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M D Houslay

Publications and source records attributed to M D Houslay.

At least 145 records · Page 8Linked to original sources
PubMed1989

The effects of p21N-ras expression in NIH-3T3 cells upon cyclic AMP metabolism.

The effects of overexpression of p21N-ras upon cyclic AMP metabolism have been examined in the inducible T15 cell line. In cells overexpressing the N-ras gene product, beta-adrenergic stimulation of cyclic AMP generation was reduced. The reduction was more pronounced the longer the ras gene was expressed and in chronically transformed cells a reduction in forskolin-stimulated cyclic AMP generation was also observed. The transformed cells exhibited a reduction in beta-adrenergic binding sites, but no change in the apparent EC50 for agonist induced cyclic AMP generation. Treatment of the cells with dibutyryl cyclic AMP induced a dose-dependent inhibition of proliferation, with the transformed cells being more sensitive than the control cells.

Animals↗
PubMed1989

Subcellular localization and hormone sensitivity of adipocyte cyclic AMP phosphodiesterase.

Treatment of intact adipocytes with either or both insulin and adrenaline stimulated membrane cyclic AMP phosphodiesterase activity only in the endoplasmic reticulum subfraction. The cyclic GMP-inhibited cyclic AMP phosphodiesterase activity was also found in this fraction. Quantitative Western blotting using a specific polyclonal antibody, raised against the homogeneous 'dense-vesicle' cyclic AMP phosphodiesterase from rat liver, identified a single 63 kDa species which was localized in the adipocyte endoplasmic reticulum fraction. The ability of adrenaline to stimulate adipocyte membrane cyclic AMP phosphodiesterase was shown to be mediated via beta-adrenoceptors and not alpha 1-adrenoceptors. Membrane cyclic AMP phosphodiesterase was stimulated by glucagon but not by vasopressin, A23187 or 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of adipocytes with either chloroquine or dansyl cadaverine failed to affect the ability of insulin to stimulate cyclic AMP phosphodiesterase activity. Treatment of an isolated adipocyte endoplasmic reticulum membrane fraction with purified protein kinase A increased its cyclic AMP phosphodiesterase activity some 2-fold. When this fraction was treated with purified protein kinase A and [32P]ATP, label was incorporated into a 63 kDa protein which was specifically immunoprecipitated with the antiserum against the liver 'dense-vesicle' cyclic AMP phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Guanine-nucleotide-binding proteins expressed in rat white adipose tissue. Identification of both mRNAs and proteins corresponding to Gi1, Gi2 and Gi3.

Considerable debate has focused on the molecular identity of the guanine-nucleotide-binding proteins (G-proteins) in adipose tissue which can be detected following pertussis-toxin-catalysed ADP-ribosylation [Rapiejko, Northup, Evans, Brown & Malbon (1986) Biochem. J. 240, 35-40; Hinsch, Rosenthal, Spicher, Binder, Gausepohl, Frank, Schultz & Joost (1988) FEBS Lett. 238, 191-196]. We have used a panel of selective anti-peptide antisera which are able to discriminate between the different pertussis-toxin-sensitive G-proteins to assess which of these are expressed in rat adipose tissue. We demonstrate that plasma membranes of rat white adipocytes contain alpha subunits corresponding to each of Gi1, Gi2 and Gi3. Furthermore, using synthetic oligonucleotides complimentary to unique regions of each of the three polypeptides, we demonstrate that the mRNAs for the three G-protein alpha subunits can also be detected in adipose tissue.

Adipose Tissue↗
PubMed1989

Insulin stimulates the tyrosyl phosphorylation and activation of the 52 kDa peripheral plasma-membrane cyclic AMP phosphodiesterase in intact hepatocytes.

