Dietary cholesterol influences cardiac beta-adrenergic receptor adenylate cyclase activity in the marmoset monkey by changes in membrane cholesterol status.

The activity of the beta-adrenergic receptor/adenylate cyclase system of the marmoset monkey heart was investigated following dietary cholesterol supplementation (0.5%). After 22 weeks, plasma cholesterol levels in the cholesterol group were more than twice that of the control group. In the cholesterol-fed group, the affinity for ICYP binding to cardiac membranes was elevated more than 2-fold, while the receptor number was decreased by 31%. Isoproterenol, norepinephrine and sodium fluoride stimulated adenylate cyclase activity was significantly higher in the cholesterol-fed group although the fold stimulation over basal levels was not affected. The most prominent change in the cardiac membrane lipids was an increase in the cholesterol to phospholipid ratio in marmoset monkeys fed cholesterol. These results indicate that in the marmoset, membrane cholesterol is an important factor in determining various properties of the cardiac beta-adrenergic receptor particularly receptor affinity which may impact on the response of the beta-adrenergic receptor/adenylate cyclase system of the heart to catecholamines. This result is in agreement with dietary fatty acid supplements designed to increase cardiac membrane cholesterol in this animal species (McMurchie, E.J. et al. (1988) Biochim. Biophys. Acta 937, 347-358). Elevated membrane cholesterol enhances beta-adrenergic receptor affinity and certain aspects of adenylate cyclase activity. This is a likely mechanism whereby atherogenic diets could promote cardiac arrhythmia in non-human primates and indeed in man.

Membrane lipid fluidity and its effect on the activation energy of membrane-associated enzymes.

1. The fatty acid composition of mitochondrial membranes from sheep and rats was altered by feeding these animals diets which were rich in unsaturated fatty acids. Changes in membrane lipid fluidity resulting from the altered membrane lipid composition were assessed by determining the upper temperature limit of the disorder-order transition (Tf) and the Arrhenius activation energy (Ea) of succinate oxidase. 2. After feeding the unsaturated fatty acid-rich diet to sheep the Ea, in the temperature range above Tf, increased from 8 to 63 kJ . mol-1 while Tf decreased from 32 to 15 degrees C. Rats fed an unsaturated fatty acid-rich diet exhibited an increase in Ea from 17 to 63 kJ . mol-1 and a decrease in Tf from 23 to 4 degrees C. 3. This decrease in Tf was related to an increase in the ratio of linoleic acid to stearic acid in the membrane lipid. Tf was not related to the proportion of unsaturated fatty acids in the membrane lipids, although an increase in unsaturation usually led to a decrease in Tf. 4. The results show that membrane lipid fluidity has a direct influence on the conformation of the active site of some membrane-associated enzymes, with the result that such enzymes display a higher Ea when the membrane lipids are comparatively more fluid. The increase in Ea of membrane-associated enzymes which accompanies changes in the physical state of membrane suggests that some proteins may phase separate with the more fluid lipids at low temperatures.

The interaction of dietary fatty acid and cholesterol on catecholamine-stimulated adenylate cyclase activity in the rat heart.

Diets supplemented with high levels of saturated or unsaturated fatty acids supplied by addition of sheep kidney fat or sunflower seed oil, respectively, were fed to rats with or without dietary cholesterol. The effects of these diets on cardiac membrane lipid composition, catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor activity associated with cardiac membranes, were determined. The fatty acid-supplemented diets, either with or without cholesterol, resulted in alterations in the proportion of the (n-6) to (n-3) series of unsaturated fatty acids, with the sunflower seed oil increasing and the sheep kidney fat decreasing this ratio, but did not by themselves significantly alter the ratio of saturated to unsaturated fatty acids. However, cholesterol supplementation resulted in a decrease in the proportion of saturated and polyunsaturated fatty acids and a dramatic increase in oleic acid in cardiac membrane phospholipids irrespective of the nature of the dietary fatty acid supplement. The cholesterol/phospholipid ratio of cardiac membrane lipids was also markedly increased with dietary cholesterol supplementation. Although relatively unaffected by the nature of the dietary fatty acid supplement, catecholamine-stimulated adenylate cyclase activity was significantly increased with dietary cholesterol supplementation and was positively correlated with the value of the membrane cholesterol/phospholipid ratio. Although the dissociation constant for the beta-adrenergic receptor, determined by [125I](-)-iodocyanopindolol binding, was unaffected by the nature of the dietary lipid supplement, the number of beta-adrenergic receptors was dramatically reduced by dietary cholesterol and negatively correlated with the value of the membrane cholesterol/phospholipid ratio. These results indicate that the activity of the membrane-associated beta-adrenergic/adenylate cyclase system of the heart can be influenced by dietary lipids particularly those altering the membrane cholesterol/phospholipid ratio and presumably membrane physico-chemical properties. In the face of these dietary-induced changes, a degree of homeostasis was apparent both with regard to membrane fatty acid composition in response to an altered membrane cholesterol/phospholipid ratio, and to down regulation of the beta-adrenergic receptor in response to enhanced catecholamine-stimulated adenylate cyclase activity.

