Regulation of arachidonic acid release by calcium influx in human endothelial cells.

In response to stimuli, endothelial cells release arachidonic acid, a lipid precursor of various vasoactive substances. We have investigated the relationships between cytosolic Ca2+ movements and arachidonic acid release in human umbilical vein endothelial cells. Histamine, a receptor-dependent agonist, and thapsigargin, a specific inhibitor of sarco-/endoplasmic Ca2+ pumps, time- and dose-dependently increased the release of [1-14C]-arachidonic acid. This release was inhibited by AACOCF3, a selective inhibitor of cytosolic phospholipase A2 (PLA2). In the absence of Ca2+ influx, arachidonic acid release was suppressed in both histamine- and thapsigargin-stimulated cells, despite marked elevations of cytosolic Ca2+ concentration ([Ca2+]i). In the presence of Ca2+ influx, arachidonic acid release was reduced in cells treated with BAPTA, an intracellular Ca2+ buffer, or with SK&F 96365, a receptor-operated Ca2+ channel blocker. Arachidonic acid release was analyzed as a function of the two successive phases of Ca2+ response to stimulation: Ca2+ peak and plateau phase, reflecting Ca2+ mobilization from internal stores and Ca2+ influx, respectively. The amount of arachidonic acid released was directly related to [Ca2+]i values measured at the influx phase with a 80 nM [Ca2+]i threshold, similar to that reported for PLA2 translocation. This suggests that Ca2+ entry from the extracellular space is essential for activating cytosolic PLA2 in human endothelial cells.

Inhibition by cholesterol oxides of NO release from human vascular endothelial cells.

Recent studies have demonstrated that, unlike cholesterol, cholesterol oxidized at position 7 can reduce the maximal endothelium-dependent relaxation of isolated rabbit aortas (Circulation. 1997;95:723-731). The aim of the current study was to determine whether cholesterol oxides reduce the release of nitric oxide (NO) from human umbilical vein endothelial cells (HUVECs). The amount of NO released by histamine-stimulated HUVECs was determined by differential pulse amperometry using a nickel porphyrin- and Nafion-coated carbon microfiber electrode. The effects of cholesterol (preserved from oxidation by butylated hydroxytoluene), 7-ketocholesterol, 7beta-hydroxycholesterol, 5alpha,6alpha-epoxycholesterol, 19-hydroxycholesterol (60 microg/mL), and alpha-lysophosphatidylcholine (10 microg/mL) were compared. Pretreatment of HUVECs with cholesterol, 5alpha,6alpha-epoxycholesterol, or 19-hydroxycholesterol did not alter histamine-activated NO production. In contrast, pretreatment with 7-ketocholesterol or 7beta-hydroxycholesterol significantly decreased NO release. The inhibitory effect of 7-ketocholesterol was time and dose dependent and was maintained in the presence of L-arginine. In the absence of serum, lysophosphatidylcholine also reduced NO production. In ionomycin-stimulated cells, pretreatment with 7-ketocholesterol did not inhibit NO release. These results demonstrate that cholesterol derivatives oxidized at the 7 position, the main products of low density lipoprotein oxidation, reduce histamine-activated NO release in HUVECs. Such an inhibitory effect of cholesterol oxides may account, at least in part, for the ability of oxidized low density lipoprotein to reduce the endothelium-dependent relaxation of arteries.

Nitric oxide production in human endothelial cells stimulated by histamine requires Ca2+ influx.

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 microM, 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.

Nitric oxide production by endothelial cells: comparison of three methods of quantification.

Vascular endothelial cells have been found to produce a relaxant mediator, identified as nitric oxide (NO) and implicated in numerous physiological functions. Subsequently, there has been an intensive search for accurate and specific detection methods to measure biological NO production. In the present study, we compared three approaches to evaluate NO production, based respectively on the Griess reaction (that quantifies nitrites and nitrates after their reduction), on the hemoglobin reaction (that quantifies oxyhemoglobin to methemoglobin transformation by NO), and on the electrochemical NO detection with a porphyrinic micro-probe. Comparison was made both under standard conditions and biological conditions, through calibration curves and measurements of histamine-induced NO production by cultured human endothelial cells and its modulation by L-arginine and N(omega)-monomethyl-L-arginine. We demonstrated that these three methods differ in terms of sensitivity and selectivity. The hemoglobin reaction and nitrate measurements suffer from a lack of specificity. Nitrite determination by the Griess reaction was hardly suitable for kinetic studies but it remains useful for the evaluation of basal NO production. The electrochemical technique, although it does not allow measurement of basal NO production, is the only one to exhibit great sensitivity and specificity and to allow instantaneous and non destructive measurements. This study brings up the potential hazards and pitfalls that may be associated with the various methods.

