Alteration of lymphocyte membrane phospholipids and intracellular free calcium concentrations in hyperlipidemic subjects.

Hypercholesterolemia has been proposed to influence cell functions via changes in membrane composition. The aim of the present study was to determine whether the membrane phospholipid composition of human lymphocytes is modified in hypercholesterolemia and whether these changes are accompanied by functional modifications. The phospholipid fatty acid contents and intracellular free calcium concentrations were determined in peripheral blood lymphocytes from 13 subjects with serum total cholesterol levels ranging from 4.6 to 8.8 mmol/l. The spontaneous basal rate of thymidine incorporation in lymphocyte of hypercholesterolemic individuals increased, while its relative stimulation by ConA was less effective. Important changes in membrane lipid composition, consisting mainly of decrease of the mass of phospholipids, and of associated polyunsaturated fatty acids were observed in hypercholesterolemia. In contrast, the cell cholesterol content was significantly increased. The intracellular free calcium concentration was enhanced and strongly associated with circulating cholesterol levels, cell cholesterol content and phospholipid fatty acids. These results indicate that hypercholesterolemia is accompanied by profound changes in lymphocyte membrane lipid composition and Ca(2+) handling.

Effect of glibenclamide on the metabolism of fatty acids in cultures of newborn rat heart cells under normoxic and hypoxic conditions.

Several deleterious biochemical alterations have been observed in myocardial cells during ischemia, including perturbations of transmembrane ion equilibria, production of noxious oxygen-derived radicals and loss of membrane phospholipids. Although the precise relationship between these alterations and the reduction of oxygen and glucose supplies is not fully understood, the decrease of intracellular ATP content appears to be a key event in the cascade. Recent evidence suggests that opening of ATP-sensitive K+ channels may constitute an endogenous protective mechanism during ischemia. We have thus tested the effects of glibenclamide, a channel blocker, and aprikalim, a channel opener, on the metabolism of membrane fatty acids in cultures of newborn rat heart cells under normoxic and hypoxic conditions. We showed that glibenclamide partially blocks the loss of membrane phospholipids induced by oxygen deprivation in contractile myocytes, whereas aprikalim fails to alter this metabolism under either normoxic or ischemic conditions. In cultures of fibroblast-like heart cells neither drug was able to modify fatty acid metabolism.

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.

Growth rate and phospholipase C activity in cardiac and aortic spontaneously hypertensive rat cells.

The cellular proliferation rate and phospholipase C sensitivity have been compared in various cardiovascular cells cultured from spontaneously hypertensive (SHR) and Wistar-Kyoto rats (WKY). Aortic smooth muscle cells from SHR showed an enhanced proliferation rate under the culture conditions (in the presence of 10% fetal calf serum) and hypersensitivity to the mitogenic action of angiotensin II and serotonin. Phospholipase C activity (determined by measuring the formation of tritiated inositol phosphates from [3H]-myoinositol) triggered by angiotensin II was also higher in these cells than in those from WKY. Cardiac fibroblasts from SHR proliferated more quickly than those from WKY, which suggests that the increased growth rate is a general feature of SHR cardiovascular cells. Furthermore, as cardiac cells were obtained from newborn animals, this impairment appears to be an intrinsic characteristic of the SHR cells. In contrast, no difference in the phospholipase C reactivity to phenylephrine was found in cardiomyocytes from SHR and WKY. The amount of tritiated inositol monophosphate measured in unstimulated cells, however, was significantly higher in cultures from WKY, which may suggest a difference in the phosphoinositide cycle between both strains of cells. Like the impairment in the proliferation rate, alterations in phosphoinositide metabolism may be a common feature of cardiovascular SHR cells.

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.

The effect of 4'-deoxypyridoxine on rat heart cell cultures under oxygen deficiency.

The purpose of this work was to study the consequences of an intracellular depletion of pyridoxal-phosphate (PLP) in rat heart cells in culture submitted to oxygen deficiency. Glucose (GLc) and 4'-deoxypyridoxine (DOP) effects were separately evaluated after 150 mn of partial oxygen deprivation (N2) with regard to enzyme leakage, beating rates, intracellular concentrations of PLP and the ratio glycogen phosphorylase activity (-5'-AMP/+5'-AMP). PLP concentrations were higher in the air-GLc group than in the air group. alpha-Hydroxybutyrate dehydrogenase (alpha-HBDH) and creatine kinase (CK) leakages were observed in the N2 group, whereas 1.17 +/- 0.17 mM GLc prevented leakage. Beatings stopped in the N2 group, slightly decreased in the N2-GLc group; the ratio (-5'-AMP/+5'--AMP) increased only in the N2 group. With a 1 mM DOP pretreatment a significant decline of PLP was observed either with or without GLc in the culture medium. The ratio (-5'-AMP/+5'-AMP) was lower in the air-DOP group than in the air group, whereas beatings, alph-HBDH and CK activities were identical to the control group. ImM DOP in the N2-DOP group partially prevented alpha-HBDH and CK leakages into the medium but did not prevent the decrease in contractile activity. Surprisingly, PLP concentration increased in the N2-DOP group/air-DOP group and the increase in ratio (-5'-AMP/+5'-AMP) was higher in the N2-DOP group than in the N2 group when compared to the respective control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

[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.

Comparison of the chronotropic effect and the cyclic AMP accumulation induced by beta 2-agonists in rat heart cell culture.

1 The chronotropic response and the variation in cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation induced by isoprenaline and six beta 2-selective agonists (fenoterol, salmefamol, soterenol, zinterol, salbutamol and formoterol) were analyzed on cultured heart cells of the rat. 2 The compounds elicited an enhancement of the frequency, but the time course of the variation of the beating rate was not identical for all of them. A rapid onset was observed for isoprenaline, zinterol and formoterol while it was slower for fenoterol, salmefamol and salbutamol. 3 In contrast with isoprenaline, the beta 2-selective agonists gave concentration-beating frequency curves which were not sigmoidal. Their effects extended up to a concentration of 5 to 6 orders of magnitude. Nevertheless, the concentration at which the maximal effect occurred and the intrinsic activities of the various compounds agrees better with the responses observed on guinea-pig atria than with those on trachea. 4 All the beta 2-selective agonists increased the accumulation of cyclic AMP in rat heart cells with a maximal effect at 10(-5)M or less. The effects of beta 2-agonists on cyclic AMP production showed some analogies with those on beating frequency of the heart cells. The increase in cyclic AMP accumulation induced by beta 2-agonists also corresponded to their chronotropic effects on guinea-pig atria. Thus, the correlation coefficient between the inverse of the log of the concentration producing the half maximal cyclic AMP accumulation in cultured heart cells and the pD2 values on guinea-pig atria was 0.93. 5 It is concluded that, in contrast to what was observed in other models, the beta 2-selective agonists induce an increase in the production of cyclic AMP in rat heart cells. Furthermore, the effects of the beta 2-agonists on cyclic AMP accumulation and on beating rate in the heart cells may correspond with their beta 1-adrenoceptor potencies.

[Optimal trypsin concentration for rat heart cell cultures]. / Concentration optimale de trypsine pour les cultures de cellules cardiaques de rat.

Three concentrations (0.5, 1 and 2 per 1000, w/v) of a single batch of trypsin were compared regarding their influence on cultured cardiac cell of newborn rats. All three allowed to obtain cardiac cells in good conditions, as evidence by beating frequencies and [16-14C]-palmitate beta-oxidation. However the 0.5 per 1000 concentration appeared to be the optimal one, inducing a smaller loss of cells during the first two days in culture.