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N J Pyne

Publications and source records attributed to N J Pyne.

68 records · Page 4Linked to original sources
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

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↗
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↗
PubMed1987

Specific antibodies and the selective inhibitor ICI 118233 demonstrate that the hormonally stimulated 'dense-vesicle' and peripheral-plasma-membrane cyclic AMP phosphodiesterases display distinct tissue distributions in the rat.

Polyclonal-antibody preparations DV1 and PM1, raised against purified preparations of rat liver insulin-stimulated 'dense-vesicle' and peripheral-plasma-membrane cyclic AMP phosphodiesterases, were used to analyse rat liver homogenates by Western-blotting techniques. The antibody DV1 identified only the 63 kDa native subunit of the 'dense-vesicle' enzyme, and the antibody PM1 only the 52 kDa subunit of the plasma-membrane enzyme. These antibodies also detected the subunits of these two enzymes in homogenates of kidney, heart and white adipose tissue from rat. Quantitative immunoblotting demonstrated that the amount of these enzymes (by wt.) varied in these different tissues, as did the expression of these two enzymes, relative to each other, by a factor of as much as 7-fold. The ratio of the dense-vesicle enzyme to the peripheral-plasma-membrane enzyme was lowest in liver and kidney and highest in heart and white adipose tissue. ICI 118233 was shown to inhibit selectively the 'dense-vesicle' cyclic AMP phosphodiesterase in liver. It did this in a competitive fashion, with a Ki value of 3.5 microM. Inhibition of tissue-homogenate cyclic AMP phosphodiesterase activity by ICI 118233 was used as an index of the contribution to activity by the 'dense-vesicle' enzyme. By this method, a tissue distribution of the 'dense-vesicle' enzyme was obtained which was similar to that found by using the immunoblotting technique. The differential expression of isoenzymes of cyclic AMP phosphodiesterase activity in various tissues might reflect a functional adaptation, and may provide the basis for the different physiological actions of compounds which act as selective inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Proteolysis of cyclic AMP phosphodiesterase-II attenuates its ability to be inhibited by compounds which exert positive inotropic actions in cardiac tissue.

Extraction of frozen canine cardiac muscle rendered soluble over 90% of the cyclic AMP phosphodiesterase activity. The residual activity was membrane-bound. Ion exchange chromatography of the soluble activity on DE-52 allowed for the resolution of three distinct cyclic AMP phosphodiesterase fractions termed PDE-I, PDE-II and PDE-III in order of elution from the column by a linear NaCl gradient. The relative ratio of cyclic AMP phosphodiesterase activity exhibited by these three peaks was 1:0.65:0.82 and of cyclic GMP phosphodiesterase activity was 1:0.52:0.05 for PDE-I, PDE-II and PDE-III respectively. PDE-II and PDE-III were further purified by re-chromatography on DE-52. Fractions PDE-II and PDE-III were thermolabile at 50 degrees, decaying as single exponentials with half lives of 180 sec and 77 sec respectively. All three species exhibited non-linear Lineweaver-Burke plots for the hydrolysis of cyclic AMP, exhibiting both high and low affinity components. Hydrolysis of cyclic GMP by all three components obeyed normal kinetics, yielding linear plots. PDE-I was a Ca2+/calmodulin-activated species which exhibited a low Km for both cyclic AMP and cyclic GMP but hydrolysed cyclic GMP with a higher Vmax than for cyclic AMP. PDE-II exhibited a much lower Km for cyclic AMP than for cyclic GMP and a much higher Vmax for the hydrolysis of cyclic AMP. PDE-III exhibited a low Km for both cyclic AMP and cyclic GMP, however, its Vmax for cyclic AMP was about 40-fold higher than for cyclic GMP. Cyclic GMP acted as a potent inhibitor (IC50 = 6.3 microM) of cyclic AMP hydrolysis catalysed by PDE-III but not of the hydrolysis of cyclic AMP by PDE-II (IC50 = 33.2 microM). The phosphodiesterase inhibitors milrinone, CI-930, UK-35,493, carbazeran and buquineran acted as potent inhibitors of cyclic AMP hydrolysis catalysed by both PDE-II and PDE-III enzymes. They did not inhibit PDE-I activity. PDE-II, when prepared in the absence of protease inhibitors exhibited a reduced potency to inhibition by these compounds. Treatment of purified PDE-II with trypsin caused a reduction in enzyme activity and reduced dramatically the sensitivity of PDE-II activity to inhibition by these various compounds. The action of proteolysis in attenuating the inhibitory effect of these compounds on PDE-II was most dramatic with CI-930, milrinone, amrinone, buquineran and UK35,493 and least dramatic with carbazeran and IBMX.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

The insulin- and glucagon-stimulated 'dense-vesicle' high-affinity cyclic AMP phosphodiesterase from rat liver. Purification, characterization and inhibitor sensitivity.

