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G M Hatch

Publications and source records attributed to G M Hatch.

At least 37 records · Page 2Linked to original sources
PubMed1997

On the mechanism of the losartan-mediated inhibition of phosphatidylcholine biosynthesis in H9c2 cells.

Phosphatidylcholine is the major phospholipid in mammalian tissues and the biosynthesis of phosphatidylcholine in H9c2 cells was previously shown to be stimulated by angiotensin II. In this study, we used the potent AT1 receptor antagonist, losartan, to determine if the angiotensin II-mediated stimulation of phosphatidylcholine biosynthesis was mediated by AT1 receptors. H9c2 cells were incubated with angiotensin II in the absence or presence of various concentrations of losartan. The cells were then incubated with [methyl-3H]choline for an additional 60 min and the radioactivity incorporated into phosphatidylcholine and its choline-containing metabolites determined. Losartan at concentrations which block AT1 receptors did not effect phosphatidylcholine biosynthesis mediated by angiotensin II. In contrast, higher concentrations of losartan inhibited radioactivity incorporated into phosphatidylcholine and its metabolites and this was due to a losartan-mediated reduction in choline uptake. Kinetic studies revealed that the losartan-mediated inhibition of choline uptake was competitive. High concentrations of losartan caused a translocation of CTP:phosphocholine cytidylyltransferase from the cytosolic (inactive) to the membrane (active) fraction likely as a compensatory mechanism for the losartan-mediated reduction in new phosphatidylcholine biosynthesis. Incubation of cells with PD123319, a potent AT2-receptor antagonist, did not block the angiotensin II-mediated stimulation of phosphatidylcholine biosynthesis. The results suggest that angiotensin II stimulates phosphatidylcholine biosynthesis independent of AT1- and AT2-receptor activation and losartan inhibits phosphatidylcholine biosynthesis by reducing choline uptake in H9c2 cells.

Angiotensin II↗
PubMed1997

Induction of free radicals in hepatocytes, mitochondria and microsomes of rats by ochratoxin A and its analogs.

Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC and O alpha; and three synthetic analogs, the ethyl amide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in hepatocytes, mitochondria and microsomes from rats. Electron paramagnetic resonance spectroscopy (EPR) using alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN) as a spin trapping agent showed an enhanced free radical generation due to the addition of NADPH to the microsomes. An EPR signal was not observed in the mitochondria and hepatocyte samples when they were treated with a variety of agents. Addition of OM-OA together with NADPH and Fe3+ to the microsomes resulted in a strong EPR signal compared with the other analogs, whereas the signal could be quenched by the addition of catalase. OM-OA does not have a dissociable phenolate group and does not chelate Fe3+. The spin adduct hyperfine splitting constants indicated the presence of alpha-hydroxyethyl radicals resulting from generated hydroxyl radicals, which were trapped by 4-POBN. The results also suggested that the production of hydroxyl radicals by OA does not require a dissociable phenolate group or the prior formation of an OA-Fe complex.

Animals↗
PubMed1997

Cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol import into mitochondria through mitochondrial membrane contact sites in permeabilized rat liver hepatocytes.

The location of cytidine-5'-diphosphate-1,2-diacylsn-glycerol import into mictochondria was examined in permeabilized rat liver hepatocytes. The model utilized the ability of intact mitochondria to freely import [14C]glycerol-3-phosphate. Import of cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol was measured by the synthesis of phosphatidyl[14C]glycerol. Phosphatidyl [14C]glycerol was synthesized in a time- and concentration-dependent manner in the presence of cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol. The presence of ATP in the incubations stimulated phosphatidyl[14C]glycerol formation. The presence of adriamycin, at concentrations that block import of proteins into mitochondria, inhibited the formation of phosphatidyl [14C]glycerol. In addition, adriamycin, inhibited in vitro biosynthesis of phosphatidyl[14C]glycerol in mitochondrial but not microsomal fractions. 1,4-Dinitrophenol, which may decrease the number of mitochondrial inner and outer membrane contact sites, inhibited formation of phosphatidyl[14C]glycerol in permeabilized hepatocytes. The results demonstrate that cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol may be imported into mitochondria through inner and outer mitochondrial membrane contact sites in hepatocytes.

Animals↗
PubMed1997

Cardiolipin remodeling in a Chinese hamster lung fibroblast cell line deficient in oxidative energy production.

