Biomedical subjects
Publications and source records attributed to M D Houslay.
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The effects on primary human skin fibroblasts of the structurally unrelated tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and teleocidin B were compared with those of epidermal growth factor and the non-promoting derivative 4-O-methyl TPA by means of two-dimensional gel electrophoresis. Both TPA and teleocidin B caused a marked increase in the synthesis of two polypeptides of molecular weights 44 kilodaltons (P44) and 55 kilodaltons (P55). P55 was complexed in cell lysates by antiactin antibody and was shown to be a component of the cytoskeleton. P44 migrated very near to actin, but it was shown not to be a variant form of actin. No such specific increase in polypeptide synthesis was observed following treatment with either epidermal growth factor or 4-O-methyl TPA. TPA inhibited the beta-adrenergic hormone response in the same human skin fibroblasts.
Insulin elicits the activation of two distinct membrane-bound cyclic AMP phosphodiesterases when incubated at 37 degrees C for 5 min with intact hepatocytes: the 'dense-vesicle' enzyme and the peripheral-plasma-membrane enzyme. In hepatocytes the lysosomotropic agents chloroquine, methylamine and NH4Cl, as well as intracellular ATP depletion elicited by fructose or incubation with insulin at 22 degrees C, blocks selectively the activation of the 'dense-vesicle' enzyme. Incubation of hepatocytes with bacitracin, leupeptin and a variety of proteinase inhibitors failed to affect insulin's activation of these two cyclic AMP phosphodiesterases by distinct routes. It is suggested that activation of the 'dense-vesicle' enzyme occurs through a pathway triggered by the endocytosis, processing and recycling of the insulin receptor. This might involve the delivery, with subsequent activation, of a latent phosphodiesterase into this fraction.
1. The lipid fluidity of three major rat liver plasma-membrane subfractions, as well as Golgi apparatus and endocytic fractions, was assessed with a fatty acid spin probe by using e.s.r. techniques. 2. The sinusoidal (blood-facing) plasma-membrane subfraction was the most fluid of the three plasma-membrane regions. Fractions originating from the bile-canalicular and contiguous (lateral) regions were most rigid. Endocytic fractions isolated (endosomes and diacytosomes) were of a similar fluidity to fractions originating from the sinusoidal plasma-membrane region. By far the most fluid fractions examined were derived from the Golgi-apparatus complex. 3. The three plasma-membrane subfractions each showed a different response to the bilayer-fluidizing effect of benzyl alcohol. 4. Arrhenius-type plots of the order parameter S and outer hyperfine splitting, 2T( parallel), identified lipid-phase separations in the plasma-membrane subfractions.
Insulin failed to exert an effect on the basal and glucagon- and guanosine 5'-[beta, gamma-imido]-triphosphate-stimulated adenylate cyclase activities of hepatocyte membranes. In the presence of high GTP (0.1 mM) concentrations, however, insulin was shown to inhibit adenylate cyclase activity. This effect was dose-dependent, exhibiting an EC50 (median effective concentration) of 3 microM for GTP. Elevated glucagon concentrations blocked the inhibitory effect of insulin in a dose-dependent fashion, with an EC50 of 1 nM. The insulin inhibition was dose-dependent (EC50 = 90 pM). The inhibitory effects of insulin were abolished using membranes from either glucagon-desensitized hepatocytes or cholera-toxin-treated hepatocytes. If either Mn2+ replaced Mg2+ in adenylate cyclase assays or Na+ was removed from the assay mixtures then insulin failed to exert any inhibitory effect. It is suggested that insulin exerts its action on adenylate cyclase through an inhibitory guanine nucleotide protein. This is integrated with the proposal [Heyworth, Rawal & Houslay (1983) FEBS Lett. 154, 87-91; Heyworth, Wallace & Houslay (1983) Biochem. J. in the press] that insulin mediates a variety of cellular effects through a specific guanine nucleotide regulatory protein and associated protein kinase(s).