SK&F 96365 inhibits intracellular Ca2+ pumps and raises cytosolic Ca2+ concentration without production of nitric oxide and von Willebrand factor.

The effects of the imidazole compound SK&F 96365 on Ca2+ movements and production of nitric oxide (NO) and von Willebrand factor (vWF) have been investigated in human endothelial cells. Changes in cytosolic Ca2+ concentration ([Ca2+]i) were measured with Fura-2. Real-time production of NO was monitored with a porphyrinic microsensor and the release of vWF with an enzyme-linked immunosorbent assay. Irrespective of the transmembrane Ca2+ gradient, 30 microM SK&F 96365 doubled [Ca2+]i suggesting a Ca2+ release from intracellular stores. The SK&F 96365-induced [Ca2+]i rise was not accompanied by detectable NO and vWF production, while 1 microM thapsigargin enhanced [Ca2+]i 2.5 times, doubled the secretion of vWF and increased the NO production to 10 +/- 4 nM (n = 5). Pretreatment with SK&F 96365 prevented thapsigargin from increasing [Ca2+]i, NO production and vWF secretion. To investigate the mechanism by which SK&F 96365 released Ca2+ from internal pools, its effect and that of thapsigargin on the ATP-dependent 45Ca2+ uptake into platelet membrane vesicles were compared. SK&F 96365 as thapsigargin, dose-dependently reduced the initial rate of 45Ca2+ uptake. In conclusion, we demonstrate that, in the absence of Ca2+ entry from the extracellular space, the [Ca2+]i increase elicited by SK&F 96365 or thapsigargin is not sufficient to initiate NO synthesis and vWF secretion. This confirms the important role of Ca2+ influx in endothelial secretion processes.

Enhanced phospholipase A2 activity in cultured cardiomyocytes from newborn spontaneously hypertensive rat.

1. Changes in membrane lipid composition and metabolism could participate in myocardial membrane dysfunction in essential or experimental hypertension. Phospholipid-bound fatty acid profile and metabolism are altered in cultured heart myocytes of newborn genetically hypertensive rats. The present study was designed to investigate the participation of phospholipase A2 in these modifications. 2. Phospholipase A2 activity of cultured cardiomyocytes of neonate spontaneously hypertensive rats and normotensive control Wistar-Kyoto rats was compared. The enzyme activity was measured using 2-[1-14C]arachidonyl-phosphatidylethanolamine as substrate. In both strains, Ca(2+)-dependent and independent phospholipase A2 activities were present. Only the Ca(2+)-dependent enzyme activity was altered in spontaneously hypertensive rat cardiomyocytes. With 0.2 mmol/l substrate and 5 mmol/l Ca2+, the phospholipase A2 activities were 79.0 +/- 13.4 and 26.0 +/- 3.6 nmol h-1 mg-1 of protein in spontaneously hypertensive and Wistar-Kyoto rat cardiomyocytes respectively (n = 10 in both cases, P = 0.001). The maximum velocity of the enzyme was three times higher in spontaneously hypertensive rat than in Wistar-Kyoto rat, without changes in the apparent affinity of the enzyme for its substrate. 3. The present results demonstrate an enhanced phospholipase A2 activity in cultured heart muscle cells of spontaneously hypertensive rats, which could be genetically determined.

Aldosterone modulates both the Na/H antiport and Cl/HCO3 exchanger in cultured neonatal rat cardiac cells.