The 52 kDa subunit of the peripheral-plasma-membrane insulin-stimulated high-affinity cyclic AMP phosphodiesterase can be specifically detected by the antibody PM1 by Western-blotting procedures and also can be immunoprecipitated from a hepatocyte extract. PM1-mediated immunoprecipitation from hepatocyte extracts showed that insulin treatment of intact 32P-labelled hepatocytes caused the rapid phosphorylation of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. Phosphoamino acid analysis and the use of a phosphotyrosine-specific antibody indicated that phosphorylation occurred on tyrosyl residue(s) of this phosphodiesterase. Prior treatment of hepatocytes with glucagon (10 nM) completely blocked the insulin-mediated tyrosyl phosphorylation of this 52 kDa protein, as detected with both the PM1 and the anti-phosphotyrosine antibodies. Treatment of hepatocytes with glucagon alone did not increase the phosphorylation state of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. The specific anti-phosphotyrosine antibody also detected the insulin-stimulated phosphorylation of proteins of 180 kDa, 95 kDa and 39 kDa. Prior treatment of hepatocytes with glucagon decreased the ability of insulin to phosphorylate the 180 kDa and 39 kDa species, but not the 95 kDa species.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Bombesin stimulation of inositol 1,4,5-trisphosphate generation and intracellular calcium release is amplified in a cell line overexpressing the N-ras proto-oncogene.

Bombesin stimulation of T15 cells in which the inducible N-ras oncogene is overexpressed caused elevated production of inositol phosphates compared to uninduced cells [Wakelam, Davies, Houslay, McKay, Marshall & Hall (1986) Nature (London) 323, 173-176]. This elevated response is shown here to result from increased generation of inositol 1,4,5-trisphosphate leading to an elevated release of intracellular stored Ca2+. Single-cell analysis of Ca2+ release showed that the elevated response is not a consequence of an increased fraction of responding cells. These amplifications are consistent with p21N-ras acting like a guanine nucleotide coupling protein in this cell line.

Bombesin↗
PubMed1989

Activation and phosphorylation of the 'dense-vesicle' high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase.

Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP-dependent protein kinase (A-kinase) selectively activated the high-affinity 'dense-vesicle' cycle AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2+. Mg2+ pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the 'dense-vesicle' phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the 'dense-vesicle' phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time-dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the 'dense-vesicle' phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Glucagon desensitization of adenylate cyclase and stimulation of inositol phospholipid metabolism does not involve the inhibitory guanine nucleotide regulatory protein Gi, which is inactivated upon challenge of hepatocytes with glucagon.

Brief exposure of hepatocytes to glucagon, angiotensin or the protein kinase C activator TPA (12-O-tetradecanoylphorbol 13-acetate) caused the inactivation of the inhibitory guanine nucleotide regulatory protein Gi. Glucagon-mediated desensitization of glucagon-stimulated adenylate cyclase activity was seen in hepatocytes from both normal rats and those made diabetic with streptozotocin, where Gi is not functionally expressed. Normal glucagon desensitization was seen in hepatocytes from young animals, 6 weeks of age, which had amounts of Gi in their hepatocyte membranes which were some 45% of that seen in mature animals (3.4 pmol/mg of plasma-membrane protein). Streptozotocin-induced diabetes in young animals abolished the appearance of functional Gi in hepatocyte plasma membranes. Pertussis-toxin treatment of hepatocytes from both normal mature animals and those made diabetic, with streptozotocin, blocked the ability of glucagon or angiotensin or TPA to elicit desensitization of adenylate cyclase. The isolated B (binding)-subunit of pertussis toxin was ineffective in blocking desensitization. Neither induction of diabetes nor treatment of hepatocytes with pertussis toxin inhibited the ability of glucagon and angiotensin to stimulate the production of inositol phosphates in intact hepatocytes. Thus (i) Gi does not appear to play a role in the molecular mechanism of glucagon desensitization in hepatocytes, (ii) the G-protein concerned with receptor-stimulated inositol phospholipid metabolism in hepatocytes appears not to be a substrate for the action of pertussis toxin, (iii) in intact hepatocytes, treatment with glucagon and/or angiotensin can elicit the inactivation of the inhibitory G-protein Gi, and (iv) pertussis toxin blocks desensitization by a process which does not involve Gi.

Adenylate Cyclase Toxin↗
PubMed1989

Binding to GDP-agarose identifies a novel 60kDa substrate for the insulin receptor tyrosyl kinase in mouse NIH-3T3 cells expressing high concentrations of the human insulin receptor.

