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M C Scrutton

Publications and source records attributed to M C Scrutton.

At least 37 records · Page 2Linked to original sources
PubMed1986

Synergistic responses in human platelets. Comparison between aggregation, secretion and cytosolic Ca2+ concentration.

Synergistic interaction between ADP, adrenaline, 5-hydroxytryptamine (5HT) and [8-arginine]vasopressin is not observed for the aggregatory response of aspirin-treated human platelets when this response is estimated directly from the decrease in the number of single platelets in the suspension. This finding is in marked contrast with prior reports of synergistic interaction between these agonists when the rate and extent of the aggregometer response is estimated from the increase in the light transmittance of the suspension, using a platelet aggregometer. We propose that the apparent synergistic response detected using the aggregometer results from the inability of this instrument to respond during the initial phase of aggregation. Significant synergistic interaction is observed for the increase in cytosolic [Ca2+] induced by addition of the ADP/5HT and, to a lesser extent, of the ADP/vasopressin agonist pairs as compared with that caused by addition of the individual agonists. This effect is not, however, typical of the system since increases in cytosolic [Ca2+] induced by addition of the ADP/thrombin or 5HT/vasopressin agonist pairs are no greater than the sum of the responses to these agonists added separately. Addition of collagen prior to ADP or 11,9-epoxymethanoprostaglandin H2 (U46619) fails to enhance the increase in cytosolic [Ca2+] induced by these latter agonists. Adrenaline, when added prior to non-saturating concentrations of U46619, thrombin, vasopressin or ADP, significantly enhances the increase in cytosolic [Ca2+] induced by these agonists in platelets suspended in media containing less than 0.1 microM or 1 mM Ca2+. However, adrenaline fails to enhance the increase in cytosolic [Ca2+] induced by the divalent cation ionophore, ionomycin. Enhancement by adrenaline of Ca2+ influx induced by U46619, thrombin and ADP has been shown by using Mn2+ as probe. Adrenaline also enhances the extent of [3H]5HT secretion induced by U46619, thrombin and vasopressin but fails to increase that induced by ADP in this aspirin-treated preparation. These results are in part consistent with the postulate that adrenaline, acting via an alpha 2-adrenoceptor, modulates receptor--phospholipase-C coupling. However, such modulation does not appear to involve inhibition of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Platelets↗
PubMed1986

Effects of guanine nucleotides on the properties of 5-hydroxytryptamine secretion from electropermeabilised human platelets.

Guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta,gamma-imido]triphosphate enhance Ca2+-dependent 5-hydroxytryptamine secretion from electropermeabilised human platelets. GTP has little such effect except when the platelets are permeabilised, and incubated with this nucleotide, at 2 degrees C and pH 7.4. The lag phase observed in the time course of 5-hydroxytryptamine secretion induced by addition of guanosine 5'-[gamma-thio]triphosphate is markedly longer than that characterising secretion induced by Ca2+ alone, by thrombin +/- GTP or by guanosine 5'-[gamma-thio]triphosphate in the presence of thrombin. GTP causes competitive inhibition of the enhancement of the Ca2+-dependent secretory response induced by guanosine 5'-[gamma-thio]triphosphate when both nucleotides are added simultaneously. The extent of inhibition is decreased if guanosine 5'-[gamma-thio]triphosphate is added prior to GTP. GTP markedly enhances the effect of thrombin on Ca2+-dependent 5-hydroxytryptamine secretion by increasing the maximal extent of the response and decreasing the thrombin concentration required to give half-maximal response. A similar effect is observed on addition of guanosine 5'-[gamma-thio]triphosphate in the presence of thrombin at short incubation times. On more prolonged incubation the effects of thrombin and guanosine 5'-[gamma-thio]triphosphate are additive. Guanosine 5'-[beta-thio]diphosphate completely inhibits the response induced by guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta,gamma-imido]triphosphate but has little effect on the response induced by Ca2+ when added alone or in the presence of thrombin. Partial inhibition is observed for the response induced by thrombin + GTP. Cyclic-AMP effectively inhibits the response induced by thrombin + GTP but has little effect on that induced by guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta,gamma]imidotriphosphate. The results provide further support for the proposal [Haslam, R.J. & Davidson, M.M.L. (1984) FEBS Lett. 170, 90-95], that receptor--phospholipase-C coupling in platelets is mediated in part by a guanine-nucleotide-binding (Np) protein but that a coupling mechanism may also exist which is independent of such a protein. The properties of guanine-nucleotide-dependent coupling resemble those previously described for receptor--adenylate-cyclase coupling.

