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B Ashby

Publications and source records attributed to B Ashby.

At least 37 records · Page 2Linked to original sources
PubMed1993

Molecular cloning and expression of a rat kappa opioid receptor.

At least three types of opioid receptors have been identified in the nervous system. In this paper we report molecular cloning and expression of a rat kappa opioid receptor. PCR was performed on double-stranded cDNA derived from poly(A)+ RNA of the rat striatum with primers similar to those of Libert and co-workers [Libert, Parmentier, Lefort, Dinsart, Van Sande, Maenhaut, Simons, Dumont and Vassart (1989) Science 244, 569-572]. One of the PCR products, which had 65% sequence similarity to the mouse delta opioid receptor, was used to screen a rat striatum cDNA library. Two positive clones were isolated and found to be identical. The clone had a 2.1-kb insert, which was termed RKOR-1. RKOR-1 has an open reading frame of 1140 bp and encodes a 380-amino-acid protein. Hydropathy analysis indicates that RKOR-1 has seven putative transmembrane domains with short intra- and extra-cellular loops. Membranes of Cos-7 cells transfected with RKOR-1 exhibited high specific binding for [3H]diprenorphine ([3H]DIP), a non-selective opioid ligand. Naloxone inhibited [3H]DIP binding with stereospecificity. [3H]DIP binding was potently inhibited by selective kappa opioid ligands, with Ki values in the nanomolar or subnanomolar range, but much less potently inhibited by drugs selective for mu or delta receptors. Thus, RKOR-1 represents an opioid receptor with kappa characteristics.

Amino Acid Sequence↗
PubMed1992

Cyclic AMP does not inhibit collagen-induced platelet signal transduction.

The adhesion of platelets to collagen and their activation is the primary event in haemostasis. Following adhesion, platelet aggregation mediated by ADP, thromboxane A2 and thrombin leads to the formation of a platelet plug. It is known that platelet activation by each of these agonists involves an increase in the cytosolic free Ca2+ concentration, and this has been thought to be controlled by cyclic AMP. However, we report here that while signal transduction induced by ADP plus a thromboxane mimetic (U46619), or by thrombin, is inhibited by stimulators of adenylate cyclase such as a prostaglandin I2 (PGI2) analogue (Iloprost), PGD2 and forskolin, elevation of cyclic AMP does not inhibit either platelet adhesion to collagen or the associated Ca2+ mobilization, phosphatidic acid formation or 5-hydroxytryptamine secretion. Furthermore, collagen did not lower elevated levels of cyclic AMP in platelets measured in the presence of both a thromboxane antagonist and an ADP-removing system. The present results are discussed in the context of previous findings.

Adenosine Diphosphate↗
PubMed1992

Comparison of Iloprost, Cicaprost and prostacyclin effects on cyclic AMP metabolism in intact platelets.

We have compared the effects of prostacyclin (PGI2) and its stable analogs, Iloprost and Cicaprost, on cyclic AMP metabolism in intact platelets. All three compounds show similar but not identical patterns of prostaglandin concentration-dependent cyclic AMP formation. All three compounds apparently stimulate and inhibit cyclic AMP formation with different concentration dependencies, indicating the presence of distinct stimulatory and inhibitory receptors. Differences in response can be accounted for by slight differences in affinity of stimulatory and inhibitory receptors for the prostaglandins, by the fact that Iloprost contains almost 50% of a relatively inactive isomer, and by the fact that PGI2 is labile in aqueous solution, with a half-life on the order of a few minutes. We conclude 1) stimulation and inhibition of adenylate cyclase is not due to separate effects of 16S- and 16R-stereoisomers of Iloprost because similar patterns were obtained with a single isomeric form of Cicaprost and with authentic PGI2; 2) prostaglandin induced inhibition of adenylate cyclase is readily reversible because inhibition disappears when PGI2 concentration decays below saturation of the inhibitory receptor; 3) the potency of prostaglandins in stimulating platelet adenylate cyclase must be viewed in terms of their effects on both stimulatory and inhibitory receptors.

Blood Platelets↗
PubMed1991

Prostaglandin-concentration-dependent desensitization of adenylate cyclase in human erythroleukaemia (HEL) cells is abolished by pertussis toxin and enhanced by induction by dimethyl sulphoxide.

