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.

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.

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.

Properties and regulation of a trans-plasma membrane redox system of perfused rat heart.

Ferricyanide was reduced to ferrocyanide by the perfused rat heart at a linear rate of 78 nmol/min per g of heart (non-recirculating mode). Ferricyanide was not taken up by the heart and ferrocyanide oxidation was minimal (3 nmol/min per g of heart). Perfusate samples from hearts perfused without ferricyanide did not reduce ferricyanide. A single high-affinity site (apparent Km = 22 microM) appeared to be responsible for the reduction. Perfusion of the heart with physiological medium containing 0.5 mM ferricyanide did not alter contractility, biochemical parameters or energy status of the heart. Perfusate flow rate and perfusate oxygen concentration exerted opposing effects on the rate of ferricyanide reduction. A net decreased reduction rate resulted from a decreased perfusion flow rate. Thus, the rate of supply of ferricyanide dominated over the stimulatory effect of oxygen restriction; the latter effect only becoming apparent when the oxygen concentration was lowered at a high perfusate flow rate. Whereas glucose (5 mM) increased the rate of ferricyanide reduction, pyruvate (2 mM), acetate (2 mM), lactate (2 mM) and 3-hydroxybutyrate (2 mM) each had no effect. Insulin (3 nM), glucagon (0.5 microM), dibutyryl cyclic AMP (0.1 mM) and the beta-adrenergic agonist ritodrine (10 microM) also had no effect, however, the alpha 1-adrenergic agonist, methoxamine (10 microM), produced a net increase in the rate of ferricyanide reduction. It is concluded that a trans-plasma membrane electron efflux occurs in perfused rat heart that is sensitive to oxygen supply, glucose, perfusion flow rate, and the alpha-adrenergic agonist methoxamine.

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.

Co-ordinated regulation of muscle glycolysis and hepatic glucose output in exercise by catecholamines acting via alpha-receptors.

Recent findings indicate that glucose uptake by contracting hindlimb #Acta Physiol. Scand. (1982) 116, 215-222 #and heart #Biochem. Biophys. Res. Commun. (1982) 108, 124-131 # of the rat is stimulated by epinephrine acting through alpha-adrenergic mechanisms. Since in exercise hepatic glucose output may be increased markedly by activation of alpha-adrenergic receptors and matched by the increase in muscle glucose uptake (maintaining blood glucose levels relatively constant), it is now proposed that a general coordination of glucose metabolism may operate via alpha-adrenergic receptor mechanisms. The basis for this proposal is discussed.

Obesity and the regulation of phosphofructokinase in heart: an apparent insensitivity to adrenergic activation in mature-age genetically obese rats.

The activity ratio of phosphofructokinase in perfused rat heart and its activation by epinephrine was examined in non-obese, fat-fed obese, and genetically obese rats. For non-obese colony rats there was an age-dependent increase in the activity ratio of phosphofructokinase from 0.2 at 40 days to 0.4 at mature age (greater than 200 days). Epinephrine (10 microM) treatment of the heart for 5 min increased the ratio at all ages but the proportional increase diminished with age. For mature-age lean Zucker rats carrying the genetic determinant for obesity the results were similar to those obtained for comparable non-obese colony rats. For fat-fed obese rats the activity ratio of phosphofructokinase at 200 days of age was 0.2 and was increased to 0.6 by epinephrine treatment. For mature-age obese Zucker rats the activity ratio was 0.2 and no significant response to epinephrine occurred. The activity ratio of glycogen phosphorylase and its response to epinephrine (beta-adrenergic receptor mediated) in heart was unaffected by age, diet or the gene for obesity. The present findings indicate a specific defect in the adrenergic regulatory mechanism for phosphofructokinase in genetically obese rats.

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)

Cilazapril and dietary gamma-linolenic acid prevent the deficit in sciatic nerve conduction velocity in the streptozotocin diabetic rat.

