low-Km Cyclic AMP Phosphodiesterase Research Articles

Sulfonylurea effects on acid and pepsinogen secretion in isolated rabbit gastric glands.

The influence of different sulfonylureas on the rate of acid and pepsinogen secretion was studied in isolated rabbit gastric glands. Neither tolbutamide (10-500 microM), chlorpropamide (10-500 microM), glibenclamide (1-50 microM) nor glipizide (1-50 microM) exerted a secretory effect. In contrast, gliquidone caused a marked and dose-dependent stimulation of acid production in gastric glands incubated under basal conditions and potentiated the stimulatory effect of both histamine and carbachol. Gliquidone also increased the rate of pepsinogen release in gastric glands incubated either under basal conditions or in the presence of cholecystokinin-octapeptide or isoproterenol. The secretory effects of gliquidone were associated with a significant increase in the glandular content of cyclic AMP, caused by a competitive inhibition of low-Km cyclic AMP phosphodiesterase. Our results indicate that, among the assayed sulfonylureas, only gliquidone, in the micromolar range, stimulates acid and pepsinogen secretion through a cyclic AMP-dependent mechanism.

Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity.

The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5'-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity.

Antithrombotic activity of BMY-43351, a new imidazoquinoline with enhanced aqueous solubility

BMY-43351 is a new broad-spectrum inhibitor of platelet aggregation with greater aqueous solubility than earlier analogs from the imidazoquinoline series. This report compares the antithrombotic activity of BMY-43351 to that of two other imidazoquinolines: BMY-20844, a simply-substituted compound, and BMY-21638, a more potent ether-linked side chain analog. All of these compounds act, at least in part, via inhibition of platelet low-Km cyclic AMP phosphodiesterase. Antithrombotic activity was assessed in the rabbit ear chamber-biolaser preparation, an animal model of small vessel thrombosis, and in the canine coronary artery stenosis-occlusion model of large vessel thrombosis. BMY-43351 was found to be remarkably potent in the biolaser model, with an ED 50 of 0.074 mg/kg p.o. In comparison, compounds such as aspirin, ticlopidine, sulfinpyrazone, and dipyridamole demonstrate little or no activity at much higher doses, (eg. 100 mg/kg p.o.). Other inhibitors of platelet low Km cyclic AMP phosphodiesterase are active but substantially weaker than BMY-43351. Similarly, in the coronary artery stenosis-occlusion model, BMY-43351 demonstrated impressive activity, significantly inhibiting arterial thrombosis at intraduodenal doses as low as 1 μg/kg. The potential use of BMY-43351 as adjunct therapy in thrombolysis was suggested in a series of experiments where this drug was used in combination with a thrombolytic regimen of stretokinase plus heparin. In this experimental setting, time to reperfusion was reduced from 42 ± 5 minutes to 11 ± 5 minutes, and reocclusion was totally inhibited.

Isolation of insulin-sensitive phosphatidylinositol-glycan from rat adipocytes. Its impaired breakdown in the streptozotocin-diabetic rat

In this study an insulin-sensitive glycophospholipid from rat adipocytes was isolated and partially characterized. A material that activated pyruvate dehydrogenase was extracted from rat adipocyte membrane supernatants. Its release was stimulated by insulin and phosphatidylinositol-specific-phospholipase C and its activity was destroyed by nitrous acid deamination. These findings suggested that insulin might stimulate breakdown of a glycophospholipid containing inositol and glucosamine, as previously reported for some other cell types [Low & Saltiel (1988) Science 239, 268-275]. A lipid that incorporated [3H]glucosamine, [3H]galactose, [3H]inositol, and [3H]myristate and whose turnover was stimulated by insulin was subsequently isolated from intact adipocytes by sequential t.l.c. using an acidic solvent system followed by a basic solvent system. The effects of insulin on turnover of the lipid in these cells were transient, with maximal effects at 1 min, and there was a typical concentration-response curve to insulin (0.07 nM-7 nM), with effects being detected over the physiological range of insulin concentrations. In contrast with studies in other cells, there was appreciable turnover of the sugar labels. The majority of the [3H]glucosamine and [3H]galactose labels were cycled through to triacylglycerol in the adipocyte. However, of that recovered in the glycophospholipid band, a major proportion (less than 40%) was recovered as the native label. Digestion of the purified molecule with phosphatidylinositol-specific phospholipase C generated a material that activated both pyruvate dehydrogenase and low-Km cyclic AMP phosphodiesterase. Impairment in insulin-stimulated breakdown of the molecule in adipocytes of streptozotocin-diabetic rats was found, consistent with the impaired insulin activation of pyruvate dehydrogenase and glucose utilization seen in this model. These findings suggest that insulin stimulates breakdown of this glycophospholipid by stimulating an insulin-sensitive phospholipase in adipocytes. This compound may serve a function as a precursor for intracellular insulin mediators.

SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae.

sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity. We have cloned SRA5 by complementation. It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase. Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP. sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.

SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae.

sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity. We have cloned SRA5 by complementation. It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase. Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP. sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.

Acute in vivo stimulation of low-Km cyclic AMP phosphodiesterase activity by insulin in rat-liver Golgi fractions.

A low-Km phosphodiesterase activity, which is acutely stimulated by insulin in vivo, has been identified in plasma membranes and Golgi fractions prepared from rat liver homogenates in isotonic sucrose. Within seconds after insulin injection (25 micrograms/100 g body weight) cAMP phosphodiesterase activity increases by 30-60% in Golgi fractions and by 25% in plasma membranes; activity in crude particulate and microsomal fractions is unaffected. The increase in activity is short-lived in the light and intermediate Golgi fractions, but persists for at least 10 min in the heavy Golgi fraction. It precedes the translocation of insulin and insulin receptors to these fractions, which is maximal at 5 min. The doses of insulin required for half-maximal and maximal activation are, respectively, 7.5 micrograms/100 g and 25 micrograms/100 g body weight. Golgi-associated cAMP phosphodiesterase activity shows non-linear kinetics; a high-affinity component (Vmax, 13 pmol min-1 mg protein-1; Km, 0.35 microM) is detectable. Insulin treatment increases the Vmax 60-70%, but does not affect the Km. Unlike the low-Km cAMP phosphodiesterase associated with crude particulate fractions, the Golgi-associated activity is not easily extractable by solutions of low or high ionic strength. On analytical sucrose density gradients, low-Km cAMP phosphodiesterase associated with the total particulate fraction equilibrates at lower densities than endoplasmic reticulum and lysosomal markers, but at a higher densities than plasma membrane, Golgi markers and insulin receptors. Insulin treatment increases the specific activity of the enzyme by 20-60% at densities below 1.12 g cm-3, and by 20-40% in the density interval 1.23-1.25 g cm-3. Such treatment also causes a slight, but significant shift in the distribution of phosphodiesterase towards lower densities. It is suggested that Golgi elements or physically similar subcellular structures are a major site of localization of insulin-sensitive cAMP phosphodiesterase in rat liver. However, internalization of the insulin-receptor complex is probably not required for enzyme activation.

The action of adenosine in relation to that of insulin on the low-Km cyclic AMP phosphodiesterase in rat adipocytes.

The adenosine-sensitive cyclic AMP phosphodiesterase of rat adipocytes was found to reside in the same subcellular fraction as the enzyme sensitive to insulin. There were several similarities between the action of adenosine and that of insulin on the enzyme. The action of adenosine on the phosphodiesterase is probably like that of insulin, both being receptor-mediated, although different sites or different receptors could be involved. Adenosine analogues with intact ribose but a modified purine moiety elicited a response similar to that of adenosine. Added Ca2+ was also not a requirement for the action of adenosine. The action of adenosine was not synergistic with that of insulin, neither was adenosine essential for insulin action. Insulin stimulated the enzyme even at low cell concentrations and in the presence of adenosine deaminase. Adenosine, however, enhanced the effect of insulin, but only at insulin concentrations that produced submaximal effects. Thus the mechanisms of action could be similar or related. The time-course effect of a suboptimal concentration of insulin was transitory, like that of adenosine, and was influenced by the presence of adenosine, whereas that of a maximally effective concentration of insulin was sustained for at least 20 min and was not affected by the presence of adenosine. Isoprenaline enhanced phosphodiesterase activity stimulated by optimal concentrations of either adenosine or insulin, suggesting that their effects were mediated through different mechanisms of action.

