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M D Houslay

Publications and source records attributed to M D Houslay.

At least 127 records · Page 7Linked to original sources
PubMed1990

Diabetes-induced changes in guanine-nucleotide-regulatory-protein mRNA detected using synthetic oligonucleotide probes.

Synthetic oligonucleotide probes were designed to detect the alpha-subunits of the guanine-nucleotide-regulatory proteins (G-proteins) Gi-1, Gi-2, Gi-3 and Gs (Gi is inhibitory and Gs is stimulatory). Each probe detected a single major mRNA species in Northern blots of RNA extracted from a variety of tissues. A probe was designed to identify the two forms of G-protein beta-subunits, beta 1 and beta 2. This probe hybridised with a single 1.8-kb transcript (beta 2) in RNA from all tissues studied except for brain, where a less-abundant 3.4-kb transcript (beta 1) was also detected. These probes were used to assess whether the induction of diabetes, using streptozotocin, altered the levels of mRNA coding for specific G-protein components. In hepatocytes, diabetes caused a significant reduction in the number of transcripts coding for alpha-Gs, alpha-Gi-2 and alpha-Gi-3; mRNA for alpha-Gi-1 was undectable. In adipocytes, diabetes increased dramatically the mRNA coding for alpha-Gi-1 and alpha-Gi-3, whilst no significant changes occurred in the fractions coding for alpha-Gi-2 and alpha-Gs. No significant changes in the mRNA coding for G-protein alpha-subunits were observed in either brain, heart, skeletal muscle or kidney. Diabetes did not cause any significant changes in the mRNA coding for beta 2 in any tissue or cell population studied. Such results on the relative levels of mRNA encoding G-protein components was obtained by comparing equal amounts of total RNA from tissues of control and diabetic animals. G-protein mRNA levels were expressed relative to ribosomal 28S RNA levels and, in some instances, relative to transcripts for a structural protein called CHO-B. The total cellular levels of both RNA and DNA were assessed in the various tissues and cells studied. Major falls in RNA levels/cell appeared to occur in hepatocytes and to a lesser extent in adipocytes and skeletal muscle. Thus major reductions in G-protein transcripts occurred in hepatocytes. The detected changes in G-protein mRNA are discussed in relation to the available evidence on G-protein expression. We suggest that diabetes causes tissue-specific changes in the levels of mRNA for particular G-protein species; this may have consequences for the functioning of cellular signal-transduction mechanisms in the affected tissues.

Animals↗
PubMed1990

Diabetes-induced alterations in the expression, functioning and phosphorylation state of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes.

Levels of the G-protein alpha-subunits alpha-Gi-2, alpha-Gi-3 and the 42 kDa, form of alpha-Gs were markedly decreased in hepatocyte membranes from streptozotocin-diabetic animals as compared with normals. In contrast, no detectable changes in alpha-Gi subunits were seen in liver plasma membranes of streptozotocin-diabetic animals, although levels of the 45 kDa form of Gs were increased. G-protein beta subunits in plasma membranes were unaffected by diabetes induction. Analysis of whole-liver RNA indicated that the induction of diabetes had little effect on transcript levels of Gi-3, caused an increase in Gs transcripts and decreased transcript number for Gi-2, albeit to a much lesser extent than was observed upon analysis of hepatocyte RNA. In both hepatocyte and liver plasma membranes, immunoblot analysis showed that levels of the catalytic unit of adenylate cyclase were increased upon induction of diabetes. Under basal conditions, alpha-Gi-2 from hepatocytes of diabetic animals was found to be both phosphorylated to a greater extent than alpha-Gi-2 isolated from hepatocytes of normal animals, and furthermore was resistant to any further phosphorylation upon challenge of hepatocytes with angiotensin, vasopressin or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. Treatment of isolated plasma membranes from normal, but not diabetic, animals with purified protein kinase C caused the phosphorylation of alpha-Gi-2. Treatment of membranes from diabetic animals with alkaline phosphatase caused the dephosphorylation of alpha-Gi-2 and rendered it susceptible to subsequent phosphorylation with protein kinase C. Low concentrations of the non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate inhibited adenylate cyclase activity in both hepatocyte and liver plasma membranes from normal, but not diabetic, animals.

