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Biomedical subjects

M M Davidson

Publications and source records attributed to M M Davidson.

At least 37 records · Page 2Linked to original sources
PubMed1994

Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+.

In human platelets, thrombin not only stimulates the phosphorylation of pleckstrin (P47) and of myosin P-light chains, but also induces the dephosphorylation of an 18-19 kDa phosphoprotein (P18) [Imaoka, Lynham and Haslam (1983) J. Biol. Chem. 258, 11404-11414]. We have now studied this protein in detail. The thrombin-induced dephosphorylation reaction did not begin until the phosphorylation of myosin P-light chains and the secretion of dense-granule 5-hydroxytryptamine were nearly complete, but did parallel the later stages of platelet aggregation. Experiments with ionophore A23187 and phorbol 12-myristate 13-acetate indicated that dephosphorylation of P18 was stimulated by Ca2+, but not by protein kinase C. Two-dimensional analysis of platelet proteins, using non-equilibrium pH gradient electrophoresis followed by SDS/PAGE, showed that thrombin decreased the amount of phosphorylated P18 in platelets by up to 70% and slightly increased the amount of a more basic unlabelled protein that was present in 3-fold excess of P18 in unstimulated platelets. These two proteins were identified as the phosphorylated and non-phosphorylated forms of the pH-sensitive actin-depolymerizing protein, cofilin, by sequencing of peptide fragments and immunoblotting with a monoclonal antibody specific for cofilin. The molar concentration of cofilin in platelets was approx. 10% that of actin. Platelet cofilin was phosphorylated exclusively on serine. Experiments with electropermeabilized platelets showed that dephosphorylation of cofilin could be stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the absence of Ca2+ or by a free Ca2+ concentration of 10 microM. This GTP[S]-induced dephosphorylation reaction was inhibited by 1-naphthyl phosphate, but not by okadaic acid. Our results add cofilin to the actin-binding proteins that may regulate the platelet cytoskeleton, and suggest that platelet cofilin can be activated by dephosphorylation reactions initiated either by a GTP-binding protein or Ca2+.

Actin Depolymerizing Factors↗
PubMed1993

Synergistic inhibitory effects of atriopeptin II and isoproterenol on contraction of rat aortic smooth muscle: roles of cGMP and cAMP.

Atriopeptin II and isoproterenol acted synergistically to inhibit the phenylephrine-induced contraction of aortic smooth muscle from Wistar-Kyoto (WKY) rats. Thus, a weakly inhibitory concentration of atriopeptin II (10 nM) caused a 5-fold decrease in the IC50 of isoproterenol from 169 nM to 32 nM, whereas a low concentration of isoproterenol (100 nM) increased the maximum inhibition attributable to atriopeptin II from 43% to 74%. Atriopeptin II (10 nM) increased the cGMP found in aortic smooth muscle and approximately doubled the accumulation of cAMP caused by isoproterenol. The results suggest that cGMP, formed by the action of atriopeptin II on receptor guanylyl cyclase (GC-A), may inhibit aortic cyclic nucleotide phosphodiesterase type III (PDE III) and that an increased accumulation of cAMP then mediates the observed synergism.

Animals↗
PubMed1993

Small round virus excretion in long-stay geriatric patients.

The role of small round viruses (SRVs) in gastroenteritis has not been evaluated. We undertook a study to evaluate SRV excretion in 40 elderly patients over 12 months in long-stay, geriatric wards. The 40 patients (11 male, 29 female) had an age range of 73-99 years. Samples were received monthly as long as the patients were alive. Samples were available from 30 patients (75%) for 6 months or more, and 20 (50%) patients completed the study. Ten grams of faeces were collected and prepared as 10-20% suspensions, and then concentrated and examined by electron microscopy. Of 348 samples examined, 116 (33%) contained SRVs. Only 5 of 40 patients did not excrete virus. However, in 103/116 (89%) positive samples, virus was present in small amounts. These results show that excretion of SRVs in asymptomatic patients was very common. Therefore, the detection of SRVs in small amounts during an outbreak of gastroenteritis suggest that they do not have a causal role.

Aged↗
PubMed1991

Louping ill in man: a forgotten disease.