Membrane fractions obtained from hepatocytes treated with glucagon exhibited a decreased glucagon (with or without GTP)-stimulated adenylate cyclase activity. A maximum effect was seen in around 5 min. No change in the rate of cyclic AMP production was observed for the basal, NaF-, p[NH]ppG (guanosine 5'-[beta, gamma-imido]-triphosphate)- and GTP-stimulated states of the enzyme. The lag observed in the p[NH]ppG-stimulated adenylate cyclase activity of native membranes was abolished when membranes from glucagon-pretreated cells were used. When Mn2+ replaced Mg2+ in the assays, the magnitude of the apparent desensitization was decreased. Mn2+ abolished the lag of onset of p[NH]ppG-stimulated activity in native membranes. The desensitization process was dose-dependent on glucagon, which exhibited a Ka of 4 X 10(-10) M. Depletion of intracellular ATP did not affect this process. It is suggested that this desensitization occurs at the level of the guanine nucleotide-regulatory protein.
Glucagon (10 nM) caused a transient elevation of intracellular cyclic AMP concentrations, which reached a peak in around 5 min, and slowly returned to basal values in around 30 min. When 1 mM-3-isobutyl-1-methylxanthine (IBMX) was present, this process yielded a Ka of 1 nM for glucagon. The addition of insulin (10 nM) after 5 min exposure to glucagon (10 nM) caused intracellular cyclic AMP concentrations to fall dramatically, attaining basal values within 10 min. The regulation of this process was dose-dependent, exhibiting a Ka of 0.4 nM for insulin. If insulin and glucagon were added together to hepatocytes, then insulin decreased the magnitude of the cyclic AMP response to glucagon. IBMX (1 mM) prevented insulin antagonizing the action of glucagon in both of these instances. A gentle homogenization procedure followed by a rapid subcellular fractionation of hepatocytes on a Percoll gradient was developed. This was used to resolve subcellular membrane fractions and to identify cyclic AMP phosphodiesterase activity in both membrane and cytosol fractions. Glucagon and insulin only affected the activity of two distinct membrane-bound species, a plasma-membrane enzyme and a 'dense vesicle' enzyme. Glucagon (10 nM), insulin (10 nM), IBMX (1 mM), dibutyryl cyclic AMP (10 microM) and cholera toxin (1 microgram/ml) all elicited the activation of the 'dense vesicle' enzyme. The plasma-membrane enzyme was not activated by glucagon, IBMX or dibutyryl cyclic AMP, although insulin and cholera toxin both led to its activation. The degree of activation of the plasma-membrane enzyme produced by insulin was increased in the presence of IBMX or dibutyryl cyclic AMP. Glucagon pretreatment (5 min) of hepatocytes blocked the ability of insulin to activate the plasma-membrane enzyme. The activity state of these phosphodiesterases is discussed in relation to the observed changes in intracellular cyclic AMP concentrations. It is suggested that insulin exerts its action on the plasma-membrane phosphodiesterase through a mechanism involving a guanine nucleotide-regulatory protein.