Differential modulation of rat heart mitochondrial membrane-associated enzymes by dietary lipid.

Diets supplemented with high levels of saturated fatty acids derived from sheep kidney (perirenal) fat or unsaturated fatty acids derived from sunflower seed oil were fed to rats and the effect on heart mitochondrial lipid composition and membrane-associated enzyme behaviour was determined. The dietary lipid treatments did not change the overall level of membrane lipid unsaturation but did alter the proportion of various unsaturated fatty acids. This led to a change in the omega 6/omega 3 unsaturated fatty acid ratio, which was highest in the sunflower seed oil fed rats. Arrhenius plots of the mitochondrial membrane associated enzymes succinate-cytochrome c reductase and oligomycin-sensitive adenosinetriphosphatase (ATPase) after dietary lipid treatment revealed different responses in their critical temperature. For succinate-cytochrome c reductase, the critical temperature was 29 degrees C for rats fed the sheep kidney fat diet and 20 degrees C for rats fed the sunflower seed oil diet. In contrast, no shift in the critical temperature for the mitochondrial ATPase was apparent as a result of the differing dietary lipid treatments. The results suggest that the discontinuity in the Arrhenius plot of succinate-cytochrome c reductase is induced by some change in the physical properties of the membrane lipids. In contrast, mitochondrial ATPase appears insensitive, in terms of its thermal behaviour, to changes occurring in the composition of the membrane lipids. However, the specific activity of the mitochondrial ATPase was affected by the dietary lipid treatment being highest for the rats fed the sheep kidney fat diet. No dietary lipid effect was observed for the specific activity of succinate-cytochrome c reductase. This differential response of the two mitochondrial membrane enzymes to dietary-induced changes in membrane lipid composition may affect mitochondrial oxidative phosphorylation.

Dietary lipid modulation of rat liver mitochondrial succinate: cytochrome c reductase.

Diets supplemented with high levels of either saturated fatty acids or unsaturated fatty acids were fed to adult rats for a period of 9 weeks and changes in the liver mitochondrial membrane phospholipid fatty acid composition and thermal behaviour of succinate: cytochrome c reductase were determined. The dietary treatment induced a change in the omega 6 to omega 3 unsaturated fatty acid ratio in the membrane lipids, with the ratio being highest with the unsaturated fatty acid and lowest with the saturated fatty acid diet. Arrhenius plots of succinate: cytochrome c reductase activity exhibited differences in both critical temperature (Tf) and Arrhenius activation energy (Ea) depending on the type of dietary treatment. The Tf was elevated from 23 degrees C in control to 32 degrees C in the saturated fatty acid-supplemented group. No significant effect on the Tf was observed in the unsaturated fatty acid-supplemented group however higher Ea values were observed due to the unsaturated fatty acid diet. The changes in succinate: cytochrome c reductase are probably due to changes in the lipid-protein interactions in the membrane, induced by the dietary lipid supplementation.

The influence of water on the phase transition of sheep lung surfactant. A possible mechanism for surfactant phase transitions in vivo.

The effect of water on the thermal properties of sheep lung surfactant lipids was determined by differential scanning calorimetry. Dry surfactant exhibited a phase transition with an upper limit of about 54 degrees C, whereas that of the fully hydrated surfactant was about 30 degrees C. The effect of water was confined to a range of hydration values from 0 to 25%. The results indicate that pulmonary surfactant lipids are capable of undergoing both thermotropic and lyotropic mesomorphism in vitro. The degree of hydration of the surfactant could influence its in vivo biophysical role in alveolar dynamics. Indeed, small changes in the surfactant to water ratio induced by regional differences in the surfactant concentration at the alveolar surface during alveolar expansion and contraction could be sufficient to trigger isothermal phase transitions in the surfactant lipids. This would allow changes to occur in the equilibrium between solidus and fluidus surfactant during the respiratory cycle.