The metabolism of cardiolipin was investigated in a Chinese hamster lung fibroblast cell line CCL16-B2 deficient in oxidative energy metabolism and its parental cell line CCL16-B1. Mitochondrial enzyme activities involved in de novo cardiolipin biosynthesis were elevated in CCL16-B2 cells compared with CCL16-B1 cells, indicating initially an elevation in cardiolipin biosynthesis. Content of all phospholipids, including cardiolipin and its precursors, and high energy nucleotides were unaltered in CCL16-B2 cells compared to CCL16-B1 cells. When cells were incubated with [1,3-(3)H]glycerol for up to 4 h radioactivity incorporated into cardiolipin in CCL16-B2 cells did not differ compared with CCL16-B1 cells. In contrast, radioactivity incorporated into phosphatidylglycerol, the immediate precursor of cardiolipin, was elevated over 2-fold in CCL16-B2 cells compared with CCL16-B1 cells. Analysis of the fatty acid molecular species in cardiolipin revealed alterations in the level of unsaturated but not saturated fatty acids in B2 compared with B1 cells. In vivo cardiolipin remodeling, that is, the deacylation of cardiolipin to monolysocardiolipin followed by reacylation back to cardiolipin, with [1-(14)C]palmitate and [1-(14)C]oleate and in vitro mitochondrial phospholipid remodeling with [1-(14)C]linoleate were altered in CCL16-B2 cells compared to CCL16-B1 cells. Since both the appropriate content and molecular composition of cardiolipin is required for optimum mitochondrial oxidative phosphorylation, we suggest that the difference in CL molecular species composition observed in CCL16-B2 cells, mediated by alterations in in vivo cardiolipin remodeling, may be one of the underlying mechanisms for the reduction in oxidative energy production in CCL16-B2 cells.

Animals↗
PubMed1997

Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis.

There is little information on the trafficking of eukaryotic lipids from a host cell to either the cytoplasmic membrane of or the vacuolar membrane surrounding intracellular pathogens. Purified Chlamydia trachomatis, an obligate intracellular bacterial parasite, contains several eukaryotic glycerophospholipids, yet attempts to demonstrate transfer of these lipids to the chlamydial cell membrane have not been successful. In this report, we demonstrate that eukaryotic glycerophospholipids are trafficked from the host cell to C. trachomatis. Phospholipid trafficking was assessed by monitoring the incorporation of radiolabelled isoleucine, a precursor of C. trachomatis specific branched-chain fatty acids, into host-derived glycerophospholipids and by monitoring the transfer of host phosphatidylserine to chlamydiae and its subsequent decarboxylation to form phosphatidylethanolamine. Phospholipid trafficking to chlamydiae was unaffected by brefeldin A, an inhibitor of Golgi function. Furthermore, no changes in trafficking were observed when C. trachomatis was grown in a mutant cell line with a nonfunctional, nonspecific phospholipid transfer protein. Host glycerophospholipids are modified by C. trachomatis, such that a host-synthesized straight-chain fatty acid is replaced with a chlamydia-synthesized branched-chain fatty acid. We also demonstrate that despite the acquisition of host-derived phospholipids, C. trachomatis is capable of de novo synthesis of phospholipids typically synthesized by prokaryotic cells. Our results provide novel information on chlamydial phospholipid metabolism and eukaryotic cell lipid trafficking, and they increase our understanding of the evolutionary steps leading to the establishment of an intimate metabolic association between an obligate intracellular bacterial parasite and a eukaryotic host cell.

Animals↗
PubMed1997

Oxidative modification of low density lipoprotein in normal and hyperlipidemic patients: effect of lysophosphatidylcholine composition on vascular relaxation.

The elevated level of plasma low density lipoprotein (LDL) in hyperlipidemic patients is an important risk factor for the production of atherosclerosis. Plasma LDL must be modified before it can produce an impairment of endothelium-dependent relaxation in aortic rings or enhancement of uptake by macrophages. The dramatic increase in lysophosphatidylcholine (lysoPC) content in oxidatively modified LDL has been touted as an important biochemical factor for the impairment of endothelium-dependent relaxation. The present study was designed to examine the lysoPC composition of oxidized LDL samples from normal and hyperlipidemic subjects, and their effects on the impairment of endothelium-dependent relaxation. Oxidatively modified LDL from hyperlipidemic patients contained a slightly higher level (17%) of lysoPC, but produced a disproportionately greater impairment of endothelium-dependent relaxation than that from normal subjects. As lysoPC is composed of many molecular species, its composition in oxidized LDL samples was analyzed. In hyperlipidemic patients, lysoPC samples were found to contain a higher proportion of long-chain acyl groups. Subsequent studies revealed that only long-chain lysoPC (C > 16:0) were effective in impairing endothelium-dependent relaxation. Experimental loading of oxidized LDL from normal subjects with long chain lysoPC to mimic levels observed in oxidized LDL from hyperlipidemic patients resulted in further impairment of endothelium-dependent relaxation. We conclude that the greater proportion of long-chain lysoPC found in the oxidized LDL of hyperlipidemic subjects is responsible for the increased impairment of endothelium-dependent vascular relaxation. We propose that the high level of LDL found in the plasma of hyperlipidemic patients, coupled with its enhanced ability to generate long chain species of lysoPC during oxidative modification, are important factors for the development of atherosclerosis in these patients.

