scholarly journals Adenylate cyclase is inhibited upon depletion of plasma-membrane cholesterol

1983 ◽  
Vol 212 (2) ◽  
pp. 331-338 ◽  
Author(s):  
A D Whetton ◽  
L M Gordon ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Spin Probe ◽  
Plasma Membranes ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Lipid Phase ◽  
Membrane Cholesterol ◽  
Cholesterol Depletion ◽  
Plasma Membrane Cholesterol

A procedure has been developed that allows for the depletion of rat liver plasma membrane cholesterol by incubation with liposomes at 4 degrees C. Upon cholesterol depletion, adenylate cyclase activity was inhibited and the membranes became more rigid, as determined by the flexibility of an incorporated fatty acid spin probe. Decreasing the cholesterol/phospholipid molar ratio elicited a pronounced drop in the net fold-stimulation of adenylate cyclase activity by glucagon. Two lipid phase separations were detected in cholesterol-depleted membranes at around 25 degrees C and 13 degrees C respectively. Breaks at these temperatures were observed in Arrhenius plots of both the mobility of the spin probe and the glucagon-stimulated adenylate cyclase activity for the range 2-40 degrees C, but only the one at the lower temperature for the fluoride-stimulated activity. It is proposed that the lipid phase separation occurring at 25 degrees C is localized in the external half of the bilayer, whereas that at 13 degrees C is due to lipids in the inner half of the bilayer. Similar structural and functional perturbations were manifest if the cholesterol-complexing polyene antibiotic amphotericin B was added to native membranes. The mechanism of adenylate cyclase inhibition achieved by cholesterol depletion and the domain structure of the plasma membrane in relation to cholesterol distribution are discussed. Native cholesterol/phospholipid ratios appear to optimize the functioning of adenylate cyclase in liver plasma membranes.

scholarly journals Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase

1983 ◽  
Vol 210 (2) ◽  
pp. 437-449 ◽  
Author(s):  
A D Whetton ◽  
L M Gordon ◽  
M D Houslay
Keyword(s):  
Fatty Acid ◽  
Adenylate Cyclase ◽  
Spin Probe ◽  
Plasma Membranes ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Cholesterol Concentration ◽  
Lipid Phase ◽  
Lipid Order ◽  
Liver Plasma Membranes

A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity. The various ligand-stimulated adenylate cyclase activities exhibit different sensitivities to inhibition by cholesterol, with inhibition of glucagon-stimulated greater than fluoride-stimulated greater than basal activity. The bilayer-fluidizing agent benzyl alcohol is able to reverse the inhibitory effect of cholesterol on adenylate cyclase activity in full. The thermostability of fluoride-stimulated cyclase is increased in the cholesterol-rich membranes. Elevated cholesterol concentrations abolish the lipid-phase separation occurring at 28 degrees C in native membranes as detected by an incorporated fatty acid spin probe. This causes Arrhenius plots of glucagon-stimulated adenylate cyclase activity to become linear, rather than exhibiting a break at 28 degrees C. It is suggested that the cholesterol contents of both halves of the bilayer are increased by the method used and that inhibition of adenylate cyclase ensues, owing to the increase in lipid order and promotion of protein-protein and specific cholesterol-phospholipid interactions.

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

1978 ◽  
Vol 174 (1) ◽  
pp. 179-190 ◽  
Author(s):  
I Dipple ◽  
M D Houslay
Keyword(s):  
Adenylate Cyclase ◽  
Benzyl Alcohol ◽  
Plasma Membranes ◽  
Break Point ◽  
Alcohol Concentration ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Lipid Phase ◽  
Glucagon Receptor ◽  
Arrhenius Plots

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.

