Diet fat influences liver plasma-membrane lipid composition and glucagon-stimulated adenylate cyclase activity.

Rats were fed diets that differed in fatty acid composition or in the proportion of energy derived from fat to determine if alteration of dietary fat intake influences the structural lipid composition of liver plasma membrane and the expression of an associated hormone-receptor-mediated function. Weanling rats were fed 9% (w/w) or 20% (w/w) low-erucic acid rape-seed oil or 9% (w/w) soya-bean oil for 24 days. Plasma membranes were isolated and the effect of diet fat on the fatty acid composition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and sphingomyelin was determined. Diet fat significantly altered total saturated and (omega-9) and (omega-6)-unsaturated fatty acid composition in addition to the (omega-6)- to (omega-3)-unsaturated fatty acid ratio in these polar lipids. Feeding the high-fat diet increased the (omega-6)- to (omega-3)-unsaturated fatty acid ratio and the (omega-9)-unsaturated fatty acid content in all lipids except sphingomyelin. Assay of glucagon-stimulated adenylate cyclase activity at both high and low glucagon concentrations indicated that high-fat intake also decreased cyclic AMP formation. In a second experiment the fat intake was held constant (40% of energy) and oleic acid was substituted for linoleic acid by blending high- and low-linoleic acid-type safflower oils. This experiment established that a dose-response relationship exists between dietary intake of fatty acid and the fatty acid composition of plasma-membrane phospholipids. Specific diet-induced transitions in membrane phospholipid fatty acid composition were paralleled by changes in glucagon-stimulated adenylate cyclase activity. This study suggests that transitions in dietary fat intake can alter a hormone-receptor-mediated enzyme function in vivo by changing the surrounding lipid environment.     

Developmental changes in the form of the Arrhenius plots of rat liver plasma membrane adenylate cyclase and 5'-nucleotidase activities

Human α-1-proteinase inhibitor is inactivated by human myeloperoxidase in the presence of hydrogen peroxide and chloride ion. Several antiarthritic drugs and related compounds, including many containing gold, were tested as inhibitors of the myeloperoxidase system. Of the twenty-six compounds used, twenty-two inhibited. The most important feature of these was the presence of a sulfhydryl group. The most effective compounds also were the most hydrophobic. The presence of gold, on the other hand, made little difference to the amount of inhibition. These drugs appear to have many effects, and their inhibition of the myeloperoxidase system suggests that this could be one of them.     

Lysophosphatidylcholines can modulate the activity of the glucagon-stimulated adenylate cyclase from rat liver plasma membranes.

1. Synthetic lysophosphatidylcholines inhibit the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes at concentrations two to five times lower than those needed to inhibit the fluoride-stimulated activity. 2. Specific 125I-labelled glucagon binding to hormone receptors is inhibited at concentrations similar to those inhibiting the fluoride-stimulated activity. 3. At concentrations of lysophosphatidylcholines immediately below those causing inhibition, an activation of adenylate cyclase activity or hormone binding was observed. 4 These effects are essentially reversible. 5. We conclude that the increased sensitivity of glucagon-stimulated adenylate cyclase to inhibition may be due to the lysophosphatidylcholines interfering with the physical coupling between the hormone receptor and catalytic unit of adenylate cyclase. 6. We suggest that, in vivo, it is possible that lysophosphatidylcholines may modulate the activity of adenylate cyclase only when it is in the hormone-stimulated state.     

Phenobarbital selectively modulates the glucagon-stimulated activity of adenylate cyclase by depressing the lipid phase separation occurring in the outer half of the bilayer of liver plasma membranes.

The pattern of incorporation of radioactivity from [1-14C]acetate and [2-14C]acetate into the polyprenyl side-chain of ubiquinones in bacteria (Azotobacter vinelandii, Pseudomonas sesami, Escherichia coli and Rhodopseudomonas capsulata) was studied. For this purpose, a new degradation method involving a modified Barbier-Wieland reaction of laevulinic acid was developed, and used along with the iodoform reaction. Both C-1 and C-2 of acetate were incorporated exclusively into C-2 of laevulinic acid suggesting that the well-known pathway through acetoacetyl-CoA ('acetoacetate pathway') was not operative in these bacteria. An alternative pathway ('acetolactate pathway'), starting with pyruvate and acetaldehyde as the distal precursors, and utilizing the reactions of leucine and valine metabolism, was postulated. It was also postulated that C-1 of acetate is incorporated not directly, but after oxidation to CO2. The pattern of incorporation of radioactivity from [U-14C]valine, [U-14C]alanine and NaH14CO3 into the side-chain of ubiquinone of R. capsulata was in agreement with the operation of the 'acetolactate pathway'.     

