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Biomedical subjects

T W Martin

Publications and source records attributed to T W Martin.

At least 37 records · Page 2Linked to original sources
PubMed1989

Long-term phorbol ester treatment dissociates phospholipase D activation from phosphoinositide hydrolysis and prostacyclin synthesis in endothelial cells stimulated with bradykinin.

Bovine pulmonary artery endothelial cells (BPAEC) were prelabeled with [3H]choline or [3H]myristic acid to selectively label endogenous phosphatidylcholine. BPAEC were stimulated with ATP and bradykinin (BK), and phospholipase D (PLD) activation was detected as a 4-fold increase in [3H]choline in cells prelabeled with [3H]choline or as a 2- to 3-fold increase in [3H]phosphatidylethanol in cells prelabeled with [3H]myristic acid and stimulated in the presence of ethanol. Pretreatment of BPAEC with 0.1 microM phorbol 12-myristate 13-acetate (PMA) for 22 hr completely inhibited agonist-induced PLD activation, whereas prostacyclin synthesis and [3H]phosphoinositide ([3H]PIns) hydrolysis were enhanced in pretreated cells. Long-term PMA treatment thus dissociates agonist-induced PLD activation from [3H]PIns hydrolysis, and agonist-induced prostacyclin synthesis is not dependent upon PLD activation.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

P2-purinergic agonists stimulate phosphodiesteratic cleavage of phosphatidylcholine in endothelial cells. Evidence for activation of phospholipase D.

The effects of purinergic agonists on phosphatidylcholine (PC) breakdown and prostacyclin synthesis were investigated in cultured bovine pulmonary artery endothelial cells (BPAEC) prelabeled with [3H]choline and [14C]myristic acid. Both labels were selectively incorporated into PC. In BPAEC prelabeled with [3H]choline, ATP stimulated a rapid 5-fold increase in intracellular free [3H]choline. [3H]Choline formation was associated with a concomitant loss of 3H from PC, and it was not preceded by an increase in the 3H content of other PC degradation products. In BPAEC prelabeled with [14C]myristic acid, ATP stimulated a rapid increase in [14C]phosphatidic acid and [14C]diacylglycerol. These changes were associated with a loss of 14C from PC but not from phosphatidylinositol. In permeabilized BPAEC prelabeled with [3H]choline, ATP and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) stimulated an increase in [3H]choline but not [3H]phosphocholine. The effects of ATP and GTP gamma S were synergistic at low GTP gamma S concentrations. Permeabilized BPAEC did not convert exogenous [3H]phosphocholine to [3H]choline. These data are consistent with the notion that purinergic agonists stimulate PC breakdown by a phospholipase D mechanism. This suggestion was supported by the observation that in the presence of ethanol, ATP stimulated the formation of [14C]phosphatidylethanol in BPAEC prelabeled with [14C]myristic acid. Several adenine nucleotides and related purine derivatives were compared for their effectiveness in stimulating PC breakdown and prostacyclin synthesis. The chemical specificity observed for the two responses indicated that both were mediated by the P2Y purinoceptor subtype. PC breakdown stimulated by ATP occurred prior to the maximum rate of release of prostacyclin into the medium. Taken together these data indicate that PC breakdown in endothelial cells stimulated with purinergic agonists is due, at least in part, to the activation of a phospholipase D that is coupled to purinoceptors by a guanine nucleotide-binding protein. In conjunction with previous data on the effects of bradykinin, these findings suggest an important role for PC breakdown in the mechanism of signal transduction in endothelial cells stimulated with Ca2+-mobilizing agonists.

Adenosine Triphosphate↗
PubMed1989

Exercise and hypoxia increase sickling in venous blood from an exercising limb in individuals with sickle cell trait.