Louping ill disease of sheep has been recognised in Scotland for centuries. It causes encephalitis and is transmitted by the sheep tick, Ixodes ricinus. Human infection was first reported in 1934. Thirty-one cases of human infection have now been described. Four clinical syndromes are seen, an influenza-like illness, a bi-phase encephalitis, a poliomyelitis-like illness and a hemorrhagic fever. Certain occupational groups, e.g. laboratory personnel working with the virus and those who kill injected sheep, are at increased risk of acquiring louping ill infection. In many instances, infection is subclinical. Eight new human cases are described. Six were in crofters or shepherds in the north and west of Scotland, one was in a general practitioner in the Western Isles and the eighth was in a butcher in Edinburgh. Louping ill disease should not be forgotten in cases of unexplained encephalitis in those whose lifestyle exposes them to the virus.

Animals↗
PubMed1991

Comparison of IgG antibody profiles by immunoblotting in patients with acute and previous Toxoplasma gondii infection.

The IgG antibody profile to Toxoplasma gondii proteins of less than 37 kilodaltons in sera from patients with acute and previous infection was studied by immunoblotting. Bands at 28, 29, and 36 kilodaltons were more common in acute infection (10 out of 10, nine out of 10, and nine out of 10, respectively) compared with previous infection (five out of 10, four out of 10, and one out of 10, respectively). The 6 kilodalton band was present in 10 out of 10 sera from patients with acute infection and five out of 10 sera from those with previous infection. A new observation is a doublet of bands of 22-25 kilodaltons present only in sera from patients with acute infection. This doublet may be a more reliable indicator of acute infection than the 6 kilodalton band.

Acute Disease↗
PubMed1990

Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.

Blood Platelets↗
PubMed1988

Effects of guanosine 5'-[gamma-thio]triphosphate and thrombin on the phosphoinositide metabolism of electropermeabilized human platelets.

Incubation of human platelets with myo-[3H]inositol in a low-glucose Tyrode's solution containing MnCl2 enhanced the labelling of phosphoinositides about sevenfold and greatly facilitated the measurement of [3H]inositol phosphates formed by the activation of phospholipase C. Labelled platelets were permeabilized by high-voltage electric discharges and equilibrated at 0 degree C with ATP, Ca2+ buffers and guanine nucleotides, before incubation in the absence or presence of thrombin. Incubation of these platelets with ATP in the presence or absence of Ca2+ ions led to the conversion of [3H]phosphatidylinositol to [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PtdInsP2). At a pCa of 6, addition of 100 microM GTP[gamma S] both prevented this accumulation of [3H]PtdInsP2 and stimulated its breakdown; the formation of [3H]inositol phosphates was increased ninefold. After 5 min these comprised 70% [3H]inositol monophosphate ([3H]InsP), 28% [3H]inositol bisphosphate ([3H]InsP2) and 2% [3H]inositol trisphosphate ([3H]InsP3). In shorter incubations higher percentages of [3H]InsP2 and [3H]InsP3 were found. In the absence of added Ca2+, the formation of [3H]inositol phosphates was decreased by over 90%. Incubation of permeabilized platelets with GTP[gamma S] in the presence of 10 mM Li+ decreased the accumulation of [3H]InsP and increased that of [3H]InsP2, without affecting [3H]InsP3 levels. Addition of unlabelled InsP3 decreased the intracellular hydrolysis of exogenous [32P]InsP3 but did not trap additional [3H]InsP3. These results and the time course of [3H]inositol phosphate formation suggest that GTP[gamma S] stimulated the action of phospholipase C on a pool of [3H]phosphatidylinositol 4-phosphate that was otherwise converted to [3H]PtdInsP2 and that much less hydrolysis of [3H]phosphatidylinositol to [3H]InsP or of [3H]PtdInsP2 to [3H]InsP3 occurred. At a pCa of 6, addition of thrombin (2 units/ml) to permeabilized platelets caused small increases in the formation of [3H]InsP and [3H]InsP2. This action of thrombin was enhanced twofold by 10-100 microM GTP and much more potently by 4-40 microM GTP[gamma S]. In the presence of the latter, thrombin also increased [3H]InsP3. The total formation of [3H]inositol phosphates by permeabilized platelets incubated with thrombin and GTP[gamma S] was comparable with that observed on addition of thrombin alone to intact platelets. However, HPLC of the [3H]inositol phosphates formed indicated that about 75% of the [3H]InsP accumulating in permeabilized platelets was the 4-phosphate, whereas in intact platelets stimulated by thrombin, up to 80% was the 1-phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Platelets↗
PubMed1985

Receptor-effector coupling in platelets: roles of guanine nucleotides.

