Spermine antagonises the effects of dexamethasone, glucagon and cyclic AMP in increasing the activity of phosphatidate phosphohydrolase in isolated rat hepatocytes

Rat hepatocytes were incubated in monolayer culture, under serum free conditions, for 8 h. Glucagon (10 nM), 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (100 microM) and dexamethasone (100 nM) increased the activity of phosphatidate phosphohydrolase by approx. 2-, 3.6- and 3.3-fold, respectively. Spermine alone had no significant effect. Spermine (2.5 mM) almost completely inhibited the glucagon induced increase in phosphohydrolase activity. It only partially inhibited the dexamethasone and cyclic AMP mediated inductions. Spermidine had no significant effect in this respect. The results are discussed in relation to the known effects of polyamines on glycerolipid synthesis, in particular, and on intermediary metabolism.     

Interactions of insulin, glucagon and dexamethasone in controlling the activity of glycerol phosphate acyltransferase and the activity and subcellular distribution of phosphatidate phosphohydrolase in cultured rat hepatocytes.

Rat hepatocytes were incubated in monolayer culture for 8 h. Glucagon (10nM) increased the total phosphatidate phosphohydrolase activity by 1.7-fold. This effect was abolished by adding cycloheximide, actinomycin D or 500 pM-insulin to the incubations. The glucagon-induced increase was synergistic with that produced by an optimum concentration of 100 nM-dexamethasone. Theophylline (1mM) potentiated the effect of glucagon, but it did not affect the dexamethasone-induced increase in the phosphohydrolase activity. The relative proportion of the phosphohydrolase activity associated with membranes was decreased by glucagon when 0.15 mM-oleate was added 15 min before the end of the incubations to translocate the phosphohydrolase from the cytosol. This glucagon effect was not seen at 0.5 mM-oleate. Since glucagon also increased the total phosphohydrolase activity, the membrane-associated activity was maintained at 0.15 mM-oleate and was increased at 0.5 mM-oleate. This activity at both oleate concentrations was also increased in incubations that contained dexamethasone, particularly in the presence of glucagon. Insulin increased the relative proportion of phosphatidate phosphohydrolase that was associated with membranes at 0.15 mM-oleate, but not at 0.5 mM-oleate. It also decreased the absolute phosphohydrolase activity on the membranes at both oleate concentrations in incubations that also contained glucagon and dexamethasone. None of the hormonal combinations significantly altered the total glycerol phosphate acyltransferase activity. However, glucagon significantly increased the microsomal activities, and insulin had the opposite effect. Glucagon also decreased the mitochondrial acyltransferase activity. There was a highly significant correlation between the total phosphatidate phosphohydrolase activity and the synthesis of neutral lipids from glycerol phosphate and 0.5 mM-oleate in homogenates of cells from all of the hormonal combinations. Phosphatidate phosphohydrolase activity is increased in the long term by glucocorticoids and also by glucagon through cyclic AMP. In the short term, glucagon increases the concentration of fatty acid required to translocate the cytosolic reservoir of activity to the membranes on which phosphatidate is synthesized. Insulin opposes the combined actions of glucagon and glucocorticoids. The long-term events explain the large increases in the phosphohydrolase activity that occur in vivo in a variety of stress conditions. The expression of this activity depends on increases in the net availability of fatty acids and their CoA esters in the liver.     

Haemocyanin mRNA from arthropod and mollusc origin. Evidence for a multi-unit structure in mollusc haemocyanin mRNA.

Hepatocytes were preincubated with 10mM-glucagon and 100 microM-corticosterone to increase phosphatidate phosphohydrolase activity. Addition of 10 nM-glucagon or 100 microM-8-bromo cyclic GMP to a second incubation mixture that contained cycloheximide increased the half-life of the phosphohydrolase activity. Dexamethasone (100 nM) had no significant effect, but insulin (500 pM) or spermine (1 mM) decreased the half-life. None of these compounds altered the general rate of degradation of proteins labelled with [3H]leucine. There appears to be a specific control of the half-life of phosphatidate phosphohydrolase activity, which could contribute to its long-term regulation in the liver.     