N 6-phenylisopropyladenosine stimulates in normal and inhibits in adrenalectomized rats the low KM cyclic AMP phosphodiesterase in the brain

In crude rat brain membranes, apparent adenylate cyclase activity tested in the absence of phosphodiesterase inhibitors was dose-dependently inhibited by the adenosine ‘R-site’ agonist N 6-phenylisopropyladenosine (N 6-PIA). In membranes from adrenalectomized rats, however, N 6-PIA induced, under the same conditions, an activation. However, in the presence of the phosphodiesterase inhibitor Ro-20-1724, brain adenylate cyclase responsiveness to N 6-PIA resulted in a dose-dependent inhibition in both sham-operated and adrenalectomized rats. Thus, the low K M cyclic AMP phosphodiesterase activity present in these brain membranes was investigated. Although this activity (tested in the presence of GTP) was unaltered by adrenalectomy, the dose-response curve of this enzyme to low concentrations of N 6-PIA showed an activation in sham-operated and an inhibition in adrenalectomized rats, two effects which were suppressed by sodium (80 mM). These results showing that N 6-PIA modulates both adenylate cyclase and cyclic AMP phosphodiesterase in the brain, provide an additional argument for a potential role of adenosine in the regulation of cyclic AMP metabolism in normal as well as in pathological brain.

Regulation of cyclic AMP phosphodiesterase from Mucor rouxii by phosphorylation and proteolysis. Interrelationship of the activatable and insensitive forms of the enzyme.

DEAE-cellulose chromatography of mycelial extracts of Mucor rouxii grown to mid-exponential phase resolves two types of low-Km cyclic AMP phosphodiesterase (EC 3.1.4.17; PDE): PDE I, highly activatable (4-6-fold) by phosphorylation or proteolysis, and PDE II, unresponsive to activation. The enzymic profile of PDE activity obtained from germlings shows only PDE I activity, whereas PDE activity from mycelia grown to stationary phase is eluted from the DEAE-cellulose column at the position of PDE II, and like PDE II is unresponsive to activation. Endogenous proteolysis or controlled trypsin treatment transforms PDE I into PDE II. The insensitive forms of PDE exhibit a slightly smaller sedimentation coefficient than the activatable forms, as judged by sucrose-gradient centrifugation. The basal activity of the highly activatable form of PDE is elevated almost to the value in the presence of trypsin on storage at 4 degrees C in the absence of proteinase inhibitors. Benzamidine, leupeptin, antipain or EGTA prevents the activation produced by storage. PDE I remains strongly activatable by phosphorylation and proteolysis after resolution by polyacrylamide-gel electrophoresis.

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone.

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.

Interaction of hydroflumethiazide and 2,4-disulfamyl-5-trifluoromethylaniline with cyclic AMP phosphodiesterase and carbonic anhydrase.

The inhibitory effect of hydroflumethiazide (HFT) and its metabolite, 2,4-disulfamyl-5-trifluoromethylaniline (DTA) on cyclic AMP phosphodiesterase and the binding of HFT and DTA to carbonic anhydrase was studied in vitro. Significant inhibition of rat kidney low-Km cyclic AMP phosphodiesterase was observed with DTA concentration above 2.5 X 10(-4) mol/l and with HFT concentration above 1 X 10(-4) mol/l. 50% inhibition was observed at a DTA concentration of 1 X 10(-3) mol/l. Binding of DTA and HFT to commercially obtained bovine erythrocyte carbonic anhydrase was demonstrated by equilibrium dialysis. Data were consistent with one class of binding sites. The product of n (number of binding sites) and Kass (association constant) was 5 X 10(5) M for DTA and 3.3 X 10(4) M for HFT at 2 degrees. In human blood in vitro at 37 degrees, the equilibrium erythrocyte/plasma concentration ratio was 18 for DTA and 1.6 for HFT. It is concluded that HFT and DTA have approximately the same potency as cyclic AMP phosphodiesterase inhibitors, whereas DTA is more extensively bound by erythrocyte carbonic anhydrase.

The antitumour agent 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DTIC) inhibits rat liver cAMP phosphodiesterase and amplifies hormone effects in hepatocytes and hepatoma cells

The antitumour agent 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) was found to inhibit competitively the low-Km cyclic AMP phosphodiesterase activity in an ammonium-sulphate-precipitable fraction of the 2,000g supernatant of rat liver. With substrate concentration at 0.25 microM, I50 was 790 microM for DTIC and 350 microM for theophylline. DTIC at 2 mM more than doubled the cAMP response to glucagon in hepatocytes and to adrenaline in MH1C1 hepatoma cells, indicating that it also exerts its inhibitory effect on the phosphodiesterase in intact cells. The possible contribution of the phosphodiesterase inhibition to the growth-inhibitory and cytotoxic effects of DTIC is discussed.