PURPOSE: The association between sickle cell trait (SCT) and complications related to exercise may be explained if exercise-induced sickling interferes with capillary blood flow and causes tissue ischemia and functional abnormalities. To test this hypothesis, we measured sickling and blood gas values in venous and arterial blood of an exercising limb in subjects with SCT and in controls. SUBJECTS AND METHODS: The study consisted of 15 subjects with hemoglobin AS (SCT group) and 15 subjects with hemoglobin AA (control group). Each performed two maximal arm crank exercise tests, one at 1,270 meters and one at simulated 4,000 meters. RESULTS: At 1,270 meters, axillary venous sickling increased significantly (p less than 0.05) from (mean +/- SD) 1.0 +/- 1.0% at rest to 2.3 +/- 2.6% during peak exercise. At simulated 4,000 meters, sickling increased significantly (p less than 0.001) from 1.5 +/- 1.2% to 8.5 +/- 7.1%. A wide range of sickling during peak exercise was observed (1% to 25%). One minute after exercise at simulated 4,000 meters, venous sickling remained elevated (7.2 +/- 7.8%) despite high levels of oxygen saturation. Arterial sickling (less than 1%) was present in only two subjects. There was no significant difference in oxygen consumption (29.4 +/- 3 versus 30.7 +/- 4 mL/kg/minute) between the subjects with SCT and the controls, nor was there a correlation between exercise performance and sickling (r less than 0.2). CONCLUSION: We conclude that exercise at 1,270 meters slightly, albeit significantly, increased sickling in blood from an exercising limb and that simulated 4,000 meters dramatically potentiated this effect. Sickling in the effluent blood of an exercising limb does not appear to measurably affect overall maximal arm crank exercise performance.

Adult↗
PubMed1988

Bradykinin stimulates phosphodiesteratic cleavage of phosphatidylcholine in cultured endothelial cells.

The ability of bradykinin to stimulate phosphodiesteratic cleavage of phosphatidylcholine (PC) was investigated in bovine pulmonary artery endothelial cells prelabeled with [3H]choline and [3H]myristic acid. Both labels were preferentially (approximately 80%) incorporated into PC. Bradykinin stimulated a rapid and parallel increase in approximately equivalent amounts of water soluble ([3H]choline plus [3H]phosphocholine) and lipid ([3H]phosphatidic acid plus [3H]diacylglycerol) phosphodiesteratic cleavage products of PC. Formation of the phosphodiesteratic cleavage products occurred prior to the maximum rate of release of prostacyclin into the medium, and ED50 values for both responses were similar (less than 1 nM) and consistent with effects mediated by a high affinity bradykinin receptor. These findings suggest that phosphodiesteratic cleavage of PC may be an important event in the process of receptor-dependent endothelial cell activation.

Animals↗
PubMed1988

Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells.

The conversion of phosphatidylcholine (PC) to diacylglycerol (DAG) was studied in sonicated endothelial cells and in subcellular fractions in the presence of 0.05% Triton X-100 and 2 mM EDTA. DAG formation occurred predominantly in an organelle fraction that sedimented at 15,000 x g. In parallel reactions with exogenous 1-oleoyl-2-[3H]oleoyl-PC (sn-2-[3H]DOPC) and phosphatidyl[3H]choline ([choline-3H]PC), [3H]DAG was formed by a reaction pathway in which [3H]choline was the only product derived from [choline-3H]PC. [3H]Choline was not formed secondarily from [3H]glycerophosphocholine or [3H]phosphocholine. Small amounts of [3H]phosphatidate ([3H]PA) were isolated from reactions with sn-2-[3H]DOPC at short incubation times, and substantial PA phosphatase activity was demonstrated. These data, taken together, supported a phospholipase D-PA phosphatase pathway of DAG formation. Kinetic data established that the low ratio of [3H]PA/[3H]DAG formed in reactions with sn-2-[3H]DOPC was due to a 15-fold higher Vmax and 7-fold lower apparent Km of the PA phosphatase. The [3H]PA/[3H]DAG product ratio was increased by addition of unlabeled PA or by selective extraction of phospholipase D with Triton X-100. The characteristics of the phospholipase D indicated a unique enzyme. Activity was optimal in the presence of EDTA and was almost totally dependent upon Triton X-100. The pH profile displayed a peak at 7.0. Of particular significance was the stringent substrate specificity. Phosphatidylinositol was not hydrolyzed, and activities towards phosphatidylethanolamine and sphingomyelin were at most 30- to 50-fold lower than those towards PC. Phospholipase D and PA phosphatase were identified in a number of rat tissues and other cells. The highest activities of phospholipase D were present in lung and endothelial cells. Phospholipase D was partially purified from rat lung by Triton X-100 extraction and anion exchange chromatography. When linked with PA phosphatase, the phospholipase D could initiate a pathway of DAG formation that is highly specific for PC.