The effect of dietary lipids on the thermotropic behaviour of rat liver and heart mitochondrial membrane lipids.

Diets supplemented with relatively high levels of either saturated fatty acids derived from sheep kidney fat (sheep kidney fat diet) or unsaturated fatty acids derived from sunflower seed oil (sunflower seed oil diet) were fed to rats for a period of 16 weeks and changes in the thermotropic behaviour of liver and heart mitochondrial lipids were determined by differential scanning calorimetry (DSC). The diets induced similar changes in the fatty acid composition in both liver and heart mitochondrial lipids, the major change being the omega 6 to omega 3 unsaturated fatty acid ratio, which was elevated in mitochondria from animals on the sunflower seed oil diet and lowered with the mitochondria from the sheep kidney fat dietary animals. When examined by DSC, aqueous buffer dispersions of liver and heart mitochondrial lipids exhibited two independent, reversible phase transitions and in some instances a third highly unstable transition. The dietary lipid treatments had their major effect of the temperature at which the lower phase transition occurred, there being an inverse relationship between the transition temperature and the omega 6 to omega 3 unsaturated fatty acid ratio. No significant effect was observed for the temperature of the higher phase transition. These results indicate that certain domains of mitochondrial lipids, probably containing some relatively higher melting-point lipids, independently undergo formation of the solidus or gel phase and this phenomenon is not greatly influenced by the lipid composition of the mitochondrial membranes. Conversely, other domains, representing the bulk of the membrane lipids and which probably contain the relatively lower melting point lipids, undergo solidus phase formation at temperatures which reflect changes in the membrane lipid composition which are in turn, a reflection of the nature of the dietary lipid intake. These lipid phase transitions do not appear to correlate directly with those events considered responsible for the altered Arrhenius kinetics of various mitochondrial membrane-associated enzymes.

The influence of dietary lipid supplementation on cardiac beta-adrenergic receptor adenylate cyclase activity in the marmoset monkey.

Dietary lipid supplements high in either saturated fat derived from sheep kidney fat or unsaturated fat derived from sunflower seed oil, and a low mixed fat reference diet were fed to marmoset monkeys for 20 months and the effects on cardiac membrane lipid composition, and myocardial catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor binding activity were investigated. For cardiac membranes enriched for beta-adrenergic binding activity, the dietary lipid treatment resulted in small changes in the proportion of saturated to unsaturated fatty acids and substantial changes in the (n - 6) to (n - 3) series of unsaturated fatty acids in the membrane phospholipids. The sheep kidney fat diet increased the cholesterol-to-phospholipid ratio in cardiac membranes in comparison to the other diets. This diet also significantly elevated basal and isoproterenol-, epinephrine- and norepinephrine-stimulated adenylate cyclase activity. The value of the dissociation constant (Kd) and the receptor number (Bmax) for the binding of [125I]ICYP to the beta-adrenergic receptor was significantly reduced in marmosets fed the sheep kidney fat diet. These results suggest that dietary lipids can influence the activity of the beta-adrenergic/adenylate cyclase system of the heart. Modulation of this transmembrane signalling system may be induced by changes in the properties of the associated membrane lipids, particularly by alteration in the membrane cholesterol-to-phospholipid ratio. This effect may be limited to those animal species in which the nature of the dietary fatty acid intake may be influencing cardiac membrane cholesterol homeostasis, which is in agreement with previous results in rats following dietary cholesterol supplementation (McMurchie et al. (1987) Biochim. Biophys. Acta 898, 137-153). ICYP, (-)-iodocyanopindolol.

Response of rat heart membranes and associated ion-transporting ATPases to dietary lipid.