Dimethylnitrosamine inhibits the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes and decreases plasma membrane fluidity

The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation.     

The activity of glucagon-stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its lipid environment.

1. The local anaesthetic benzyl alcohol progressively activated glucagon-stimulated adenylate cyclase activity up to a maximum at 50 mM-benzyl alcohol. Further increases in benzyl alcohol concentration inhibited the activity. The fluoride-stimulated adenylate cyclase activity was similarly affected except for an inhibition of activity occurring at low benzyl alcohol concentrations (approx. 10 mM. 2. The fluoride-stimulated adenylate cyclase activity of a solubilized enzyme preparation was unaffected by any of the benzyl alcohol concentrations tested. 3. Increases in 3-phenylpropan-1-ol and 5-phenylpentan-1-ol concentrations progressively activated both the fluoride- and glucagon-stimulated adenylate cyclase activities up to a maximum, above which further increases in alcohol concentration inhibited the activities. 4. The 'break' points in Arrhenius plots of glucagon-stimulated adenylate cyclase activity in native plasma membranes, and in plasma membranes fused with synthetic dimyristoyl phosphatidylcholine so as to constitute 60% of the total lipid pool, were decreased by approx. 6 degrees C by addition of 40 mM-benzyl alcohol. This was accompanied by a fall in the associated activation energies. 6. Arrhenius plots of fluoride-stimulated adenylate cyclase activity in the presence and absence of 40 mM-benzyl alcohol were linear, although addition of benzyl alcohol caused a dramatic decrease in the associated activation energy of the reaction. 7. 5'-Nucleotidase activity was stimulated by benzyl alcohol, and the 'break' point in the Arrhenius plot of its activity was decreased by about 6 degrees C by addition of 40 mM-benzyl alcohol to the assay. 8. It is suggested that benzyl alcohol effects a fluidization of the bilayer, which is clearly demonstrated by its ability to lower the temperature of a lipid phase separation occurring at 28 degrees C in the outer half of the bilayer to around 22 degrees C. The increase in bilayer fluidity relieves a physical constraint on the membrane-bound adenylate cyclase, activating the enzyme. 9. The various inhibition phenomena are discussed in detail, together with the suggestion that the interaction between the uncoupled catalytic unit of adenylate cyclase and the lipids of the bilayer is altered on its physical coupling to the glucagon receptor.     

Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. I. Biochemical determination

The distribution of adenylate cyclase (AC) in Golgi and other cell fractions from rat liver was studied using the Golgi isolation procedure of Ehrenreich et al. In liver homogenate the AC activity was found to decay with time, but addition of 1 mM EGTA reduced the rate of enzyme loss. The incorporation of 1 mM EGTA into the sucrose medium used in the initial two centrifugal steps of the Golgi isolation method stabilized the enzyme activity throughout the entire procedure and resulted in good enzyme recovery. In such preparations, AC activity was demonstrated to be associated not only with plasma membranes but also with Golgi membranes and smooth microsomal membranes as well. Furthermore, under the conditions used, enzyme activity was also associated with the 105,000 g x 90 min supernatant fraction. The specific activity of the liver homogenate was found to be 2.9 pmol-mg protein-1-min-1, the nonsedimentabel and microsomal activity was of the same order of magnitude, but the Golgi and plasma membrane activities were much higher. The specific activity of plasma membrane AC was 29 pmol-mg proten-1-min-1. The Golgi activity varied in the three fractions, with the highest activity (14 pmol) in GF1 lowest activity (1.8) in GF2, and intermediate activity (5.5) in GF3, when the Golgi activity was corrected for the presence of content protein, the activity in GF1 became much higher (9 x) than that of the plasma membrane while the activities in GF2 and GF3 were comparable to that of plasma membrane. In all locations studied, the AC was sensitive to NaF stimulation, especially the enzyme associated with Golgi membranes. The activities in plasma and microsomal membranes were stimulated by glucagon, whereas the Golgi and nonsedimentable AC were not.     

