Biomedical subjects

G M Hatch

Publications and source records attributed to G M Hatch.

At least 55 records · Page 3Linked to original sources

PubMed1995

Inhibition of cardiolipin biosynthesis in the hypoxic rat heart.

Cardiolipin is the principal polyglycerophospholipid in the heart. The effect of hypoxia on cardiolipin biosynthesis was investigated in isolated rat hearts perfused in the Langendorff mode. Hearts were pulsed-labeled for 60 min with 0.1 mM [1,(3)-3H]glycerol in Krebs Henseleit buffer saturated with either 95% O2/5% CO2 (control) or 95% N2/5% CO2 (hypoxic). Radioactivity incorporated into phosphatidylglycerol and cardiolipin were reduced 88% (P < .05) and 79% (P < .05), respectively, in hypoxic hearts compared to controls. In other experiments, hearts were pulse-labeled for 15 min with 1.4 mM [32P]Pi in Krebs Henseleit buffer saturated with 95% O2/5% CO2 and subsequently perfused for 60 min under control or hypoxic conditions. The radioactivity incorporated into CDP-1,2-diacyl-sn-glycerol, phosphatidylglycerol, and cardiolipin were reduced 61% (P < .05), 71% (P < .05), and 70% (P < .05), respectively, in the hypoxic hearts compared to controls, indicating a decreased formation of CDP-1,2-diacyl-sn-glycerol in the hypoxic heart. The activities of the enzymes involved in cardiolipin biosynthesis and the cardiac pool sizes of cardiolipin, phosphatidylglycerol, and CDP-1,2-diacyl-1,2-diacyl-sn-glycerol were unaltered between hypoxic and control hearts. In contrast, cardiac adenosine-5'-triphosphate and CPT levels were decreased 94% (P < .05) and 92% (P < .05), respectively, in hypoxic hearts compared to controls. We postulate that the biosynthesis of the cardiac polyglycerophospholipid cardiolipin may be inhibited by a decreased adenosine-5'-triphosphate and cytidine-5'-triphosphate level in the heart.

Adenosine Triphosphate↗

PubMed1994

Dephosphorylation of CTP-phosphocholine cytidylyltransferase is not required for binding to membranes.

The sequence of the reversible phosphorylation and activation of CTP:phosphocholine cytidylyltransferase was investigated. Treatment of primary rat hepatocytes with oleic acid or phospholipase C caused a significant increase in the activity and amount of particulate cytidylyltransferase which correlated with decreased cytidylyltransferase activity and protein in the cytosol. The increase in membrane-associated cytidylyltransferase is accompanied by a decrease in the phosphorylation of the enzyme. Reversal of membrane association resulted in an increased amount of phosphorylated cytidylyltransferase in the cytosol. We wished to determine if dephosphorylation of the enzyme were a prerequisite for its translocation from the cytosol to the membranes. In vitro studies with membranes from oleic acid- or phospholipase C-treated cells showed that phosphorylated cytosolic cytidylyltransferase associated with these membranes with negligible dephosphorylation. Incubation of hepatocytes with oleic acid for different periods of time demonstrated that cytidylyltransferase associated with membranes in an active, phosphorylated form and was subsequently dephosphorylated. This result was supported by comparison of phosphopeptide maps of 32P-labeled cytidylyltransferase obtained from cytosolic, as well as membrane fractions of control, oleic acid-treated, or phospholipase C-treated cells. These studies revealed dephosphorylation on some sites and phosphorylation on other sites. Our data strengthen the hypothesis that a change in the lipid composition of membranes can mediate the initial binding of cytidylyltransferase to the membrane and that subsequently the enzyme becomes dephosphorylated.

Animals↗

PubMed1994

Cardiolipin biosynthesis in the isolated heart.