Platelet-activating factor (PAF), which is thought to cause platelet aggregation and degranulation via a receptor-mediated activation of phospholipase C, had no direct action on PGE1-stimulated cyclic AMP formation in intact human platelets, although it caused a GTP and Na+-dependent inhibition of the adenylate cyclase activity of human platelet particulate fractions. Studies with PAF analogues indicated that the receptors mediating this inhibition of adenylate cyclase had structural specificity very similar or identical to that of the receptors mediating platelet aggregation. These results suggest that the PAF receptors linked to the activation of phospholipase C in intact platelets may, in membrane preparations, become coupled to the inhibition of adenylate cyclase via the guanine nucleotide-binding protein, Gi. Studies with permeabilized human platelets that secrete 5-HT on addition of low concentrations of Ca2+ showed that addition of either PAF or a guanine nucleotide decreased the Ca2+ required for secretion. When added together, PAF and GTP promoted secretion synergistically at low Ca2+ concentrations. Enhanced secretion of 5-HT was associated with increased formation of diacylglycerol. These results show that PAF can stimulate phospholipase C by both GTP-dependent and independent mechanisms. In intact human platelets, PAF receptors may interact preferentially with a guanine nucleotide-binding protein that promotes phosphoinositide breakdown by phospholipase C, rather than with Gi.

Adenylyl Cyclase Inhibitors↗
PubMed1984

Potentiation by thrombin of the secretion of serotonin from permeabilized platelets equilibrated with Ca2+ buffers. Relationship to protein phosphorylation and diacylglycerol formation.

After human platelets have been rendered permeable to small molecules by high voltage electric discharges, addition of buffered micromolar concentrations of Ca2+ causes an ATP-dependent secretion of dense granule serotonin [Knight & Scrutton (1980) Thromb. Res. 20, 437-446]. In the present study, platelets permeabilized by this technique were found to show an up to 10-fold increase in their sensitivity to Ca2+ after exposure to thrombin. In permeabilized platelets, as in the intact cells, release of serotonin was associated with the Ca2+-dependent phosphorylation of 47 000 and 20 000 Da polypeptides (P47 and P20). Thrombin markedly increased the phosphorylation of P47 in the presence of 0.1-1.0 microM-Ca2+ free but had a much smaller effect on phosphorylation of P20. Thrombin also stimulated the formation of 1,2-diacylglycerol in the presence of 0.1 microM-Ca2+ free and was even more effective with 1.0 microM-Ca2+ free, suggesting that receptor-activated hydrolysis of phosphoinositides to 1,2-diacylglycerol was preserved in permeabilized platelets and was potentiated by low intracellular concentrations of Ca2+. The increase in phosphorylation of P47 on addition of thrombin may therefore be accounted for by the stimulatory action of 1,2-diacylglycerol on Ca2+-activated, phospholipid-dependent protein kinase. However, in both the presence and absence of thrombin, higher Ca2+ concentrations were required for optimal secretion than for maximal phosphorylation of both P47 and P20, indicating that additional actions of Ca2+ and thrombin, perhaps also mediated by 1,2-diacylglycerol formation, may be involved in the release of serotonin.

Blood Platelets↗
PubMed1984

Guanine nucleotides decrease the free [Ca2+] required for secretion of serotonin from permeabilized blood platelets. Evidence of a role for a GTP-binding protein in platelet activation.

