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.     

Quinidine and melittin both decrease the fluidity of liver plasma membranes and both inhibit hormone-stimulated adenylate cyclase activity

Increasing concentrations of either quinidine or melittin gave a dose-dependent inhibition of both the glucagon- and fluoride-stimulated activities of adenylate cyclase in the liver plasma membranes. At similar concentrations these agents increased the order of liver plasma membranes as detected by a fatty acid ESR probe, doxyl stearic acid. This increase in bilayer order (decrease in 'fluidity') is suggested to explain the inhibitory action of quinidine on adenylate cyclase activity but only in part contributes to the inhibitory action of melittin on adenylate cyclase. Arrhenius plots of fluoride-stimulated activity became non-linear in the presence of either quinidine or melittin, with a single well-defined break occurring at around 12 degrees C in each instance. Arrhenius plots of the glucagon-stimulated activity also exhibited such a novel break at around 12 degrees C when either quinidine or melittin were present as well as exhibiting a break at around 28 degrees C, as was seen in the absence of these ligands. The fatty acid spin probe inserted into liver plasma membranes detected a novel lipid phase separation occurring at around 12 degrees C when either quinidine or melittin was present and showed that the lipid phase separation occurring at around 28 degrees C in native membranes was apparently unaffected by these ligands.     

Glucagon-stimulated adenylate cyclase detects a selective perturbation of the inner half of the liver plasma-membrane bilayer achieved by the local anaesthetic prilocaine.

Prilocaine can increase the fluidity of rat liver plasma membranes, as indicated by a fatty acid spin-probe. This led to the activation of the membrane-bound fluoride-stimulated adenylate cyclase activity, but not the Lubrol-solubilized activity, suggesting that increased lipid fluidity can activate the enzyme. With increasing prilocaine concentrations above 10 mM, the membrane-bound fluoride-stimulated activity was progressively inhibited, even though bilayer fluidity continued to increase and the activity of the solubilized enzyme remained unaffected. Glucagon-stimulated adenylate cyclase was progressively inhibited by increasing prilocaine concentrations. Prilocaine (10 mM) had no effect on the lipid phase separation occurring at 28 degrees C and attributed to those lipids in the external half of the bilayer, as indicated by Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and the order parameter of a fatty acid spin-probe. However, 10 mM-prilocaine induced a lipid phase separation at around 11 degrees C that was attributed to the lipids of the internal (cytosol-facing) half of the bilayer. It is suggested that prilocaine (10 mM) can selectively perturb the inner half of the bilayer of rat liver plasma membranes owing to its preferential interaction with the acidic phospholipids residing there.     

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.     

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.     

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.     

Perturbations of liver plasma membranes induced by Ca2+ are detected using a fatty acid spin label and adenylate cyclase as membrane probes

Ca2+ decreased the lipid fluidity of rat liver plasma membranes labeled with 5-nitroxide stearate, I(12,3), as indicated by the order parameter (S). These effects form a reversible, saturable process with an association constant of 1 x 10(3) M-1. Arrhenius-type plots of S indicated that the lipid phase separation, present in the external leaflet of native membranes between 28 and 19 degrees C, is perturbed by mM Ca2+ such that the high temperature onset is elevated to 32-34 degrees C. Fluoride-stimulated adenylate cyclase was similarly inhibited by Ca2+ (ID50 = 1 mM) for the enzyme in membrane-bound or solubilized states. The glucagon-stimulated activity was more sensitive to Ca2+ inhibition with an ID50 of 0.2 mM. These inhibitory effects are due neither to perturbations of glucagon binding to its receptor nor to fluidity changes, but are instead attributed to direct Ca2+-enzyme interactions. Such binding desensitizes the enzyme to fluidity alterations induced by temperature elevation or benzyl alcohol addition. With Ca2+, Arrhenius plots of glucagon-stimulated activity indicated breaks at 32 and 16 degrees C, whereas those of fluoride-stimulated activity showed one break at 17 degrees C. Without Ca2+, Arrhenius plots exhibited one break at 28 degrees C for glucagon-stimulated activity, whereas fluoride-stimulated plots were linear. We propose that Ca2+ achieves these effects through asymmetric perturbations of the membrane lipid structure.     

