Eicosapentaenoic acid reduces thrombin-evoked release of endothelin-1 in cultured bovine endothelial cells.

Endothelial cells were isolated from bovine thoracic aorta and cultured. Bovine aortic endothelial cells (BAEC) were incubated with radiolabeled arachidonic acid (3H-AA) or eicosapentaenoic acid (14C-EPA) (1 microM) for 3 hr. Both fatty acids were predominantly incorporated into phosphatidylcholine (57 +/- 2% and 62 +/- 2% respectively) and slightly into phosphatidylethanolamine (11 +/- 0.5% and 12 +/- 0.6% respectively). phosphatidylinositol (26 +/- 1.5% and 10 +/- 0.5% respectively) and neutral lipids (6 +/- 0.5% and 15 +/- 1% respectively). After BAEC incubation with 3H-AA for 24 hr with or without EPA (1 microM), the release of radioactive metabolites of AA induced by thrombin (5.5 U/ml) was strongly reduced by the preliminary treatment with EPA (72 +/- 5%). After BAEC incubation with AA, EPA or vehicle (control), endothelin-1 levels were measured by RIA in the culture medium and we observed that: 1) the basal production of endothelin-1 was not modified after either AA or EPA treatment, 2) the thrombin-evoked release of endothelin-1 was significantly reduced by EPA (5.8 +/- 0.82 and 3.8 +/- 0.50 pg/microg proteins in control and EPA-treated cells, respectively); 3) by contrast, AA had no significant effect on the thrombin-evoked release of endothelin-1. In conclusion, EPA reduces strongly the endothelin-1 release but AA is ineffective. This reduction of endothelin-1 release may account partly for some of the vascular effects of EPA.

The elastic modulus of conductance coronary arteries from spontaneously hypertensive rats is increased.

OBJECTIVE: To assess the alterations of morphological and functional properties of conductance coronary and mesenteric resistance arteries in spontaneously hypertensive rats (SHR). DESIGN: The in-vitro intrinsic elastic properties of the wall material in SHR coronary arteries were determined in comparison with those of Wistar-Kyoto (WKY) rats. Mesenteric resistance arteries from rats of both strains were also studied. METHODS: Arterial segments were cannulated at both ends using an arteriograph system and subjected to pressure increments with simultaneous measurements of the wall thickness and internal diameter. The strain, stress and incremental elastic modulus (Einc) were calculated from diameter-pressure curves. RESULTS: Over the full range of pressures tested (10-160 mmHg), the internal diameters of SHR coronary arteries were not significantly different from those of WKY rat arteries, whereas we observed that SHR mesenteric resistance arteries had a significantly smaller diameter. The stress-strain curve for coronary arteries was shifted significantly to the left-hand side for the SHR group indicating more stress per unit strain, whereas the opposite was found for mesenteric resistance arteries. When Einc was determined under isobaric conditions, we found no difference between SHR and WKY rat coronary arteries, whereas this parameter was decreased significantly for SHR mesenteric resistance arteries. When Einc was estimated at the respective operating pressures, it was 1.7- to 2.8-fold greater for SHR than it was for WKY rat mesenteric resistance and coronary arteries. Moreover, the total collagen area: lumen area ratio was significantly greater for the SHR than it was for the WKY rat coronary artery wall, but this ratio was similar for mesenteric preparations from the two strains. CONCLUSION: These results show that, at a given stress or operating pressure level, the material of SHR coronary artery wall is characterized by an increase in Einc, whereas there is no increase in Einc for in mesenteric resistance arteries. This functional alteration is accompanied by an increase in the relative proportion of collagen, a component with a high elastic modulus, in the wall. In contrast, we found no change in elastic modulus and in the relative proportion of collagen for the SHR mesenteric resistance arteries. Furthermore, the present results support the hypothesis that alterations in distensibility differ among the components of the SHR vasculature.

Reduced basal NO-mediated dilation and decreased endothelial NO-synthase expression in coronary vessels of spontaneously hypertensive rats.