Animals↗
PubMed1988

Effect of Army basic training in sickle-cell trait.

In order to determine if sickle-cell trait (SCT) represents an inherent adverse effect on response to training, we objectively evaluated exercise performance in 22 healthy, black men with SCT (hemoglobin AS) and 15 controls (hemoglobin AA) before and after seven weeks of army basic training at an altitude of 1270 m. An incremental exercise test to exhaustion on a cycle ergometer was used. Before basic training, peak exercise measurements did not reveal significant differences between groups other than a slightly lower, albeit significant, value for oxygen uptake (VO2) per kilogram (42 +/- 1 vs 45 +/- 1.4 mL/min per kilogram) for the SCT group. Both groups experienced modest overall cardiovascular improvement reflected in both peak and submaximal exercise responses. No statistically significant difference was observed between the SCT and the control groups at the end of basic training for any of the measured variables at peak exercise, including power (258 +/- 6 vs 266 +/- 9W), VO2 (3.24 +/- .06 vs 3.36 +/- .16 L/min), VO2 per kilogram (46 +/- 0.7 vs 46 +/- 1.2), minute ventilation (138 +/- 4 vs 147 +/- 8 L/min), heart rate (185 +/- 2 vs 184 +/- 3 beats per minute), oxygen pulse (17.6 +/- .3 vs 18.4 +/- 1 mL/min per beat), as well as anaerobic threshold (1.81 +/- .04 vs 1.80 +/- .06 L/min), respectively. No medical problems directly attributed to SCT were reported; it remains uncertain, however, whether a seizure experienced by one of the other SCT basic trainees after a two-mile run was SCT related. The results of this study would, therefore, suggest that for the majority of individuals who possess SCT, the response to the moderate training regimen provided by army basic training is not impaired.

Adult↗
PubMed1987

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells.

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.

Animals↗
PubMed1987

Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C.

The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.

Animals↗
PubMed1985

Activation of human platelets by N-substituted aminophospholipids.

N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphatidylserine (NBD-PS), a fluorescent phospholipid synthesized from phosphatidylserine by reaction with NBD-chloride, caused platelet shape change and aggregation when added at micromolar concentrations to suspensions of washed human platelets in the absence of added fibrinogen. Platelet aggregation by NBD-PS was accompanied by thromboxane synthesis and secretion of contents from dense, alpha-, and lysosomal granules in the absence of appreciable platelet damage. Indomethacin completely inhibited NBD-PS-induced thromboxane synthesis, but platelet aggregation and [14C]serotonin secretion were only slightly inhibited. Neither inhibition of the ADP-dependent pathway with creatine phosphate/creatine kinase plus ATP, alone or in combination with indomethacin, nor maximum elevation of cyclic AMP by treatment with prostaglandin I2 and theophylline completely inhibited NBD-PS-induced platelet aggregation or [14C]serotonin secretion. Platelet effects of NBD-PS were specific in that neither phosphatidylserine nor lyso-NBD-PS were similarly active. The activation of platelets by NBD-PS is not attributable to the NBD moiety exclusively since acylation of the amino group with 5-dimethylaminonaphthalene-1-sulfonyl-chloride yielded a similarly active derivative. Dansylated phosphatidylethanolamine was also active. The findings indicate that NBD-PS and other N-substituted aminophospholipids can activate a central pathway of platelet secretion and aggregation that is independent of released ADP and thromboxane formation and is only partially controlled by platelet cyclic AMP.

4-Chloro-7-nitrobenzofurazan↗
PubMed1982

Rat mast cell phospholipase A2: activity toward exogenous phosphatidylserine and inhibiton by N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylserine.