Prostaglandin-regulated cyclic AMP metabolism in human erythroleukaemia (HEL) cells was similar to that previously described in platelets [Ashby (1989) Mol. Pharmacol. 36, 866-873], displaying prostaglandin-concentration-dependent desensitization that could be explained by the presence of separate stimulatory and inhibitory prostaglandin receptors. Pertussis toxin abolished prostaglandin-concentration-dependent desensitization, indicating that the process is mediated through a pertussis toxin-sensitive GTP-binding protein. Treatment of HEL cells for 4 days with the inducer dimethyl sulphoxide enhanced prostaglandin-concentration-dependent desensitization, but did not alter the initial rate of cyclic AMP synthesis or the amount of Gi2 alpha measured by immunoblotting, suggesting that the inhibitory receptor was selectively induced by changing the cells to a more platelet-like form.

Adenylate Cyclase Toxin↗
PubMed1991

Baseline corrections in experimental and quasi-experimental clinical trials.

Three methods of correcting for baseline differences are evaluated for use with randomized experimental designs and nonrandomized quasi-experimental designs. Cases where baseline means differ significantly in spite of random assignment and cases where observed baseline means do not differ significantly in quasi-experimental designs are given special attention. For comparison of treatment-induced change in randomly constituted treatment groups, analysis of covariance (ANCOVA) provides appropriate type I error protection and superior power regardless of the apparent significance of chance baseline differences. Correcting for baseline differences by expressing outcome scores as percentage of the baseline value for each individual produces results that closely approximate ANCOVA under specified conditions. Tests on simple pre-post difference scores are nonconservative when baseline means differ significantly in spite of randomization. In quasi-experimental research where treatment groups represent samples from different predefined populations, ANCOVA and percentage change provide inadequate baseline corrections even when the observed baseline means do not differ significantly. Although no method of baseline correction is entirely satisfactory in the absence of random assignment, tests on simple pre-post difference scores are generally superior for quasi-experimental designs in which treatment groups represent samples from different populations.

Analysis of Variance↗
PubMed1990

Ligands to the platelet fibrinogen receptor glycoprotein IIb-IIIa do not affect agonist-induced second messengers Ca2+ or cyclic AMP.

Previous studies have suggested that the platelet glycoprotein complex GPIIb-IIIa, which is the putative fibrinogen receptor, regulates Ca2+ influx into platelets, possibly operating as a Ca2+ channel. We have used RGD-peptides (peptides containing the sequence Arg-Gly-Asp; disintegrins), isolated from snake venoms, that have a high affinity and specificity for the fibrinogen-binding site of GPIIb-IIIa to address the question of whether blocking this site inhibits Ca2+ movement from the extracellular medium to the cytosol. Using fura-2-loaded human platelets, we found that neither disintegrins nor a monoclonal antibody (M148) to the GPIIb-IIIa complex altered the level of cytosolic Ca2+ obtained when the cells were stimulated with various agonists in the presence of either nominal or 1 mM extracellular Ca2+. In the presence of Mn2+, an ion that quenches fura-2 fluorescence, fura-2-loaded platelets were stimulated with thrombin or ADP. Neither disintegrins nor the monoclonal antibody altered the kinetics or the amount of quenching of fura-2 fluorescence by Mn2+. These data indicate that the binding of ligands to the fibrinogen receptor is not associated with an inhibition of Ca2+ movement through a receptor-operated channel. Furthermore, the disintegrins have no effect on platelet cyclic AMP metabolism in either the presence or the absence of phosphodiesterase inhibitors.

Antibodies, Monoclonal↗
PubMed1990

Kinetics of coagulation factor X activation by platelet-bound factor IXa.

