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Biomedical subjects

J D Vickers

Publications and source records attributed to J D Vickers.

16 recordsLinked to original sources
PubMed1990

Inositol phospholipid metabolism in human platelets stimulated by ADP.

ADP-induced changes in inositol phospholipids, phosphatidic acid and inositol phosphates of human platelets have been studied in detail, using not only 32P labelling, but also by examining changes in amounts of the phospholipids, their labelling with [3H]glycerol and their specific radioactivities; changes in the labelling of inositol phosphates in platelets prelabelled with [3H]inositol were also measured. During the early (10 s) stage of reversible ADP-induced primary aggregation in a medium containing fibrinogen and with a concentration of Ca2+ in the physiological range (2 mM), the amounts of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol 4-phosphate (PtdInsP) decreased (by 11.2 +/- 4.9% and 11.3 +/- 5.3%, respectively) while the labelling, but not the amount, of phosphatidic acid increased. The decreases do not appear to be attributable to the action of phospholipase C because the specific radioactivity of phosphatidic acid labelling with [3H]glycerol was not significantly increased at 10 s (although the initial specific radioactivities of the inositol phospholipids and PtdCho were more than double that of phosphatidic acid), and no increases in the labelling of inositol trisphosphate (InsP3), inositol bisphosphate (InsP2) or inositol phosphate (InsP) were detectable at 10 s. Shifts in the interconversions between PtdInsP2 and PtdInsP, and PtdInsP and PtdIns may occur. By 30 to 60 s, when deaggregation was beginning, the amounts of PtdInsP2, PtdInsP and phosphatidic acid were not different from those in unstimulated platelets, but large increases in the 32P-labelling and [3H]glycerol labelling of phosphatidic acid were observed. Formation of [3H]inositol-labelled InsP3 was not detectable at any time in association with ADP-induced primary aggregation, indicating that degradation of PtdInsP2 by phospholipase C is not appreciably stimulated by ADP. These findings were compared with those obtained when platelets were aggregated by ADP in a medium without added of Ca2+ in which secondary aggregation associated with thromboxane A2 (TXA2) formation and release of granule contents occurs. At 10 s (during primary aggregation) the changes were similar in the two media. At 30 s and 60 s (during secondary aggregation in the low-Ca2+ medium), the increases in PtdInsP2, PtdInsP and phosphatidic acid in platelets suspended in the absence of added Ca2+ were larger than those in platelets suspended in the presence of 2 mM Ca2+. In the absence of added Ca2+, ADP-induced increases in the labelling of InsP3, InsP2 and InsP which were probably due to the effects of TXA2 since they were abolished by aspirin.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1990

Catching information technology by the tail for problem-based learning.

While computer-based resources are appealing, there is currently little evidence that they are superior to less expensive, traditional educational technologies and thus their introduction will be slow until we learn how to benefit from them. The lesson learned from the orientation of problem-based learning is that communications aspects of the new technologies may be much more important and should be, at least currently, the focus of attention. Thus, rather than concerning ourselves with the tiger, since communications do not require particularly sophisticated equipment, the important approaches now appear to be to give students experience with the means to access and exchange information; with those skills they should be reasonably prepared to survive in the Information Age and take advantage of what technology has to offer in the future.

Canada↗
PubMed1990

Thrombin-induced inositol phosphate production by platelets from rats with diet-induced or genetically determined hypercholesterolemia.

Platelets from rats with diet-induced or genetically determined hypercholesterolemia are hypersensitive to thrombin through a pathway that is independent of the effects of released ADP or formation of thromboxane A2. We examined production of inositol phosphates by platelets from these hypercholesterolemic rats to determine whether the enhanced responsiveness to thrombin is associated with increased production of inositol trisphosphate (IP3). The opportunity to study rats with hypercholesterolemia determined genetically or induced by diet makes it possible to determine whether any differences in inositol phosphate production are caused by hypercholesterolemia alone rather than to any other effect of the diet used to induce hypercholesterolemia. Platelets were prelabeled with [3H]inositol so that increases in inositol phosphates (IP, IP2, and IP3) upon stimulation with thrombin could be assessed by measuring the amount of label in these compounds. Platelets were preincubated with CP/CPK, to inhibit effects of released ADP, and aspirin, to inhibit formation of thromboxane A2/endoperoxides. In platelets from rats with either form of hypercholesterolemia, the percentage increase in labeling of IP3 was significantly greater 30 seconds after stimulation with low concentrations of thrombin than in platelets from control rats. Increased IP3 formation in platelets from hypercholesterolemic rats indicates that there is increased activity of a pathway(s) leading to IP3 formation and that this may be a mechanism responsible for the thrombin-induced hypersensitivity of these platelets.