The pathway for the biosynthesis of new cardiolipin was investigated in the isolated perfused intact rat heart. Isolated rat hearts were perfused in the Langendorff mode for up to 60 min with Krebs-Henseleit buffer containing 0.1 microM [U-14C]glycerol. Analysis of radioactivity incorporated into phospholipids in the organic phase revealed an increase in radioactivity incorporated into phosphatidylglycerol, cardiolipin and other phospholipids with time of perfusion. This was associated with a loss of radioactivity from phosphatidic acid. In contrast, perfusion of hearts for up to 60 min with 0.1 mM [1,(3)-3H]glycerol in the perfusate revealed an increased radioactivity associated with phosphatidic acid as well as cardiolipin, phosphatidylglycerol and other phospholipids. Perfusion of hearts for up to 60 min with [32P]Pi in the perfusate revealed a time-dependent increase in radioactivity associated with all phospholipids. Perfusion of hearts for up to 60 min with 0.1 microM or 0.1 mM glycerol in the perfusate did not affect the concentration of phosphatidic acid, cardiolipin or phosphatidylglycerol. To determine the rate-limiting step of cardiolipin biosynthesis, hearts were pulsed for 5 min with 0.1 microM [1,(3)-3H]glycerol and chased for up to 60 min with 0.1 microM glycerol in the perfusate. Radioactivity was maximum at the start of the chase in phosphatidic acid (and 1,2-diacylglycerol), and was subsequently chased into phosphatidylinositol, phosphatidylglycerol and cardiolipin (and other phospholipids). Significant radioactivity in phosphatidylglycerol phosphate was not detected. Radioactivity in CDP-sn-1,2-diacylglycerol remained constant throughout the chase. The activities of the enzymes of the Kennedy pathway for cardiolipin biosynthesis in the heart were determined. On the basis of continuous-pulse and pulse-chase labelling studies it is postulated that the cardiac polyglycerophospholipids phosphatidylglycerol and cardiolipin are actively synthesized from newly synthesized phosphatidic acid via the Kennedy pathway. In addition, the results suggest that the rate-limiting step of cardiolipin biosynthesis in the intact heart is probably the conversion of phosphatidic acid into CDP-sn-1,2-diacylglycerol.

Animals↗

PubMed1994

Stimulation of phosphatidylglycerolphosphate phosphatase activity by unsaturated fatty acids in rat heart.

Phosphatidylglycerolphosphate (PGP) synthase and PGP phosphatase catalyze the sequential synthesis of phosphatidylglycerol from cytidine-5'-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG) and glycerol-3-phosphate. PGP synthase and PGP phosphatase activities were characterized in rat heart mitochondrial fractions, and the effect of fatty acids on the activity of these enzymes was determined. PGP synthase was observed to be a heat labile enzyme that exhibited apparent Km values for CDP-PG and glycerol-3-phosphate of 46 and 20 microM, respectively. The addition of exogenous oleic acid to the assay mixture did not affect PGP synthase activity. PGP phosphatase was observed to be a heat labile enzyme, and addition of oleic acid to the assay mixture caused a concentration-dependent stimulation of PGP phosphatase activity. Maximum stimulation (1.9-fold) of enzyme activity was observed in the presence of 0.5 mM oleic acid, but the stimulation was slightly attenuated by the presence of albumin in the assay. The presence of oleic acid in the assay mixture caused the inactivation of PGP phosphatase activity to be retarded at 55 degrees C. Stimulation of PGP phosphatase activity was also observed with arachidonic acid, whereas taurocholic, stearic and palmitic acids did not significantly affect PGP phosphatase activity. The activity of mitochondrial phosphatidic acid phosphohydrolase was not affected by inclusion of oleic acid in the incubation mixture. We postulate that unsaturated fatty acids stimulate PGP phosphatase activity in rat heart.

Albumins↗

PubMed1994

Alteration of lysophosphatidylcholine content in low density lipoprotein after oxidative modification: relationship to endothelium dependent relaxation.

OBJECTIVE: The aim was to examine the formation of lipid peroxidation products and the alteration in phospholipid content in low density lipoprotein (LDL) after oxidative modification by CuSO4, and subsequently, to determine the ability of the modified LDL to impair endothelium dependent relaxation in rat aortic rings. METHODS: Blood samples were obtained from normal human volunteers. LDL was prepared by sequential ultracentrifugation and it was oxidatively modified in the presence of 5 microM CuSO4. Lipid peroxidation products (thiobarbituric acid reactive substances, TBARS), and alterations in electrophoretic mobility and phospholipid content were determined in normal (native) and oxidised LDL. Endothelium dependent relaxation was produced by acetylcholine (10(-8)-10(-5) M) in phenylephrine precontracted rat aortic rings. RESULTS: LDL incubated for 24 h with 5 microM CuSO4 at 20 degrees C and 37 degrees C with constant agitation displayed higher amounts of TBARS than the respective native LDL. While the amounts of TBARS in LDL modified at 20 degrees C and 37 degrees C were similar, the former condition resulted in statistically smaller changes of phospholipid contents. LDL with higher lysophosphatidylcholine content showed greater impairment of endothelium dependent relaxation in rat aortic rings than LDL with lower lysophosphatidylcholine content. CONCLUSIONS: The raised lysophosphatidylcholine level in oxidatively modified LDL was related to the ability of the LDL to impair endothelium dependent relaxation. However, lipid peroxidation products assessed by TBARS did not relate to the phospholipid changes in LDL and therefore cannot be used to predict the vascular effects of LDL after oxidative modification.