Blood Platelets↗
PubMed1986

Particle volume changes associated with light transmittance changes in the platelet aggregometer: dependence upon aggregating agent and effectiveness of stimulus.

The increase in light transmittance of aspirin-treated platelet-rich plasma caused by addition of non-saturating doses of ADP and, at earlier times, of adrenaline is correlated with formation of aggregates having a volume in the range 490-8580 fl. and containing 100-2000 platelets. The disappearance of single platelets and the formation of aggregates having volumes less than 490 fl. makes no significant contribution to the increase in light transmittance. Similar relationships are observed on addition of saturating doses of ADP and adrenaline except that the formation of aggregates larger than 8580 fl. contributes significantly to the initial phase of the increase in light transmittance and is more closely correlated with the overall change in this parameter.

Adenosine Diphosphate↗
PubMed1985

Effect of various excitatory agonists on the secretion of 5-hydroxytryptamine from permeabilised human platelets induced by Ca2+ in the presence or absence of GTP.

Addition of GTP markedly enhances the ability of thrombin to cause a leftward shift in the Ca2+ dose/response curve for 5-hydroxytryptamine secretion from permeabilised human platelets. Little effect is observed on addition of GTP in the absence of thrombin. Neither ADP nor adrenaline, in the presence or absence of GTP, causes such a shift, whereas 5-hydroxytryptamine does so to a small extent but only in the presence of GTP. The leftward shift in the Ca2+ dose/response curve induced by 12-O-tetradecanoyl-phorbol-13-acetate or 1-oleyl-2-acetylglycerol is not enhanced by addition of GTP. The thrombin concentration required for half-maximal enhancement of the response to Ca2+ is markedly reduced by addition of GTP. The results support the postulate that the effects of excitatory agonists in this system correlate with their ability to activate phospholipase C and provide further evidence for a role for GTP in signal transduction between the receptor and phospholipase C.

Adenosine Diphosphate↗
PubMed1985

Inhibition by luciferin-luciferase reagents of aggregatory responses to excitatory agonists in washed platelet suspensions.

Addition of two commercial luciferin-luciferase reagents caused marked inhibition of the aggregatory response of washed human platelets to thrombin, ADP, vasopressin and platelet-activating factor (PAF). Analysis of the effects of the individual components of one of these reagents revealed that Mg2+, and to a lesser extent bovine serum albumin, was responsible for the observed inhibition. A modified luciferin/luciferase reagent has been designed on the basis of these data for use in washed platelet suspensions which causes minimal inhibition of the aggregatory and secretory responses to thrombin but which gives a near maximal luminescence yield.

Adenosine Diphosphate↗
PubMed1985

Intracellular calcium fluxes in human platelets.

Fluorescence changes and secretory responses have been measured on addition of various excitatory agonists to platelets loaded with the cytosolic Ca2+ probe, Quin 2 or with chlortetracycline as a probe for membrane-associated Ca2+. When extracellular [Ca2+] is decreased to less than 0.1 microM by addition of EGTA a linear correlation is observed between the extent of increase in cytosolic [Ca2+] and the extent of mobilisation of membrane-associated Ca2+ on stimulation by maximal doses of five excitatory agonists. A similar linear correlation between the increase in cytosolic [Ca2+] and the extent of ATP secretion is observed over the thrombin dose/response curve. Similar EC50 values are observed for ATP secretion, the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by thrombin. However, the decrease in chlortetracycline fluorescence shows a sigmoidal relationship with the increase in cytosolic [Ca2+] and a hyperbolic relationship with ATP secretion over this dose/response curve. Addition of prostaglandin D2 prior to thrombin causes parallel inhibition of the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by this agonist. However, addition of prostaglandin D2 after thrombin reverses the increase in cytosolic [Ca2+] induced by this agonist but fails to cause a similar reversal of the decrease in chlortetracycline fluorescence. The data provide further evidence supporting the proposal that chlortatracycline can be used as a probe to monitor mobilisation of membrane-associated Ca2+ but suggest that, in platelets stimulated in the effective absence of extracellular Ca2+, both Ca2+ mobilisation and Ca2+ removal can under some conditions involve sites which are not monitored by this probe.

