Study in pig coronary smooth muscle cell subcellular fractions of the activity of various enzymes involved in lipid metabolism and of the beta-receptor adenylate cyclase couple (original) (raw)
Related papers
Journal of Molecular and Cellular Cardiology, 1987
19, 465-475. Three groups of male, weanling, Sprague--Dawley rats were fed diets containing 7% hydrogenated coconut oil, 6.6% hydrogenated coconut oil + 0.4% corn oil, or 7% corn oil for 8-17 weeks. These diets provided 0% (EFAD group), 0.5% (MEFAD group) or 5% (CONTROL group) of the total energy as linoleic acid, respectively. Crude plasma membranes were prepared from heart and assayed for adenylate cyclase activity. Both basal and fluoride-stimulated activity was lower in the membranes from EFAD and MEFAD rats than that of the controls. The double bond index of total lipids and phospholipids, and fluorescence polarization of 1,6-diphenyl-l,3,5-hexatriene (DPH) were not appreciably different in the membranes from the three dietary groups. The fatty acid composition of total phospholipids of the membranes, however, was quite different and indicative of biochemical changes typical of an EFA deficiency. Feeding of the control diet to the EFAD or MEFAD rats for up to 6 weeks did not alleviate completely the changes in adenylate cyclase activity although the fatty acid patterns were restored to the normal levels. There was also a decrease in the number of [3H]-DHA binding sites in heart of EFAD rats as compared with their controls. The results suggest that the changes induced by EFA deficiency in the acyl group composition of membrane phospholipids and in the number of beta-adrenergic receptors may be important in regulating adenylate cyclase activity in the heart. activity, [SH]-Dihydroalprenolol binding and fatty acid composition of rat heart membranes in essential fatty acid deficiency [Abstract] Fed Proc 45, 1026 (1986) #5069.
Effect of dietarytrans fatty acids on some membrane-associated enzymes and receptors in rat heart
Lipids, 1989
Three groups of male weanling Sprague~Dawley rats were fed diets containing 20% corn oil, 20% partially hydrogenated soybean oil (PHSBO) or 18% PHSBO + 2% corn oil. PHSBO contained about 48% of its total fatty acids as trans-octadecenoate. Rats were killed after 16-18 weeks of feeding the various diets, hearts were dissected and crude sarcolemma was prepared by differential centrifugation. The activities of ouabain-sensitive (Na + + K+)ATPase were significantly lower in membranes of rats fed 20% PHSBO than the control rats fed 20% corn oil. The feeding of 2% corn oil with 18% PHSBO resulted in partial restoration of the enzyme activity. The maximum number of [3H]ouabain-binding sites {Bmax) was also lower in cardiac membranes of rats fed 20% PHSBO than those fed 20% corn oil. Similar to {Na + + K+) -ATPase activity, some restoration of the number of [3H]ouabain-binding sites was observed when 2% corn oil was fed with 18% PHSBO-containing diet. There was no difference in the binding affinity of the radioligand for the receptor among the 3 dietary groups. Adenylate cyclase activities (fluoride-, isoproterenol-and forskolinstimulated) were lower in membranes of rats fed 20% PHSBO or 18% PHSBO + 2% corn oil than in the control group fed 20% corn oil. Density of the f3-adrenergic receptor was the lowest in cardiac membranes of rats fed 20% PHSBO. The feeding of 2% corn oil with 18% PHSBO resulted in partial restoration of the maximum number of [3H]dihydroalprenolol (DHA)-binding sites. The affinity of the binding sites was, however, not affected by the type of the dietary fat. The results of this study suggest that dietary trans fatty acids can affect the activities of (Na + + K+)ATPase and adenylate cyclase and the density of digitalis and ~-adrenergic receptors in rat heart.
Journal of Molecular and Cellular Cardiology, 1985
G. P. HEATHERS, N. AL-MUHTASEB AND R. V. BRUNT. The Effect of Adrenergic Agents on the Activities of Glycerol 3-phosphate Acyltransferase and Triglyceride Lipase in the Isolated Rat Heart. Journal of Molecular and Cellular Cardiology (1985) 17, 785-796. Glycerol 3-phosphate acyltransferase (GPAT) activity and triglyceride lipase (TGL) activity were measured in homogenates from hearts perfused with adrenergic agonists and antagonists. Perfusion with adrenalin or the fl-agonist isoprenaline produced an increase in TGL activity and a fall in GPAT activity. These changes could be imitated by incubation of heart homogenates with cAMPdependent protein kinase. The ~2-agonist clondine produced the opposite effect, thus it increased GPAT activity and decreased TGL activity. Methoxamine, an cq-agonist, had no effect on TGL activity but reduced GPAT activity. Continuous perfusion of the fl-antagonist atenolol reduced TGL activity to half that found in controls but also reduced GPAT activity. No change was seen on continuous perfusion ofc~ 1-or ~z-antagonists. Changes in GPAT activity were localized mainly in the microsomal enzyme. These changes are consistent with both enzymes being regulated via a cyclic-AMP dependent protein kinase system and via ~-adrenergic mechanisms.