Acetylcholine↗

PubMed1993

Rat liver mitochondrial phospholipase A2 is an endotoxin-stimulated membrane-associated enzyme of Kupffer cells which is released during liver perfusion.

A novel fluorescence assay for phospholipase A2 [Wilton (1990) Biochem. J. 266, 435-439] has been used to study the Group-II rat liver mitochondrial enzyme, and a number of novel properties of this enzyme were identified. (1) The enzyme activity was located in the liver macrophages (Kupffer cells) while negligible activity was associated with hepatocytes. (2) Although subcellular fractionation of whole liver confirmed the predominantly mitochondrial location of this enzyme activity, the analysis of the hepatocyte-free Kupffer-cell-enriched fraction revealed a different enzyme distribution, with the majority of activity being associated with the microsomal membrane fraction. (3) Bacterial endotoxin has been previously shown to be scavenged by Kupffer cells in rats. Treatment of rats with bacterial lipopolysaccharide (endotoxin) resulted in a dramatic time- and dose-dependent increase in liver phospholipase A2 activity. (4) It is known that injection of endotoxin into rodents results in elevated serum phospholipase A2 activity, while a similar phenomenon is seen in the condition of septic shock in man. The source of this serum enzyme was unknown. In this study perfusion of livers from rats pretreated with lipopolysaccharide with physiological saline demonstrated a 6-fold increase in phospholipase A2 activity in the perfusate compared with sham-treated controls, with only minor release of hepatic lipase. (5) Western-blot analysis confirmed an increased release of this Group-II phospholipase A2 into the perfusate of lipopolysaccharide-treated rats compared with sham-treated controls. These results suggest that liver Kupffer cells are a major source of the endotoxin-induced serum Group-II phospholipase A2 activity associated with bacterial infection and trauma.

Animals↗

PubMed1993

Evidence that binding of CTP:phosphocholine cytidylyltransferase to membranes in rat hepatocytes is modulated by the ratio of bilayer- to non-bilayer-forming lipids.

The mechanism by which phospholipase C (PLC) digestion of cultured cells mediates binding of CTP:phosphocholine cytidylyltransferase to cellular membranes was investigated. Incubation of choline-depleted rat hepatocytes with PLC caused a translocation of enzyme from cytosol to membranes concomitant with a decrease in the concentration of phosphatidylcholine with no effect on the concentration of other phospholipids. Removal of PLC and supplementation with choline restored the amount of phosphatidylcholine in the cells and translocated cytidylyltransferase to the cytosol. However, when phosphatidylcholine levels were decreased by incubation with phospholipase A2 (PLA2), there was no significant redistribution of cytidylyltransferase activity. With PLA2 the concentration of phosphatidylethanolamine, as well as of phosphatidylcholine, was significantly decreased. Since PLC, but not phospholipase A2, raised the cellular concentration of diacylglycerol, possibly diacylglycerol mediated the binding of cytidylyltransferase to membranes. This possibility was examined, but is unlikely, since addition of lysophosphatidylcholine to PLC-treated cells restored the concentration of phosphatidylcholine and released cytidylyltransferase into the cytosol, but did not lower diacylglycerol levels to normal values. Studies in vitro, incubations of cells with choline analogues and a survey of the literature suggested that the over-riding common factor in regulation of cytidylyltransferase binding to membranes may be the ratio of bilayer to non-bilayer lipids in that membrane.

Animals↗

PubMed1993

Involvement of the lysosome in the catabolism of intracellular lysophosphatidylcholine and evidence for distinct pools of lysophosphatidylcholine.