Adenylyl Cyclase Inhibitors↗
PubMed1990

Changes in the phosphorylation state of the inhibitory guanine-nucleotide-binding protein Gi-2 in hepatocytes from lean (Fa/Fa) and obese (fa/fa) Zucker rats.

Treatment of intact, 32Pi-labelled hepatocytes from lean Zucker rats with a range of agents including 12-O-tetradecanoyl-phorbol 13-acetate (TPA), vasopressin, and angiotensin II elicited substantial increases in the phosphorylation of the alpha-subunit of the inhibitory G protein of adenylate cyclase (alpha Gi-2). These agonist-induced phosphorylations of alpha Gi-2 were associated with loss of Gi function as assessed by the ability of low concentrations of guanylyl 5'-[beta,gamma imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity. Hepatocytes from obese Zucker rats displayed a resistance to both agonist-induced phosphorylation of alpha Gi-2 and to p[NH]ppG-mediated inhibition of adenylate cyclase. The basal level of alpha Gi-2 phosphorylation in hepatocytes from obese Zucker rats was considerably greater at 1.06 +/- 0.09 mol phosphate/mol alpha Gi-2 than in hepatocytes from lean animals which gave 0.54 +/- 0.09 mol phosphate/mol alpha Gi-2. Incubation with TPA (10 ng/ml, 15 min) approximately doubled the level of phosphorylation of alpha Gi-2 in the hepatocytes from lean animals but had little effect on the phosphorylation of alpha Gi-2 in hepatocytes from obese animals. Incubation of hepatocytes from lean animals with ligands which lead to the phosphorylation of alpha Gi-2 abolished the ability of low concentrations of p[NH]ppG to inhibit adenylate cyclase expressed in isolated membranes. Treatment of hepatocyte plasma membranes from lean but not obese Zucker rats with pure protein kinase C led to the phosphorylation of alpha Gi-2. The resistance to protein-kinase-C-mediated phosphorylation in hepatocyte membranes from obese animals could be overcome by treatment of the membranes with alkaline phosphatase. These results indicate that the defect in guanine-nucleotide-mediated 'Gi function' seen in obese Zucker rats may be due to an inactivating phosphorylation of alpha Gi-2.

Adenylyl Cyclases↗
PubMed1990

Identification and selective inhibition of four distinct soluble forms of cyclic nucleotide phosphodiesterase activity from kidney.

Homogenization of rat kidney under isotonic conditions and in the presence of protease inhibitors showed that some 92% of the cyclic AMP phosphodiesterase activity and some 83% of the cyclic GMP phosphodiesterase activity was released into the soluble fraction. Analysis of soluble phosphodiesterase activity by FPLC on a Mono-Q column resolved four distinct fractions expressing cyclic nucleotide phosphodiesterase activity. Lineweaver-Burk plots for the hydrolysis of both cyclic GMP and cyclic AMP yielded linear results. The first two peaks (KPDE-MQ-II, KPDE-MQ-III) showed higher activities towards cyclic GMP than cyclic AMP with the ratio of their Vmax values for the hydrolysis of cyclic AMP/cyclic GMP being 0.66 and 0.16, respectively. For the second two peaks (KPDE-MQ-IV, KPDE-MQ-V) the Vmax ratios for the hydrolysis of cyclic AMP/cyclic GMP were 6.4 and 16.7, respectively. All enzymes exhibited similar low Km values for both cyclic AMP and cyclic GMP but had very different Vmax values. KPDE-MQ-II was activated by Ca2+/calmodulin. The cyclic AMP phosphodiesterase activity of KPDE-MQ-III was augmented by the presence of low concentrations of cyclic GMP. Thermal denaturation studies showed that the phosphodiesterase activity of each fraction decayed as a single exponential indicating that each phosphodiesterase fraction contained but a single phosphodiesterase activity. The inhibitors IBMX, zaprinast, milrinone, amrinone, buquineran, carbazeran, ICI 118233, ICI 63197 exerted selective effects on the activities of these enzymes. We compared the action of these compounds on cyclic GMP phosphodiesterases from bovine retina. Over the concentration ranges used, the bovine retinal enzyme was only inhibited by IBMX, zaprinast and carbazeran. The cytosolic isoenzymes of cyclic AMP phosphodiesterases play a much more important role in metabolizing cyclic AMP in kidney compared with liver, where the activity of membrane-bound isoenzymes predominate.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1990