Archives of Biochemistry and Biophysics, 1978
Subcellular fractions of rat skeletal muscle enriched in plasma membrane (sarcolemma) were used to characterize properties of the fl-adrenergie receptor and adenylate cyelase. The equilibrium binding of (-)-[:~H]dihydroalprenolol to the sarcolemmal membrane was characterized by a high affinity, K = 3.6 • 10 ~ liters/tool and 1.2 • 10 ~ mol/g of membrane protein of homogeneous noninteracting sites. The binding was reversible, saturable, and stereospecific. Displacement of labeled dihydroalprenolol by adrenergic ligands revealed an order of potency corresponding to the fie-type response: (-)-isoproterenol > (-)-epinephrine > norepinephrine. The (+)-stereoisomers and the a-adrenergic antagonist phentolamine showed little ability to compete with (-)-[:~H]dihydroalprenolol for occupancy of the binding sites. In the membrane fractions, a substantial basal activity of adenylate cyclase activity was demonstrated (35-50 pmol of cyclic AMP/mg/5 min). This activity was stimulated 40-to 50-fold by sodium fluoride, 7-to 8-fold by isoproterenol, and 5-to 6-fold by guanylyl 5'-imidodiphosphate [Gpp(NH)p]. Maximum activation depended upon optimal Mg ~+ ion concentration (I0 mM). In the presence of 0.1 mM Gpp(NH)p, the isoproterenol activation of adenylate cyclase was increased twofold. In addition, the concentration of hormone required for half-maximal stimulation of enzyme activity was shifted from 2.2 • 10-~ M in the absence of Gpp(NH)p to 2.5 • I0 ~ M in the presence of Gpp(NH)p. Gpp(NH)p also stimulated epinephrine and norepinephrine activation without altering the fl2-type potency series for the enzyme. Gpp(NH)p had no effect on either the number or the affinity of (-)-[:JH]dihydroalprenolol binding sites, but it did increase the dissociation constant for isoproterenol by approximately fivefold. Deceased. This paper represents the last investigations carried out by Dr. Stuart P. Grefrath. His warmth, friendship, and critical acumen will be missed.
Subcellular fractionation of pig coronary artery smooth muscle
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1985
A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the ]proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is dE;scribed. A number of marker enzymes and Ca 2 ÷ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide-or ruthenium red-insensitive Ca2 + uptake, and the plasma membrane markers K+-activated ouabain-sensitive pnitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) ,which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and t,a a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca 2+ uptake. 173 fraction (28-40% sucrose) was maximally enriched in the ATP-and oxalate-dependent azide-insensitive Ca 2÷ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide-or ruthenium red-sensitive ATP-dependent Ca 2÷ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca 2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca 2 ÷ uptake studies to understand the physiology of coronary artery vasodilation.
Modulation byn-alkanols of rat cardiac adenylate cyclase activity
The Journal of Membrane Biology, 1986
n-Alkanols (from methanol to decanol) have a biphasic effect on rat cardiac adenylate cyclase either basal or stimulated by GTP, GppNHp, NaF or hormones (isoproterenol, glucagon, secretin) in the presence of GTP. At high concentration, all the enzyme activities are inhibited. At low concentration, adenylate cyclase activity is either unchanged or potentiated depending on both the stimulus and the alkanols involved. Potentiation is due to an increase of maximum velocity with no change in the activation constant of the enzyme. Basal activity is unchanged as well as the isoproterenol-and glucagon-stimulated enzyme. The secretin-stimulated enzyme is potentiated. It is the guanyl nucleotide regulatory protein-mediated stimulation of adenylate cyclase which is mainly affected. An attempt was made to relate these effects on adenylate cyclase with physical parameters of the alkanols (partition coefficient). From the data obtained as a function of the alkanol chain-length and of temperature on the adenylate cyclase stimulated by GTP, GppNHp, NaF and permanently activated, it is concluded that the increase in efficacy observed in the presence of alkanol is due to an interaction with the protein moeity particularly with the guanyl nucleotide regulatory protein.