The hormone-stimulated 'dense-vesicle' cyclic AMP phosphodiesterase was solubilized as a proteolytically 'clipped' species, and purified to apparent homogeneity from rat liver with a 2000-3000-fold purification and a 13-18% yield. It appeared to be a dimer (Mr 112,000), of two Mr-57,000 subunits. Solubilization of either a liver or a hepatocyte membrane fraction, with sodium cholate in the presence of the protein inhibitor benzamidine, identified three protein bands which could be immunoprecipitated by a polyclonal antibody raised against the pure enzyme. The major band at Mr 62,000 is suggested to be the native 'dense-vesicle' enzyme, having a Mr-5000 extension which serves to anchor this enzyme to the membrane and which is cleaved off during proteolytic solubilization; the Mr-200,000 band is an aggregate of the Mr-62,000 species, and the Mr-63,000 species is possibly a precursor. The purified 'clipped' enzyme hydrolysed cyclic AMP with kinetics indicative of apparent negative co-operativity, with a Hill coefficient (h) of 0.43 and limiting kinetic constants of Km1 = 0.3 +/- 0.05 microM, Km2 = 29 +/- 6 microM, Vmax.1 = 0.114 +/- 0.015 unit/mg of protein and Vmax.2 = 0.633 +/- 0.054 unit/mg of protein. It hydrolysed cyclic GMP with Michaelis kinetics, Km = 10 +/- 1 microM and Vmax. = 4.1 +/- 0.2 units/mg of protein. Cyclic GMP was a potent inhibitor of cyclic AMP hydrolysis, with an IC50 (concn. giving 50% inhibition) of 0.20 +/- 0.01 microM-cyclic GMP when assayed at 0.1 microM-cyclic AMP. This enzyme was inhibited potently by several drugs known to exert positive inotropic effects on the heart, was extremely thermolabile, with a half-life of 4.5 +/- 0.5 min at 40 degrees C, and was shown to be distinct from the rat liver insulin-stimulated peripheral-plasma-membrane cyclic AMP phosphodiesterase [Marchmont, Ayad & Houslay (1981) Biochem. J. 195, 645-652].

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

The phorbol ester TPA inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes.

Treatment of intact hepatocytes with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) potentiated the ability of glucagon to increase intracellular cyclic AMP concentrations. This effect was dose-dependent upon TPA, exhibiting an EC50 of 0.39 ng/ml and such activation was observed at both saturating and sub-saturating concentrations of glucagon. However, this stimulatory effect of TPA was completely abolished by the presence of the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine, when TPA now inhibited the glucagon-stimulated increase in intracellular cyclic AMP concentrations. It is suggested that, as well as inhibiting glucagon-stimulated adenylate cyclase activity, TPA also inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes. Treatment of either hepatocyte homogenates or purified cyclic AMP phosphodiesterase with TPA failed to show any direct inhibitory effect of TPA on activity showing that TPA did not exert any direct inhibitory action on phosphodiesterase activity. However, homogenates made from hepatocytes that had been pre-treated with TPA did show a reduced cyclic AMP phosphodiesterase activity. It is suggested that TPA might inhibit cyclic AMP phosphodiesterase activity through phosphorylation by C-kinase.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver.

Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a 'particulate enzyme' found as an integral membrane protein associated with the plasma membrane, and a 'soluble' enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMedDate not supplied

Delayed cardioprotection is associated with the sub-cellular relocalisation of ventricular protein kinase C epsilon, but not p42/44MAPK.