Adenosine Triphosphate↗
PubMed1985

The sub-cellular localisation and regulatory properties of pyruvate carboxylase from Rhizopus arrhizus.

Cell-free extracts of Rhizopus arrhizus contain exclusively cytosolic pyruvate carboxylase and NAD-glutamate dehydrogenase, a single mitochondrial isoenzyme of NADP-isocitrate dehydrogenase, and both mitochondrial and cytosolic isoenzymes of NADP-malate dehydrogenase (decarboxylating). Other enzymes examined have sub-cellular localisations similar to those characteristic of mammalian liver. Purified preparations of R. arrhizus pyruvate carboxylase are subject to partial regulatory inhibition by L-aspartate and 2-oxoadipate. L-Glutamate acts as a less effective analogue of L-aspartate while 2-oxoglutarate is ineffective. Competition studies indicate the presence of separate inhibitory sites for L-aspartate and 2-oxoadipate. Under routine assay conditions R. arrhizus pyruvate carboxylase shows significant activation by acyl derivatives of coenzyme A with long chain acyl CoA being more effective than acetyl-CoA. This activation is no longer observed in the presence of high concentrations of pyruvate, MgATP2- and HCO-3. The concentrations of L-aspartate and 2-oxoadipate required to give 50% inhibition ([I]0.5), and the maximal extents of inhibition, are increased by addition of acetyl-CoA. Acetyl-CoA increases the sigmoidal character of the relationship: initial rate/[L-aspartate], but decreases this parameter for the relationship: initial rate/[2-oxoadipate]. The studies indicate that R. arrhizus possesses an entirely cytosolic pathway for the conversion of glucose to fumaric acid and that both the organisation of pyruvate metabolism and the regulation of pyruvate carboxylase differ significantly in this organism as compared to that proposed previously for Aspergillus nidulans.

Acetyl Coenzyme A↗
PubMed1984

Thrombin and activators of protein kinase C modulate secretory responses of permeabilised human platelets induced by Ca2+.

Addition of thrombin enhances secretion of both [14C]serotonin and beta-N-acetylglucosaminidase induced by Ca2+ in human platelets rendered permeable by exposure to intense electric fields. Enhancement of beta-N-acetylglucosaminidase secretion by thrombin results from an increase in the maximal extent of the response with no significant change in the median effective concentration EC50 for Ca2+. In contrast, thrombin shifts the dose/response curve for Ca2+-induced [14C]serotonin secretion to the left and has little effect on the maximal extent of this response even when this extent is reduced by use of a non-saturating concentration of MgATP2-. The relationship between extent of response and [MgATP2-] is similar for secretion of [14C]serotonin and of beta-N-acetylglucosaminidase in the presence or absence of thrombin. Similar nucleotide specificities are also observed. Activators of protein kinase C have previously been shown to mimic quantitatively the effect of thrombin on [14C]serotonin secretion induced by Ca2+ [D. E. Knight & M. C. Scrutton (1984) Nature (Lond.) 309, 66-68]. Such activators have the same qualitative effect as thrombin on the properties of beta-N-acetylglucosaminidase secretion induced by Ca2+ but are less effective. The EC50 for thrombin observed for enhancement of [14C]serotonin and beta-N-acetylglucosaminidase secretion is in the same range as that obtained for intact platelets under comparable conditions [D. E. Knight, T. J. Hallam & M. C. Scrutton (1982) Nature (Lond.) 296, 256-257]. The EC50, and the specificity of response, observed for activators of protein kinase C in these systems are consistent with those reported previously for the purified enzyme. Addition of 1-10 microM Ca2+ to permeabilised platelets in the presence of [gamma-32P] ATP causes marked enhancement of 32P incorporation into polypeptides of molecular mass 20 kDA, 45 kDA and 66 kDA. No additional polypeptides become phosphorylated in this system when thrombin is added together with 10 microM Ca2+, but some increase is observed in the extent of phosphorylation of the 45-kDa polypeptide. Addition of 1-oleyl-2-acetylglycerol + 1 - 2 microM Ca2+ causes enhanced phosphorylation of the 45-kDa polypeptide and to a lesser extent of the 20-kDa polypeptide. The dose/response curves for Ca2+-dependent phosphorylation of the 45-kDa polypeptide in the presence and absence of 1-oleyl-2-acetylglycerol are similar to those observed for Ca2+-dependent [14C]serotonin secretion under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylglucosaminidase↗
PubMed1984