Adult↗
PubMed1996

Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblasts by cytidine 5'-triphosphate.

The regulation of cardiolipin biosynthesis by CTP in H9c2 cardiac myoblasts was investigated. H9c2 cells were incubated in the presence of cyclopentenylcytosine which is converted to cyclopentenylcytosine-triphosphate, a potent and specific inhibitor of CTP synthetase. Incubation of cells for 12 h with cyclopentenylcytosine reduced the cellular pool size of CTP to less than 10% of control cells but did not influence the pool size of other nucleotides. The de novo biosynthesis of phosphatidylcholine from [methyl-3H]choline, phosphatidylethanolamine from [1-3H]ethanolamine, and biosynthesis of all glycerol containing phospholipids from [U-14C]glycerol or [1,3-3H]glycerol were reduced approximately 50% after preincubation of the cells with cyclopentenylcytosine. In contrast, radioactive glycerol accumulated in phosphatidic acid, diacylglycerol, and triacylglycerol in cyclopentenylcytosine-treated cells compared with controls suggesting a re-routing of phospholipid biosynthesis away from CTP utilizing reactions toward neutral lipid synthesis. The de novo biosynthesis of all phospholipids was restored to control levels by addition of cytidine to the medium which elevated CTP levels. Cyclopentenylcytosine did not affect the in vitro enzyme activities involved in cardiolipin biosynthesis in these cells. In addition, the resynthesis of cardiolipin and most phospholipids from [1-14C]linoleic acid was not affected by cyclopentenylcytosine. Our findings indicate that the cellular CTP level may regulate cardiolipin biosynthesis in H9c2 cardiac myoblasts and support the notion that the cellular CTP level may be a universal signal/switch for all phospholipid biosynthesis in eukaryotic cells.

Animals↗
PubMed1996

The acylation of lysophosphatidylglycerol in rat heart: evidence for both in vitro and in vivo activities.

The reacylation of lysophospholipids back to their parent molecules is important for attaining the appropriate fatty acyl composition in many phospholipids and for preventing the accumulation of arrhythmia generating lysophospholipids in the heart. In this study, we report the presence of an active acyltransferase activity for lysophosphatidylglycerol reacylation to phosphatidylglycerol in rat heart membrane preparations. The activity of acyl-Coenzyme A:1-acylglycerophosphorylglycerol acyltransferase in rat heart subcellular fractions was in the order of microsomal > mitochondrial > cytosol. The activity in the membrane fractions were characterized and found to have a pH optimum in the alkaline range. However, significant enzyme activity was observed at physiological pH. With oleoyl-Coenzyme A as substrate, the microsomal activity had a preference for lysophosphatidylglycerol substrates in the order of myristoyl > palmitoyl > oleoyl > stearoyl. The apparent K(m) values for 1-palmitoylglycerophosphorylglycerol and oleoyl-Coenzyme A were 9.4 and 7.1 microM, respectively. In contrast, the mitochondrial activity had a preference for lysophosphatidylglycerol substrates in the order of oleoyl > myristoyl = stearoyl = palmitoyl. The apparent K(m) values for 1-oleoylglycerophosphorylglycerol and oleoyl-Coenzyme A were 17.8 and 18.0 microM, respectively. Both membrane activities were heat labile as pre-incubation at 55 degrees C for 1 min completely abolished the activity. However, pre-incubation at 50 degrees C resulted in different profiles of inactivation in both microsomal and mitochondrial fractions. Both membrane activities were inhibited by high concentrations of lysophosphatidylglycerol and affected to a similar extent by various detergents. To demonstrate whether reacylation of lysophosphatidylglycerol to phosphatidylglycerol occurred in vivo, isolated rat hearts were perfused for 60 min in the Langendorff mode with 0.1 microM 1-palmitoylglycerophosphoryl[14C]glycerol bound to albumin. 1-Palmitoylglycerophosphoryl[14C]glycerol was readily taken up by the isolated perfused rat heart and significant synthesis of phosphatidyl[14C]glycerol was observed. The findings indicate the presence of an acyl-Coenzyme A:1-acylglycerophosphorylglycerol acyltransferase activity in the rat heart subcellular membranes which is capable of catalyzing lysophosphatidylglycerol acylation to phosphatidylglycerol in vitro and in vivo.

