Turnover of arachidonic acid in the major diacyl and ether phospholipids of human platelets

In this work, the uptake and release of [3H]arachidonic acid by the diacyl and ether species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human platelets were studied. Uptake of [3H]arachidonic acid into 1,2-diacyl-PC and 1,2-diacyl-PE was much greater than into the ether phospholipids of the same class. In [3H]arachidonoyl-labeled platelets stimulated by thrombin, there was a decrease in total [3H] arachidonoyl-PC. This was accounted for mostly by a decrease in 1-acyl-2-[3H]arachidonoyl-PC while the level of 1-O-alkyl-2-[3H]arachidonoyl-PC (a precursor for platelet-activating factor) increased slightly. However, in ionophore A23187-stimulated platelets, the reduction of total [3H]arachidonoyl-PC was due to a decrease in both 1-acyl-2-[3H]arachidonoyl-PC and 1-O-alkyl-2-[3H] arachidonoyl-PC, suggesting that ionophore should yield more platelet-activating factor than thrombin. In both thrombin- and ionophore-stimulated platelets, there was a net increase in total [3H]arachidonoyl-PE. This consisted of a decrease in 1,2-diacyl-PE, which was essentially complete by 1 min, followed by an increase in 1-O-alk-1'-enyl-2-[3H]arachidonoyl-PE, which was slower and not apparent until 3-5 min after thrombin. During reincubation of labeled platelets with saline, the 1-O-alkyl-2-[3H]arachidonoyl-PC increased by a factor of 2, between 0 and 4 h, with no significant change in the radioactivity of any other phospholipid. Thus, upon stimulation of human platelets, arachidonic is released from both 1,2-diacyl-PC and 1,2-diacyl-PE for metabolism by platelet cyclooxygenase and lipoxygenase, while certain ether pools of PC and PE also collect arachidonic acid.     

Chronic exposure to inhaled anesthetics increases cholesterol content in Acholeplasma laidlawii

Acholeplasma laidlawii cells were grown in cholesterol-enriched medium and exposed continuously to either air (control), 4.0 vol.% halothane in air at 1 atm pressure (4% atm halothane), or 80% cyclopropane in oxygen for 24 h at 37°C. Cells grown in the presence of 4% atm halothane or 80% cyclopropane had approximately twice as much membrane cholesterol content/mg protein as the control cells. Cells grown in an anesthetic environment also tended to have a higher membrane cholesterol/phospholipid molar ratio compared to control cells. Membranes isolated from halothane-exposed cells grown in a cholesterol-enriched medium were more ordered at 37°C (measurements were made with no anesthetic present) than membranes from control cells grown in an identically enriched medium. This difference in membrane physical state between control and anesthetic-exposed cells decreased as the temperature decreased, and disappeared at approx. 23°C. Continuous exposure of A. laidlawii to 4% atm halothane or 80% cyclopropane for 24 h did not markedly affect membrane fatty acid composition, either in cells grown on an unsupplemented medium or in cells grown in a medium enriched in myristic, palmitic or stearic acids. These results further support the hypothesis that an increased membrane cholesterol content may play a role in the tolerance or dependence that develops after chronic exposure to anesthetic agents.     

Histochemical study of calcium on T-tubule membranes and in the sarcoplasmic reticulum, in frog twitch muscle fibres at rest and during activity

The trigger calcium hypothesis of signal transmission between T-tubules and terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in twitch muscle fibres implies the presence of calcium along T-tubule membranes at rest and its release upon excitation. To test this hypothesis, calcium was immobilised using a fixing and precipitating solution of glutaraldehyde in phosphate buffer at pH 8.0 and the calcium was substituted for by lead. Simultaneous tension recordings revealed the occurrence of contractions or a burst of twitches upon perfusion with the fixative. Procaine or tetrodotoxin (TTX) was used to inhibit this activity. In fibres without fixative-induced activity, precipitates were observed along T-tubules and in adjoining parts of TC. In activated fibres, tubular and TC precipitates were absent. These results are consistent with the trigger calcium hypothesis. In fibres activated by depolarisation, calcium returned to TC after passing successively through different parts of the SR.     