Insulin increased dramatically the tyrosyl phosphorylation of the insulin receptor beta-subunit in mouse NIH-3T3 fibroblasts transfected with the human insulin receptor. Insulin also increased the phosphorylation, on tyrosine residues, of an endogenous 60kDa protein. This protein was identified after being eluted from a GDP-agarose support by GDP. It is suggested that the 60kDa species may be a novel guanine nucleotide binding protein which is specifically phosphorylated by the insulin receptor.

Animals↗
PubMed1989

Treatment of intact hepatocytes with either the phorbol ester TPA or glucagon elicits the phosphorylation and functional inactivation of the inhibitory guanine nucleotide regulatory protein Gi.

The antiserum AS7 can specifically immunoprecipitate alpha-Gi from membrane extracts as well as from a mixture of purified alpha-Gi and alpha-Go as ascertained using [32P]ADP-ribosylated G-proteins. Using this antiserum to immunoprecipitate alpha-Gi from hepatocytes labelled with 32P it was evident that alpha-Gi was phosphorylated under basal (resting) conditions. Challenge of hepatocytes with the tumour promoting phorbol ester TPA, however, elicited a marked enhancement of the phosphorylation state of alpha-Gi. This was accompanied by the loss of inhibitory effect of Gi on adenylate cyclase, as judged by the inability of low concentrations of p[NH]ppG to inhibit forskolin-stimulated adenylate cyclase activity. Such actions were mimicked by treatment of hepatocytes with either glucagon or TH-glucagon, an analogue of glucagon which is incapable of activating adenylate cyclase and elevating intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with either glucagon, TPA or insulin did not affect the ability of pertussis toxin to cause the NAD+-dependent, [32P]ADP-ribosylation of alpha-Gi in membrane fractions isolated from such pre-treated hepatocytes. We suggest that protein kinase C can elicit the phosphorylation and functional inactivation of alpha-Gi in intact hepatocytes. As pertussis toxin only causes the ADP-ribosylation of the holomeric form of Gi, it may be that phosphorylation leaves alpha-Gi in its holomeric state.

Adenylyl Cyclases↗
PubMed1989

Multiple defects occur in the guanine nucleotide regulatory protein system in liver plasma membranes of obese (fa/fa) but not lean (Fa/Fa) Zucker rats: loss of functional Gi and abnormal Gs function.

Hepatocyte membranes from both lean and obese Zucker rats exhibited adenylate cyclase activity that could be stimulated by glucagon, forskolin, NaF and elevated concentrations of p[NH]ppG. In membranes from lean animals, functional Gi was detected by the ability of low concentrations of p[NH]ppG to inhibit forskolin-activated adenylate cyclase. This activity was abolished by treatment of hepatocytes with either pertussis toxin or the phorbol ester TPA, prior to making membranes for assay of adenylate cyclase activity. In hepatocyte membranes from obese animals no functional Gi activity was detected. Quantitative immunoblotting, using an antibody able to detect the alpha subunit of Gi, showed that hepatocyte plasma membranes from both lean and obese Zucker rats had similar amounts of Gi-alpha subunit. This was 6.2 pmol/mg plasma membrane for lean and 6.5 pmol/mg plasma membrane for obese animals. Using thiol pre-activated pertussis toxin and [32P]-NAD+, similar degrees of labelling of the 40 kDa alpha subunit of Gi were found using plasma membranes of both lean and obese Zucker rats. We suggest that liver plasma membranes from obese Zucker rats express an inactive Gi alpha subunit. Thus lesions in liver Gi functioning are seen in insulin-resistant obese rats and in alloxan- and streptozotocin-induced diabetic rats which also show resistance as regards the acute actions of insulin. Liver plasma membranes of obese animals also showed an impairment in the coupling of glucagon receptors to Gs-controlled adenylate cyclase, with the Kd values for activation by glucagon being 17.3 and 126 nM for lean and obese animals respectively. Membranes from obese animals also showed a reduced ability for high concentration of p[NH]ppG to activate adenylate cyclase. The use of [32P]-NAD+ and thiol-preactivated cholera toxin to label the 43 kDa and 52 kDa forms of the alpha-subunit of Gs showed that a reduced labelling occurred using liver plasma membranes from obese animals. It is suggested that abnormalities in the levels of expression of primarily the 52 kDa form of alpha-Gs may give rise to the abnormal coupling between glucagon receptors and adenylate cyclase in liver membranes from obese (fa/fa) Zucker rats.