The role of a guanine nucleotide-binding protein in the activation of rat liver plasma-membrane adenylate cyclase by forskolin.

The effects of the photoreactive GTP analogue GTP-gamma-azidoanilide on rat liver plasma-membrane adenylate cyclase are described. U.v. irradiation in the presence of the analogue abolished activation by any effector or combination of effectors that function via the activatory G protein. Partial protection against this inhibition was given by F- and guanosine 5'-[gamma-thio]triphosphate. It is concluded that GTP-gamma-azidoanilide acts by a light-induced covalent reaction with the G protein. In the dark the effects of the analogue were similar to those of GTP. Irradiation in the presence of GTP-gamma-azidoanilide was found to reduce but not to abolish activation of rat liver plasma membrane adenylate cyclase by forskolin. The activation by forskolin and GTP together were greater than the sum of the individual activations. Forskolin doubled adenylate cyclase activity in the presence of glucagon and guanosine 5'-[beta, gamma-imido]triphosphate, which might be expected to activate to the maximum possible extent via the G protein. It is concluded that there are two components to the forskolin activation, a guanine nucleotide-dependent and a guanine nucleotide-independent component.     

The effect of adenylate cyclase inhibitor (ACI) on guanylate cyclase, phosphodiesterase and other enzymes in heart.

The effect of an inhibitor of adenylate cyclase (ACI) was measured on some enzymes associated with cyclic nucleotide-regulated metabolism. Soluble guanylate cyclase was inhibited; both soluble and particulate cyclic GMP-phosphodiesterases were stimulated. Cyclic AMP phosphodiesterases were unaffected. In contrast, the activities of Na, K-ATPase, protein kinase, phosphorylase kinase, glycogen synthetase and a number of glycosidases were not altered by equipotent amounts of the inhibitor. It is concluded that this substance acts as a modulator of both cyclic AMP and cyclic GMP metabolism in heart and other tissues.     

Effect of freezing-thawing on the adenylate cyclase activity of the rat liver

Various regimes of freezing and thawing as well as adrenaline and fluoride ions are studied for their effect on the adenylate cyclase activity in liver tissue preparations. The reduction of basal and fluoride-stimulating adenylate cyclase activity and a decrease in the adrenaline-stimulating activity of the enzyme after freezing and thawing are shown. Freezing and thawing are studied for molecular mechanisms of their damaging effect on adenylate cyclase.     

Endocytosis of beta-adrenergic ligands by rat liver. Comparison of beta-adrenergic receptor and adenylate cyclase distribution in endosome and plasma-membrane fractions.

The internalization of beta-adrenergic receptors was investigated in rat livers perfused with an agonist ([3H]isoprenaline) or an antagonist ([125I]iodocyanopindolol). Analytical centrifugation of liver homogenates indicated that the ligands were transferred rapidly to endosomal and lysosomal positions in sucrose gradients. Endosome fractions contained beta-adrenergic binding sites, but adenylate cyclase activity was low and poorly activated by isoprenaline. The results indicate that the receptor-regulatory-protein-adenylate cyclase complex was disassembled during uptake of beta-adrenergic ligands, with the adenylate cyclase being retained at the plasma membrane.     

Effect of serum lipoproteins on the adenylate cyclase activity of rat liver plasma membranes.

Four rat lipoprotein classes [lymph chylomicrons, VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins] were tested for their ability to affect basal adenylate cyclase (EC 4.6.1.1) activity of rat liver plasma membranes. All the lipoproteins, with the exception of lymph chylomicrons, effectively increase the enzyme activity. VLD lipoproteins are the most active class (67% maximal increase), followed by HD lipoproteins (33%) and LD lipoproteins (23%). The effect of VLD lipoproteins is additive to that elicited by GTP or GTP plus glucagon (at least within a certain concentration range). VLD lipoproteins affect only the Vmax. of the enzyme, not the Km.     