Animals↗
PubMed1989

Eicosapentaenoic acid interferes with U46619-stimulated formation of inositol phosphates in washed rabbit platelets.

Eicosapentaenoic acid (EPA) inhibits platelet responsiveness to aggregating agents. To investigate the reactions that are affected by EPA, we examined the effect of preincubating aspirin-treated rabbit platelets with EPA on stimulation of inositol phosphate formation in response to the TXA2 analogue U46619. Stimulation of platelets with U46619 (0.5 microM) caused aggregation and slight release of dense granule contents; aggregation and release were inhibited by preincubation of the platelets with EPA (50 microM) for 1 h followed by washing to remove unincorporated EPA. Incubation with EPA (50 microM) for 1 h did not cause a detectable increase in the amount of EPA in the platelet phospholipids. When platelets were prelabelled with [3H]inositol, stimulation with U46619 of control platelets that had not been incubated with EPA significantly increased the labelling of inositol phosphates. The increases in inositol phosphate labelling due to U46619 at 10 and 60 s were partially inhibited by preincubation of the platelets with 50 microM EPA. Since the activity of cyclo-oxygenase was blocked with aspirin, inhibition of inositol phosphate labelling in response to U46619 indicates either that there may be inhibition of signal transduction without a detectable change in the amount of EPA in platelet phospholipids, that changes in signal transduction require only minute changes in the fatty acid composition of membrane phospholipids, or that after a 1 h incubation with EPA, activation of phospholipase C is affected by a mechanism that is not directly related to incorporation of EPA.

Animals↗
PubMed1988

Thrombin-induced inositol trisphosphate production by rabbit platelets is inhibited by ethanol.

Ethanol has an inhibitory effect on some platelet functions, but the mechanisms by which it exerts this effect are not known. Using suspensions of washed platelets, we observed that ethanol (1-9 mg/ml) did not affect the aggregation of rabbit platelets stimulated with ADP (0.5-10 microM). When platelets were prelabelled with 5-hydroxy[14C]tryptamine, aggregation and secretion of granule contents in response to thrombin (0.01-0.10 unit/ml) were not inhibited by ethanol, but these responses to thrombin at lower concentrations (less than 0.01 unit/ml) were inhibited by ethanol (2-4 mg/ml). Platelets were prelabelled with [3H]inositol so that increases in inositol phosphates upon stimulation could be assessed by measuring the amount of label in these compounds. ADP-induced increases in IP (inositol phosphate) and IP2 (inositol bisphosphate) were not affected by ethanol. IP3 (inositol trisphosphate) was not changed by ADP or ethanol. Although ethanol did not affect the increases in IP, IP2 and IP3 caused by stimulation of platelets with thrombin at concentrations greater than 0.01 unit/ml, ethanol did inhibit the increases observed at 2 and 3 min in these inositol phosphates caused by lower concentrations of thrombin (less than 0.01 unit/ml). Since ADP did not cause formation of IP3 in rabbit platelets, and since no thromboxane B2 was detected in platelets stimulated with the lower concentrations of thrombin, it is unlikely that the inhibitory effect of ethanol in IP3 formation was due to effects on further stimulation of platelets by released ADP or by thromboxane A2. Ethanol may inhibit platelet responses to thrombin by inhibiting the production of the second messenger, IP3.

Adenosine Diphosphate↗
PubMed1987

Phosphatidylinositol 4,5-bisphosphate is selectively retained by platelet-fibrin clots formed by thrombin.

Stimulation of human or rabbit platelets with thrombin in the presence of fibrinogen caused a large decrease, compared with unstimulated controls, in the amount of phosphatidylinositol 4,5-bisphosphate (PIP2) that could be extracted with acidified chloroform/methanol (60% at 60 s). In contrast, stimulation in the absence of added fibrinogen increased the amount of PIP2. The decrease was specific for PIP2, because similar decreases could not be demonstrated for other phosphoinositides or phospholipids. The interaction of polymerizing fibrin with stimulated platelets was required for the decrease in PIP2, since polymerized fibrin formed by reptilase did not cause the decrease in the amount of extractable PIP2, and inhibition by glycyl-L-prolyl-L-arginyl-L-proline of polymerization of fibrin formed by the action of thrombin prevented the large decrease in extractable PIP2. The decrease in extractable PIP2 could not be explained by increased degradation of PIP2, since sufficient degradation products were not formed. Thus, when platelets are stimulated with thrombin in the presence of fibrinogen, an association of polymerizing fibrin with the stimulated platelets occurs that leads to decreased extractability of PIP2. This may mean that PIP2 forms a specific association with platelet proteins that are involved in clot retraction.