Effects of cyclic AMP, glucocorticoids and insulin on the activities of phosphatidate phosphohydrolase, tyrosine aminotransferase and glycerol kinase in isolated rat hepatocytes in relation to the control of triacylglycerol synthesis and gluconeogenesis.

Rat hepatocytes were incubated in monolayer culture in modified Leibovitz L-15 medium containing either 10% (v/v) newborn-calf serum or 0.2% (w/v) fatty-acid-poor bovine serum albumin. The addition of 100 nM-dexamethasone increased the activities of both phosphatidate phosphohydrolase and tyrosine aminotransferase by about 3.5-fold after 8h, and these activities continued to rise until at least 24h. Incubating the hepatocytes in the albumin-containing medium with 10 microM- or 100 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate increased the activities of the phosphohydrolase and aminotransferase by 2.6- and 3.4-fold respectively after 8h. These increases were blocked by actinomycin D. The increases in the activities that were produced by the cyclic AMP analogue and dexamethasone were independent and approximately additive. Insulin when added alone did not alter the phosphohydrolase activity, but it increased the aminotransferase activity by 34%. The dexamethasone-induced increase in the phosphohydrolase activity was completely blocked by 7-144 microM-insulin, whereas that of the aminotransferase was only partly suppressed. Insulin had no significant Effects on the increases in the activities of phosphatidate phosphohydrolase and tyrosine aminotransferase that were produced by the cyclic AMP analogue, but this may be because the analogue is fairly resistant to degradation by the phosphodiesterase. The activity of glycerol kinase was not significantly changed by incubating the hepatocytes with insulin, dexamethasone and the cyclic AMP analogue alone or in combinations. It is proposed that high concentrations of cyclic AMP and glucocorticoids increase the total activity of phosphatidate phosphohydrolase in the liver and provide it with an increased capacity for synthesizing triacylglycerols and very-low-density lipoproteins, which is expressed when the availability of fatty acids is high. There appears to be a co-ordinated hormonal control of triacyglycerol synthesis and gluconeogenesis in diabetes and in metabolic stress to enable the liver to supply other organs with energy.     

Growth factor-mediated regulation of aromatase activity in human skin fibroblasts.

We investigated the effects of various hormones and growth factors on aromatase activity in cultured human skin fibroblasts. Several potential trophic factors were tested for their ability to modify basal aromatase activity or the response to dibutyryladenosine 3',5'-cyclic monophosphate and dexamethasone because (i) no endogenous ligand has been identified that is responsible for stimulating aromatase activity in the periphery, and (ii) dexamethasone and cAMP analogs can increase this enzyme's activity in fibroblasts. The effect of insulin and insulin-like growth factors were examined in closer detail because of the clinical association between insulin and hyperandrogenism. Pituitary hormones and hypothalamic releasing factors, such as human ACTH (10 nM), beta-endorphin (10 nM), beta-lipotropin (10 nM), alpha-MSH (10 nM), gamma 3-MSH (10 nM), ovine luteinizing hormone (10 ng/ml), ovine follicle-stimulating hormone (10 ng/ml), ovine thyroid-stimulating hormone (10 ng/ml), rat growth hormone (10 ng/ml), rat prolactin (10 ng/ml), rat corticotropin-releasing factor (10 nM), luteinizing hormone-releasing factor (10 nM), thyrotropin-releasing factor (10 nM), human growth hormone-releasing factor (10 nM), and somatostatin (10 nM), have no significant effects on aromatase activity. Porcine inhibin A (10 ng/ml) and porcine activin AB (10 ng/ml), two ovarian hormones with structural transforming homology to transforming growth factor-beta, also have no effect on aromatase activity. Although basic fibroblast growth factor (1-100 ng/ml), acidic fibroblast growth factor (1 ng/ml), epidermal growth factor (1 ng/ml), platelet-derived growth factor (1 ng/ml), tumor necrosis factor (1 ng/ml), and transforming growth factor-beta 1 (1 ng/ml) have no effect on basal aromatase activity in human skin fibroblasts, all of these growth factors inhibited the ability of dibutyryladenosine 3',5'-cyclic monophosphate to stimulate aromatase activity. In contrast, both insulin (100 pg/ml-10 ng/ml) and insulin-like growth factor-1 (1-100 ng/ml) had no effect on cAMP-stimulated aromatase but potentiated the action of dexamethasone (100 nM). Thus, there is a clear distinction between the effects of dexamethasone and cAMP on peripheral aromatase. On the basis of the results presented here, it is interesting to speculate that the hyperandrogenism that is often associated with insulin resistance may be due to a combination of growth factor-mediated inhibition of aromatase activity and the failure of peripheral tissues to respond to insulin and metabolize androgens to estrogens.     