Thrombin-activated human platelets, in the presence of factors VIIIa and X, have specific, high-affinity (Kd approximately 0.5 nM), saturable binding sites for factor IXa that are involved in factor X activation [Ahmad, S.S., Rawala-Sheikh, R., & Walsh, P.N. (1989) J. Biol. Chem. 264, 3244-3251]. To determine the functional consequences of factor IXa binding to platelets, a detailed kinetic analysis of the effects of platelets, phospholipids, and factor VIII on factor IXa catalyzed factor X activation was done. In the absence of platelets, phospholipids, or factor VIII, the Michaelis constant (Km = 81 microM) was greater than 500-fold higher than the factor X concentration in human plasma. Unactivated platelets and thrombin-activated factor VIII, alone or in combination, had no effect on the kinetic parameters, whereas thrombin-activated platelets caused a major decrease in Km (0.39 microM) with no significant effect on kcat (0.052 min-1) and allowed factor VIIIa to decrease the Km further to a concentration (0.16 microM) near that of factor X in plasma and to increase the kcat 24,000-fold to 1240 min-1. Sonicated mixed phosphatidylserine/phosphatidylcholine vesicles (25/75, mol/mol) had kinetic effects similar to those of activated platelets. When factor IXa binding to thrombin-activated platelets and rates of factor X activation were measured simultaneously at saturating concentrations of factor X and factor VIIIa, the kcat was independent of factor IXa concentration, and the mean kcat value was 2391 min-1. The increase in catalytic efficiency (kcat/Km) in the presence of thrombin-activated platelets and factor VIIIa was (17.4 x 10(6))-fold.

Factor IX↗
PubMed1990

Novel mechanism of heterologous desensitization of adenylate cyclase: prostaglandins bind with different affinities to both stimulatory and inhibitory receptors on platelets.

Prostaglandins E1, I2, and D2 (PGE1, PGI2, and PGD2) all stimulate and desensitize platelet adenylate cyclase, giving rise to peak and plateau effects in the time course of cyclic AMP metabolism in the intact cell. The peak and plateau effects vary with prostaglandin concentration to a different extent for each prostaglandin. However, at high concentrations, all prostaglandins give rise to the same time course of cyclic AMP formation. Differences in the extent of activation and desensitization can be modeled in terms of distinct stimulatory and slow-acting inhibitory receptors that differ in affinity for each prostaglandin but lead to the same maximum extent of activation and desensitization for all prostaglandins. The affinity for the stimulatory receptor is in the order PGI2 greater than PGE1 much greater than PGD2; the affinity for the inhibitory receptor is in the order of PGE1 greater than PGI2 much greater than PGD2. In addition, the inhibitory receptor binds PGE1 more tightly than the stimulatory receptor, whereas in the case of PGI2 or PGD2, the stimulatory receptor binds agonist more tightly than the inhibitory receptor. It is shown that the model gives rise to heterologous desensitization such that PGE1 readily inhibits PGI2- and PGD2-stimulated cyclic AMP formation, because it has high affinity for the inhibitory receptor. At the same time, because the final steady state concentration of cyclic AMP depends on the fractional occupancy of both the stimulatory and inhibitory receptors, PGE1 can cause either a rise or fall in cyclic AMP level, depending on the concentration of PGI2 or PGD2 used to challenge the platelets before PGE1 addition. The presence of a distinct inhibitory receptor may represent a general mechanism of autocoid desensitization, buffering cellular response against transient localized increases in agonist concentration that may occur when agonists are produced close to their sites of action.

Adenylyl Cyclases↗
PubMed1990

Mechanisms of platelet activation and inhibition.

Mechanisms of stimulus-response coupling in platelets are as complex and varied as the compounds that elicit the responses. The complexities are compounded by feedback mechanisms from substances released or synthesized by platelets as well as by "cross-talk" between signal transduction pathways. Examples of cross-talk include the ability of epinephrine to inhibit platelet adenylate cyclase through a G protein-mediated mechanism while causing platelet aggregation by some other mechanism and the ability of cAMP to inhibit thrombin-stimulated diacylglycerol formation. Despite the complexities, certain common threads are beginning to emerge, such as the involvement of G proteins in transducing many receptor-mediated processes, the involvement of relatively few second messenger pathways and the role of calcium in many of events leading to platelet responses, and the common involvement of protein kinases in carrying out second messenger function. The latter offers a useful assay for the effect of many agonists because they lead to the phosphorylation of specific proteins that can readily be detected by radioautography. Indeed, the emphasis has shifted in the past 10 years from relatively crude measurements of platelet function such as aggregation to precise, quantifiable measurement of processes such as protein phosphorylation and calcium release, which are indicators of the fundamental mechanisms involved in platelet function and thus serve as assays of these processes. On the other hand, there are other pathways and regulators yet to be discovered, notably regarding the action of epinephrine and the regulation of phospholipase A2. In addition, certain receptors remain elusive, including those for ADP and eicosanoids. The mechanisms of action of thrombin and cathepsin G, which involve their proteolytic activities, also remain an enigma. The combination of new insights into second messenger function and the techniques of molecular biology will allow many of these problems to be resolved, providing new approaches to therapy of thromboembolic disorders.