The Journal of Nutritional Biochemistry, 1994
The purpose of the present investigation was to determine if the diet-induced changes in cardiac adenylate cyclase activity and membrane lipids can be reversed. Three groups of male weanling Sprague-Dawley rats were fed purified diets containing different fats: 9% butter (Btr) + 1% corn oil (CO) (group 1), 10% CO (group II), and 9% ethyl ester concentrate of n-3 fatty acids (EEC) + 1% CO (group 111). After 5 weeks of feeding, rats from each group were killed. Cardiac membranes were prepared and assayed for adenylate cyclase activity. The fatty acid composition of membrane total phospholipids was also determined. The remaining rats in group 1 were divided into four subgroups and fed the following diets for the reversal study: 9% Btr + 1% CO (group la), 9% EEC + 1% CO (group lb), 10% CO (group Ic), and 7% Btr + 2% EEC + 1% CO (group Id). Rats in groups H and 111 were maintained on their original diets. Rats were killed 5 weeks after changing the dietary regimes, and membranes were prepared from heart and analyzed for their fatty acid composition of total phospholipids. Adenylate cyclase activity (basal, fluoride-and forskolin-stimulated) was also measured. The enzyme activity was lower in membranes of rats in group la than those in groups H or IlL Whereas the diet-induced changes in fatty acid composition were essentially reversed by dietary modification (groups Ib and lc ), the changes in adenylate cyclase activity were only partially reversed. (J. Nutr. Biochem. 5:106-112, 1994.)
Altered function of adenylate cyclase in the myocardium of the spontaneously hypertensive rat
Biochemical Pharmacology, 1978
The effects of Me, Mn'+, Ca'+, guanylylimidodiphosphate [Gpp(NH)p] and isoproterenol on adenylate cyclase activity in myocardial membrane preparations from spontaneously hypertensive rats (SHR) and Kyoto Wistar normotensive rats (WKY) were compared. The isoproterenol-st~ulated adenylate cyclase activity of myocardial membranes from WKY rats responded at a lower threshold (10 vs 200 nM) and with a lower EC 50 (300 nM vs 1 @M) and a higher maximal velocity. When the isoproterenol effect was studied in the presence of 1OOnM Gpp(NH)p, the threshold for isoproterenol was similar in SHR and WKY. However, the activity at each dose was significantly lower (P 2: 0.05) in SHR. In the presence of Gpp(NH)p alone, no differences were observed between SHR and WKY. Increasing Mg2+ and Mn*+ concentrations stimulated adenylke cyclase activity but no differences were observed between SHR and WKY for Mn'+ stimulation. However, the SHR myocardial activity was significantly reduced at Mg*+ concentrations ranging from 6 to 2.5 mM. The effect of varying the Mp concentration was further tested in the prsence of 1 FM isoproterenol. The adenylate cyclase activity of myocardia of SHR was significantly reduced at Me concentrations between 4 and 24 mM. These o. >rvations suggest that sensitivity of the adenylate cyclase to isoproterenol has been decreased in SdR.
Differential effects of fatty acids on glycolysis and glycogen metabolism in vascular smooth muscle
Biochimica Et Biophysica Acta-molecular Cell Research, 1991
The effects of fatty acids of different chain lengths on aerobic glycolysis, lactic acid production, glycogen metabolism and contractile function of vascular smooth muscle were investigated. Porcine carotid artery segments were treated with 50/LM iodoacetate and perchloric acid tissue extracts were then analyzed by 3tP-NMR spectroscopy to observe the accumulation of phosphorylated glycolytic intermediates so that the activity of the Embden-Myerhof pathway could be tracked under various experimental paradigms. Aerobic glycolysis and lactate production in resting arteries were almost completely inhibited with 0.5 mM octanoate, partially inhibited with 0.5 mM acetate and unaffected by 0.5 mM palmitate. Inhibition of glycolysis by octanoate was not attributable to inhibition of glucose uptake or glucose phosphorylation. Basal glycogen synthesis was unchanged with palmitate and acetate, but was inhibited by 52% with octanoate incubation. The characteristic glycogenolysis which occurs upon isometric contraction with 80 mM KCi in the absence of fatty acid in the medium was not demonstrable in the presence of any of the fatty acids tested. Glycogen sparing was also demonstrable in norepinephrine contractions with octanoate and acetate, but not with palmitate. Additionally, norepinephrine-stimulated isometric contraction was associated with enhanced synthesis of glycogen amounting to 6-times the basal rate in medium containing octanoate. Contractile responses to norepinephrine were attenuated by 20% in media containing fatty acids. Thus, fatty acids significantly alter metabolism and contractility of vascular smooth muscle. Fatty acids of different chain lengths affect smooth muscle differentially; the pattern of substrate utilization during contraction depends on the contractile agonist and the fatty acid present in the medium°