Beta-adrenoceptor antagonists and human platelets: relationship of effects to lipid solubility.

Six beta-adrenoceptor antagonists (propranolol (+ and - isomers); ICI-118,551; oxprenolol; timolol; metoprolol; and practolol (+ and - isomers), chosen to represent a spectrum of physicochemical and pharmacological properties, inhibited the response of human platelets to all aggregating agents tested. For any given aggregating agent the extent of inhibition correlated with the lipid solubility of the beta-adrenoceptor antagonist and showed no relation to other properties of these compounds. Inhibition of aggregation by the beta-adrenoceptor antagonists was manifested as a parallel shift to the right in the dose-response curve. Analysis of these data according to Arunlakshana and Schild (Br. J. Pharmac. 14, 48-58 (1969] showed a dependence of the apparent pA2 on the agonist employed and gave a slope approximating unity when ADP, 9,11-epoxymethanoprostaglandin H2 (U-46619), adrenaline, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) or arachidonate were used as agonists. Slopes significantly greater than unity, and approaching a value of 2, were obtained when this analysis was applied to data obtained using collagen in the presence or absence of aspirin, 12-O-tetradecanoylphorbol-13-acetate (TPA), or a divalent cation ionophore (A-23187) as agonists. Inhibition by (+/-) propranolol of secretion induced by collagen was manifested as a parallel shift to the right in the dose-response curve for collagen. The Schild plot of these data has a slope of unity. (+/-)-Propranolol inhibited thromboxane B2 production induced by collagen but over a similar concentration range had little effect on conversion of arachidonate to thromboxane B2. (+/-)Propranolol had no significant effect on the level of cyclic-3',5'-AMP (cAMP) in unstimulated platelets or on the increase in the level caused by addition of forskolin, but caused partial inhibition of the increase in platelet cAMP induced by prostaglandin E1. It also completely abolished inhibition by ADP of the increase in [cyclic-3',5'-AMP] induced by prostaglandin E1. These data are interpreted on the basis of a model in which interaction of propranolol with phosphatidylserine and phosphatidylinositol causes inhibition of phospholipases C and A2, inhibition of protein kinase C and alteration of membrane receptor properties as a consequence of distortion of their microenvironment.

Adenosine Diphosphate↗
PubMed1984

The role of cytoplasmic free calcium in the responses of quin2-loaded human platelets to vasopressin.

Responses to vasopressin were studied in human platelets loaded with the fluorescent Ca2+ indicator, quin2. In the presence of 1 mM external Ca2+, vasopressin caused a transient rise in [Ca2+]i from the basal level near 100nM to about 700 nM; peak [Ca2+]i was reached in a few seconds and the level then declined towards resting over several minutes. In the absence of external Ca2+ there was a much smaller rise of similar time-course, suggesting that vasopressin increases [Ca2+]i mainly by stimulated-influx across the plasma membrane but also by partly releasing internal Ca2+. Inhibition of thromboxane A2 formation somewhat reduced the peak [Ca2+]i in the presence of external Ca2+, but had no effect on the response attributed to release of internal Ca2+. With external Ca2+, vasopressin stimulated shape-change, secretion and aggregation. Secretion and aggregation were decreased by about half following blockage of thromboxane production. The ability of vasopressin to induce shape-change and secretion even at near basal [Ca2+]i suggests that activators other than Ca2+ are involved.

Aminoquinolines↗
PubMed1984

The role of Ca2+ uptake in the response of human platelets to adrenaline and to 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor).