The effects of different dietary fat intake on the lipid composition and enzyme behaviour of sarcolemmal (Na+ + K+)ATPase and sarcoplasmic reticulum Ca2+-ATPase from rat heart were investigated. Rat diets were supplemented with either sunflower seed oil (unsatd./satd. 5.6) or sheep kidney fat (unsatd./satd. 0.8). Significant changes in the phospholipid fatty acid composition were observed in both membranes after 9 weeks dietary lipid treatment. For both membranes, the total saturated/unsaturated fatty acid levels were unaffected by the dietary lipid treatment, however the proportions of the major unsaturated fatty acids were altered. Animals fed the sunflower seed oil diet exhibited an increase in n-6 fatty acids, including linoleic (18:2(n-6] and arachidonic (20:4(n-6] while the sheep kidney fat dietary rats were higher in n-3 fatty acids, principally docosahexaenoic (22:6), with the net result being a higher n-6/n-3 ratio in the sunflower seed oil group compared to sheep kidney fat dietary animals. Fluorescence polarization indicated that the fluidity of sarcoplasmic reticular membrane was greater than that of sarcolemmal membrane, with a dietary lipid-induced decrease in fluidity being observed in the sarcoplasmic reticular membrane from sheep kidney fat dietary animals. Despite these significant changes in membrane composition and physical properties, neither the specific activity nor the temperature-activity relationship (Arrhenius profile) of the associated ATPases were altered. These results suggest that with regard to the parameters measured in this study, the two ion-transporting ATPases are not modulated by changes which occur in the membrane lipid composition as a result of the diet.

Incorporation and effects of dietary eicosapentaenoate (20:5(n-3)) on plasma and erythrocyte lipids of the marmoset following dietary supplementation with differing levels of linoleic acid.

The effect of dietary eicosapentaenoic acid (EPA, 20:5(n-3), as the ethyl ester) on plasma lipid levels and the incorporation of EPA into erythrocyte and plasma lipids were investigated in the marmoset monkey. Marmosets were fed high mixed-fat diets (14.5% total fat) supplemented with or without 0.8% EPA for 30 weeks. Markedly elevated plasma cholesterol (16.4 mmol/l) was induced by an atherogenic-type diet but with EPA supplementation, plasma cholesterol increased to only 6.6 mmol/l. Plasma triacylglycerol levels were not elevated with an atherogenic type diet. Substantial EPA incorporation was evident for plasma phospholipid, triacylglycerol and cholesterol ester fractions. The proportion of docosapentaenoic acid (22:5(n-3)) but not docosahexaenoic acid (22:6(n-3)) was also elevated in these plasma lipid fractions. Greatest incorporation of EPA occurred when it was administered with an atherogenic type diet having a P:M:S (polyunsaturated:monounsaturated:saturated) fatty acid ratio of about 0.2:0.6:1.0 in comparison to the control diet of 1.0:1.0:1.0. Incorporation of EPA and 22:5(n-3)) into erythrocyte phospholipids was also apparent and this was at the expense of linoleic acid (18:2(n-6)). These results in the marmoset highlight both the cholesterol-lowering properties of EPA and the extent of its incorporation into plasma lipids and erythrocyte membrane phospholipids with far greater incorporation occurring when the level of dietary linoleic acid was reduced.

Prevention of nerve conduction deficit in diabetic rats by polyunsaturated fatty acids.

The influence of diets containing gamma-linolenic acid (GLA; 18:3n-6) on sciatic nerve conduction velocity (NCV) was determined in diabetic rats. NCV was lower in diabetic rats fed diets supplemented with olive oil or sunflower seed oil than in nondiabetic rats; rats supplemented with GLA during a 5-wk diabetic period, however, did not exhibit significantly lower NCV. The mean proportion of the phospholipid fatty acid linoleic acid (18:2n-6) was higher in the sciatic nerves of diabetic rats than in the nondiabetic groups irrespective of dietary lipid treatment. Additionally, the proportion of linoleic acid was higher in the diabetic rats fed sunflower oil than in all other groups. Dietary GLA supplementation did not significantly influence the fatty acid composition of nerve membrane phospholipids and there was no obvious correlation between the fatty acid composition of nerve membrane phospholipids and NCV. The content of fructose and glucose in sciatic nerves was higher, whereas that of myo-inositol was lower, in diabetic rats than in nondiabetic rats; however, this was not significantly influenced by dietary GLA. GLA administration did not significantly influence Na(+)-K(+)-exchanging ATPase or ouabain binding activity in sciatic nerve preparations, both of which remained nonsignificantly different in the diabetic and nondiabetic groups. The results suggest that dietary GLA can prevent the deficit in NCV induced by diabetes and that this effect is independent of the nerve phospholipid fatty acid profile, sugar and polyol content, Na(+)-K(+)-exchanging ATPase activity, and ouabain binding. GLA may prevent the deficit in NCV indirectly, possibly by its role as a precursor of vasodilatory prostaglandins. These results confirm that GLA is the active component of evening primrose oil.