Adenosine Diphosphate↗
PubMed1987

Involvement of phosphoinositide metabolism in potentiation by adrenaline of ADP-induced aggregation of rabbit platelets.

Changes in phosphoinositide metabolism were examined in washed rabbit platelets stimulated with 0.5 microM-ADP, 50 microM-adrenaline, or ADP and adrenaline in combination. Adrenaline does not stimulate platelet aggregation when used alone, but does potentiate aggregation stimulated by ADP. In platelets prelabelled with [32P]Pi and [3H]glycerol, adrenaline was found to potentiate the ADP-induced changes in platelet phospholipids, causing larger increases in the amount and labelling of phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid than was observed with ADP alone. The combination of ADP and adrenaline did not produce a greater decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) than was produced by ADP alone. In platelets prelabelled with [3H]inositol, adrenaline potentiated the increases in labelling of inositol phosphate and inositol bisphosphate stimulated by ADP; no increase in inositol trisphosphate labelling was detected with ADP alone or with the combination of ADP and adrenaline. Phentolamine, an alpha-adrenergic-receptor antagonist, blocked potentiation by adrenaline of ADP-induced changes in phosphoinositide metabolism. Propranolol and sotalol, beta-adrenergic-receptor antagonists, augmented the potentiation; this is consistent with the concept that the effect of adrenaline is mediated by beta-adrenergic receptors. The effect of adrenaline on phosphoinositide metabolism appears to be to potentiate the mechanisms by which ADP causes turnover of PIP and possibly degradation of PI, rather than the mechanism by which PIP2 is decreased.

Adenosine Diphosphate↗
PubMed1986

The phosphoinositides exist in multiple metabolic pools in rabbit platelets.

The labelling of the phosphoinositides and phosphatidic acid in washed rabbit platelets incubated with [32P]phosphate or [3H]glycerol was studied in the presence of isotope and after unincorporated isotope had been removed. With both isotopes the increase in the specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2) lagged behind that of phosphatidylinositol 4-phosphate (PIP) but the specific radioactivity remained higher after unincorporated isotope had been removed. This result was consistent with the presence of a second pool of PIP2, which interconverted slowly with the pool of PIP2 which was in direct equilibrium with PIP, proposed to explain the increase in specific radioactivity of PIP2 which accompanies the decrease in amount of PIP2 at 10 s in ADP-stimulated platelets. In platelets labelled with [3H]glycerol, the specific radioactivity of PIP2 became higher than that of PIP and the specific radioactivity of PIP became higher than that of phosphatidylinositol (PI). These results were interpreted to indicate that there were two pools of PIP; of these the pool with the higher specific radioactivity was the precursor of PIP2. Similarly, two pools of PI were proposed. The presence of pools of the phosphoinositides with different specific radioactivities necessitates the measurement of chemical amount of these compounds when studying the effect of stimulation of the platelets, since changes in labelling may not accurately reflect changes in the amount of the phosphoinositides.

Adenosine Diphosphate↗
PubMed1986

The decrease in phosphatidylinositol 4,5-bisphosphate in ADP-stimulated washed rabbit platelets is not primarily due to phospholipase C activation.