Mineralocorticoid hormones regulate many physiological functions in the cardiovascular system. Although high affinity binding sites for aldosterone have been found in myocardium, aldosterone effects on pHi regulatory systems in cardiac cells have not been described. We have addressed this issue by using microspectrofluorimetric monitoring of intracellular pH in developing neonatal rat cardiomyocytes cultured for 2 weeks. Developmental changes in cell morphology were controlled by anti-myosin light chain antibody staining of the sarcomeric units using confocal laser scanning microscopy. The data obtained demonstrate that from early stages of the development, pHi in neonatal cardiac cells is regulated by three ion transporting mechanisms, namely, Na/H antiport, Na- and HCO3-dependent transporter and Cl/HCO3 exchanger. A 24-h treatment of the cells with aldosterone increases the activity of the Cl/HCO3 exchanger at day 6 of cell culture while the Na/H antiport activity is enhanced in the cells treated with the hormone at days 9 and 13 of culture. Thus, by affecting the activity of ionic transporters, aldosterone modulates acid-base balance in cardiac cells.

Altered phospholipid fatty acid content and metabolism in heart cell cultures from newborn spontaneously hypertensive rats.

Genetic hypertension has been proposed to be associated with impaired lipid metabolism. To investigate whether lipid metabolism is altered in young rats of the spontaneously hypertensive Okamoto strain (SHR), we have compared the phospholipid fatty acid content and metabolism in cultured heart myocytes and fibroblasts from SHR and normotensive Wistar-Kyoto (WKY) newborn rats. The phospholipid-bound fatty acid profile and metabolism were altered in SHR cardiomyocytes and unchanged in SHR fibroblasts. In SHR myocytes, the fatty acid composition of the phospholipid fraction was modified, with a lowered proportion of linoleic (P < .05) and eicosapentaenoic acid (P < .001), resulting in a decreased polyunsaturated to saturated fatty acid ratio (1.16 +/- 0.08 in SHR v 1.44 +/- 0.08 in WKY, P < .02). The metabolism of radioactive arachidonate (C20:4) and linoleate (C18:2) also differed between SHR and WKY myocytes. Their release was increased (P < .004 and .05 for C20:4 and C18:2, respectively). The labeled phospholipid species also differed between the two strains, suggesting an altered phospholipid turnover in SHR. This study demonstrates modifications of phospholipid fatty acid profile and metabolism in spontaneously contractile cardiac cells from newborn prehypertensive SHR, in the absence of neural, hormonal, and hemodynamic influences.

Regulation of membrane phospholipid metabolism in heart cell culture.

We have isolated, from newborn rats, heart cultures enriched in contractile muscle cells (M) and cultures of fibroblast-like cells (F). M cultures respond to simulated ischemia by an arrest of beating activity, by a decrease in beta oxydation rate, ATP and phosphocreatine content and by a loss of membrane phospholipids associated with neutral lipids accumulation. F cells in contrast do not respond to oxygen deprivation. Firstly, we observed that cocultures of M and F cells respond to oxygen deprivation by an arrest of beating activity and a decrease in cellular ATP content, but failed to exhibit any significant loss of membrane phospholipids. Secondly, we demonstrated that culture medium conditioned by F cells is able to inhibit the reaction of M cells to simulated ischemia thus suggesting that fibroblasts produce a diffusible factor able to block phospholipase activation. Heat treatment and trypsinisation failed to abolish this activity, indicating that the phospholipase inhibitory factor is probably not a polypeptide.

Cytosolic pH in cultured cardiac myocytes and fibroblasts from newborn spontaneously hypertensive rats.