The role of the lysosome in the metabolism of lysophosphatidylcholine was investigated in isolated rat hepatocytes. Chloroquine, primaquine, and ammonium chloride caused a 2.5-fold increase in radioactive lysophosphatidylcholine in [methyl-3H]choline-labeled cells. This effect was confirmed by a 1.7-fold increase in lysophosphatidylcholine mass in chloroquine-treated hepatocytes. Chloroquine caused a 2.7-fold increase in radioactive lysophosphatidylethanolamine in [1-3H]ethanolamine-labeled cells and a 2.3-fold increase in radioactive lysophosphatidylcholine in [methyl-3H]methionine-labeled cells. Chloroquine did not affect formation of choline-containing aqueous metabolites or the level of radioactivity in phosphatidylcholine (PC). The effect of chloroquine on radioactive lysophosphatidylcholine accumulation was concentration-dependent and occurred within 10 min, consistent with rapid inhibition of lysosomal function. As there was no observed decrease in the 3H in PC, the accumulation of lysophosphatidylcholine was likely due to the inhibition of acid lysophospholipase activity in chloroquine-treated cells. The accumulation of lysophosphatidylcholine in the presence of chloroquine was observed in both short-term- (30 min) and equilibrium-(24 h) [methyl-3H]choline-labeled cells. Simultaneous incubation of hepatocytes with both albumin and chloroquine increased the radioactivity in lysophosphatidylcholine in the medium independently of the accumulation of radioactive lysophosphatidylcholine in the cells. The results suggest that there are separate pools of lysophosphatidylcholine in the hepatocyte and that the pool donated to an extracellular acceptor is different from the lysosomal pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗

PubMed1992

On the mechanism of the okadaic acid-induced inhibition of phosphatidylcholine biosynthesis in isolated rat hepatocytes.

The mechanism of inhibition of phosphatidylcholine biosynthesis by okadaic acid was investigated in suspension cultures of isolated rat hepatocytes. Cells were pulsed with [methyl-3H]choline and chased in the absence or presence of 1 microM okadaic acid for up to 120 min. Phosphatidylcholine biosynthesis was inhibited after 15 min of chase. To see if okadaic acid altered the degree of phosphorylation of cytidylyltransferase (CT), hepatocytes were incubated with 32P(i) and chased in the absence or presence of okadaic acid. Okadaic acid caused a rapid (within 15 min) increase in the phosphorylation state of the cytosolic enzyme. Two-dimensional peptide map analysis revealed an increase in the phosphorylation of several peptides in okadaic acid-treated hepatocytes compared with controls. After 15 min of incubation of hepatocytes with okadaic acid, membrane CT activity was decreased and a corresponding increase in cytosolic CT activity was observed. In hepatocytes incubated with okadaic acid and oleate a correlation between membrane CT activity, diacylglycerol level, and phosphatidylcholine biosynthesis was observed. These data suggest that the concentration of diacylglycerol is responsible for the increase in membrane CT activity and subsequently phosphatidylcholine biosynthesis in oleate-treated cells. We postulate that the okadaic acid-induced decrease in phosphatidylcholine biosynthesis is due to an increase in the phosphorylation state of CT which promotes a translocation of CT activity from the membranes to the cytosol.

Animals↗

PubMed1992

Stimulation of sphingomyelin biosynthesis by brefeldin A and sphingomyelin breakdown by okadaic acid treatment of rat hepatocytes.