Adenosine Diphosphate↗
PubMed1989

Platelet receptor-mediated factor X activation by factor IXa. High-affinity factor IXa receptors induced by factor VIII are deficient on platelets in Scott syndrome.

We have studied factor IXa binding and factor X activation with normal platelets and with platelets obtained from a patient with a bleeding disorder and an isolated deficiency of platelet procoagulant activity termed Scott syndrome. In the absence of factor VIIIa and factor X, normal, thrombin-treated platelets exposed 560 +/- 35 sites for factor IXa with a Kd of 2.75 +/- 0.27 mM, compared with 461 +/- 60 sites per patient platelet with Kd of 3.2 +/- 0.33 nM. The addition of factor VIIIa and factor X resulted in a decrease in the Kd for normal platelets to 0.68 nM but had no effect on the Kd for patient platelets. The concentrations of factor IXa required for half-maximal rates of factor X activation for normal (0.52 nM) and patient platelets (2.5 nM) were similar to those determined from equilibrium binding studies. Kinetic parameters for factor X activation by factor IXa showed that the Km and Kcat were identical for normal and patient platelets in the absence of factor VIIIa. In the presence of factor VIIIa, and kcat for patient platelets (163 min-1) was only 33% of that for normal platelets (491 min-1): This result can be explained by the difference in affinity for factor IXa between normal and patient platelets in the presence of factor VIIIa, suggesting impaired factor VIIIa binding to Scott syndrome platelets.

Blood Coagulation Tests↗
PubMed1989

Model of prostaglandin-regulated cyclic AMP metabolism in intact platelets: examination of time-dependent effects on adenylate cyclase and phosphodiesterase activities.

The kinetics of prostaglandin-regulated cyclic AMP formation by intact human platelets were studied in the presence and absence of phosphodiesterase inhibitors. In the case of iloprost, a chemically stable analogue of prostaglandin I2, the shape of the time course varied with prostaglandin concentration. In the presence of phosphodiesterase inhibitors, low concentrations of iloprost gave a linear rate of cyclic AMP formation. At higher concentrations of iloprost, the initial rate increased as a saturable function of prostaglandin concentration but the curves decayed with time to give new linear rates of cyclic AMP formation with a different prostaglandin concentration dependence from the initial rates. Time courses were simulated using KINSIM [Anal. Biochem. 130: 134-145 (1983)], a kinetic simulation program that employs numerical integration, over a wide range of iloprost concentration (3 nM to 30 microM) by use of a simple model involving rapid activation of adenylate cyclase, followed by slow reversible transition of adenylate cyclase to an inactive form (desensitization) through a distinct inhibitory receptor. The model requires that the affinity for prostaglandins of both the stimulatory and inhibitory receptors declines with prostaglandin concentration, which may be related to the existence of high and low affinity receptor forms depending on the activation state of the appropriate GTP-binding protein. The same two-receptor model can be used to describe cyclic AMP metabolism in the absence of phosphodiesterase inhibitors, giving rise to characteristic peak and plateau effects in the time courses. The putative inhibitory receptor has an apparent affinity for prostaglandin lower than the stimulatory receptor in the case of iloprost and a higher affinity than the stimulatory receptor in the case of prostaglandin E1. The contribution of phosphodiesterase activation to the time course of cyclic AMP formation through phosphorylation by cyclic AMP-dependent protein kinase was assessed. It was shown that phosphodiesterase activation must be rapid. A plausible and perhaps complete description of prostaglandin-regulated cyclic AMP metabolism in platelets is presented.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Cyclic AMP turnover in response to prostaglandins in intact platelets: evidence for separate stimulatory and inhibitory prostaglandin receptors.