Insulin alters the target size of the peripheral cyclic AMP phosphodiesterase but not the integral cyclic GMP-stimulated cyclic AMP phosphodiesterase in liver plasma membranes.

Radiation inactivation of the two high affinity cyclic AMP phosphodiesterases (PDE) found in liver plasma membranes afforded an estimation of their molecular target sizes in situ. The activity of the peripheral plasma membrane PDE decayed as a single exponential with a target size corresponding to a monomer of circa 54 kDa. The integral, cyclic GMP-stimulated PDE decayed as a dimer of circa 125 kDa. Preincubation of plasma membranes with insulin (10nM), prior to irradiation, caused the target size of only the peripheral plasma membrane PDE to increase. We suggest that insulin addition causes the peripheral plasma membrane PDE to alter its coupling to an integral plasma membrane protein with a target size of circa 90 kDa.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

Hormonal regulation of Gi2 alpha-subunit phosphorylation in intact hepatocytes.

Hepatocytes contain the Gi2 and Gi3 forms of the 'Gi-family' of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity ('Gi'-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a 'Gi-like' G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Diabetes abolishes the GTP-dependent, but not the receptor-dependent inhibitory function of the inhibitory guanine-nucleotide-binding regulatory protein (Gi) on adipocyte adenylate cyclase activity.

Adipocyte membranes from control rats exhibited a functional Gi (inhibitory guanine-nucleotide-binding protein) activity which could be assessed either by the inhibitory action of low concentrations of guanosine 5-[beta gamma-imido]triphosphate (p[NH]ppG) upon forskolin-stimulated adenylate cyclase activity or by the inhibitory action of high concentrations of GTP upon isoprenaline-stimulated adenylate cyclase activity. When membranes from animals made diabetic with streptozotocin were used, then both such inhibitory functions of Gi were abolished. In contrast, receptor-mediated inhibitory responses of Gi, effected by N6-phenylisopropyl (adenosine), prostaglandin E2 or nicotinate, were either unchanged or even apparently more effective in membranes from diabetic animals. Induction of diabetes did not cause any change in the adipocyte plasma membrane levels of the alpha, GTP-binding subunits of either Gi1 or Gi2 or of Gs (stimulatory guanine-nucleotide-binding protein), but elicited an increase in the level of alpha-Gi3. The induction of diabetes reduced the specific activity of adenylate cyclase in adipocyte membranes and enhanced the stimulatory effect of isoprenaline. It is suggested that diabetes causes selective changes in the functioning of Gi in adipocyte membranes which removes the tonic GTP-dependent inhibitory function of this G-protein.

Adenylyl Cyclases↗
PubMed1990

The use of selective inhibitors and computer modelling to evaluate the role of specific high affinity cyclic AMP phosphodiesterases in the hormonal regulation of hepatocyte intracellular cyclic AMP concentrations.