Young adult male Hooded Wistar rats were rendered diabetic by administration of streptozotocin and maintained for 5 weeks on a diet containing either 6% olive oil as the total source of fat (OO diet), or purified gamma-linolenic acid (GLA) at a concentration of 0.5% with the remaining 5.5% provided by olive oil (GLA diet). Rats were treated with the angiotensin converting inhibitor, cilazapril, administered in the drinking water at a dose of 20 mg kg-1 body weight day-1. For the OO diet groups, sciatic nerve conduction velocity (NCV) in diabetic rats was reduced by 32% (p < 0.01) in comparison with nondiabetic (vehicle-treated) rats and 27.5% (p < 0.05) in comparison with diabetic rats treated with cilazapril. Diabetic, cilazapril-treated rats showed no reduction in NCV. For the nondiabetic, diabetic, and diabetic plus cilazapril groups fed GLA, the NCV was not significantly different, indicating that dietary GLA also prevented the deficit in the NCV induced by the diabetic state. Analysis of the sciatic nerve endoneurial phospholipid fatty acids revealed a significant reduction in the proportion of GLA and an elevation in the proportion of linoleic acid in the diabetic groups compared with the nondiabetic groups and this was independent of the cilazapril treatment or the dietary lipid supplement. Sciatic nerve myo-inositol content was unaltered while mannose, fructose, glucose, and sorbitol levels were elevated in the diabetic groups and these changes were independent of the cilazapril treatment or the dietary lipid supplement. These results indicate that in the rat, cilazapril treatment or dietary GLA, at the doses tested, are effective in preventing the deficit in the NCV induced by diabetes.

The effect of dietary supplementation with eicosapentaenoic acid on the phospholipid and fatty acid composition of erythrocytes of marmoset.

Adult male marmoset monkeys were fed eicosapentaenoic acid (20:5n-3) as the ethyl ester in diets containing either 32% (reference diet, no added cholesterol) or 7% (atherogenic diet with 0.2% added cholesterol) linoleic acid (18:2n-6) for 30 wk. No changes were seen in the level of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) but minor changes were observed in both the sphingomyelin (SPM) and phosphatidylinositol plus phosphatidylserine (PI+PS) fractions of erythrocyte lipids. The extent of total n-3 fatty acid incorporation into membrane lipids was higher in atherogenic diets (polyunsaturated/monounsaturated/saturated (P/M/S) ratio 0.2:0.6:1.0) than reference diets (P/M/S ratio 1:1:1) and this was true for both PE (33.4 +/- 1.0% vs 24.3 +/- 1.1%) and PC (9.3 +/- 0.5% vs 4.9 +/- 0.3%). Although suitable controls for cholesterol effects were not included in the study, earlier results obtained with marmosets lead us to believe such effects were probably small. Regardless of basic diet (atherogenic, reference), 20:5n-3 was preferentially incorporated into PE (10.8 +/- 0.2%, 6.0 +/- 0.02%) while smaller amounts were incorporated into PC (6.9 +/- 0.4%, 3.2 +/- 0.2%). The major n-3 polyunsaturated fatty acid found in PE in response to dietary 20:5n-3 was the elongation metabolite 22:5n-3 in both the atherogenic (17.7 +/- 0.7%) and reference (14.3 +/- 1.0%) dietary groups; 22:6n-3 levels were less affected by diet (4.7 +/- 0.3% and 3.9 +/- 0.2%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+-mediated activation of phosphofructokinase in perfused rat heart.

Perfusion of the isolated rat heart with Ca2+ concentrations exceeding 3 mM activated phosphofructokinase and phosphorylase, and decreased the concentration of cyclic AMP. Half-maximal activation of phosphofructokinase occurred at 5 mM-CaCl2; significant activation of phosphorylase did not occur until the concentration of CaCl2 exceeded 12 mM. The time course for the activation of phosphofructokinase at 12 mM-CaCl2 indicated that maximal activation occurred within 2 min; when the perfusion-medium Ca2+ concentration was re-adjusted to 3 mM, the phosphofructokinase activity returned to pre-activation values within 30 s. The addition of Ca2+ to extracts of heart did not activate phosphofructokinase. The activation of phosphofructokinase by sub-maximal doses of adrenaline and Ca2+ were not additive. The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol and by 1 microM-isoprenaline was inhibited by high concentrations of K+ (22-56 mM). The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol, 12 mM-CaCl2 and by 1 microM-isoprenaline was blocked by the slow Ca2+-channel blocker nifedipine. These findings suggest that both the beta- and alpha-adrenergic mechanisms for the activation of rat heart phosphofructokinase involve an increase in the myoplasmic Ca2+ concentration. This increase may result from an inhibition of Ca2+ efflux or a stimulation of Ca2+ influx.