Acylation↗
PubMed1996

Regulation of cardiolipin biosynthesis in the heart.

Cardiolipin is one of the principle phospholipids in the mammalian heart comprising as much as 15-20% of the entire phospholipid phosphorus mass of that organ. Cardiolipin is localized primarily in the mitochondria and appears to be essential for the function of several enzymes of oxidative phosphorylation. Thus, cardiolipin is essential for production of energy for the heart to beat. Cardiac cardiolipin is synthesized via the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway. The properties of the four enzymes of the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway have been characterized in the heart. The rate-limiting step of this pathway is catalyzed by the phosphatidic acid: cytidine-5'-triphosphate cytidylyltransferase. Several regulatory mechanisms that govern cardiolipin biosynthesis in the heart have been uncovered. Current evidence suggests that cardiolipin biosynthesis is regulated by the energy status (adenosine-5'-triphosphate and cytidine-5'-triphosphate level) of the heart. Thyroid hormone and unsaturated fatty acids may regulate cardiolipin biosynthesis at the level of three key enzymes of the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway, phosphatidylglycerol phosphate synthase, phosphatidyl-glycerolphosphate phosphatase and cardiolipin synthase. Newly synthesized phosphatidic acid and phosphatidylglycerol may be preferentially utilized for cardiolipin biosynthesis in the heart. In addition, separate pools of phosphatidylglycerol, including an exogenous (extra-mitochondrial) pool not derived from de novo phosphatidylglycerol biosynthesis, may be utilized for cardiac cardiolipin biosynthesis. In several mammalian tissues a significant number of studies on polyglycerophospholipid biosynthesis have been documented, including detailed studies in the lung and liver. However, in spite of the important role of cardiolipin in the maintenance of mitochondrial function and membrane integrity, studies on the control of cardiolipin biosynthesis in the mammalian heart have been largely neglected. The purpose of this review will be to briefly discuss cardiolipin and cardiolipin biosynthesis in some selected model systems and focus primarily on current studies involving the regulation of cardiolipin biosynthesis in the heart.

Animals↗
PubMed1996

Susceptibility of exogenous surfactant to phospholipase A2 degradation.

The inhibition of surfactant biophysical activity in vivo is potentially mediated by many factors, including serum proteins, particularly enzymatic proteins such as phospholipases. In the present study, we investigated the susceptibility of the phosphatidylcholine component of two exogenous surfactants, Exosurf and Survanta, to secretory-type phospholipase A2 (PLA2) deacylation in vitro. Lyophilized Exosurf and Survanta preparations were incubated at 37 degrees C for 120 min in the presence of bovine pancreatic PLA2, and the production of lysophosphatidylcholine was determined as a measure of the magnitude of phosphatidylcholine deacylation. The phosphatidylcholine component of Survanta was readily deacylated by PLA2, whereas the dipalmitoylphosphatidycholine (DPPC) component of Exosurf was resistant over the entire duration of the assay. To further evaluate this observed resistance the individual and combined effects of tyloxapol and hexadecanol, components of Exosurf, upon PLA2 deacylation of Survanta and DPPC were investigated. In both Survanta and DPPC preparations, PLA2-mediated deacylation was significantly inhibited in the presence of tyloxapol. We conclude that the presence of tyloxapol in the Exosurf preparation inhibits secretory type PLA2 mediated DPPC deacylation. This unique feature of Exosurf may be of clinical significance when this preparation is utilized in the treatment of surfactant-deficient infants.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Thyroxine stimulates phosphatidylglycerolphosphate synthase activity in rat heart mitochondria.