Newborn spontaneously hypertensive rats (SHR) develop cardiac hypertrophy before a rise in blood pressure. Cytosolic pH (pHi) has been discovered to modulate cell growth and proliferation; therefore, we have investigated pHi in myocytes and fibroblasts from 3- to 4-day-old SHR and normotensive Wistar (W) and Wistar-Kyoto controls (WKY). The ratio of heart to body weight was higher in SHR than in W and WKY (7.56 +/- 0.10 v 6.21 +/- 0.10 and 5.98 +/- 0.14 mg/g in 10, 5, and 7 groups of 20 to 40 animals; P less than .001 for both). Cytosolic pH, determined with the fluorescent probe BCECF, was measured from the sixth to the eighth day in culture on confluent cells. The mean pHi was higher in myocytes from SHR than in those from W or WKY rats (7.19 +/- 0.03, N = 30, v 7.09 +/- 0.03 and 7.11 +/- 0.02, N = 25 and 30; P = .008 and .024, respectively). In contrast, pHi was similar in fibroblasts from the three strains (7.21 +/- 0.03, 7.18 +/- 0.03, and 7.19 +/- 0.02, N = 15, 15, and 14, in SHR, W, and WKY rats, respectively). External acidification induced similar decreases in pHi from SHR and WKY myocytes, maintaining higher pHi values in SHR myocytes along the entire external pH (pHo) range studied. The inhibition of Na+/H+ exchange by the amiloride derivative, ethylisopropylamiloride, decreased the steady-state pHi of myocytes independently of the initial pHi values. This study demonstrated a cytosolic alkalinization in contractile cardiac cells from SHR before a significant rise in blood pressure and in the absence of hemodynamic influences and specific plasma factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of isoproterenol on lipid metabolism and prostaglandin production in cultures of newborn rat heart cells, under normoxic and hypoxic conditions.

Catecholamines are known to exert deleterious effects on heart cells and to provoke biochemical alterations similar to those observed during myocardial infarction. In order to investigate the mechanisms of these effects, we have studied in cultures of muscle (M) and fibroblast-like (F) cells derived from newborn rat hearts, the action of isoproterenol on membrane lipid metabolism and on prostaglandin production. We showed in F cells that beta-agonist stimulation produced a striking loss of membrane phospholipids and a moderate hydrolysis of cell triglycerides. In addition, isoproterenol treatment induced a significant stimulation of the secretion of prostacyclin but not of prostaglandin E2 by F cells. None of these effects were potentiated by oxygen deprivation. In contrast, M cells, which are sensitive to ischemia, failed to respond to isoproterenol treatment. These results suggest that catecholamines and hypoxia may exert combined deleterious effects on heart tissue by acting separately on the different target cells in vivo.

Abnormal fatty acid utilization by cultured cardiac cells from 7-day-old obese Zucker rats.

Fatty acid utilization by muscle and nonmuscle heart cells in culture has been investigated in the 7-day-old Zucker rat to determine if this tissue could contribute to the lower energy expenditure reported in obese rats at the onset of obesity. The partitioning of oleate to oxidation and esterification products and the effect of genotype on this partitioning according to cell types were studied. Results showed that the fatty acid beta-oxidation and its esterification in neutral lipid was decreased by 30% in beating muscle cells from obese animals when compared with those from lean animals. In contrast, nonmuscle cells exhibited a decreased beta-oxidation alone. A similar fatty acid composition of the phospholipids was found in non-muscle cells of obese animals and their lean litter mates. In muscle cultures, palmitic and oleic acids are lower in cells of obese rats than in those of lean rats. The present study indicates that a defect in energy metabolism could be found in heart cells at the onset of obesity, suggesting that this defect is determined by intrinisic factor(s).

Effect of oxygen deprivation on metabolism of arachidonic acid by cultures of rat heart cells.

To investigate the mechanisms responsible for the impairment of phospholipid metabolism observed in ischemic cells, we have studied the effect of conditions simulating ischemia on the metabolism of arachidonic acid (AA) by muscle (M-) and nonmuscle (F-) cells isolated from newborn rat hearts and cultured separately. In muscle cells, oxygen deprivation induces a significant stimulation of the release of [14C]AA from prelabeled cells associated with a preferential redistribution of [14C]AA into cell triglycerides but not formation of radioactive prostaglandins. Moreover, the fatty acid content of phospholipids, as measured by capillary gas chromatography, appears markedly reduced in ischemic myocardial cells. This fact may be related to phospholipase stimulation during ischemia as suggested by the antagonistic effect of mepacrine or p-bromophenacyl bromide. In contrast, oxygen deprivation failed to induce any significant alteration of AA metabolism in fibroblast-like heart cells. Our results indicate that these cultures of newborn rat heart cells, which exhibit many of the features observed in intact organ during ischemia, may represent a useful experimental model to investigate the pharmacological control of the membrane phospholipid turnover.

Effect of glucocorticoids on arachidonic acid metabolism and prostaglandin secretion by cultures of newborn rat heart cells.