Adenylate cyclase and a fatty acid spin probe detect changes in plasma membrane lipid phase separations induced by dietary manipulation of the cholesterol:phospholipid ratio

Rats fed with a cholesterol supplement to their diet exhibited an increase in their plasma membrane cholesterol phospholipid (C/P)-lipid molar ratio from 0.72 to 0.98, whereas those fed the hypocholesterolaemic drug clofibrate in their diet exhibited a decrease in this ratio to 0.62. The properties of these membranes were analysed with regard to ligand-stimulated adenylate cyclase activity and the mobility of a fatty acid spin probe which allowed lipid phase separations to be identified. Membranes with elevated C/P ratios exhibited two distinct lipid phase separations, one at around 36 degrees C that was attributed to the external half of the bilayer and one at around 22 degrees C which was attributed to the inner half of the bilayer. Membranes with lowered C/P ratios exhibited a single lipid phase separation occurring at around 21 degrees C which was attributed to the lipids of the inner half of the bilayer. These results were compared with those obtained by manipulation of C/P ratios in vitro using liposome-cholesterol exchange techniques. Dietary manipulation of the C/P ratio of plasma membranes in vivo led to alterations in the fold stimulation of adenylate cyclase by various stimulatory ligands.     

Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma-membrane enzymes occurring on hibernation.

1. Arrhenius plots of the glucagon-stimulated adenylate cyclase, 5'-nucleotidase, (Na+ + K+)-stimulated adenosine triphosphatase and Mg2+-dependent adenosine triphosphatase activities of control hamster liver plasma membranes exhibited two break points at around 25 and 13 degrees C, whereas Arrhenius plots of their activities in hibernating hamster liver plasma membranes exhibited two break points at around 25 and 4 degrees C. 2. A single break occurring between 25 and 26 degrees C was observed in Arrhenius plots of the activities of fluoride-stimulated adenylate cyclase, basal adenylate cyclase and cyclic AMP phosphodiesterase of liver plasma membranes from both control and hibernating animals. 3. Arrhenius plots of phosphodiesterase I activity showed a single break at 13 degrees C for membranes from control animals, and a single break at around 4 degrees C for liver plasma membranes from hibernating animals. 4. The temperature at which break points occurred in Arrhenius plots of glucagon- and fluoride-stimulated adenylate cyclase activity were decreased by about 7--8 degrees C by addition of 40 mm-benzyl alcohol to the assays. 5. Discontinuities in the Arrhenius plots of 4-anilinonaphthalene-1-sulphonic acid fluorescence occurred at around 24 and 13 degrees C for liver plasma membranes from control animals, and at around 25 and 4 degrees C for membranes from hibernating animals. 6. We suggest that in hamster liver plasma membranes from control animals a lipid phase separation occurs at around 25 degrees C in the inner half of the bilayer and at around 13 degrees C in the outer half of the bilayer. On hibernation a change in bilayer asymmetry occurs, which is expressed by a decrease in the temperature at which the lipid phase separation occurs in the outer half of the bilayer to around 4 degrees C. The assumption made is that enzymes expressing both lipid phase separations penetrate both halves of the bilayer, whereas those experiencing a single break penetrate one half of the bilayer only.     

Thermotropic lipid phase separations in human platelet and rat liver plasma membranes