Basal vasomotor tone in coronary vessels is, in part, maintained by nitric oxide (NO) production by endothelial constitutive NO synthase (ecNOS). Alteration of coronary circulation observed in left ventricular hypertrophy secondary to hypertension could be associated with a decrease in NO production. The aim of this study was to measure: (1) coronary flow in the Langendorff-perfused heart model at baseline, after maximum vasodilation in response to adenosine (10(-5) M), after endothelium-dependent vasodilation in response to bradykinin (10(-8) M) and after ecNOS inhibition by nitro-L-arginine methyl ester (L-NAME) (10(-4) M); (2) medial thickening of coronary microvessels and perivascular collagen on histological heart sections; and (3) ecNOS expression by immunohistochemical staining in these vessels using 20-week-old spontaneously hypertensive (SHR) and Wistar-Kyoto control rats (WKY). These measurements were determined by computer-directed color analysis. When SHR were compared with WKY rats, we found: (1) a decrease in basal flow (10.1+/-0.6 v 15.3+/-1.2 ml/min/g, n=10, P<0.0001), in maximum flow (15.4+/-0.7 v 24.3+/-1.3 ml/min/g, n=10, P<0.001), in bradykinin-induced flow increment (1.5+/-0.3 v 2.6+/-0.3 ml/min/g, n=5, P<0.05) and in L-NAME-sensitive flow (3.3+/-0.6 v 6.3+/-0.9 ml/min/g, n=7, P<0.05); (2) an increase in medial thickness (9.4+/-0.6 v 5.4+/-0.3 microm, n=8, P<0.001) and in perivascular collagen area (1509+/-311 v 462+/-120 microm2, n=8, P<0.01) of coronary arterioles; and (3) a decrease in ecNOS expression in the endothelium (ecNOS-stained cross-sectional area in arterioles: 40.0+/-9.1 v 84.6+/-9.0 microm2, n=7, P

Characterization of angiotensin II receptors in cultured adult rat cardiac fibroblasts. Coupling to signaling systems and gene expression.

Cardiac hypertrophy is largely due to cardiac fibroblast growth and increased synthesis of extracellular matrix. This study has investigated the contribution of the vasoactive hormone, angiotensin II, toward this hypertrophic process. We have demonstrated that cultures of adult rat cardiac fibroblasts express AT1 but not AT2 receptors for angiotensin II. The ability of angiotensin II to stimulate phosphoinositide catabolism and to elevate intracellular calcium concentrations in these cells was blocked by losartan, a specific AT1 receptor antagonist, but not by the AT2 receptor antagonist CGP 42112. Exposure of adult cardiac fibroblasts to angiotensin II resulted in the induction of several growth-related metabolic events including c-fos protooncogene expression and increased synthesis of DNA, RNA, and protein. Angiotensin II was also found to induce collagen type I, alpha 1 chain transcript expression in cardiac fibroblasts as well as the synthesis and secretion of collagen by these cells. The data demonstrate that angiotensin II, via AT1 receptors, can stimulate cardiac fibroblast growth and increase collagen synthesis in cardiac tissue. Thus, angiotensin II may contribute toward the development of cardiac hypertrophy in conditions of hypertension that are associated with elevated concentrations of angiotensin II.

Transcriptional activation of low density lipoprotein receptor gene by angiotensin-converting enzyme inhibitors and Ca(2+)-channel blockers involves protein kinase C isoforms.