Addition of 10 micron-ADP to washed rabbit platelets caused platelet shape change and aggregation without release of the contents of the amine-storage granules, and caused a transient decrease (8.8% at 10 s) in the amount of phosphatidylinositol 4,5-bisphosphate (PIP2). By 20 s the decrease in PIP2 was no longer apparent, but by 60 s the amount of PIP2 was again decreased. Addition of thrombin (1 unit/ml), which causes platelet shape change, aggregation and the release of the contents of the amine-storage granules, caused a decrease in the amount of PIP2 (8.0% at 10 s); at 60 s the amount of PIP2 was not significantly different from that in controls. In platelets prelabelled with [3H]glycerol, the specific radioactivity of PIP2 was increased at 10 s in ADP-stimulated platelets, and unchanged in thrombin-stimulated platelets. In platelets prelabelled with [3H]inositol and incubated with 20 mM-Li+ to inhibit the degradation of the inositol phosphates to inositol, there was no increase in the labelling of inositol trisphosphate (IP3) upon stimulation with ADP. In contrast, stimulation with thrombin caused a significant increase in the labelling of IP3 at 10 s. These differences in the changes in polyphosphoinositide metabolism in ADP- and thrombin-stimulated platelets are consistent with the hypothesis that the decrease in PIP2 in ADP-stimulated platelets may be due not to degradation of PIP2 by phospholipase C, but rather to a shift in the equilibrium between PIP2 and phosphatidylinositol 4-phosphate (PIP). Increases in the labelling of phosphatidic acid at 10 s and of inositol bisphosphate and inositol phosphate after 20 s are consistent with phospholipase C being stimulated through some other mechanism that leads to the degradation of PIP and phosphatidylinositol; one possibility is that ADP causes an increase in cytoplasmic Ca2+.

Adenosine Diphosphate↗
PubMed1979

Phosphorylation of casein by human erythrocyte membrane-bound protein kinase: competition of casein with endogenous substrates.

The possibility that spectrin and band-3 protein are phosphorylated by the same membrane-bound protein kinase was investigated by adding casein to unsealed erythrocyte ghosts and examing competition of the three proteins for phosphorylation. The extent of spectrin and band-3 protein phosphorylation was reduced by up to approximately 55%. This indicated that casein was competing with these endogenous substrates for phosphorylation and was most probably phosphorylated by the same protein kinase(s). Furthermore, the extent of inhibition of the phosphorylation of the two endogenous substrates was indistinguishable over the range of casein concentrations tested (0.1 to 5 mg/ml). This indicates that spectrin and band-3 protein may be phosphorylated by the same protein kinase. In contrast, casein was found to have no effect on the cAMP-dependent phosphorylation of band 4.5. This result indicates that casein only competes with the endogenous proteins phosphorylated by the cAMP-independent protein kinase(s). The extent of reduction of endogenous substrate phosphorylation in the presence of casein was found to be constant over incubation periods of 1 to 15 min, indicating that this reduction was not due to consumption of ATP. Since the spectrin and band-3 protein phosphorylations were specifically and identically reduced by casein and these reductions were not due to the ATP consumption or to a general alteration of the membrane, we conclude that the two substrates are likely phosphorylated by one kinase which also phosphorylates casein.

Caseins↗
PubMed1979

Myotonic muscular dystrophy: abnormal temperature response of membrane phosphorylation in erythrocyte membranes.

The activities of the membrane-bound protein kinases of the human erythrocytes membrane that phosphorylate spectrin, band-3 protein, and phospholipids were compared in patients with myotonic muscular dystrophy and normal age- and sex-matched controls. These activities tended to be lower in the patients, but the differences were not statistically significant. In contrast, the temperature responses (the increase in activity in response to an increase in temperature from 30 degrees C to 37 degrees C) of the spectrin and band-3 protein kinase activities were significantly lower in the patients. Although they do not eliminate an alteration of one of the substrates, these results are consistent with the proposal that differences in erythrocytes from myotonic muscular dystrophy (MyD) patients are due to a membrane lipid change. Cholesterol is unlikely to be the altered lipid, as no difference in membrane cholesterol content was found.

Adenosine Triphosphate↗
PubMed1978

Alterations of membrane phosphorylation in erythrocyte membranes from patients with Duchenne muscular dystrophy.

The phosphorylation of spectrin, band 3 protein, and the phospholipids of erythrocyte membranes (ghosts) was examined in 10 patients with Duchenne muscular dystrophy (DMD) and in healthy age- and sex-matched controls. The rates of phosphorylation of spectrin and band 3 protein were significantly higher in ghosts prepared from patient blood than from control blood at both 30 degrees C and 37 degrees C. However, the mean increases in the rate of phosphorylation of both spectrin and band 3 protein in response to a temperature change from 30 degrees C to 37 degrees C were identical in ghosts from patient and controls. Phosphorylation of phospholipid and its temperature response did not differ between patients and controls. These results complement previous observations of differences in erythrocytes from patients with DMD. The similarity of the changes in phosphorylation of both spectrin and band 3 protein indicates a common cause, possibly their lipid environment.

Adult↗