Studies on sphingomyelin metabolism in rat hepatocytes were facilitated by the use of choline-deficient cells which allowed for the rapid labeling of phosphatidylcholine and as a result sphingomyelin. Pulse and pulse-chase studies with [methyl-3H]choline and [methyl-3H]methionine demonstrated that both compounds were effectively used for sphingomyelin biosynthesis and that newly made and pre-existing phosphatidylcholine could be used for sphingomyelin biosynthesis. When hepatocytes were incubated with brefeldin A, there was a 2.4-fold stimulation of the conversion of phosphatidylcholine into sphingomyelin. Since brefeldin A causes collapse of the cis/medial Golgi into the endoplasmic reticulum the stimulation of sphingomyelin biosynthesis could be due to more rapid access of the labeled phosphatidylcholine in the endoplasmic reticulum to sphingomyelin synthase in the collapsed Golgi. Forskolin inhibited the brefeldin A-induced stimulation of sphingomyelin biosynthesis. To investigate whether or not phosphorylation reactions regulate sphingomyelin metabolism, hepatocytes were incubated with okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A. Rather than stimulating sphingomyelin biosynthesis, okadaic acid enhanced the catabolism of sphingomyelin. In contrast, a cyclic AMP analogue and forskolin had no effect on sphingomyelin biosynthesis or catabolism. Surprisingly, other pulse-chase studies demonstrated that okadaic acid stimulated the catabolism of only newly made sphingomyelin. The brefeldin A and okadaic acid effects were independent of lysosomal involvement. Subcellular fractionation studies revealed that brefeldin A and okadaic acid effects were generalized in all sphingomyelin containing membranes. The brefeldin A studies suggest that the rate of transfer of phosphatidylcholine from the endoplasmic reticulum to the Golgi might be limiting for sphingomyelin biosynthesis. The okadaic acid studies indicate that the catabolism of sphingomyelin by a sphingomyelinase is regulated by an unidentified protein kinase and by either protein phosphatase 1 and/or 2A activity in hepatocytes.

Animals↗

PubMed1992

Effects of okadaic acid on the activities of two distinct phosphatidate phosphohydrolases in rat hepatocytes.

Incubation of hepatocytes with okadaic acid displaced the N-ethylmaleimide-sensitive phosphatidate phosphohydrolase from the membrane fraction into the cytosol and partially prevented the oleate-induced movement of phosphohydrolase from cytosol to membranes. However, higher concentrations of oleate still caused translocation and activation of the phosphohydrolase. This enzyme is stimulated by Mg2+, and is probably involved in glycerolipid synthesis. Okadaic acid also decreased the concentration of diacylglycerol within the hepatocytes. Okadiac acid had no observable effect on the activity of an N-ethylmaleimide-insensitive phosphatidate phosphohydrolase which remained firmly attached to membranes. This activity is not stimulated by Mg2+ and is probably involved in signal transduction by the phospholipase D pathway.

Animals↗

PubMed1991

Effects of premature weaning and diet on lung growth and appearance of adenylate cyclase activator in rat lung.

Early weaning of rat pups on day 16 to semi-ground Purina chow food and drinking water, ad libitum, delayed growth of body and lungs, and the appearance of adenylate cyclase activator (ACA) in lung after day 22. However, early weaning of pups to either milk or a gel diet containing semi-ground Purina chow food, agarose gel, water (30:1:69, w/w), and drinking water, restored lung and body growth and the appearance of ACA to control values. Early weaning of pups to dry semi-ground Purina chow food and drinking water also induced a transient rise in ACA on day 19. This early rise in ACA was completely absent in pups weaned on day 16 to milk, whereas it persisted in pups weaned similarly to a gel diet. Interestingly, lung glycogen decreased on day 19 in pups weaned early to dry semi-ground Purina chow food without (group 2) or with triiodothyronine administration (group 3), and on day 25 after normal weaning on day 22 (Nijjar, M.S. Biochim. Biophys. Acta 586: 464-472, 1979). These data indicate 1) that reduced food intake (starvation) in pups weaned on day 16 to dry semi-ground Purina chow food was responsible for the delayed growth of body and lung, and the delayed appearance of ACA in lung after day 22, and 2) that a change in diet from milk to Purina chow food and associated alterations in hormones, possibly cortisol and insulin, were responsible for the appearance of ACA in rat lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1991

The protein phosphatase inhibitor, okadaic acid, inhibits phosphatidylcholine biosynthesis in isolated rat hepatocytes.