Membrane-Associated Phospholipase D Activity in Rat Sciatic Nerve

Rat sciatic nerve contains a membrane-bound phospholipase D that catalyzes the hydrolysis of exogenous phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. The enzyme is associated with a particulate fraction consisting primarily of microsomes and myelin. This fraction also contains phosphatidate phosphohydrolase activity leading to the production of diacylglycerols (DAG). The phosphohydrolase activity can be completely inhibited by NaF. Hydrolysis of exogenous PC requires detergent and is linear up to about 40 micrograms of protein at a pH optimum of 6.5. In the absence of NaF, the sum of PA and DAG increases linearly for 40 min, whereas in its presence, PA production is linear for only 15 min. At optimum conditions, PC hydrolysis proceeds at 15 nmol/h/mg of protein. Addition of increasing amounts of ethanol to the incubation system leads to the generation of increasing amounts of phosphatidylethanol, indicating transphosphatidylation activity. At an ethanol concentration of 0.4 M, phosphatidylethanol represents about one-half of the reaction products generated at approximately the same rate of enzymic activity observed in the absence of ethanol. Higher ethanol concentrations are inhibitory.     

The role of Mg2+-dependent phosphatidate phosphohydrolase in pulmonary glycerolipid biosynthesis

Rat lung microsomes washed with increasing concentrations of NaCl show a displacement of protein from microsomes to the wash supernatant. Among the proteins removed from the microsomal surface was the Mg2+-dependent phosphatidate phosphohydrolase, while the Mg2+-independent activity remained associated with the microsomes. The Mg2+-dependent activity could be quantitatively assayed in the wash supernatant. Microsomes washed with increasing concentrations of NaCl showed a progressive impairment in the synthesis of labelled neutral lipid and phosphatidylcholine from [14C]glycerol 3-phosphate with a concomitant increase in the labelling of phosphatidic acid. The impairment was sigmoidal and correlated highly with the decrease in Mg2+-dependent phosphatidate phosphohydrolase activity. When Mg2+-dependent phosphatidate phosphohydrolase from wash supernatant was incubated with microsomes previously washed with high salt concentrations, the labelling of neutral lipid and phosphatidylcholine was returned to control levels. Labelling of neutral lipids and phosphatidylcholine could be restored upon addition of a cytosolic Mg2+-dependent phosphatidate phosphohydrolase isolated by gel filtration. Mg2+-independent phosphatidate phosphohydrolase isolated from cytosol was incapable of restoring the labelling of neutral lipids and phosphatidylcholine. These findings confirm that the Mg2+-dependent phosphatidate phosphohydrolase of rat lung is involved in pulmonary glycerolipid biosynthesis. The role of the Mg2+-independent phosphatidate phosphohydrolase activity remains unknown.     

Gonadotropin-releasing hormone activates phospholipase D in ovarian granulosa cells. Possible role in signal transduction