Responsiveness to glucagon by isolated rat hepatocytes controlled by the redox state of the cytosolic nicotinamide—adenine dinucleotide couple acting on adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1. The effects of changes in the cytoplasmic [NADH]/[NAD+] ratio on the efficacy of glucagon to alter rates of metabolism in isolated rat hepatocytes were examined. 2. Under reduced conditions (with 10mM-lactate), 10nM-glucagon stimulated both gluconeogenesis and urea synthesis in isolated hepatocytes from 48h-starved rats; under oxidized conditions (with 10mM-pyruvate), 10nM-glucagon had no effect on either of these rates. 3. The ability of glucagon to alter the concentration of 3':5'-cyclic AMP and the rates of glucose output, glycogen breakdown and glycolysis in cells from fed rats were each affected by a change in the extracellular [lactate]/[pyruvate] ratio; minimal effects of glucagon occurred at low [lactate]/[pyruvate] ratios. 4. Dose-response curves for glucagon-mediated changes in cyclic AMP concentration and glucose output indicated that under oxidized conditions the ability of glucagon to alter each parameter was decreased without affecting the concentration of hormone at which half-maximal effects occurred. 5. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.05 mM) significantly reversed the inhibitory effects of pyruvate on glucagon-stimulated glucose output. 6. For exogenously added cyclic [3H]AMP(0.1 mM), oxidized conditions decreased the stimulatory effect on glucose output as well as the intracellular concentration of cyclic AMP attained, but did not alter the amount of cyclic [3H]AMP taken up. 7. The effects of lactate, pyruvate, NAD+ and NADH on cyclic AMP phosphodiesterase activities of rat hepatocytes were examined. 8. NADH (0.01--1 MM) inhibited the low-Km enzyme, particularly that which was associated with the plasma membrane. 9. The inhibition of membrane-bound cyclic AMP phosphodiesterase by NADH was specific, reversible and resulted in a decrease in the maximal velocity of the enzyme. 10. It is proposed that regulation of the membrane-bound low-Km cyclic AMP phosphodiesterase by nicotinamide nucleotides provides the molecular basis for the effect of redox state on the hormonal control of hepatocyte metabolism by glucagon.

HYPERREACTIVE ARTERIAL ENDOTHELIAL CELLS: A CLUE FOR THE TREATMENT OF ATHEROSCLEROSIS

Arterial endothelial cells, which are capable of phagocytizing carbon particles of the same size as beta- and pre-beta-lipoprotein, were found only in endothelial cells of arterial segments susceptible to atheromatous changes in susceptible animal species, and the distribution closely corresponded to the susceptibility. The distribution of such endothelial cells is dense in large arteries, in the openings to their branches, especially in downstream portions, of rabbits, hens, and cocks; however, the distribution is relatively scanty in arteries of rhesus monkeys and is very scanty in dogs. Carbon particles were also rare in the suckling rabbit and tended to increase with age. They were not found in Wistar rats but were found in spontaneously hypertensive rats, which showed a characteristically diffuse distribution, even in relatively small arteries. The carbon particles, phagocytized, were released to the subendothelial space but were difficult to pass through the internal elastic lamina and tended to stagnate there for more than one month. The authors therefore call these cells hyperreactive endothelial cells. Various vasoactive substances, such as angiotensin II, histamine, and serotonin, significantly enhanced the phagocytic activities of arotic endothelial cells in rabbits; epinephrine and norepinephrine also slightly enhanced these activities. Various smooth muscle relaxants, such as ATP, pyridinol carbamate (ATP synthesis-enhancing substance), cycli-AMP, dibutyryl cycli-AMP, phthalazinol (cyclic-AMP phosphodiesterase inhibitor), iproveratril (calcium entry-inhibiting substance), colchicine, and vinblastine, with their different modes of action, commonly inhibited phagocytic activities, a finding that suggests a significant role for contractile protein in the permeability problem of atherogenesis. The atheromatous lesions of cholesterol-fed rabbits exhibited a striking increase in hyperreactive endothelial cells, accompanied by a marked rise in the activity of low-Km cyclic-AMP phosphodiesterase activity in atheromatous lesions and adjacent muscular layers, especially in rabbits with rapidly progressing atheroma.