Dietary-induced changes in the fatty acid composition of human cheek cell phospholipids: correlation with changes in the dietary polyunsaturated/saturated fat ratio.

Healthy normotensive volunteers aged 20 to 59 yr were randomly allocated either to a control group or to one of two experimental groups. The control group ate a low P/S ratio diet for 12 wk while the first experimental group ate a high P/S ratio diet for 6 wk followed by a low P/S ratio diet for the next 6 wk. The second experimental group ate a low P/S ratio diet in the first 6 wk followed by a high P/S ratio diet for the next 6 wk. Dietary P/S ratio, plasma linoleic acid (18:2), and cheek cell phospholipid 18:2 levels were compared in each dietary group at the end of the 1st and 2nd 6 wk. On change from a low to a high P/S ratio diet, there was a 36% increase in the proportion of 18:2 in the cheek cell phospholipids in comparison with the proportion existing before the change. This was associated with an increase in the proportion of 18:2 in the plasma lipids of this group. No reduction in the proportion of 18:2 in the cheek cell phospholipids was apparent in the control group or the group which changed from a high to a low P/S ratio diet, although in the latter group there was a reduction in the proportion of 18:2 in the plasma lipids. As the phospholipid fatty acid composition of human cheek cells reflects dietary lipid status under certain conditions, this observation may be useful in dietary and nutritional studies, particularly as human cheek cells can be obtained in a noninvasive manner.

Effect of a high fat/cholesterol diet with or without eicosapentaenoic acid on plasma lipids, lipoproteins and lipid transfer protein activity in the marmoset.

Marmosets fed a diet supplemented with 0.2% cholesterol and 10% sheep fat (by weight) developed hypercholesterolemia with a 4-fold increase in plasma cholesterol (4.28 +/- 0.57-16.38 +/- 4.22 mmol/l, mean +/- SD, P less than 0.001). This was due mainly to a 5-fold increase in the intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) fraction (d = 1.006-1.063 g/ml). The proportion of plasma cholesterol in high density lipoproteins (HDL) decreased from 56% to 25% although HDL cholesterol increased from 2.40 +/- 0.42 to 4.09 +/- 0.92 mmol/l (P less than 0.001), and HDL particle radius increased from 5.10 +/- 0.18 nm to 6.06 +/- 0.73 nm (P less than 0.05). Plasma lipid transfer protein (LTP) activity increased 2.5-fold in whole plasma and 2-fold in lipoprotein-deficient plasma. The atherogenic lipoprotein profile was attenuated by adding 0.8% eicosapentaenoic acid (EPA, 20:5 n - 3, as the ethyl ester) to the atherogenic diet. Plasma cholesterol increased only 55% to 6.64 +/- 2.55 mmol/l with only an 80% increase in lipoproteins in the d = 1.006-1.063 g/ml fraction and a more favourable proportion of plasma cholesterol in HDL (44%) than without EPA. LTP activity was reduced to 1.7-fold above control in whole plasma by addition of EPA to the atherogenic diet. There was a positive correlation between plasma cholesterol and LTP activity in whole plasma (r = 0.89, P less than 0.001) and in lipoprotein-deficient plasma (r = 0.67, P less than 0.001). EPA therefore attenuated some of the adverse effects of a 0.2% cholesterol, 10% sheep fat diet on plasma lipids and lipoproteins and induced a less atherogenic profile.

Species variation in the ouabain sensitivity of cardiac Na+/K+-ATPase. A possible role for membrane lipids.

The role of membrane lipid composition on the modulation of ouabain sensitivity of cardiac Na+/K+-ATPase has been studied in vitro using several animal species. The animals can be grouped as ouabain-sensitive and ouabain-insensitive species. Ouabain-sensitive species (I50; 0.5-2.2 microM) include sheep, marmoset, pig and the guinea pig, whilst rat and mouse form the ouabain-insensitive group (I50; 100-105 microM). Although no species variation in the distribution of major phospholipid classes was observed, significant differences were apparent in the proportions of certain saturated and unsaturated phospholipid fatty acids. Thus, there was a marked increase in the relative proportion of docosahexaenoic (22:6, omega-3) acid in the Na+/K+-ATPase preparations from the rat and mouse compared to ouabain-sensitive species. Despite these differences, all animals had similar proportions of total saturated (sigma SAT) and total unsaturated (sigma Unsat) fatty acids. On the other hand, a good correlation between the unsaturation index of membrane lipids and I50 value for ouabain was observed. It is proposed that acyl chain characteristics (unsaturation and/or chain length) rather than the head group of the phospholipid molecule play a major role in the modulation of Na+/K+-ATPase to inhibition by ouabain.