Epinephrine activation of phosphofructokinase in perfused rat heart independent of changes in effector concentrations.

Phosphofructokinase was determined at low substrate concentration using a new isotopic assay in extracts of perfused rat heart. Epinephrine treatment of the perfused heart resulted in an activation of the enzyme. Half-maximal activation of phosphofructokinase occurred at 5 X 10(-7) M epinephrine, which was approximately that required to produce half-maximal activation of phosphorylase. Time course studies indicated that epinephrine-mediated changes in beating rate, cyclic AMP concentration, and phosphorylase a activity were maximal at 1 to 2 min and preceded maximal activation of phosphofructokinase by approximately 3 min. the activated form of the enzyme as expressed in heart extracts was sensitized to the activators, cyclic AMP, AMP, glucose 1,6-bisphosphate, and fructose 1,6-bisphosphate. Passage of control extract that was untreated, activated by AMP, or inhibited by citrate through Sephadex G-25 columns gave eluate activities approaching control extract values. The epinephrine-activated form of the enzyme remained activated following similar treatment. The data suggest that epinephrine mediates a modification of phosphofructokinase that is independent of changes in intracellular effector concentration.

Adrenergic regulation of glucose metabolism in rat heart. A calcium-dependent mechanism mediated by both alpha- and beta-adrenergic receptors.

Epinephrine treatment of the perfused rat heart led to an increase in glucose uptake, detritiation of [5-3H] glucose, glycogenolysis, and the formation of lactate. The change in the rate of formation of 3H2O from [5-3H]glucose was slower to develop (commencing at approximately 30 s) than changes in cyclic AMP concentration, hexose-6-P concentration, and the phosphorylase a/(a + b) ratio which were maximal at 24 s. Epinephrine plus propranolol (alpha-adrenergic combination) treatment of the perfused heart also led to increases in glucose uptake, detritiation of [5-3H]glucose, and the formation of lactate, but these occurred without significant changes in cyclic AMP concentration, hexose-6-P concentration, or the phosphorylase a/(a + b) ratio. Half-maximal stimulation of glucose uptake occurred at 0.2 microM epinephrine, 1.5 microM methoxamine, and 1 microM isoproterenol. The increase in glucose uptake mediated by 1 microM epinephrine was blocked by 10 microM prazosin but unaffected by 10 microM propranolol. The increase in glucose uptake mediated by 10 microM epinephrine plus 10 microM propranolol was partly blocked by yohimbine and completely blocked by prazosin. A role for Ca2+ in the adrenergic regulation of glucose uptake was indicated by the sensitivity of the epinephrine dose curve to Ca2+ and the dependence of epinephrine on Ca2+. In addition the increases in glucose uptake mediated by 1 microM epinephrine, 1 microM epinephrine plus 10 microM propranolol, 1 microM isoproterenol, and by 10 mM CaCl2 were each blocked by the Ca2+ channel blocker nifedipine (1 microM). It is concluded that Ca2+-dependent alpha- and beta-adrenergic receptor mechanisms are present in rat heart for controlling glucose uptake. At submicromolar levels of epinephrine the predominant receptors utilized appear to be alpha 1.

Effects of dietary eicosapentaenoate (20:5 n-3) on cardiac beta-adrenergic receptor activity in the marmoset monkey.

Eicosapentaenoic acid (EPA; 20:5 n-3; ethyl ester) in combination with atherogenic or non-atherogenic high fat diets was fed to young adult male marmoset monkeys for a period of 30 weeks. EPA markedly reduced the raised plasma cholesterol level evident when feeding an atherogenic diet but did not influence the cardiac membrane cholesterol-to-phospholipid ratio. EPA and its elongation product 22:5 n-3 was incorporated into cardiac membrane phospholipids at the expense of linoleic and arachidonic acids. Dietary EPA increased cardiac beta-AR affinity and reversed the decreased beta-AR affinity evident when feeding an atherogenic diet.