The shape of the time-course of cyclic AMP formation by intact human platelets in response to the stable prostaglandin I2 analogue iloprost varied with the concentration of the prostaglandin. At low concentrations of iloprost, the time-course showed a rise to a plateau with little subsequent decrease in cyclic AMP level. At high concentrations of iloprost, the initial rate of cyclic AMP formation was more rapid than at low concentrations, but the curves showed a marked time-dependent fall in cyclic AMP level to values below those observed at lower prostaglandin concentration. By contrast, PGE1 gave a rise and marked fall in cyclic AMP level at all concentrations of the prostaglandin and the curves did not cross. The time- and concentration-dependent fall in cyclic AMP level in response to iloprost was still apparent in the presence of phosphodiesterase inhibitors, indicating that inhibition of adenylate cyclase, rather than activation of cyclic AMP phosphodiesterases, was responsible for the fall in cyclic AMP level. Activators of protein kinase C, which phosphorylates platelet Ni and impairs its function, abolished the time-dependent fall in cyclic AMP level, indicating that Ni may be involved in prostaglandin-induced inhibition of adenylate cyclase. Time-courses were analyzed using an equation derived by Barber et al. (Adv. Cyc. Nuc. Res. 9, 507-516 (1978)) to yield rate constants for activation and inhibition of adenylate cyclase. Because of the difference in prostaglandin dependence of the activation and inhibition rate constants we propose that activation of adenylate cyclase in platelets is mediated by a rapid-acting stimulatory receptor, while time-dependent inhibition (desensitization) is mediated through a separate, slow-acting inhibitory receptor. The stimulatory receptor has an affinity for prostaglandin greater than the putative inhibitory receptor in the case of iloprost (as well as PGI2 and PGD2), and a lower affinity than the inhibitory receptor in the case of PGE1 (and PGE2). Prostaglandin-induced inhibition may be mediated through Ni.

Blood Platelets↗
PubMed1986

Kinetic evidence indicating separate stimulatory and inhibitory prostaglandin receptors on platelet membranes.

Progress curves of cyclic AMP production by human platelet lysates in the presence of 10 microM GppNp were upward curving, reaching a steady-state rate after about 20 min. Increasing concentrations of prostaglandin E1 reduced the lag time required to reach steady-state and increased the steady-state rate in a dose-dependent manner. At 0.3 microM PGE1 the progress curve was linear for at least 40 min. At higher concentrations of PGE1 the initial rate of cyclic AMP formation was linear and similar to that obtained at 1.0 microM PGE, however, the progress curve showed a downward curvature after several minutes, reaching a new steady-state rate after about 10 min. The extent of downward curvature was dose-dependent, and at 10 microM PGE1 the final steady-state rate had almost returned to that observed in the presence of GppNp alone. Analysis of initial and final steady-state rates as a function of prostaglandin concentration revealed two apparently saturable processes that were interpreted as binding to a stimulatory receptor (EC50 = 130 nM), followed by binding to a lower affinity inhibitory receptor (EC50 = 1200 nM) that showed a slow response to receptor occupancy. Qualitatively similar results were obtained with PGD2 and the stable PGI2 analog ZK36374.

Adenylyl Cyclases↗
PubMed1985

Differences in the mode of action of 1-oleoyl-2-acetyl-glycerol and phorbol ester in platelet activation.

The ADP-hydrolyzing enzyme apyrase inhibited platelet aggregation and phosphorylation of a 40,000 dalton platelet protein (P40) induced by 1-oleoyl-2-acetyl glycerol (OAG), indicating a dependence on secreted ADP. Apyrase also enhanced OAG-induced potentiation of forskolin or prostaglandin I2 activation of cyclic AMP formation in platelets. Cyclic AMP formation induced by OAG alone could be demonstrated in the presence of a phosphodiesterase inhibitor. Elevation of cyclic AMP level inhibits platelet aggregation so that secreted ADP may be required to inhibit OAG-activated adenylate cyclase for aggregation to proceed. In contrast, apyrase only partially affected phosphorylation of P40 and aggregation induced by the tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA). TPA caused marked inhibition of forskolin-stimulated cyclic AMP formation. TPA inhibition of cyclic AMP formation was largely reversed by apyrase, indicating that it was mainly due to release of ADP.