Using experimentally derived data for the activities and kinetic constants of hepatocyte cyclic AMP phosphodiesterase isoenzymes together with the derived changes in adenylate cyclase activity, due to stimulation and subsequent desensitization by glucagon, a computer model was established to simulate hepatocyte cyclic AMP metabolism. The established ability of glucagon to activate the 'dense-vesicle' cyclic AMP phosphodiesterase by eliciting its cyclic AMP-dependent phosphorylation was shown on the model to be capable of eliciting a profound reduction in the glucagon-stimulated increase in intracellular cyclic AMP. This was consistent with experimentally derived observations using the compound ICI 118233 which was used to inactivate the 'dense-vesicle' enzyme selectively. The non-hydrolysable adenosine agonist N6 (phenylisopropyl)-adenosine (PIA), which prevents glucagon pre-treatment of hepatocytes blocking the ability of insulin to stimulate the peripheral plasma membrane cyclic AMP phosphodiesterase, is shown here to accentuate the ability of insulin to decrease glucagon-elevated intracellular cyclic AMP concentrations. This effect was obliterated using the compound ICI 63197, a selective inhibitor of the peripheral plasma membrane phosphodiesterase. Computer modelling studies, taking into account experimentally derived actions in insulin in activating the peripheral plasma membrane phosphodiesterase, confirmed the potential of this enzyme to decrease intracellular cyclic AMP concentrations. Modelling of the putative effect of an insulin 'mediator' in activating the two cyclic GMP-stimulated cyclic AMP phosphodiesterase isoenzymes was shown to elicit a decrease in intracellular cyclic AMP concentrations which was comparable to that caused by insulin's action on intact hepatocytes. The relative contribution of each phosphodiesterase form to the metabolism of hepatocyte intracellular cyclic AMP, together with an assessment of the potential effect of inhibition and activation of specific species, was evaluated using the computer model. These experimental and stimulation studies indicate that alterations in the phosphodiesterase activity of the 'dense-vesicle' enzyme, the peripheral plasma membrane enzyme, the cyclic GMP-stimulated cyclic AMP isoforms and the IBMX-insensitive PDE-MQ-II can elicit profound effects upon hepatocyte intracellular cyclic AMP concentrations.

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

The local anaesthetic benzyl alcohol attenuates the alpha 2-adrenoceptor-mediated inhibition of human platelet adenylate cyclase activity when stimulated by prostaglandin E1, but not that stimulated by forskolin.

Treatment of human platelets with concentrations of benzyl alcohol up to 50 mM augmented adenylate cyclase activity when it was assayed in the basal state and when stimulated by prostaglandin E1 (PGE1), isoprenaline or NaF. Benzyl alcohol antagonized the stimulatory effect exerted on the catalytic unit of adenylate cyclase by the diterpene forskolin. Benzyl alcohol did not modify the magnitude of the inhibitory response when the catalytic unit of adenylate cyclase was inhibited by using either low concentrations of guanosine 5'-[beta gamma-imido]triphosphate, which acts selectively on the inhibitory guanine nucleotide-regulatory protein Gi, or during alpha 2-adrenoceptor occupancy, by using adrenaline (+ propranolol). Some 34% of the potent inhibitory action of adrenaline on PGE1-stimulated adenylate cyclase was obliterated in a dose-dependent fashion (concn. giving 50% inhibition = 12.5 mM) by benzyl alcohol, with the residual inhibitory action being apparently resistant to the action of benzyl alcohol at concentrations up to 50 mM. Treatment of membranes with benzyl alcohol did not lead to the release of either the alpha-subunit of Gi or G-protein subunits. The alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase was abolished when assays were performed in the presence of Mn2+ rather than Mg2+ and, under such conditions, dose-effect curves for the action of benzyl alcohol on PGE1-stimulated adenylate cyclase activity were similar whether or not adrenaline (+propranolol) was present. We suggest that (i) alpha 2-adrenoceptor- and Gi-mediated inhibition of PGE1-stimulated adenylate cyclase may have two components, one of which is sensitive to inhibition by benzyl alcohol, and (ii) the Gi-mediated inhibition of forskolin-stimulated adenylate cyclase exhibits predominantly the benzyl alcohol-insensitive component.

Adenylyl Cyclases↗
PubMed1989

Insulin affects the ability of Gi to be ADP-ribosylated but does not elicit its phosphorylation in intact hepatocytes.