The effect of administration of exogenous thyroxine on mitochondrial phosphatidylglycerol content and biosynthesis was investigated in rat heart ventricles. Rats were treated for 5 consecutive days with thyroxine (250 mg/kg body weight) and on the sixth day after an overnight fast the mass of ventricular mitochondrial phosphatidylglycerol and cardiolipin content were determined. Saline-treated animals served as controls. Thyroxine treatment did not affect body weight but increased heart weight 30% compared with controls. In addition, the ratio of heart weight/body weight (x 1000) was increased from 0.69 in controls to 0.89 in thyroxine-treated rats consistent with this model. Thyroxine-treatment resulted in a 34% increase (P < 0.05) in phosphatidylglycerol and a 23% increase (P < 0.05) in cardiolipin content in ventricular mitochondrial fractions compared with controls. The mechanism for the increase in ventricular mitochondrial phosphatidylglycerol was investigated. Phosphatidic acid:cytidine-5'-triphosphate-1,2-diacylglycerol cytidylyltransferase and phosphatidylglycerolphosphate phosphatase activities were unaltered in the ventricular mitochondria of thyroxine-treated rats. In contrast, phosphatidylglycerolphosphate synthase activity was increased 3.5-fold (P < 0.05) in these mitochondrial fractions compared with controls. As a control for the effectiveness of thyroxine on mitochondria, cardiolipin synthase activity was determined. A 2.8-fold increase (P < 0.05) in cardiolipin synthase activity was observed in ventricular mitochondrial fractions of thyroxine-treated rats compared with controls. We postulate that thyroxine-treatment of rats produces an increase in the pool size of ventricular mitochondrial phosphatidylglycerol and that the mechanism is an increase in phosphatidylglycerolphosphate synthase activity.

Animals↗
PubMed1995

Cardiolipin is synthesized from exogenous phosphatidylglycerol in rat heart.

De novo biosynthesis of phosphatidylglycerol and cardiolipin in the isolated intact rat heart was shown to occur from newly synthesized phosphatidic acid via the formation of cytidine-5'-diphosphate-1,2-diacylglycerol (Hatch, G.M. (1994) Biochem. J. 297, 201-208). The biosynthesis of new cardiolipin was investigated in isolated rat hearts perfused with exogenous phosphatidylglycerol. Phosphatidylglycerol was rapidly (within 5 min) incorporated into the heart when hearts were perfused with either phosphatidyl-[14C]glycerol or NBD-phosphatidylglycerol. In hearts perfused with phosphatidyl-[14C]glycerol for up to 30 min the amount of radioactivity observed in phosphatidylglycerol was maximum by 5 min of perfusion and remained constant throughout the perfusion period. In the presence of 1-50 microM phosphatidylglycerol, the amount of radioactive phosphatidylglycerol incorporated into the heart was not affected. There was a time-dependent accumulation of radioactivity incorporated into cardiolipin. In addition, radioactivity was incorporated with time into lysophosphatidylglycerol. No significant amount of radioactivity was associated with other phospholipids involved in the biosynthesis of cardiolipin, including phosphatidic acid and cytidine-5'-diphosphate-1,2-diacylglycerol, precursors of cardiolipin biosynthesis via the CDP-DG pathway. We postulate that cardiac cardiolipin may be synthesized from exogenous phosphatidylglycerol independent of phosphatidylglycerol synthesized within the heart.

Animals↗
PubMed1995

Decrease in cardiac phosphatidylglycerol in streptozotocin-induced diabetic rats does not affect cardiolipin biosynthesis: evidence for distinct pools of phosphatidylglycerol in the heart.

Biosynthesis of phosphatidylglycerol (PG) and cardiolipin (CL) were investigated in perfused hearts of diabetic rats 4 days or 28 days after streptozotocin injection. Sham-injected and insulin-treated diabetic rats were used as controls. In addition, another group of rats fasted for 54 h was examined. Isolated rat hearts from these groups were perfused for 30 min with [32P]P(i), and the radioactivity incorporated into PG and CL and their pool sizes were determined in heart ventricles. There was no difference in the amount of radioactivity incorporated into CL, PG or other phospholipids between all groups. In addition, the pool sizes of CL and other phospholipids were unaltered. However, a striking decrease in the pool size of PG was observed in both diabetic and fasted rats compared to sham- and insulin-treated controls at 4 days after streptozotocin injection. The decrease in PG mass in diabetic rats was rapid (within 24-48 h) and was localized to cardiac membranes. Diabetes did not affect the activity of the enzymes of PG and CL biosynthesis in the mitochondrial fraction, or phospholipase A activity in subcellular fractions prepared from rat heart homogenates. In addition, pulse-chase experiments confirmed that diabetes did not affect the rate of new PG or CL biosynthesis. Since radioactivity associated with PG was unaltered in continuous-pulse perfusion experiments, a calculated 1.8-fold increase in the specific radioactivity of cardiac PG was observed in the hearts of acute diabetic rats compared with controls. Since the radioactivity incorporated into PG and CL, and the rate of CL biosynthesis, were unaltered in diabetic-rat hearts compared with controls, new CL was probably synthesized from newly synthesized PG. We postulate the existence of distinct pools of PG in the heart, and that the pool of newly synthesized PG used for CL biosynthesis does not appear to mix immediately with the pre-existing pool of PG in the isolated intact rat heart.

Animals↗