The presence of phospholipase A2 in intact rat peritoneal mast cells was investigated by using two synthetic radiolabeled phosphatidylserine (PS) substrates. Incubation of intact cells with 1-oleoyl-2-[3H]oleoyl-PS resulted in the release of a considerable quantity of [3H]oleic acid from the substrate. To establish that [3H]oleic acid release was mediated via direct enzymatic attack at the sn-2 position, we measured release of the [3H]serine moiety from the glycerol backbone of 1,2-dimyristoylphosphatidyl[3H]serine. This activity, which represents the combined actions of phospholipases C and D, was 10-fold lower than [3H]oleic acid release, indicating that neither of these enzymes is required for the release of the preponderance of [3H]oleic acid. These results establish the existence in intact rat mast cells of a phospholipase A2 active toward exogenous PS. Over the concentration range at which exogenous PS activates mast cell secretion, intact mast cells and broken cells possessed nearly equal levels of phospholipase A2 activity, and enzyme activity was 3--4-fold higher toward PS than phosphatidylcholine. Several agents were tested for their ability to inhibit phospholipase A2 in intact mast cells. Of the agents tested, an N-substituted derivative of PS previously identified as an inhibitor of mast cell secretion was shown to be a particularly potent and efficacious inhibitor of mast cell phospholipase A2. The concentration dependence of enzyme inhibition paralleled inhibition of histamine secretion, providing a strong positive correlation between the level of phospholipase A2 in mast cells and the capacity for secretion.

4-Chloro-7-nitrobenzofurazan↗
PubMed1979

Inhibition of mast cell histamine secretion by N-substituteed derivatives of phosphatidylserine.

The structural basis for the highly specific action of phosphatidylserine in enhancing mast cell histamine secretion induced by concanavalin A was investigated by studying the activities of three N-substituted derivatives: N-acetyl phosphatidylserine, N-1-dimethylaminonaphthalene-5-sulfonly phosphatidylserine, and N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylserine. None of the derivatives was capable of activating concanavalin A-induced histamine secretion at concentrations two to three times that required for maximal activation by phosphatidylserine. Instead, the derivatives were found to inhibit the secretory response of mast cells to the calcium ionophore A23187 as well as to concanavalin A. The inhibition was noncytotoxic, partially reversible by washing, and associated with binding of N-substituted phosphatidylserine to the mast cell.

Animals↗
PubMed1978

Interaction of phosphatidylserine with mast cells.

Phosphatidylserine (PtdSer) potentiates histamine secretion from mast cells exposed to concanavalin A and Ca2+. In order to identify the form of PtdSer that is responsible for its effect on mast cell secretion, PtdSer containing a tritium-labeled serine moiety (3H-PtdSer) was synthesized from egg yolk phosphatidylcholine. The critical micelle concentration (CMC) of 3H-PtdSer and the binding isotherm for 3H-PtdSer interaction with mast cells were determined. The midpoints of the binding isotherm and the dose-response curve for potentiation of secretion coincide and are 2 orders of magnitude greater than the CMC. The shape of the binding curve is explicable either in terms of simple binding of preformed PtdSer micelles or of cooperative binding of monomeric PtdSer in which the number of molecules cooperatively associating with a mast cell binding site is equal to the number of monomers in a PtdSer micelle. In either case, at equilibrium, PtdSer micelles are bound to the mast cells. The number of PtdSer molecules bound to a single mast cell at equilibrium was estimated to be 3.7 X 10(9).

Animals↗
PubMed1975

A review of reproductive patterns in new world monkeys.

A review of the literature indicated that most of the information of reproductive patterns in New World monkeys has been gained from studies on the squirrel monkey. This species has been successfully bred in both indoor and outdoor colonies. Both sexes exhibit seasonal cyclicity that appears to be influenced by a number of environmental conditions. Reports indicated that most of the other New World species have been bred in captivity, most commonly in zoos. The scientific data on these species are sparse, usually obtained from small groups or individual animals.

Alouatta↗