Calcium uptake by isolated nerve endings: evidence for a rapid component mediated by the breakdown of phosphatidylinositol

Several physiological stimuli, including neuronal depolarization, increase the production of phosphatidate (PA) from phosphatidylinositol (PI) and increase calcium fluxes across cell membranes. To determine if breakdown of PI is required for neuronal calcium uptake, we tested inhibitors of PI-specific phospholipase C on depolarization-dependent uptake of calcium by isolated brain synaptosomes. At a concentration of 0.1 mM these inhibitors reduced calcium uptake produced by depolarization for 1 to 3 sec, but did not affect uptake due to more prolonged depolarization. Exogenous PA also stimulated calcium accumulation by synaptosomes and this uptake was not reduced by the enzyme inhibitors. These results suggest that the rapid calcium influx produced by neuronal depolarization may be mediated by the breakdown of PI.     

Effect of prostaglandins on lipid transfer between high- and low-density lipoproteins in human plasma

Prostaglandin (PG) E1 was shown to stimulate the transfer of phosphatidylcholine and cholesterol esters from human high density lipoproteins to low density lipoproteins. The enhancement of the interlipoprotein lipid transfer by PGE1 was observed both at low prostaglandin concentrations under conditions of spontaneous exchange as well as in the presence of the lipoprotein-depleted plasma and the partly purified lipid transfer plasma protein. At the same time PGE2 showed no significant influence on the interlipoprotein lipid transfer. It is supposed that the effect of PGE1 is due to the PGE1-induced reorganization of the high density lipoprotein surface and that the PG-lipoprotein interaction is a factor which regulates cholesterol homeostasis.     

Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously. The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R[( 2KB]/[pyruvate]), was found to be 40 +/- 3 under a wide variety of conditions. Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme. At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration. On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions. In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids. 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate. However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism. The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paleoceanographic interpretations of coccoliths and oxygen-isotopes from the sediment surface of the southwest Indian Ocean

The distribution of forty-four coccolithophore species in one hundred deep-sea core-tops from the southwest Indian Ocean is described. Three coccolith assemblages have been recognised (Maputo, Agulhas Current and deep water) by the relative abundances of four ecologically significant coccolithophore species (Gephyrocapsa oceanica, Emiliania huxleyi, Calcidiscus leptoporus and Umbilicosphaera sibogae). Their biogeographical distribution appears to be related to water temperature, nutrient concentration and dissolution.The degree of preservation of coccoliths and foraminifera indicates that the carbonate lysocline lies somewhere between 3500 and 4000 m, resulting in the concentration of dissolution-resistant microfossils below this depth.Stable oxygen isotope ratios of the planktonic foraminiferal species Globigerinoides sacculifer range between −1.5 to −1.0‰ PDB (equal to 22.8–25.1°C) and occur in a narrow band on the sea floor beneath the “A” route of the Agulhas Current.These values are about 0.5 per mil PDB lighter than samples analyzed on either side of this band and can be explained by the Agulhas Current's elevated temperature at the ocean surface of 2–3°C. Thus an oxygen isotope imprint of the Agulhas Current exists beneath it on the sea floor.The Agulhas Current is probably the major factor influencing sedimentation, sediment-distribution patterns and geological features in the study area. At present it is voluminous and fast flowing, possibly eroding sediments up to 2500 m below the surface.The oxygen-isotope ratios and nannoplankton counts obtained in this study indicate, however, that the majority of samples are most probably recent or at least not older than 85,000 years. This implies that sediments are accumulating on the ocean floor and that the Agulhas Current does not have a pronounced erosional influence, at least in areas from which cores were retrieved for this study.