Adenosine Diphosphate Ribose↗
PubMed1989

Molecular basis of insulin receptor function.

Insulin regulates a wide spectrum of metabolic processes in a variety of tissues. In addition, insulin is a potent growth factor, promoting normal cell growth and division in cultured cells, though the physiological importance of such actions in man is less certain. Some of the effects of insulin are very rapid and apparent within seconds, for example those on transport processes and the activity of regulatory enzymes. Other effects, such as those on enzyme concentrations and cell growth, may be manifest only over a period of many minutes or even hours. Such differences in time dependence may be more a reflection of the nature of the processes themselves than an indication of different signalling mechanisms. However, it is still unclear whether the regulation of diverse metabolic processes by insulin involves just one or multiple distinct primary intracellular signals. We will review here what is at present known about the structure and activity of the receptor, and current ideas on signalling mechanisms.

Cloning, Molecular↗
PubMed1989

Metformin treatment of lean and obese Zucker rats modulates the ability of glucagon and insulin to regulate hepatocyte adenylate cyclase activity.

Glucagon stimulated adenylate cyclase activity some 21-fold in liver membranes from lean (Fa/Fa) and some 20-fold in membranes from obese (fa/fa) Zucker rats, with constants yielding half-maximal activation (Ka values) of 12.6 and 120.1 nmol/l respectively. Treatment of animals with the biguanide drug metformin (N',N'-dimethylbiguanide) decreased the ability of glucagon to stimulate this enzyme to some 16-fold for both the lean and obese animals and reduced the Ka values for activation of this enzyme by glucagon to 6.3 and 60.9 nmol/l respectively. Insulin inhibited glucagon-stimulated adenylate cyclase activity by some 24% in liver membranes from lean animals and some 17% in liver membranes from obese animals, with constants yielding half-maximal inhibition (Ki values) of 110 and 160 nmol/l respectively. The ability of insulin to inhibit the adenylate cyclase activity, from obese but not lean animals, was attenuated when insulin concentrations over 5 nmol/l were employed. Treatment of animals with metformin profoundly altered the sensitivity of adenylate cyclase to inhibition by insulin, with inhibition being increased to some 32% using liver membranes from either lean or obese animals. Values of Ki for this inhibitory action of insulin were 520 and 500 nmol/l using membranes from the lean and obese animals respectively, and no reduction in the ability of insulin, at concentrations over 5 nmol/l, to inhibit adenylate cyclase activity was observed using membranes from obese animals. Metformin also changed the kinetics of inhibition of adenylate cyclase by insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1989

Identification of the pertussis and cholera toxin substrates in normal and N-ras transformed NIH3T3 fibroblasts and an assessment of their involvement in bombesin-stimulation of inositol phospholipid metabolism.

Cholera and pertussis toxin-sensitive G-proteins were examined using specific immunological probes in wild type NIH3T3 cells and in clones of these cells containing the N-ras gene attached to a promotor where expression either was (T15+) or was not (T15-) induced. The major pertussis toxin sensitive-polypeptide had the immunological characteristics of Gi2. Two distinct forms of Gs alpha (45 and 42 kDa) were identified. Long term over-expression of p21N-ras (T15+ cells) did not alter the levels of Gi2 alpha or of Gs alpha. Pretreatment of NIH3T3 or T15 cells with either pertussis toxin or cholera toxin led to the complete in situ ADP-ribosylation of the respective G-proteins. Modification of Gi2 by pertussis toxin, however, had no inhibitory effect on the ability of bombesin to stimulate the production of inositol phosphates in any of these cells lines. Treatment of these cells with cholera toxin elicited a potent inhibition of the bombesin-stimulated production of inositol phosphates. This could be mimicked, however, by other agents which increase intracellular cyclic AMP concentrations. Cholera toxin treatment did not produce a significant alteration in the number of bombesin receptors on the cell surface. These results suggest that, in the T15 cell line, enhanced coupling of bombesin receptors to a phospholipase C-mediated hydrolysis of inositol phospholipids is either produced directly by p21N-ras or that overexpression of this gene product leads to the enhanced expression or function of a cholera and pertussis toxin-insensitive G-protein which then mediates the effect.