Both noradrenaline administration to rats and rapid cardiac pacing in dogs induces delayed protection of the heart against ischaemia-induced ventricular arrhythmias. In an attempt to establish molecular mechanisms underlying the delayed cardioprotection, we have examined the potential role of two kinases, PKC epsilon and p42/44MAPK. These protein kinases are expressed in the ventricles of the heart and are characterised by their ability to regulate ion-flux and gene transcription. In the rat p42MAPK is predominantly localised in the high-speed supernatant fraction of the ventricle homogenate, whereas p44MAPK is enriched in the nuclear low speed pellet. A small proportion of the p42MAPK is activated even in hearts from control animals. However, neither kinase is relocalised or activated by noradrenaline administration and this provides preliminary evidence the p42/44MAPK may not play a significant role in delayed protection in this species. In contrast, noradrenaline does induce the translocation of PKC epsilon to cell membranes, a response that is sustained for up to 4 h. However, PKC epsilon is down-regulated from the cytoplasm after 24 h post noradrenaline treatment. PKC epsilon is also translocated to the membrane in dogs that have been classically pre-conditioned and cardiac paced. In the latter case, translocation of PKC epsilon from the cytoplasm to the cell membrane is evident 24 h after pacing. These results indicate that the release of endogenous mediators may either inhibit down-regulation or elicit an increase in PKC epsilon mRNA expression. Therefore, in dog heart the subcellular relocalisation of PKC epsilon persists into the 'second window' and may play a central role in the molecular mechanism governing delayed cardioprotection. It is important in the future to identify either the gene products that are induced or the target protein(s) that are phosphorylated by PKC epsilon.

Animals↗
PubMedDate not supplied

Ischaemic preconditioning in the rat heart: the role of G-proteins and adrenergic stimulation.

UNLABELLED: Since recent findings indicate the involvement of G-proteins in the mechanism of ischaemic preconditioning (PC), the present study was aimed to investigate the role of adrenergic mechanisms, such as G-proteins and stimulation of adrenergic receptors, in this phenomenon. For this purpose, isolated Langendorff-perfused rat hearts were subjected to regional ischaemia (30 min occlusion of LAD) followed by reperfusion. The effect of PC (a single 5 min occlusion/reperfusion before a long occlusion) on ischaemia- and reperfusion induced arrhythmias was studied in conjunction with an assessment of G-proteins in the myocardial tissue by means of Western blotting and ADP-ribosylation with bacterial toxins. To follow the link between G-proteins and adrenergic receptors, their stimulation by exogenous norepinephrine (NE) was applied to test whether it can mimic the effect of PC on arrhythmias. Thirty min ischaemia and subsequent reperfusion induced high incidence of ventricular tachycardia (VT) and fibrillation (VF). PC significantly reduced a total number of extrasystoles, incidence of VT and abolished VF. It was, however, insufficient to suppress reperfusion-induced sustained VF. Measurement of G-proteins revealed that PC led to a reduction of stimulatory Gs proteins, whereas inhibitory Gi proteins were increased. NE (50 nmol) introduced a manner of similar to PC (5 min infusion, 10 min normal reperfusion) reduced ischaemic arrhythmias in the same way, as PC. In addition, in NE-pretreated hearts reperfusion induced mostly transient VF, which was spontaneously reverted to normal sinus rhythm. A transient increase in heart rate and perfusion pressure during NE infusion completely waned before the onset of ischaemia, indicating that antiarrhythmic effect was not related to haemodynamic changes and to conditions of myocardial perfusion. CONCLUSION: antiarrhythmic effect of PC may be mediated by a stimulation of adrenergic receptors coupled to appropriate G-proteins. Consequently, the inhibition of adenylate cyclase activity and reduction in cAMP level, as well as the activation of protein kinase C may be considered as two possible pathways leading to a final response.

Adenosine Diphosphate Ribose↗
PubMedDate not supplied

Platelet-derived growth factor activates a mammalian Ste20 coupled mitogen-activated protein kinase in airway smooth muscle.

We have investigated the mechanisms regulating p38MAPK in airway smooth muscle cells. Incubation of cells with platelet-derived growth factor (PDGF) stimulated MAPKA kinase-2 activity, a kinase immediately down-stream of P38MAPK. Preincubation of the cells with forskolin (10 microM), which stimulated a 20-fold increase in intracellular cAMP formation, inhibited this response. An antibody raised against subdomain VI of yeast Ste20 kinase co-immunoprecipitated p38MAPK from cell lysates. The immunoprecipitated kinase(s) was shown to catalyse the phosphorylation of myelin basic protein (MBP) and to activate purified MAPKAP kinase-2. Incubation of cells with PDGF did not increase the amount of p38MAPK isolated in the anti-Ste20 immunoprecipitate. However, the kinase phosphorylated MBP and stimulated purified MAPKAP kinase-2 activity more effectively than kinase from control cells. The preincubation of cells with forskolin (10 microM) reduced the amount of P38MAPK in the immunoprecipitate and this correlated with a decrease in kinase activity. We conclude the PDGF induces the activation of p38MAPK, whereas forskolin elicits its dissociation from the complex with Ste20. Therefore, Ste20/p38MAPK may form part of a signal transduction pathway linked to activation of MAPKAP kinase-2 in ASM cells.

Animals↗