Electron spin resonance (ESR) studies were conducted on human platelet plasma membranes using 5-nitroxide stearate, I(12,3). The polarity-corrected order parameter S and polarity-uncorrected order parameters S(T parallel) and S(T perpendicular) were independent of probe concentration at low I(12.3)/membrane protein ratios. At higher ratios, S and S(T perpendicular) decreased with increasing probe concentration while S(T parallel) remained unchanged. This is the result of enhanced radical interactions due to probe clustering. A lipid phase separation occurs in platelet membranes that segregates I(12,3) for temperatures less than 37 degrees C. As Arrhenius plots of platelet acid phosphatase activity exhibit a break at 35 to 36 degrees C, this enzyme activity may be influenced by the above phase separation. Similar experiments were performed on native [cholesterol/phospholipid ratio (C/P) = 0.71] and cholesterol-enriched [C/P = 0.85] rat liver plasma membranes. At 36 degrees C, cholesterol loading reduces I(12,3) flexibility and decreases the probe ratio at which radical interactions are apparent. The latter effects are attributed to the formation of cholesterol-rich lipid domains, and to the inability of I(12,3) to partition into these domains because of steric hinderance. Cholesterol enrichment increases both the high temperature onset of the phase separation occurring in liver membranes from 28 degrees to 37 degrees C and the percentage of probe-excluding, cholesterol-rich lipid domains at elevated temperatures. A model is discussed attributing the lipid phase separation in native liver plasma membranes to cholesterol-rich and -poor domains. As I(12,3) behaves similarly in cholesterol-enriched liver and human platelet plasma membranes, cholesterol-rich and -poor domains probably exist in both systems at physiologic temperatures.     

Effects of 4-hydroxynonenal on adenylate cyclase and 5'-nucleotidase activities in rat liver plasma membranes

Adenylate cyclase and 5'-nucleotidase activities in rat liver plasma membranes were assayed in vitro in the presence of 4-hydroxy-2,3-trans-nonenal (HNE), a major end-product of microsomal lipid peroxidation. Both basal and glucagon-stimulated adenylate cyclase were inhibited in a dose-dependent manner, even at micromolar HNE concentrations, whereas fluoride-stimulated activity increased. A biphasic, dose- and time-dependent effect was noted when the basal activity was monitored at increasing doses. 5'-Nucleotidase activity was also decreased by HNE, but only at millimolar concentrations. These findings are related to the view that aldehydes, especially HNE, may act as diffusible cytotoxic compounds when lipid peroxidative derangement of membrane lipids is provoked by toxic conditions.     

Role of cellular redox state and glutathione in adenylate cyclase activity in rat adipocytes.

Adenylate cyclase in rat adipocyte membranes was inactivated as a result of treatment with sulfhydryl oxidants or with p-chloromercuribenzoate as well as by S-alkylating agents. The inhibition of the basal and isoproterenol- or glucagon-stimulated enzyme activity by the oxidants or the mercurial could be reversed by adding thiols to the isolated membranes. The activity of the enzyme paralleled the cellular glutathione (GSH) content. Lowering of intracellular glutathione by incubating the cells with specific reactants resulted in the inhibition of both basal and hormone-stimulated adenylate cyclase activity in the isolated membranes. Activity could be partly restored by supplying glucose to the incubation medium of intact cells. The fluoride-stimulated adenylate cyclase was also inhibited by the oxidants or the sulfhydryl inhibitors. The results suggest that adenylate cyclase may be partly regulated by oxidation-reduction. Thus, a direct relationship between both basal and hormone-stimulated adenylate cyclase activity and the cellular redox potential, determined by the cellular level of reduced glutathione, may be ascribed to the protection of the catalytic -SH groups of the enzyme from oxidative or peroxidative reactions and maintenance of the redox optimum for the reaction.     

The selective effects of charged local anaesthetics on the glucagon- and fluoride-stimulated adenylate cyclase activity of rat-liver plasma membranes

The cationic local anaesthetics carbocaine and unpercaine were found to increase the fluoride-stimulated adenylate cyclase up to a maximum level; above this maximum level further increases in drug concentration inhibited the enzyme. At concentrations where this activity was stimulated, a fatty acid spin label detected an increase in bilayer fluidity, which, it is suggested, is responsible for the activation of the enzyme. A solubilized enzyme was unaffected by the drugs, a finding consistent with this proposal. These cationic drugs began to inhibit the glucagon-stimulated activity at concentrations where they activated the fluoride-stimulated activity. It is suggested that this is due to their effect on the coupling interaction between the receptor and catalytic unit. The anionic drugs, phenobarbital, pentobarbital, and salicylic acid, all inhibited the fluoride-stimulated enzyme. This may be due in part to a direct effect on the protein and in part to the interaction of the drugs with the bilayer. The drugs had small inhibitory effects on the lubrol-solubilized enzyme. The glucagon-stimulated enzyme was initially inhibited by the anionic drugs at low concentrations, then activated, and finally inhibited with increasing drug concentration. The reasons for such changes are complex, but there was no evidence from electron spin resonance studies to suggest that the elevations in activity were due to increases in bilayer fluidity.     