The pharmacological potency of angiotensin-converting enzyme (ACE) inhibitors (lisinopril and enalaprilat) on the transcription of low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase genes was examined in human vascular smooth muscle cells and compared with the action of Ca(2+)-channel blockers (manidipine, verapamil, and diltiazem). Analogous to Ca(2+)-channel blockers, nanomolar concentrations of enalaprilat or lisinopril stimulated the synthesis of low density lipoprotein receptor mRNA and amplified the transcription induced by recombinant platelet-derived growth factor BB. In contrast to Ca(2+)-channel blockers, ACE inhibitors did not alter the transcription of the 3-hydroxy-3-methylglutaryl-CoA reductase gene. Platelet-derived growth factor BB stimulated the translocation of delta and epsilon isoforms of protein kinase C. Similar to Ca(2+)-channel blockers, ACE inhibitors reduced the translocation of delta and epsilon isoforms of protein kinase C. Furthermore, ACE inhibitors and Ca(2+)-channel blockers inhibited platelet-derived growth factor BB-induced transcription of c-fos and c-jun genes. The findings suggest that increased de novo synthesis of mRNA low density lipoprotein receptor apparently involves the participation of delta and epsilon isoforms of protein kinase C and transcription factors c-Fos and c-Jun.

Effect of tumour-promoting phorbol ester, thrombin and vasopressin on translocation of three distinct protein kinase C isoforms in human platelets and regulation by calcium.

Protein kinase C (PKC) acts in synergy with Ca2+ mobilization for the activation of platelets. Three different PKC subtypes that specifically react with antibodies to alpha- beta- and zeta-PKC have been detected in human platelets. We have compared the subcellular redistribution of these isoforms in platelets after exposure to the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and to two physiological agonists, thrombin and vasopressin. In the presence of PMA, beta-PKC is most rapidly translocated to membranes, followed by zeta-PKC and alpha-PKC [membrane contents of 39 +/- 6, 31 +/- 4 and 24 +/- 4% (means +/- S.E.M.) respectively after 2 min incubation]. In contrast, both thrombin and vasopressin induced a biphasic translocation of PKC isoforms. For both agonists, the first phase of translocation occurred within 1 min and was identical for the three isoforms. However, during the second phase, the translocation of zeta-PKC by thrombin and vasopressin differed [membrane contents (mean +/- S.E.M.) of 24 +/- 3 and 46 +/- 4% respectively after 10 min]. These results suggest a differential activation of zeta-PKC by vasopressin and thrombin. PMA-induced translocation of alpha-PKC was decreased from 278 +/- 27 to 198 +/- 24 (mean +/- S.E.M., P = 0.02; percentage increase over control value) in the presence of 1 mM-EDTA, whereas chelation of intracellular Ca2+ by Quin2-AM does not influence this response. These results suggest that the PMA-induced translocation of alpha-PKC depends on the presence of 1 mM concentration of extracellular Ca2+. In addition, the chelation of either extracellular or intracellular Ca2+ inhibited both vasopressin- and thrombin-induced translocation of all three isoforms, suggesting that Ca2+ is an important requirement for the translocation of alpha-, beta- and zeta-PKC by physiological agonists. In conclusion, the translocation of PKC varies between different isoforms and between different agonists.

The calcium binding protein tropomyosin in human platelets and cardiac tissue: elevation in hypertensive cardiac hypertrophy.

Intracellular calcium transients play a major role in the control of cellular contraction and act through binding to target proteins and inducing subsequent conformational changes and activation of enzymes. Abnormalities of intracellular calcium handling are involved in the pathophysiology of essential hypertension and cardiac hypertrophy. In this study we report on the isolation, purification and calcium binding of a 33 kDa protein from human platelets and of a 38 kDa protein from cardiac tissue, both of which are identified as tropomyosin. The calcium binding properties of these human tropomyosin isoforms indicate a putative role for these proteins in the fine tuning of the cellular contraction. Elevated tropomyosin level is demonstrated in platelets from untreated essential hypertensive patients with left ventricular hypertrophy (tropomyosin/actin: 45.1 +/- 3.5, n = 12) relative to essential hypertensive patients without cardiac hypertrophy (tropomyosin/actin: 33.8 +/- 2.3). These findings suggest an association between the enhanced expression of tropomyosin in platelets and the development of cardiac hypertrophy which may relate to the cellular calcium overload of this disease.