Membrane fluidity changes are associated with the antiarrhythmic effects of docosahexaenoic acid in adult rat cardiomyocytes.

Previous studies using neonatal rat cardiomyocytes have reported antiarrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs). In this study, we examined the effects of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) on the spontaneous contractile activity and membrane fluidity of adult rat ventricular myocytes. Cardiomyocytes were induced to contract spontaneously by continuous superfusion of a solution containing the arrhythmogenic agents isoproterenol (a beta-adrenergic receptor agonist) or lysophosphatidylcholine. The percentage of cardiomyocytes displaying spontaneous contractions induced by isoproterenol when pretreated with the saturated fatty acid docosanoic acid was 48.1 +/- 7.7%; the percentage for cardiomyocytes pretreated with DHA was 7.1 +/- 2.4% (P < 0.01). DHA significantly prevented lysophosphatidylcholine-induced spontaneous contractions (17.7 +/- 6.5%) compared with treatment with the saturated fatty acid stearic acid (78.0 +/- 7.3%, P < 0.01). The membrane fluidizing agent benzyl alcohol also significantly prevented spontaneous contractions in cardiomyocytes. Membrane fluidity was determined by steady-state fluorescence anisotropy (r(ss)) using the fluorescent probe N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl) trimethyl-ammonium p-toluene-sulfonate (TMAP-DPH). DHA and benzyl alcohol dose-dependently decreased the r(ss); however, saturated fatty acids were without effect. These results suggest that the antiarrhythmic mechanisms of the n-3 PUFAs such as DHA may involve changes in membrane fluidity.

Antiarrhythmic fatty acids and antioxidants in animal and cell studies.

From the animal and cellular studies that will be discussed in this review, it is apparent that dietary fatty acids and antioxidants play an important role in influencing the development of ventricular tachycardia and potentially lethal ventricular fibrillation. It is this latter disturbance to the rhythmic beating of the heart that is responsible for much of the mortality from coronary heart disease. It is now recognized that diets high in certain polyunsaturated fatty acids (PUFAs) and diets containing antioxidants can afford considerable protection to the heart with regard to the generation of disorders of contractile rhythmicity. The mechanism by which such dietary components confer their cardioprotective effects are now being intensively investigated, particularly with respect to their possible effects on the molecular mechanisms underlying the excitation-contraction coupling process of the myocardial cell. This overview will cover recent studies that have focused on the antiarrhythmic role of PUFAs, particularly those of the n-3 (or omega 3) class with emphasis on experiments performed using laboratory animals, isolated heart preparations, and isolated heart cells (cardiomyocytes). The role of free radicals (reactive oxygen species) and antioxidants in disorders of cardiac rhythm also will be addressed within the perspective of reperfusion injury to the myocardium following ischemia. Emphasis will be placed on the cardioprotective role of nutritional factors and components and the possible cellular mechanisms by which such components may act.

Effects of dietary n-3 fatty acids on contractility, Na+ and K+ currents in a rat cardiomyocyte model of arrhythmia.

The n-3 polyunsaturated fatty acids (PUFAs) have been reported to prevent ventricular fibrillation in human clinical studies and in studies involving experimental animals and isolated cardiomyocytes. This study aimed to determine whether dietary n-3 PUFAs could prevent isoproterenol and free radical-induced arrhythmic (asynchronous) contractile activity in adult rat cardiomyocytes and whether whole-cell Na(+) and K(+) currents measured by patch-clamp techniques were affected. Dietary supplementation with fish oil for 3 weeks significantly increased the proportion of total n-3 PUFAs in ventricular membrane phospholipids compared with saturated fat supplementation (18.8 +/- 0.6% vs. 8.1 +/- 1.0%, respectively). Cardiomyocytes from the fish oil group were less susceptible to isoproterenol-induced asynchronous contractile activity than were those from the saturated fat group [EC(50) values: 892 +/- 130 nM, n = 6 and 347 +/- 91 nM, n = 6 (P < 0.05), respectively]. Fish oil supplementation also prolonged the time taken to develop asynchronous contractile activity induced by superoxide and hydrogen peroxide. The voltage dependence of inactivation of Na(+) currents were significantly altered (-73.5 +/- 1.2 mV, n = 5 vs. -76.7 +/- 0.7 mV, n = 5, P < 0.05, for saturated fat and fish oil treated groups, respectively). The voltage dependence of activation of Na(+) and K(+) currents was not significantly affected by the dietary fish oil treatment. These results demonstrate the antiarrhythmic effects of dietary fish oil in a cardiomyocyte model of arrhythmia.