Thrombin induces Ca2+ uptake into both stirred and unstirred human platelets in the presence or absence of acetylsalicylate. This Ca2+ uptake is closely correlated with adenine nucleotide secretion in accord with previous observations [Massini, P. and Lüscher, E.F. (1974) Biochim. Biophys. Acta 372, 109-121] but a low level of secretion is observed in the absence of significant Ca2+ uptake. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkylAcGEPC) also induces Ca2+ uptake into both stirred and unstirred human platelets in the presence and absence of acetylsalicylate. Aggregation and adenine nucleotide secretion induced by 1-O-alkylAcGEPC can be observed in the absence of added fibrinogen, but addition of fibrinogen causes a very marked shift to the left in the dose/response curves for aggregation and Ca2+ uptake induced by 1-O-alkylAcGEPC. In the absence of added fibrinogen a close correlation is observed between Ca2+ uptake and adenine nucleotide secretion induced by 1-O-alkylAcGEPC. In the presence of added fibrinogen significant aggregation can be observed in the absence of detectable Ca2+ uptake. Adrenaline induces Ca2+ uptake only into stirred human platelets in the presence of added fibrinogen. Blockade of secretion, e.g. by addition of acetylsalicylate, also prevents Ca2+ uptake. Addition of adrenaline fails to cause breakdown of phosphatidylinositol or phosphatidylcholine in acetylsalicylate-treated platelets under conditions where such a response is observed on addition of thrombin. We conclude that Ca2+ uptake into human platelets induced by thrombin, 1-O-alkylAcGEPC and adrenaline is closely associated with the secretory response and in some circumstances, e.g. stimulation by thrombin, is clearly a consequence of this latter response. Previous reports of Ca2+ uptake as an initiating event in the response of human platelets to adrenaline [Owen, N.E., Feinberg, H. and Le Breton, G.C. (1981) Am J. Physiol. 239, H483-488] have not been confirmed in this study.

Adenine Nucleotides↗
PubMed1984

Pyruvate carboxylase from Aspergillus nidulans. Effects of regulatory modifiers on the structure of the enzyme.

A method is described for purification of pyruvate carboxylase from Aspergillus nidulans by affinity chromatography on monomeric avidin-Sepharose. The purified enzyme is homogeneous as judged by electrophoretic and immunochemical analysis. The sub-unit Mr determined by electrophoresis in the presence of sodium dodecyl sulphate is 133000 +/- 5000. Electron microscopic analysis of purified A. nidulans pyruvate carboxylase after negative staining with uranyl acetate reveals the presence of molecules showing rhomboid and triangular projections as well as a projection showing two intensity maxima. A cleft running along the longitudinal axis of the sub-unit is observed in the rhomboid and triangular projections. Interconversion between all three projections can be obtained in a tilt series. Significantly better preservation of molecular structure is obtained if A. nidulans pyruvate carboxylase is prepared for electron microscopy in the presence of acetyl-CoA, 2-oxoglutarate or as the enzyme-avidin complex. L-Aspartate has no significant effect when added alone but markedly decreases the enhanced preservation given by acetyl-CoA. No marked alterations in molecular dimensions are caused by any of these additions. L-Aspartate, but not 2-oxoglutarate, enhances the rate of inactivation observed on incubation of A. nidulans pyruvate carboxylase at 4 degrees C in the presence of 0.5 M KCl. Addition of L-aspartate in low concentrations enhances the effectiveness of inhibition by 2-oxoglutarate by causing a decrease in the value of [I]0.5 without affecting the Hill coefficient h or the extent of activity observed at saturating 2-oxoglutarate concentrations. Conversely addition of low concentrations of 2-oxoglutarate decreases the concentration of L-aspartate required to give 50% inhibition but also causes a fall in h and an absolute increase in the extent of activity observed in the presence of saturating L-aspartate concentrations. The data are consistent with the proposal that A. nidulans pyruvate carboxylase is a tetrameric molecule in which the four sub-units are located at the corners of a tetrahedron. Metabolites which regulate the activity of the enzyme do not cause major alterations in this molecular structure but may alter its stability.(ABSTRACT TRUNCATED AT 400 WORDS)

Aspartic Acid↗