis-like compound(s) in human plasma and point to its possible association with hypertension.

The specific binding of angiotensin II and des-Asp1-angiotensin II ("angiotensin III") III") to rat adrenals was studied with the use of the tritiated peptides. The binding sites having maximal affinity for angiotensin II were characterized by an equilibrium dissociation constant of 3.3 to 5.2 X 10(-9) M. Angiotensin III was able to interact with these sites, and also with a class of sites with very high affinity, characterized by an equilibrium dissociation constant of 1 to 2 X 10(-10) M. These sites exhibited a greater affinity for the heptapeptide angiotensin III than for the octapeptide angiotensin II. These findings, together with the known potent aldosterone stimulating effect of angiotensin III and its presence in rat plasma, suggest that this heptapeptide could be the physiologically important steroidogenic angiotensin in this species.

The causal relationships between cytosolic free-Ca2+ concentration ([Ca2+]i) increases and production of nitric oxide (NO) have been investigated mostly with indirect methods and remain unclear. Here we demonstrate, by direct real-time measurements of [NO] with a porphyrinic microsensor, that Ca2+ entry, but not an increase in [Ca2+]i, is required for triggering of NO production in human endothelial cells. Histamine, ranging from 0.1 to 100 μM, increased both NO production and [Ca2+]i when given in a single dose. However, histamine caused increased NO release but induced progressively smaller [Ca2+]i changes when cumulatively added. In the absence of a transmembrane Ca2+ gradient, no significant NO release was detectable, despite the marked Ca2+ peak induced by histamine. Inhibition of Ca2+ entry by SK&F 96365 abolished histamine-elicited NO production but only reduced the transient [Ca2+]i rise. The suppression of the sustained [Ca2+]i response under these two conditions suggests that NO release was closely associated with Ca2+ entry from the extracellular space. In addition, membrane depolarization, achieved by increasing the extracellular K+ concentration from 5 to 130 mM, reduced both the amplitude of histamine-induced sustained [Ca2+]i elevation and NO production. These results lead us to propose that the availability of numerous Ca2+ ions around the internal side of the plasma membrane would promote the association between nitric oxide synthase and calmodulin, thereby activating the enzyme.

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 Nadiet increases the number of receptors, while a high Nadiet 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.

Sodium pumps of cardiac plasma membranes were studied in young, spontaneously hypertensive rats (SHR) and in their normotensive controls (Wistar-Kyoto; WKY) using the two following methods. The enzymatic activity and its sensitivity to ouabain were measured as the Na+, K +-dependent ATP hydrolysis, and the number of pumps was estimated by [3H]ouabain binding. The main results of this study were the observations that (a) concentrations of ouabain as low as 10-10Minhibited 10–15 of the enzyme activity in both strains; (b) Na +, K +-adenosine triphosphatase (ATPase) activity in membranes from SHR was double that in membranes from WKY (16.5 ± 3.2 μmol Pi/h/mg protein vs. 8.2 ± 1.2 μmol Pi/h/mg protein for 10-7Mouabain; p < 0.01); (c) sensitivity to three different cardiac glycosides, ouabain, digoxin, and digitoxigenin, was identical in SHR and WKY vesicles; and (d) the binding capacity of [3H]ouabain was significantly higher in SHR than in WKY vesicles, but the dissociation constant (KD)did not appear to differ between the two substrains. These studies, performed on 3-week-old rats before the appearance of hypertension, showed, on the one hand, the existence of a Na +, K +-ATPase of very high affinity in the rat heart, and, on the other, that cardiac sarcolemmal membranes from SHR had a greater number of sodium pumps than those from WKY and thus a greater ability to extrude sodium.

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Circulating digitalis-like compounds have been proposed to be raised in volume expanded hypertension and to participate in Na+ homeostasis. We have investigated the temporal relationships between the activity of these circulating digitalis-like compounds, blood pressure and body fluid volume variations during a chronic NaCl load in the Wistar rat.Characteristics of salt-loaded rats were compared to those of weight-matched controls. At one week, when extracellular fluid volume (ECFV) was elevated, the capacity of plasma extracts to inhibit the Na+K+ATPase activity begun to rise. At two weeks, ECFV remained elevated, and plasma volume, blood pressure and the activity of plasma digitalis-like compounds increased. After 13 weeks, the continuous rise in plasma digitalis-like activity and in blood pressure was accompanied by the return of body fluid volumes towards control values. These changes in plasma digitalis-like activity, body fluid volumes, and systolic blood pressure during a high NaCl diet are compatible with the proposed role of circulating digitalis-like compounds as natriuretic and hypertensive factors.

Binding of natural antibodies to endothelial cell plays an important role in hyperacute xenograft rejection between discordant species. Human intravenous immunoglobulins (IVIg) delay this hyperacute rejection, but their mechanisms of action on endothelial cells have to be defined. Here we demonstrate that IVIg dose-dependently prevent thrombin from eliciting cytosolic Ca2+ movements and nitric oxide (NO) production in aortic endothelial cells from guinea pig. The Ca2+ response to thrombin was similarly affected by IVIg whether they were removed or not from the incubation medium before stimulation. Pretreatment by rat natural antibodies also suppress the thrombin-induced Ca2+ peak corresponding to Ca2+ release from intracellular stores but stimulate the subsequent sustained increase in [Ca2+]i and the release of NO. The action of human intravenous immunoglobulins seems to be selective for the thrombin receptor because they do not affect [Ca2+]i and NO responses to endothelin-1 or thapsigargin. However, these antibodies also suppress the first phase of the cytosolic Ca2+ response to ATP, which does not release NO under our experimental conditions. These observations raise the possibility that IVIg selectively interact with targets localized on plasma membrane of endothelial cells for controlling receptor-activated Ca2+ pathways and NO release.