A microsomal fraction from rat liver was subfractionated into three rough endoplasmic reticulum fractions RIII, RII and RI, together with a smooth endoplasmic reticulum plus Golgi fraction. Cyclic nucleotide phosphodiesterase activity was found in all fractions. Subsequently it was shown that Golgi fractions were essentially devoid of cyclic AMP phosphodiesterase activity and the activity resided in the smooth endoplasmic reticulum fraction. The activity of the endoplasmic reticulum constituted some 20% of the homogenate activity, with the major fraction of this being associated with the RII fraction and the least with the RI fraction. With the exception of the activity of the RI fraction, which was a peripheral enzyme, all of the other enzyme activities were integral, requiring detergent or repeated freeze-thawing to effect solubilization. All of the activities appeared to be exposed at the external surface of the endoplasmic reticulum, as they were inactivated by trypsin under conditions where glucose 6-phosphatase was not. All of these activities displayed distinct sensitivities to both thermal and trypsin inactivation, yielding activity decays consistent with a single enzyme species being present in each case. The freeze-thaw-solubilized enzymes yielded single symmetrical peaks on sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The sedimentation coefficients for the enzymes in the smooth-endoplasmic-reticulum-plus-Golgi, RIII, RII and RI fractions were 3.2S, 4.2S, 4.5S and 4.5S respectively. Whereas the activity in the smooth-endoplasmic-reticulum-plus-Golgi fraction exhibited normal Michaelis kinetics, those in the other fractions yielded kinetics indicative of apparent negative co-operativity. All of the enzymes exhibited low Km values towards cyclic AMP. The enzymes did not appear to be regulated by Ca2+ or calmodulin. ZnCl2 was found to be a potent non-competitive inhibitor of the enzyme in all fractions. NaF was a weak non-competitive inhibitor. The bilayer fluidizing agent benzyl alcohol exerted dissimilar effects on the enzyme activities. It is concluded that the endoplasmic reticulum displays lateral heterogeneity, with single, rather distinct, cyclic AMP phosphodiesterases being found in the different fractions.
Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.
The cholesterol content of rat liver plasma membranes was manipulated using either cholesterol-free or cholesterol-enriched liposomes. Removal of cholesterol from the membranes led to a marked increase in 5'-nucleotidase activity. However, increase in cholesterol content failed to exert any significant effect on 5'-nucleotidase activity. Arrhenius plots of the activity of the native enzyme exhibited a break at around 28 degrees C with the activation energy of the reaction less above this temperature than below. In cholesterol-depleted membranes a single break at around 26 degrees C was observed with activation energies greater above this temperature than below it. In cholesterol-enriched membranes Arrhenius plots were linear over the range examined. It is suggested that the lipid environment of the external half of the bilayer only influences 5'-nucleotidase activity in these membranes and that cholesterol exerts controlling effects on both the activity and conformation of the enzyme in native membranes.
Both forskolin and ethanol elicit the activation of basal and ligand-stimulated adenylate cyclase activities in rat liver plasma membranes. Ethanol is most potent at activating the fluoride- and glucagon-stimulated activities whilst having little effect on basal activity. In contrast forskolin exerts its greatest effect on basal activity. Over the concentration range that ethanol activates adenylate cyclase, it also increases bilayer fluidity as indicated by a decrease in the values of the order parameters for an incorporated fatty acid spin probe. At high concentrations forskolin does increase bilayer fluidity. However, it only begins to do so at concentrations above those where forskolin has already exerted its maximal effect in activating adenylate cyclase. Forskolin can still activate, albeit to a reduced extent, detergent-solubilized adenylate cyclase whereas ethanol cannot. Forskolin elicits a pronounced rise in hepatocyte intracellular cyclic AMP concentrations, whereas ethanol does not. Both forskolin and ethanol reduce the temperature of onset of the lipid phase separation occurring in rat liver plasma membranes. This is detected in Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and order parameters of an incorporated fatty acid spin probe, where we find that forskolin is particularly potent in decreasing the temperature at which this lipid phase separation occurs. Our results are consistent with the notion that forskolin exerts its effect on adenylate cyclase primarily by a direct action on the catalytic unit of the enzyme. However, as forskolin is a potent perturber of the organisation of the lipid bilayer it is possible that this could modulate its effect on adenylate cyclase and might be expected to affect the activity of other membrane enzymes.
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.
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.
The insulin-stimulated cyclic AMP phosphodiesterase from liver plasma membranes is shown to be activated upon incubation with guanine nucleotides in the presence of ATP. The non-hydrolysable analogue of ATP, adenylyl imidodiphosphate failed to substitute for ATP in achieving activation. GTP, its non-hydrolysable analogues p[NH]ppG and GTP-gamma-S, as well as GDP, all elicited activation. It is suggested that guanine nucleotides, and probably insulin, exert their effect on this enzyme through a distinct species of guanine nucleotide regulatory protein.
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.
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.
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.