Different translocation of three distinct PKC isoforms with tumor-promoting phorbol ester in human platelets.

Protein kinase C (PKC), a family of related but distinct enzymes whose cellular functions are poorly understood, acts in synergy with Ca2+ mobilization for the activation of platelets. Using specific antibodies for the different isoforms, immunoblot analysis revealed the presence in human platelets of three different PKC subtypes which specifically react with alpha, beta and zeta-PKC antibodies. Whereas the subcellular distribution of the alpha PKC remained unaffected, incubation of platelets with 1 microM PMA for 2 min resulted in a significant subcellular distribution from cytosol to membrane of beta-PKC (25%) and zeta (15%). The beta-PKC isoform is more sensitive than alpha and zeta-PKC to PMA, since 100 nM PMA resulted in a translocation of 85%, 64% and 66% respectively of a maximum translocation observed with 1 microM PMA.

[Physiologic role of the sodium pump. Implications for the study of arterial hypertension]. / Rôle physiologique de la pompe à sodium. Implications pour l'étude de l'hypertension artérielle.

Mammalian cells have the same hydroelectrolytic composition (high K, low Na), highly different from that of their surrounding. Constancy of cellular composition is insured by the balance between ionic leaks and (Na, K)-pump activity. Ionic leaks, specially sodium, are fundamental. They allow cells to perform a majority of their general and special functions (import of aminoacids, glucose, phosphates; export of acids; nerve influx; muscular contraction; glandular secretion; intestinal and renal reabsorption and secretion). (Na, K)-pump is essential to life. It is a kind of general motor that creates and maintains ionic concentrations differences whose potential energy is dissipated by leaks to perform cellular functions. Constancy of hydroelectrolytic intracellular composition hides that leak and pump rates, equivalent between them, are extremely variable among cell types (more than 200 times), and can increase 4 times in less than one minute within a cell type with cellular activity. In a cell, (Na, K)-pump rate is far from maximum velocity. This rate is adjusted nearly instantaneously to balance variations in leak rates; it may undergo short term modulation by endo or exocellular factors; it may undergo long term changes through synthesis of new enzyme molecules. Studies on whole cells of the balance between leaks and pump rates is necessary to understand these cells physiology and pathology. Balance between leaks and (Na, K)-pump activity is altered in renal cells from hypertensive rats, spontaneously (SHR) or genetically selected (Milan Hypertensive Strain: MHS). Strikingly, sodium activation of (Na, K)-pump of inner medullary collecting duct cells of MHS rats is greatly blunted compared to controls.

Atrial natriuretic peptide regulates release of Na+-K+-ATPase inhibitor from rat brain.

Tissue culture media from incubations of fragments of rat brain were collected and partially purified. These supernatants were effective in inhibiting the Na+-K+ pump as indicated by a 77% reduction of ouabain-sensitive 86Rb+ uptake into human erythrocytes. Release of the Na+-K+-ATPase inhibitor depended on the amount of tissue, the temperature, and the length of incubation. Atrial natriuretic peptide (ANP) injected intravenously, or included (10(-8) M) in the in vitro incubation of brain tissue, decreased the release of the Na+-K+-ATPase inhibitor by 74 and 42%, respectively. Control experiments using the neuropeptide arginine vasopressin showed no effect on release of the inhibitor. These studies indicate that ANP is capable of regulating the release from brain of a Na+-K+-ATPase inhibitor with similar chromatographic characteristics to the one previously obtained from extraction of bovine hypothalamus and raise the possibility that the two factors are interrelated in the regulation of fluid and electrolyte balance.

An endogenous digitalis-like compound extracted from human urine: biochemical and chemical studies.