There is evidence that phosphatidylcholine (PC) biosynthesis in hepatocytes is regulated by a phosphorylation-dephosphorylation mechanism. The phosphatases involved have not been identified. We, therefore, investigated the effect of okadaic acid, a potent protein phosphatase inhibitor, on PC biosynthesis via the CDP-choline pathway in suspension cultures of isolated rat hepatocytes. Okadaic acid caused a 15% decrease (P less than 0.05) in [Me-3H]choline uptake in continuous-pulse labeling experiments. After 120 min of treatment, the labeling of PC was decreased 46% (P less than 0.05) with a corresponding 20% increase (P less than 0.05) in labeling of phosphocholine. Cells were pulsed with [Me-3H]choline for 30 min and subsequently chased for up to 120 min with choline in the absence or presence of okadaic acid. The labeling of phosphocholine was increased 86% (P less than 0.05) and labeling of PC decreased 29% (P less than 0.05) by 120 min of chase in okadaic acid-treated hepatocytes. The decrease of label in PC was quantitatively accounted for in the phosphocholine fraction. Incubation of hepatocytes with both okadaic acid and CPT-cAMP did not produce an additive inhibition in labeling of PC. Choline kinase and cholinephosphotransferase activities were unaltered by treatment with okadaic acid. Hepatocytes were incubated with digitonin to cause release of cytosolic components. Cell ghost membrane cytidylyltransferase (CT) activity was decreased 37% (P less than 0.005) with a concomitant 33% increase (P less than 0.05) in released cytosolic cytidylyltransferase activity in okadaic acid-treated hepatocytes. We postulate that CT activity and PC biosynthesis are regulated by protein phosphatase activity in isolated rat hepatocytes.

Animals↗

PubMed1991

CPT-cAMP and okadaic acid enhance phosphatidylcholine catabolism in choline-deficient rat hepatocytes.

The effect of CPT-cAMP and okadaic acid on phosphatidylcholine catabolism in suspension cultures of choline-deficient rat hepatocytes was investigated. Choline-deficient hepatocytes were pulse-labeled for 30 min with [methyl-3H]choline and subsequently chased for up to 60 min with choline in the absence or presence of 0.5 mM CPT-cAMP or 0.5 microM okadaic acid. Radioactivity in phosphatidylcholine and lysophosphatidylcholine were unchanged during the chase. However, the radioactivity incorporated into glycerophosphocholine was significantly increased (P less than 0.05) 59 and 77% after 60 min of chase in hepatocytes incubated with either okadaic acid or CPT-cAMP, respectively. Incubation of choline-deficient hepatocytes with both okadaic acid and CPT-cAMP produced an additive effect on radioactivity incorporated ino glycerophosphocholine. Crude mitochondrial, microsomal, and cytosolic phospholipaselysophospholipase activities, assayed in the presence of exogenously labeled phosphatidylcholine, were unchanged in both CPT-cAMP and okadaic acid treated hepatocytes compared with control. Phospholipase-lysophospholipase activity, assayed with endogenously labeled phosphatidylcholine, was increased 28 and 47% (P less than 0.05) in the crude mitochondrial fraction of hepatocytes treated with either okadaic acid or CPT-cAMP, respectively, compared with the control. Incubation of choline-deficient hepatocytes, labeled with L-[methyl-3H]methionine, with CPT-cAMP or okadaic acid caused a 31 and 20% increase (P less than 0.05) in the radioactivity incorporated into glycerophosphocholine, respectively, compared with the control. We postulate that phosphatidylcholine catabolism in choline-deficient hepatocytes may be regulated by a phosphorylation-dephosphorylation mechanism mediated through cAMP-dependent protein kinase and phosphoprotein phosphatase activities.

Animals↗

PubMed1990

Effect of hypoxia on phosphatidylcholine biosynthesis in the isolated hamster heart.

In hamster heart, the majority of the phosphatidylcholine is synthesized via the CDP-choline pathway, and the rate-limiting step of this pathway is catalysed by CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15). We have shown previously [Choy (1982) J. Biol. Chem. 257, 10928-10933] that, in the myopathic heart, the level of cardiac CTP was diminished during the development of the disease. In order to maintain the level of CDP-choline, and consequently the rate of phosphatidylcholine biosynthesis, cardiac cytidylyltransferase activity was increased. However, it was not clear if the same compensatory mechanism would occur when the cardiac CTP level was decreased rapidly. In this study, hypoxia of the hamster heart was produced by perfusion with buffer saturated with 95% N2. The heart was pulse-labelled with radioactive choline and then chased with non-radioactive choline for various periods under hypoxic conditions. There was a severe decrease in ATP and CTP levels within 60 min of hypoxic perfusion, with a corresponding fall in the rate of phosphatidylcholine biosynthesis. Analysis of the choline-containing metabolites revealed that the lowered ATP level did not affect the phosphorylation of choline to phosphocholine, but the lower CTP level resulted in the decreased conversion of phosphocholine to CDP-choline. Determination of enzyme activities revealed that hypoxic treatment resulted in the enhanced translocation of cytidylyltransferase from the cytosolic to the microsomal form. This enhanced translocation was probably caused by the accumulation of fatty acids in the heart during hypoxia. We postulate that the enhancement of translocation of the cytidylyltransferase to the microsomal form (a more active form) is a mechanism by which the heart can compensate for the decrease in CTP level during hypoxia in order to maintain phosphatidylcholine biosynthesis.