Insulin inhibited the ability of activated pertussis toxin to catalyse the ADP-ribosylation of alpha-Gi in isolated plasma membranes in either the absence of added guanine nucleotides or in the presence of GTP. In contrast, when the non-hydrolysable GTP analogue guanylyl-5'-imido-diphosphate (p[NH]ppG) was added to ribosylation mixtures, to inhibit the action of pertussis toxin in catalysing the ADP-ribosylation of alpha-Gi, then the addition of insulin attenuated the action of p[NH]ppG causing an increase in alpha-Gi ribosylation. Pre treatment of intact hepatocytes with insulin had no effect on the subsequent ability of thiol-preactivated pertussis toxin to cause the ADP-ribosylation of alpha Gi using isolated membranes from such cells. The ability of p[NH]ppG to inhibit forskolin-stimulated adenylate cyclase activity was attenuated in the presence of insulin. Insulin did not cause the phosphorylation of alpha-Gi in either intact hepatocytes or in isolated membranes.

Adenosine Diphosphate Ribose↗
PubMed1989

Resolution of soluble cyclic nucleotide phosphodiesterase isoenzymes, from liver and hepatocytes, identifies a novel IBMX-insensitive form.

DEAE chromatography of a high speed supernatant fraction from a homogenate of rat liver, prepared under isotonic conditions in the presence of protease inhibitors, yielded three peaks of cyclic nucleotide phosphodiesterase activity (PDE activity). The first peak could be resolved on Affi-gel Blue chromatography to yield a Ca2+/calmodulin stimulated cyclic GMP specific PDE and a cyclic AMP and cyclic GMP hydrolysing PDE whose activity was insensitive to Ca2+/calmodulin. These two activities could also be clearly resolved by Mono-Q chromatography of soluble extracts from both liver and hepatocytes. These had different molecular weights, kinetics of substrate utilization, thermostabilities, dependence on Mg2+ and inhibitor sensitivities. The cyclic AMP and cyclic GMP utilizing PDE resolved in these procedures appears to be a novel enzyme form (PDE-MQ-I) which is insensitive to inhibition by the so-called non-selective PDE inhibitor IBMX and displays catalytic activity in the absence of Mg2+. None of the inhibitors tested were capable of inhibiting this form showing that the catalytic activity of this species could be distinguished from all the other soluble activities. This novel enzyme hydrolysed both cyclic AMP and cyclic GMP with Km values of 25 microM and 237 microM, respectively. The Vmax ratio of hydrolysis of cyclic GMP/cyclic AMP was above unity (1.4). It accounted for 30% of the soluble cyclic AMP PDE activity and 10% of the cyclic GMP PDE activity assessed at 1 microM substrate. Gel filtration of PDE-MQ-I indicated a size of 33,150 Da, in contrast to the size of 237,500 Da observed for the Ca2+/calmodulin PDE-MQ-II. Thermal inactivation of PDE-MQ-I and PDE-MQ-II yielded single exponential decays with t1/2 values of 6.33 min and 0.7 min at 60 degrees respectively. In the presence of saturating Ca2+, PDE-MQ-II was activated by calmodulin with an EC50 of ca. 30 ng/ml. In the presence of calmodulin, PDE-MQ-II was activated by Ca2+ with an EC50 of ca. 20 microM. Chromatography of homogenates on Mono-Q also identified a cyclic GMP-activated cyclic nucleotide PDE (PDE-MQ-III) and two cyclic AMP specific activities (PDE-MQ-IV and PDE-MQ-V). These exhibited very different inhibitor sensitivities and could be readily distinguished using the compound Ro-20-1724 which yielded IC50 values for inhibition of greater than 500 microM, 13 microM and 1.5 microM, respectively, for the hepatocyte enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1989

Adipocyte plasma-membrane Gi and Gs in insulinopenic diabetic patients.

Changes in the amounts and functions of G-proteins have been reported in diabetic rats. We determined the Gi/Gs ratio of adipocyte plasma membranes in six insulinopenic diabetic patients and matched controls by immunoblotting with antisera against synthetic peptides corresponding to regions of G-proteins. No consistent changes in the Gi/Gs ratio were observed. It is still possible that the functional status of the G-proteins may be altered in diabetes.

Adipose Tissue↗