Bombesin↗
PubMed1988

Tosyl-lysyl chloromethylketone detects conformational changes in the catalytic unit of adenylate cyclase induced by receptor and G-protein stimulation.

Incubation of striatal membranes with tosyl-lysyl chloromethylketone (TLCK) led to the irreversible inactivation of adenylate cyclase. However, under conditions where an interaction between the catalytic unit of adenylate cyclase and the alpha-subunit of the stimulatory G-protein GS were promoted, then the ability of TLCK to inhibit adenylate cyclase was markedly attenuated. The potency of stimulatory ligands, functioning through GS, to attenuate the sensitivity of adenylate cyclase to inactivation by TLCK was paralleled by their potency to activate adenylate cyclase. The local anaesthetic and membrane-fluidizing agent benzyl alcohol amplified GS-mediated stimulation of adenylate cyclase activity, whilst diminishing the ability of GS-mediated coupling to attenuate inactivation of adenylate cyclase by TLCK. In the absence of GS-mediated coupling, benzyl alcohol exerted only a small stimulatory effect on adenylate cyclase activity and had little effect on the ability of TLCK to inactivate this enzyme. We suggest that TLCK modifies a reactive group at or near the active site of adenylate cyclase which causes the functional inactivation of this enzyme. The reactivity of this group appears to be markedly affected by conformational changes elicited through coupling of adenylate cyclase to GS.

Adenylyl Cyclase Inhibitors↗
PubMed1988

An insulin mediator preparation serves to stimulate the cyclic GMP activated cyclic AMP phosphodiesterase rather than other purified insulin-activated cyclic AMP phosphodiesterases.

An insulin mediator preparation was obtained from rat hepatocytes which had been treated with insulin. This preparation inhibited adenylate cyclase activity. It stimulated the activity of homogeneous preparations of both the cytosolic and membrane-bound forms of rat liver cyclic GMP-activated cyclic AMP phosphodiesterase. It failed to activate homogeneous preparations of both the peripheral plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases. The insulin mediator preparation stimulated cyclic GMP-activated cyclic AMP phosphodiesterase activity in a dose-dependent fashion with a hill coefficient of 0.46. Insulin caused the dose-dependent production of mediator activity in intact hepatocytes with a Ka of 9 pM, although concentrations of insulin greater than 10 nM progressively reduced stimulatory activity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Treatment of intact hepatocytes with the calcium ionophore A23187 perturbs both the synthesis and the degradation of the second messenger cyclic AMP. Actions on adenylate cyclase and cyclic AMP phosphodiesterase activities.

The presence of the calcium ionophore A23187 augmented glucagon's ability to elevate intracellular cyclic AMP concentrations in intact hepatocytes. However, when the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine (IBMX) was added to prevent the degradation of cyclic AMP then the presence of A23187 attenuated the ability of glucagon to increase intracellular cyclic AMP concentrations. Treatment of intact hepatocytes with A23187 led to a dose-dependent persistent inhibition of the glucagon-stimulated adenylate cyclase activity expressed by a membrane fraction isolated from such ionophore-treated hepatocytes. In hepatocytes where glucagon-stimulated adenylate cyclase activity was desensitized then A23187-treatment of hepatocytes failed to exert any inhibitory action on adenylate cyclase. Treatment of isolated membranes directly with A23187 did not elicit any changes in glucagon-stimulated adenylate cyclase activity. Such actions of A23187 were blunted when Ca2+ (2.5 mM) was not added to the extracellular medium. It is suggested that treatment of hepatocytes with A23187 leads to the functional uncoupling of glucagon-stimulated adenylate cyclase activity in a manner which appears to mimic the desensitization process. A23187-treatment also exerted an overall inhibitory effect on the cyclic AMP phosphodiesterase activity displayed by intact hepatocytes. Thus treatment of hepatocytes with A23187 exerted a profound effect on cyclic AMP metabolism in these cells.

1-Methyl-3-isobutylxanthine↗