Human platelets containing granule-bound [14C]serotonin were permeabilized, equilibrated at 0 degrees C with ATP and with various Ca2+ buffers and guanine nucleotides, and then incubated at 25 degrees C with or without a stimulatory agonist. Ca2+ alone induced the ATP-dependent secretion of [14C]serotonin (50% at a pCa of 5.1) but the sensitivity of secretion to Ca2+ was greatly enhanced by guanine nucleotides [6-fold by 100 microM GTP, 100-fold by 100 microM guanyl-5'-yl imidodiphosphate and greater than 500-fold by 100 microM guanosine 5'-O-(3-thiotriphosphate)] or by stimulatory agonists (10-fold by 2 units thrombin/ml and 4-fold by 1 microM 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine). When both GTP and a stimulatory agonist were added, they had synergistic effects on secretion. Cyclic GMP and GMP acted similarly to GTP. The effects of all these guanine nucleotides were inhibited by guanosine 5'-O-(2-thiodiphosphate), whereas those of stimulatory agonists were not. Our results demonstrate the presence in platelets of guanine nucleotide-dependent and independent mechanisms regulating the sensitivity of secretion to Ca2+.

Blood Platelets↗
PubMed1984

Receptor-induced diacylglycerol formation in permeabilized platelets; possible role for a GTP-binding protein.

Exposure of human platelets to 10 discharges from a 4.5 microF capacitor charged at 3 kV permitted isolation of a stable preparation of permeabilized platelets that, after equilibration with Ca2+ buffers (pCa less than 6) for 15 min at O degrees C, secreted 5-hydroxytryptamine (5-HT) at 25 degrees C. Thrombin enhanced the sensitivity to Ca2+ of the secretion of 5-HT by about 10-fold, whereas Arg -vasopressin and the prostaglandin endoperoxide analogue, U-46619, increased sensitivity to Ca2+ by 3 to 4-fold. This action of thrombin was associated with stimulation of diacylglycerol formation, a marked increase in phosphorylation of protein P47 and a smaller increase in phosphorylation of the P-light chain of myosin. Thrombin exerted these effects at a [Ca2+ free] of 0.1 microM, suggesting that the receptor-activated breakdown of platelet phosphoinositides to diacylglycerol may not require prior Ca2+ mobilization in intact platelets. In both the presence and absence of thrombin, a higher [Ca2+ free] was required for optimal secretion than for maximal phosphorylation of P47 and myosin light-chain, indicating that Ca2+ and possibly diacylglycerol have roles in the secretory mechanism additional to activation of the enzymes that phosphorylate these proteins. Stable GTP analogues such as guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), and to a lesser extent GTP itself, enhanced the Ca2+ sensitivity of the secretion of 5-HT from permeabilized platelets. Moreover, GTP potentiated the stimulatory action of thrombin. These effects of GTP gamma S and GTP were associated with increased diacylglycerol formation and were inhibited by guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) suggesting that a GTP-binding protein may play a role in the receptor-activated breakdown of phosphoinositides. However, as GDP beta S did not inhibit the potentiation of secretion caused by thrombin alone, a GTP-independent pathway of platelet activation may also exist.

Blood Platelets↗
PubMed1978

Cyclic nucleotides in platelet function.

Inhibition of adenylate cyclase in intact platelets by addition of compounds such as 2', 5' - dideoxyadenosine prevented the inhibition of platelet aggregation by PGE1 but did not affect the responses of platelets to aggregating agents in the absence of PGE1. This confirms that cyclic AMP mediates the effects of PGE1 but indicates that the level of cyclic AMP in unstimulated platelets is too low to affect the actions of aggregating agents. Studies on the phosphorylation of proteins in intact 32P-labelled platelets showed that PGE1 increased the phosphorylation of a membrane-bound polypeptide (P24) and prevented the increased phosphorylation of other polypeptides (P47 and P20) that occurred on addition of inducers of the release reaction. It is suggested that the cyclic AMP-dependent phosphorylation of P24 stimulates the active transport of Ca(2+) out of the platelet cytosol, so preventing phosphorylation of P47 and P20, reactions which may be involved in the release mechanism. As increases in platelet cyclic GMP could be dissociated from both platelet aggregation and the release reaction, it is proposed that the bidirectional regulation of platelet function is achieved primarily by the opposing actions of increases in the concentrations of Ca(2+) and cyclic AMP.

Blood Platelets↗
PubMed1978

Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.

Adenosine↗