Activation by GTP of liver adenylate cyclase in the presence of high concentrations of ATP

Effect of GTP on adenylate cyclase of liver plasma membrane was examined using ATP which was extensively purified by DEAE-cellulose column chromatography. In the incubation containing 2mM purified ATP as substrate, GTP enhanced basal and glucagon- or fluoride-stimulated activities. When the unpurified ATP at 2mM was used, all the activities were high and the stimulatory effect of GTP was not detected. The substance(s) which was recovered from a small but significant peak on DEAE-cellulose column was equivalent to 10–100μM GTP in stimulating adenylate cyclase. These results indicate that, if highly purified ATP is used as substrate, GTP can enhance adenylate cyclase activity in the presence of millimolar concentration of ATP and that GTP enhances not only the glucagon-stimulated adenylate cyclase but also the basal as well as fluoride-stimulated adenylate cyclase activities.     

Mechanism of glucagon activation of adenylate cyclase in the presence of Mn2+

For a variety of ligand states, adenylate cyclase activity in the presence of Mn2+ was greater than with Mg2+. Trypsin treatment of intact hepatocytes, under conditions which destroy cell surface glucagon receptors, led to a first order loss of glucagon-stimulated adenylate cyclase activity in isolated membranes assayed in the presence of Mn2+ whether or not GTP (100 microM) was present in the assays. Arrhenius plots of basal activity exhibited a break at around 22 degrees C, those with NaF were linear and those with glucagon +/- GTP (100 microM) were biphasic with a break at around 28 degrees C. It is suggested that Mn2+ perturbs the coupling interaction between the glucagon receptor and catalytic unit of adenylate cyclase at the level of the guanine nucleotide regulatory protein. This appears to take the form of Mn2+ preventing GTP from initiating glucagon's activation of adenylate cyclase through a collision coupling mechanism.     

Involvement of calcium in the inhibition by insulin of the glucagon-stimulated adenylate-cyclase activity.

1. The inhibitory effect of adenosine on the glucagon-stimulated adenylate cyclase activity of liver plasma membranes, prepared from PVG/c rats, was potentiated by insulin. In the presence of EGTA, such potentiating effect of insulin was lost. 2. Calcium (10 microM) potentiated the inhibitory effects of both adenosine and insulin on the glucagon-stimulated cyclase activity. The synergestic effect of calcium + insulin required the presence of adenosine as judged from the use of adenosine deaminase. 3. Insulin had no significant inhibitory effect on the glucagon-stimulated cyclase activity of liver plasma membranes, prepared from young Wistar rats, unless both adenosine (50 microM) and calcium (10 microM) were added externally. 4. Results demonstrate an interaction of calcium and insulin at membrane level that, in the presence of adenosine, results in the inhibition of the glucagon-stimulated adenylate cyclase activity.     

Effects of temperature and benzyl alcohol on the structure and adenylate cyclase activity of plasma membranes from bovine adrenal cortex