An apparatus to assay opioid activity in the infused lumen of the intact isolated guinea pig ileum.

A modified apparatus is described that provides for the simultaneous bathing of the serosa of an intact piece of isolated guinea pig ileum while allowing infusion of the isolated lumen. The comparative compartmental potency of the opioid agonists morphine, casomorphins, and enkephalins to inhibit electrically driven contractions are described in this system. The rank-order potency for serosally applied opioid agonists was (IC(50) values, nM): [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO) (15)>[D-Ala(2),D-Leu(5)]-enkephalin (DADLE) (35)> or =morphine (46)> or =[D-Ala(2)]-met-enkephalinamide (55)>[D-Ala(2)]-beta-casomorphin[1--4] amide (122)>beta-casomorphin[1--4] amide (940)>met- and leu-enkephalin (>6000). This contrasted to the rank-order potency for the luminally applied opioid agonists: DADLE (63)>DAMGO (135)>[D-Ala(2)]-met-enkephalinamide=morphine (4700)>[D-Ala(2)]-beta-casomorphin[1--4] amide (29000). beta-Casomorphin[1--4] amide, leu-enkephalin and met-enkephalin are mostly inactive when applied luminally. Furthermore, the opioid antagonists, casoxin 4 and [D-Ala(2)]-casoxin 4, when infused into the lumen, significantly overcame the inhibitory effect of morphine added to the serosal side. This model provides an assay and screening system to differentiate between the effects of chemical agents applied via the blood stream (serosa) or food side (lumen) on quiescent or electrically driven gut activity of the nervous plexi or receptor systems of the ileum.

Sodium transport activity in cheek epithelial cells from adolescents at increased risk of hypertension.

Sodium transport including amiloride-sensitive Na+/H+ antiporter activity was measured in cheek epithelial cells of adolescents displaying either high or low BP tracking characteristics and in a subgroup of high BP tracking adolescents exhibiting a positive family history of hypertension. From the BP tracking behaviour of over 500 adolescents measured over a period of three years, 24 low BP tracking and 29 high BP tracking adolescents were recruited for the study. Cheek cells were collected from these subjects and proton-dependent, amiloride-sensitive Na+/H+ antiporter activity and the response of this antiporter to a proton gradient were measured. Cheek cell Na+/H+ antiporter activity was 50% lower (P = 0.0004) in the high BP tracking group (1.02 +/- 0.15 nmol Na+/mg protein/5 min (mean +/- SEM) compared with the activity in the low BP tracking group (2.05 +/- 0.24). A significantly lower Na+/H+ antiporter activity (69%; P < 0.01) was also apparent in the high BP tracking adolescents with family history of hypertension (n = 7) compared with the low BP tracking group. The graded response of cheek cell Na+/H+ antiporter activity to the proton gradient was 58% lower (P = 0.0039) for adolescents in the high BP tracking group compared with the low BP tracking group. Passive Na+ influx was also significantly lower in the cheek cells of the high BP tracking group. Our results therefore show that the activity of the Na+/H+ antiporter in cheek cells and the passive Na+ transport activity are lower in those adolescents considered at greatest risk of future development of essential hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of lung lamellar body phospholipids from premature and term infants: is sphingomyelin a contaminant of surfactant?

Lamellar bodies were isolated from homogenates of lungs obtained at autopsy from premature infants (n = 9), and compared to lamellar bodies from three full term reference groups including 55 infants with sudden infant death syndrome (SIDS), 14 control infants, and 3 who succumbed to intra-uterine death (IUD) at term. Analysis of the phospholipids was expected to reveal a high proportion of sphingomyelin (Spm) in the lamellar bodies isolated from the lungs of premature infants (24 to 33 weeks gestational age) since at this stage of their development the percentage of Spm in the amniotic fluid is high. However, Spm was either absent or a small proportion of the total phospholipid in lamellar body surfactant from these premature infants. Spm was also a small percentage of phospholipid in the lamellar body surfactant of infants with SIDS and from control and IUD specimens. It is suggested that Spm may not be a normal constituent of lamellar body surfactant but rather a contaminant from membranes.