Plasma and urine levels of an endogenous digitalis-like compound (EDLC) are increased in low renin Na+-dependent experimental hypertension, in some normotensive offspring of hypertensive patients and in some essential hypertensive patients. Urine-drived EDLC was purified from 550 L of urine from essential hypertensive patients (n = 8) and from normotensive subjects with a family history of hypertension (n = 27), using flash chromatography on C18 reversed-phase, anion exchange chromatography and various reversed-phase high performance liquid chromatographies. The mechanism of Na+-K+ ATPase inhibition and the related effects of semipurified urine-derived EDLC were studied and compared with those of ouabain. Its action was similar to that of ouabain in 8 out of 10 of the tests applied. The main effects of such a compound were the depression of Na+-K+ pump activity of human erythrocytes, the inhibition of 5-hydroxytryptamine reuptake by human platelets, and the induction of natriuresis in urethanized rats. Therefore, EDLC may be considered as one of the natriuretic hormones whose mechanism of action closely resembles that of ouabain.

Endogenous Na+ transport inhibitor in human hypertension: further biochemical and chemical studies.

Endogenous digitalis-like compound(s) (endalin) has(ve) been reported to be involved in some diseases. Endalin activity is increased in plasma and urine of some essential hypertensives, and in Na+-dependent experimental hypertension. The aims of this study are to compare the biological properties of one endalin extracted from urine of hypertensive patients and of normotensive offspring of hypertensive subjects to those of ouabain and to determine the chemical nature of such an urine-derived endalin. The donors were selected on the basis of the highest Na+,K+-ATPase inhibition produced by extract from their 24-h urine. They consisted of 8 hypertensive patients, 21 normotensive subjects with family history of hypertension and 6 normotensive subjects with no known family history of hypertension. Endalin was semi-purified from 500 liters of pooled urine by flash chromatography on RP 18 packing (40 microns) followed by anion exchange chromatography and two HPLCs on RP 18 reversed phase. Endalin was traced by its capability of inhibiting dog kidney Na+,K+-ATPase activity and 3H-ouabain binding to the enzyme, by its cross-reaction with anti-digoxin antibodies and by its natriuretic effect in rat bioassay. The mechanism of Na+,K+-ATPase inhibition by a semi-purified urine-derived endalin and its consequences on Na-transport were studied and compared to those of ouabain. Semi-purified urine-derived endalin was similar to ouabain in that: it reversibly and specifically inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Further biochemical characterization of an Na+ pump inhibitor purified from human urine.

An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.

Recent progress on an endogenous digitalislike factor in hypertension.

Evidence exists that demonstrates the relationship between a natriuretic factor, or Na+, K+-ATPase inhibitor, and volume expansion in man. Patients having extracellular volume expansion have been studied for the effect of their plasma on erythrocyte [3H]ouabain binding. High levels of ouabainlike activity were 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 the urine of hypertensive patients. The various steps of purification achieved a 400,000-fold purified compound of apparent homogeneity. The inhibitor was extracted from 140 liters of urine of 21 donors (hypertensive patients and normotensive offspring of hypertensive patients). The purification steps included flash chromatography, anionic exchange, and reversed-phase HPLC on RP 18, diphenyl and phenyl packings. Nuclear magnetic resonance and mass spectrometry indicated a nonpeptidic compound, which was possibly a steroid with a low molecular mass (less than 500 daltons).

High yield-purification of a urinary Na+-pump inhibitor.

A Na+-pump inhibitor was purified from 140 liters of human urine to an apparent homogeneity. Tracing of the inhibitor during the different steps of purification was achieved by simultaneous determination of its capacity to inhibit the activity of Na+,K+-ATPase and ouabain binding, and to cross-react with antidigoxin antibodies. The final purification achieved a 400,000 fold. The purification steps included flash chromatography, anionic exchange chromatography, and reversed-phase HPLC on RP18, diphenyl and phenyl packings. NMR studies indicated that the final product was a non-peptidic, possibly steroidal compound. Its molecular weight as determined by mass spectrometry was 431.

Chemical, experimental, and clinical studies on endogenous ouabain-like substance in hypertension.

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.

Recent advances on endogenous Na+,K+-ATPase inhibitors: clinical investigation and purification.

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 semi-purified 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.