Inhibition of the soluble form of testis adenylate cyclase by catechol estrogens and other catechols

The soluble form of rat germ cell adenylate cyclase was inhibited by compounds with a catechol moiety. Among the naturally occurring catechols tested, catechol estrogens were the most potent inhibitors. Catechol estrogens at 2-6 microM inhibited enzyme activity by 50% and almost completely at 30-100 microM concentration. The inhibitory activity of catechol estrogens depends on the catechol moiety of the molecule. Catechol per se also inhibited the activity of this enzyme, 50% inhibition being achieved at about 11 microM. The two hydroxyls of the catechol moiety are essential for the inhibitory interaction with the enzyme. Thus, aromatic compounds containing only one hydroxyl group in the benzene ring, such as tyrosine, phenylephrine, estradiol, and 6 alpha-hydroxyestradiol were either completely inactive or had marginal inhibitory activity at concentrations up to 0.3-1 mM. Moreover, methylation of the hydroxyl groups of the catechol moiety of the catechol estrogens as in 2-methoxyestradiol 3-methyl ether rendered the catechol estrogens inactive. The inhibitory potency of these compounds varied greatly depending on the structure associated with the catechol ring. Thus, compounds in which catechol is associated with an aliphatic side chain, such as dopamine, L-dopa, norepinephrine, and isoproterenol, were about 11- to 34-fold less potent than catechol. On the other hand, compounds in which catechol is associated either with a hydroaromatic ring system, as in apomorphine, or with an alicyclic ring system, as in catechol estrogens, were about 2- to 5-fold more potent than catechol. The inhibitory effect of dopamine, apomorphine, and catechol estrogens was not affected by specific D-1 or D-2 antagonist, indicating that they do not act via receptors for dopamine.     

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in syrian hamster hypothalamus

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [125I]iodomelatonin, was examined using an incubation temperature (30 degrees C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [125I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.     

Solubilization and other studies on adenylate cyclase of baker''s yeast.

1. Adenylate cyclase of Saccharomyces cerevisiae was sedimented from mechanically disintegrated preparations of yeast over an unusually wide range of centrifugal forces. 2. The enzyme was readily solubilized by Ficoll and by Lubrol PX. Lubrol caused a 2-fold activation. 3. Both particle-bound and Lubrol-solubilized enzyme had an apparent Km for ATP of 1.6 mM in the presence of 0.4 mM-cyclic AMP and 5 mM-MnCl2 at pH 6.2 and 30 degrees C. 4. The Lubrol-solubilized enzyme behaved on gel filtration as a monodisperse protein with an apparent mol.wt. of about 450000.     

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.     

Effects of Mn2+ and other divalent cations on adenylate cyclase activity in rat brain.

Abstract— Mn²⁺ caused an 8-to 16-fold stimulation of adenylate cyclase activity in homogenates as well as synaptosomcs. isolated synaptic membranes, and slices prepared from rat brain. The stimulation occurred at low concentrations of Mn²⁺. with a doubling of activity at 50-60μM. and was unaffected by a 60-fold excess of Mg²⁺. Whether or not Mg²⁺ was added, inclusion of a low concentration of Mn²⁺ reduced, but did not prevent the stimulation of adenylate cyclase caused by dopaminc in homogenates of corpus striatum. In contrast, Ca²⁺. at a concentration that had little effect on basal cyclase activity, completely prevented the stimulation by dopamine. The increase of cyclase activity produced by Mn²⁺ in brain homogenates was potentiated by F^?. Other ions, notably Hg²⁺. Pb²⁺. Cu²⁺ and Zn²⁺. in order of decreasing potency, inhibited both basal and Mn^2--stimulated cyclase activity. It is proposed that the effect of Mn²⁺ on adenylate cyclase activity may involve only the catalytic subunit of the enzyme, and that the mechanism is different from that by which either dopamine or F^? stimulates the enzyme. These results suggest that the effects of low concentrations of Mn²⁺ and certain other divalent metal ions on adenylate cyclase activity may be involved in their neuropsychiatrie or other toxic effects, and that such ions may also participate in normal physiological mechanisms involving cyclic nucleotides.