Adenylyl Cyclases↗
PubMed1983

Platelet AMP deaminase. Regulation by Mg-ATP2- and inorganic phosphate and inhibition by the transition state analog coformycin.

Kinetic studies with platelet AMP deaminase, at pH 7.0 and 100 mM NaC1, gave cooperative initial velocity curves with AMP as substrate, with Mg-ATP2- as an activator, and with Pi as an inhibitor. In the absence of Mg-ATP2-, the s0.5 for AMP was 4.5 mM with a Hill coefficient approaching 2.0. In the presence of saturating Mg-ATP2-, the s0.5 for AMP was reduced to 0.18 mM, the maximum velocity was increased by about 35%, and the Hill coefficient was 1.0. The half-activation constant for Mg-ATP2- varied from 0.7 to 0.07 mM as the concentration of AMP was varied from 0.1 to 5.0 mM and the Hill coefficient for Mg-ATP activation changed from 2.0 to 1.0 over the same range. Phosphate inhibition was competitive with AMP and with Mg-ATP2- (Ki = 2.0 mM) and reversed the activation by Mg-ATP2-. Coformycin inhibited the Mg-ATP-activated enzyme with a Ki less than 0.25 microM. Coformycin inhibition was slow, with a second order rate constant of 6.0 X 10(4) M-1 min-1, suggesting that the compound acts as a transition state analog according to Frieden, C., Kurz, L. C., and Gilbert, H. R. (1980) Biochemistry 19, 5303-5309. The kinetic properties of the enzyme indicate that substantial regulation can occur through changes in AMP concentration acting synergistically to enhance Mg-ATP2- binding and displace Pi from a single type of regulatory site.

AMP Deaminase↗
PubMed1983

Coformycin inhibition of platelet AMP deaminase has no effect on thrombin-induced platelet secretion nor on glycolysis or glycogenolysis.

Thrombin-stimulated platelet secretion is accompanied by a 30% reduction in the steady state level of cytosolic ATP, a breakdown that proceeds through ADP, AMP, IMP, and inosine to hypoxanthine. The ATP to hypoxanthine conversion could be blocked at the stage of AMP deamination by incubation of platelet-rich plasma for 6 h with 200 microM coformycin, a transition-state analog inhibitor of AMP deaminase. Abolition of AMP deaminase activity had no effect on thrombin-induced secretion from the dense granules, alpha-granules, or acid hydrolases measured in gel-filtered platelets. Coformycin treatment had no effect on thrombin-stimulated lactate production, even when oxidative phosphorylation was blocked by antimycin A, nor on the rate of thrombin-stimulated glycogenolysis. In addition, although it was clear that the adenylate energy charge was maintained by activation of AMP deaminase following thrombin treatment, the adenylate energy charge was also maintained in coformycin-treated platelets, albeit after a short lag, by stimulated ATP production and equilibration through the adenylate kinase reaction. Hydrogen peroxide brings about similar adenylate degradation which could also be inhibited by coformycin. The results indicate that AMP deamination and secretion, although temporally related, are not coupled. The role of AMP deaminase appears to be to maintain the adenylate energy charge in the absence of stimulation of ATP production or to buffer the adenylate charge before ATP production is stimulated.

AMP Deaminase↗
PubMed1981

Platelet AMP deaminase. Purification and kinetic studies.

AMP deaminase has been purified to homogeneity from human platelets by phosphocellulose chromatography. Kinetic studies showed sigmoidal behavior as a function of AMP concentration with the midpoint of the saturation curve (S0.5) at 3.5 and 4.0 mM in NaCl and KCl, respectively, at pH 6.5. Activation by saturating ATP converted the velocity versus substrate plot to hyperbolic with a Michaelis constant of 1.2 mM and the same maximum velocity in either salt. Addition of increasing concentrations of GTP in the presence of NaCl led to activation followed by inhibition whereas GTP in the presence of KCl gave inhibition with no apparent activation.

AMP Deaminase↗