Adenylate cyclase activity in Y1 mouse adrenocortical tumor cells: some properties of the enzyme associated with purified plasma membrane fractions

1983 ◽  
Vol 61 (7) ◽  
pp. 547-552 ◽  
Author(s):  
Bernard P. Schimmer
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Tumor Cell Line ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Adenylate Cyclase System ◽  
Adrenocortical Tumor ◽  
Intact Cells ◽  
Order Of Magnitude

Fractions enriched in plasma membranes were prepared from the Y1 mouse adrenocortical tumor cell line and were characterized with respect to adenylate cyclase activity. Optimal requirements of the adenylate cyclase system for guanyl nucleotides, Mg2+, ATP, and corticotropin (ACTH) were determined. The sensitivity of the adenylate cyclase system to ACTH1–24 in plasma membrane fractions was comparable with that observed in isolated intact cells. Polycations such as poly-L-arginine and histone competitively inhibited the action of ACTH1–24, supporting the view that the affinity of ACTH for the adenylate cyclase system is determined by the basic core of amino acids at residues 15–18. ACTH1–24 was at least one order of magnitude more potent than ACTH1–39 in stimulating adenylate cyclase activity in plasma membrane fractions.

scholarly journals Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of non-nuclear adenylate cyclase

1977 ◽  
Vol 162 (3) ◽  
pp. 473-482 ◽  
Author(s):  
D E Snider ◽  
C W Parker
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Prostaglandin E1 ◽  
Plasma Membranes ◽  
Subcellular Fractions ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Marker Enzyme ◽  
Discontinuous Sucrose Gradient

Human peripheral lymphocytes were broken in a Dounce homogenizer and subcellular fractions enriched in plasma membranes or microsomal particles and mitochondria were isolated by centrifugation through a discontinuous sucrose gradient. Various agents that promote cyclic AMP accumulation in intact lymphocytes were compared in their ability to stimulate adenylate cyclase activity in the individual fractions. Plasma-membrane-rich fractions that were essentially free of other subcellular particles as judged by electron microscopy and marker enzyme measurements responded to fluoride, but weakly or not at all to prostaglandin E1 and other prostaglandins. Microsomal and mitochondrial-rich fractions responded markedly to both prostaglandin E1 and fluoride. In some, but not all, experiments phytohaemagglutinin produced a modest increase in enzyme activity in plasma-membrane-rich fractions. Catecholamines, histamine, parathyrin, glucagon and corticotropin produced little or no response. In the absence of theophylline, adenosine (1-10 micronM) stimulated basal enzyme activity, although at higher concentrations the responses to prostaglandin E1 and fluoride were inhibited. GTP (1-100 micronM) and GMP(5-1000 micronM) respectively inhibited or stimulated the response to fluoride, whereas the converse was true with prostaglandin E1.

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

1986 ◽  
Vol 235 (1) ◽  
pp. 237-243 ◽  
Author(s):  
M D Houslay ◽  
L Needham ◽  
N J Dodd ◽  
A M Grey
Keyword(s):  
Phase Separation ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Lipid Phase ◽  
Liver Plasma Membranes ◽  
Native Liver ◽  
Rat Liver Plasma Membranes ◽  
Lipid Phase Separation

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.

Modulation of receptor-mediated gonadotropin action in rat testes by dietary fat

1988 ◽  
Vol 254 (6) ◽  
pp. E708-E712
Author(s):  
E. Sebokova ◽  
M. L. Garg ◽  
M. T. Clandinin
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Linolenic Acid ◽  
Plasma Membranes ◽  
Saturated Fatty Acids ◽  
Phospholipid Composition ◽  
Cholesterol Content ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Hcg Receptor

The effect of feeding diets enriched with 18:2 omega 6, 18:3 omega 3, or saturated fatty acids on lipid composition and receptor-mediated action of luteinizing hormone/human chorionic gonadotropin (LH/hCG) in rat testicular plasma membranes was investigated. Linoleic and alpha-linolenic acid treatments reduced total phospholipid and cholesterol content of the testicular plasma membrane and altered membrane phospholipid composition. Change in phospholipid and cholesterol content after feeding the polyunsaturated fats decreased cholesterol to phospholipid ratios and binding capacity of the LH/hCG receptor in the testicular plasma membrane. LH-stimulated adenylate cyclase activity was decreased in animals fed the linolenic acid-rich diet. NaF-stimulated adenylate cyclase activity was decreased in animals fed diets high in either polyunsaturated fatty acid. Decreased plasma membrane LH/hCG receptor content was associated with decreased testosterone production in Leydig cells in response to LH in the linolenic acid-fed group. It is suggested that change in cholesterol-to-phospholipid ratios alters the physical properties of testicular plasma membranes in a manner that influences accessibility of LH/hCG receptors in testicular tissue.