We described that oxygen deprivation induced in cultures of heart muscle cells, biochemical events similar to those described in ischemic tissue: arachidonic acid liberation, loss of membrane phospholipids and increase in neutral lipids. Since glucocorticoids have been described to inhibit phospholipase activity and to exert beneficial effects during myocardial infarction, we studied in our experimental model the action of dexamethasone on the metabolism of arachidonic acid and on the synthesis of immunoreactive prostaglandins. Our results show that heart muscle cells produce prostaglandin E2 and 6-keto-prostaglandin-F1 alpha. This synthesis, inhibited by dexamethasone (70% inhibition), decreased after oxygen-deprivation (-45%). The effect of oxygen deprivation and dexamethasone (-60%) are not additive. Moreover, steroid treatment failed to counteract the loss of polyunsaturated fatty acids from the phospholipids, the increase in neutral lipids and the liberation of arachidonic acid induced by oxygen deprivation in muscle cells. These results may indicate that the cardiovascular effects of glucosteroids are not the consequence of a direct effect on heart metabolism at cellular level.

Effects on the sodium channel of some new cardiotonic drugs: the 4-, 5-, and 6-pyridyl-2(1H)-quinolone derivatives.

To study the action of some new cardiotonic drugs, the 4-, 5-, and 6-pyridyl-2(1H)-quinolone series, on the fast Na+ channel, we compared the effects of eight compounds of this series and milrinone on 22Na uptake in rat brain synaptosomes and in rat heart muscle cells in culture. The action of tetrodotoxin, a specific Na+ channel blocker, on the positive inotropic effect of these compounds on guinea pig atria was also examined. The new positive inotropic agents enhance 22Na uptake in synaptosomes in a dose-dependent manner. The activities, expressed as percentage of the maximum activity of protoveratrine B, a classic Na+ channel agonist, reached 70% for milrinone, 60% for compound 7, 57% for compound 6, and less than 50% for the other drugs. For compound 8, but not for milrinone, it was possible to observe a stimulatory effect of the 22Na uptake on heart muscle cells in culture. Tetrodotoxin (1 and 100 microM) inhibited the stimulatory effects of the inotropic drugs on both preparations. The positive inotropic activities of protoveratrine B, milrinone, and compounds 5 and 8, in guinea pig atria, were inhibited by tetrodotoxin. The affinity and the activity of the other compounds were unchanged in the presence of tetrodotoxin. Our results showed that the stimulation of Na+ influx through the fast Na+ channel might represent a part of the mechanism of action of the inotropic effect of some new cardiotonic drugs.

Effects of various antianginal drugs on sodium influx in rat brain synaptosomes and in rat heart muscle cells in culture.

This paper describes the inhibitory effects of several antianginal drugs on 22Na uptake of the fast Na+ channel in rat brain synaptosomes and in rat heart muscle cells in culture. Calcium antagonists like verapamil, flunarizine, perhexiline, two perhexiline derivatives IPS 629 and IPS 672, and beta-adrenoceptor antagonists like propranolol and practolol were tested. IPS 672 was the most active compound on synaptosomes and heart muscle cells (IC50 = 2.0 X 10(-6) and 2.4 X 10(-6) M respectively). The relative potencies of the Ca2+ antagonists tested on heart muscle cells were found to be IPS 672 greater than IPS 629 greater than perhexiline greater than flunarizine greater than verapamil. Verapamil was 55 and 10 times less active than IPS 672 on synaptosomes and heart cells respectively. Propranolol had an inhibitory activity comparable to that of flunarizine and was 100 times more active than practolol. It can be concluded that several antianginal drugs seems to interfere with the Na+ fast channel on rat brain and heart.

[Indices of partial oxygen deprivation in the culture medium of cardiac cells]. / Etude de quelques indices de la privation partielle d'oxygène dans le milieu de culture de cellules cardiaques.

Three indexes of partial oxygen deprivation, i.e. hypoxanthine, alpha HBDH and CK, were investigated in rat heart cell cultures, 7 day-old. Enzyme release in the medium and hypoxanthine uptake by the cells pointed out both oxygen and glucose deprivation, which modelized ischemia. Conversely, hypoxanthine release pointed out oxygen deprivation, in the presence of glucose however, which modelized hypoxia, whereas there was no enzyme leakage in the latter condition.