Adenosine Triphosphate↗

PubMed1990

Effect of NaF and okadaic acid on the subcellular distribution of CTP: phosphocholine cytidylyltransferase activity in rat liver.

The effect of preincubation of rat liver post-mitochondrial supernatant with NaF and okadaic acid on the subcellular distribution of CTP: phosphocholine cytidylyltransferase activity was investigated. NaF (20 mM) inhibited the time-dependent activation of cytidylyltransferase activity in post-mitochondrial supernatant. Subcellular fractionation of the post-mitochondrial supernatant revealed that cytidylyltransferase activity in the microsomal fraction was decreased and activity in the cytosolic fraction increased with time of preincubation with NaF compared to controls. Okadaic acid is a specific and potent inhibitor of type 1 and 2A phosphoprotein phosphatases. Preincubation of cytosol with 5 microM okadaic acid inhibited the time-dependent activation of cytosolic cytidylyltransferase activity. Preincubation of post-mitochondrial supernatants with 5 microM okadaic acid inhibited the time-dependent activation of cytidylyltransferase activity by 13% at 45 min and 16% at 60 min of preincubation compared to controls. Microsomal cytidylyltransferase activity was decreased 27% at 45 min and 31% at 60 min with a corresponding retention of cytosolic cytidylyltransferase activity of 21% at 45 min and 37% at 60 min of preincubation with okadaic acid compared to controls. We postulate that the activity of the type 1 and/or type 2A phosphoprotein phosphatases affect the subcellular distribution of CTP: phosphocholine cytidylyltransferase activity in rat liver.

Alkaline Phosphatase↗

PubMed1988

Relationship between the cytoplasmic activator of adenylate cyclase and glycogen metabolism in rat lung.

The role of cytoplasmic activator of adenylate cyclase in rat lung metabolism was investigated. Mouse adrenal tumor (MAT) cells undergo differentiation in response to choleratoxin which acts through cyclic AMP. The activator of adenylate cyclase from rat lung also produced cyclic AMP in a disrupted MAT cell preparation. However, unlike choleratoxin, it did not induce MAT cell differentiation in whole cells. These results suggest impermeability of MAT cells, and possibly other cells, to the activator. Thus, means of altering activator activity in lung cytoplasm were sought, and changes in activator activity were related to lung glycogen. Adrenalectomy (ADX) in rats led to a reduction in activator activity that was accompanied by an elevation in lung glycogen. Dexamethasone treatment of adrenalectomized rats reversed both of these effects. Streptozotocin-induced diabetes in rats elevated activator activity and lowered lung glycogen. Insulin treatment of the diabetic rats restored activator activity to the normal control values. Preweaning of rats on day 16 instead of day 22 increased activator activity on the 19th day over the controls and there was a concomitant decrease in lung glycogen. Feeding the separated pups with homogenized milk restored glycogen and activator activity to the control values. These results indicate that activator activity in rat lung cytoplasm was dependent on the circulating levels of cortisol and insulin, and that there appeared to be an inverse relationship between activator activity and glycogen level in rat lungs.

Adenylyl Cyclases↗

PubMed1988

Effect of diethyl ether on phosphatidylcholine biosynthesis in hamster organs.

The effect of diethyl ether anesthesia on phosphatidylcholine biosynthesis in hamster organs was investigated. Ether administration did not affect the incorporation of radioactive choline into phosphatidylcholine in the liver, heart, lung, brain and spleen. A significant (29%) decrease in the labeling of phosphatidylcholine was detected in the kidney of ether-treated hamsters. Reduction in phosphatidylcholine labeling was not due to a diminished radioactive choline uptake but a decrease in the conversion of phosphocholine to CDP-choline. The accumulation of labeled phosphocholine was caused by the translocation of CTP:phosphocholine cytidylyltransferase from microsomal (more-active) form to cytosolic (less-active) form. Ether administration appears to modulate the cytidylyltransferase in hamster kidney differently than that in other hamster organs.

Animals↗