scholarly journals Guanosine 5′-triphosphate and guanosine 5′-[βγ-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase

1980 ◽  
Vol 186 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Miles D. Houslay ◽  
Irene Dipple ◽  
Keith R. F. Elliott
Keyword(s):  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Regulatory Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Lipid Phase ◽  
Trypsin Treatment ◽  
Phase Separations ◽  
Catalytic Unit

1. GTP, but not p[NH]ppG (guanosine 5′-[βγ-imido]triphosphate), abolishes the sensitivity of glucagon-stimulated adenylate cyclase to the lipid-phase separations occurring in the outer half of the bilayer in liver plasma membranes from rat. 2. When either GTP or p[NH]ppG alone stimulate adenylate cyclase, the enzyme senses only those lipid-phase separations occurring in the inner half of the bilayer. 3. Trypsin treatment of intact hepatocytes has no effect on the basal, fluoride-, GTP- or p[NH]ppG-stimulated adenylate cyclase activity. However, 125I-labelled-glucagon specific binding decays with a half-life matching that of the decay of glucagon-stimulated adenylate cyclase activity. 4. When GTP or p[NH]ppG are added to assays of glucagon-stimulated activity, the half-life of the trypsin-mediated decay of activity is substantially increased and the decay plots are no longer first-order. 5. Trypsin treatment of purified rat liver plasma membranes abolishes basal and all ligand-stimulated adenylate cyclase activity, and 125I-labelled-glucagon specific binding. 6. Benzyl alcohol activates the GTP- and p[NH]ppG-stimulated activities in an identical fashion, whereas these activities are affected differently when glucagon is present in the assays. 7. We suggest that guanine nucleotides alter the mode of coupling between the receptor and catalytic unit. In the presence of glucagon and GTP, a complex of receptor, catalytic unit and nucleotide regulatory protein occurs as a transient intermediate, releasing a free unstable active catalytic unit. In the presence of p[NH]ppG and glucagon, the transient complex yields a relatively stable complex of the catalytic unit associated with a p[NH]ppG-bound nucleotide-regulatory protein.

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

1979 ◽  
Vol 178 (1) ◽  
pp. 217-221 ◽  
Author(s):  
M D Houslay ◽  
R W Palmer
Keyword(s):  
Adenylate Cyclase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Hormone Receptors ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes ◽  
Increased Sensitivity

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.

scholarly journals Ultrastructural localization of adenylate cyclase activity in chicken osteoclasts.

1991 ◽  
Vol 39 (9) ◽  
pp. 1207-1213 ◽  
Author(s):  
O Fukushima ◽  
T Yamamoto ◽  
C V Gay
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Cell Function ◽  
Ultrastructural Localization ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Marrow Cavity ◽  
Calcium Diet ◽  
Low Calcium Diet ◽  
Ruffled Border

Using lead citrate as a capture reagent and adenylate-(beta, gamma-methylene) diphosphate (AMP-PCP) as a substrate, we localized adenylate cyclase activity on the non-ruffled border plasma membrane of approximately half of the osteoclasts on trabecular bone surfaces in the tibial metaphyses of chickens fed a low (0.3%)-calcium diet. The enzyme was not detectable in osteoclasts when chickens were fed a normal calcium diet. Activity was observed on the entire plasma membrane of detached osteoclasts that were situated between osteoblasts on the bone surface and blood vessels in the marrow cavity. Detection of activity on detached osteoclasts required the presence of an activator, implying lower levels in these cells than in those with ruffled borders. Staining was greater on the lateral sides of osteoblasts and osteoclasts when they were in contact with each other. Reaction specificity was indicated by the demonstration of stimulation by forskolin, guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP), dimethylsulfoxide, and NaF, inhibition by alloxan and 2',5'-dideoxyadenosine, and absence of activity when sections were incubated in substrate-free medium or when GMP-PCP replaced AMP-PCP as a substrate. The finding of adenylate cyclase in osteoclast plasma membrane provides structural evidence that the adenylate cyclase-cyclic AMP system has a role in regulation of osteoclast cell function. The low-calcium diet appears to have resulted in increased amounts of adenylate cyclase in osteoclasts.

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