Adenylate cyclase activation by corticotropin (ACTH), fluoride and forskolin was studied as a function of membrane structure in plasma membranes from bovine adrenal cortex. The composition of these membranes was characterized by a very low cholesterol and sphingomyelin content and a high protein content. The fluorescent probes 1,6-diphenylhexa-1,3,5-triene (DPH) and a cationic analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) were, respectively, used to probe the hydrophobic and polar head regions of the bilayer. When both probes were embedded either in the plasma membranes or in liposomes obtained from their lipid extracts, they exhibited lifetime heterogeneity, and in terms of the order parameter S, hindered motion. Under all the experimental conditions tested, S was higher for TMA-DPH than for DPH but both S values decreased linearly with temperature within the range of 10 to 40 degrees C, in the plasma membranes and the liposomes. This indicated the absence of lipid phase transition and phase separation. Addition to the membranes of up to 100 mM benzyl alcohol at 20 degrees C also resulted in a linear decrease in S values. Membrane perturbations by temperature changes or benzyl alcohol treatment made it possible to distinguish between the characteristics of adenylate cyclase activation with each of the three effectors used. Linear Arrhenius plots showed that when adenylate cyclase activity was stimulated by forskolin or NaF, the activation energy was similar (70 kJ.mol-1). Fluidification of the membrane with benzyl alcohol concentrations of up to 100 mM at 12 or 24 degrees C produced a linear decrease in the forskolin-stimulated activity, that led to its inhibition by 50%. By contrast, NaF stabilized adenylate cyclase activity against the perturbations induced by benzyl alcohol at both temperatures. In the presence of ACTH, biphasic Arrhenius plots were characterized by a well-defined break at 18 degrees C, which shifted at 12.5 degrees C in the presence of 40 mM benzyl alcohol. These plots suggested that ACTH-sensitive adenylate cyclase exists in two different states. This hypothesis was supported by the striking difference in the effects of benzyl alcohol perturbation when experiments were performed below and above the break temperature. The present results are consistent with the possibility that clusters of ACTH receptors form in the membrane as a function of temperature and/or lipid phase fluidity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of adenylate cyclase activity of fibroblasts by free fatty acids and phospholipids

The effect of certain lipids on adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from fibroblasts in culture has been investigated. The unsaturated fatty acids, as well as lysolecithin, were found to act as potent inhibitors of fibroblast adenylate cyclase activity. Increasing the degree of unsaturation increases the extent of inhibition noted at a given fatty acid concentration. The inhibitory effect of the unsaturated fatty acids or lysolecithin is not selective for a specific function of the adenylate cyclase system since basal, and hormone- or fluoride-stimulated cyclase activities are inhibited to the same extent. The fatty acid-inactivated state of fibroblast adenylate cyclase is not readily reversed for enzyme activity is not restored when arachidonate-treated membranes are washed with Tris buffer containing 10 mm EDTA, 0.15 mm albumin, or 0.15 m KCl. Previous studies have shown that the adenylate cyclase system from Moloney sarcoma virus-transformed NRK (MNRK) cells is not stimulated by the addition of GTP or hormones. Of interest is the present finding that the addition of unsaturated fatty acids, or lysolecithin, over a narrow concentration range (0.1 – 0.2 mm) leads to partial restoration of GTP activation of MNRK cyclase activity. Hormonal responsiveness to l-epinephrine or prostaglandin E₁ is not restored to the MNRK enzyme with fatty acid or lysolecithin treatment.     

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.     

Modification of TSH-stimulated adenylate cyclase activity of bovine thyroid by manipulation of membrane phospholipid composition with a nonspecific lipid transfer protein

The lipid composition of bovine thyroid plasma membranes was modified using the nonspecific lipid transfer protein from bovine liver. Incubation of plasma membranes with transfer protein and phosphatidylinositol-containing liposomes caused a strong, concentration dependent, inhibition of TSH-stimulated adenylate cyclase activity. Other phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid were two to four times less effective as inhibitors of TSH-stimulation. The phosphatidylinositol-induced inhibition was not reversed when more than 80% of phosphatidylinositol incorporated was removed using phosphatidylinositol-specific phospholipase C. Incorporation of phosphatidylinositol in plasma membranes provoked no significant change in the fluorescence anisotropies of the fluorophores 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(14-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), indicating that the inhibition was not due to changes in membrane fluidity. At phosphatidylinositol concentrations causing a 66% reduction in TSH-stimulated adenylate cyclase activity cholera toxin- and forskolin-stimulated activity as well as basal activity were decreased by maximally 10%. Since TSH binding to bovine thyroid plasma membranes was not affected it is suggested that phosphatidylinositol can act as a negative modulator of the TSH activation of adenylate cyclase and this probably by interfering with the coupling between the occupied TSH receptor and the stimulatory GTP-binding regulatory protein of the adenylate cyclase complex.