We have investigated the stimulation of phospholipase D activity by the gonadotropin-releasing hormone receptor agonist [D-Ala6, des-Gly10]GnRH N-ethylamide (GnRH-A) in preovulatory, cultured granulosa cells. GnRH-A stimulated up to 10-fold accumulation of phosphatidylethanol, produced by phospholipase D phosphatidyl transferase activity when ethanol acts as the phosphatidyl group acceptor. The effect of GnRH-A was concentration dependent (EC50 = 1 nM) and was inhibited by a specific GnRH receptor antagonist. Low GnRH-A concentrations (less than 10 nM) stimulated also accumulation of phosphatidic acid, but at higher concentrations this response was attenuated. Propranolol, which inhibits phosphatidic acid phosphohydrolase, increased both basal and GnRH-A-stimulated production of phosphatidic acid. A protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM), increased up to 30-fold phosphatidylethanol levels. The effects of supramaximal concentrations of GnRH-A (50 nM) and TPA (1 microM) on the accumulation of phosphatidylethanol were additive, suggesting that the two agents may not act via the same mechanism. This is supported by the fact that 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the effect of TPA 50%, but not that of GnRH-A. However, 24 h pretreatment with TPA abolished cellular response to subsequent treatment with either TPA or GnRH-A. The stimulatory action of GnRH on steroidogenesis could be mimicked by elevating endogenous phosphatidic acid levels in granulosa cells. Exogenous phospholipase D (from Streptomyces chromofuscus, 10 IU/ml) significantly increased (2.7-fold) progesterone production by the cells; under the same conditions, GnRH-A and FSH stimulated progesterone production 3- and 2.6-fold, respectively. Similarly, propranolol stimulated progesterone production 2.2-fold. These results suggest that, in granulosa cells, GnRH receptors are coupled to a phospholipase D whose activation may participate in transducing the GnRH signal for accelerated steroidogenesis. Phospholipase D activity can be independently regulated also by protein kinase C. The possible interrelationships between phospholipase D and other phospholipases which may be activated by GnRH in these ovarian cells are discussed.     

Some characteristics of sn-glycero-3-phosphocholine diesterases from rat brain.

1. sn-Glycero-3-phosphocholine diesterase activities, glycerophosphohydrolase (EC 3.1.4.2) and choline phosphohydrolase (EC 3.1.4.38) from rat brain have been partially purified and characterized using sn-glycere-3-[32P]phosphocholine as substrate and separating the reaction products by anion-exchange chromatography and ionophoresis. 2. Rat brain contained particulate (75%) and soluble (25%) activity from both diesterases. No difference in pH optimum or metal ion requirement for the particulate compared to the soluble enzymes was observed. 3. Glycerophosphohydrolase (EC 3.1.4.2) was purified 60-fold, choline phosphohydrolase (EC E.1.4.38) 120-fold from rat brain supernatant fraction by DEAE-cellulose ion-exchange chromatography and sucrose density gradient centrifugation. The density gradient results in conjunction with dodecyl sulphate-polyacrylamide gel disc electrophoresis yielded molecular weight estimates of 230 000 (monomer 62 000) for choline phosphohydrolase and 120 000 (monomer 70 000) for glycerophosphohydrolase (EC 3.1.4.2). 4. Glycerophosphohydrolase (EC 3.1.4.2) has a pH optimum of 8.9 and a Km for sn-glycero-3-phosphocholine of 0.6 mM. The enzyme is inhibited by EDTA and reactivated by Ca2+. Choline phosphohydrolase (EC 3.1.4.38) has pH optimum 10.5, a Km of 2 mM and is unaffected by EDTA. Both enzymes require Ca2+ for maximum activity.     

Presence of membrane-associated phosphatidate phosphohydrolase activity in cultured islets and its stimulation by glucose

The cellular location at which exogenous phosphatidic acid is hydrolysed in cultured neonatal rat islets was examined. Phosphatidate phosphohydrolase activity could be demonstrated in both whole cell sonicates and isolated plasma membranes. In the whole cell fraction phosphatidic acid hydrolysis to diacylglycerol was stimulated 43% by the presence of Mg2+. The activity present in isolated membranes was totally dependent on the presence of Mg2+ and was increased in plasma membranes from glucose-stimulated islets. Following exposure of islets to low glucose concentrations, raising the Ca2+ concentration from 150 nM to 40 microM in the presence of Mg2+ did not affect the formation of diacylglycerol in whole cell fractions or plasma membranes. These results indicate the presence within the islet of membrane-bound phosphatidate phosphohydrolase activity and demonstrate its activation by glucose.     

Pulmonary phosphatidic acid phosphohydrolase: further studies on the activities in rat lung responsible for the hydrolysis of membrane-bound and aqueously dispersed phosphatidate

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5-10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10-20 min), the rates were comparable.     

Cycloheximide blocks insulin-like growth factor I but not somatostatin inhibition of growth hormone secretion

Growth hormone secretion is controlled by the two hypothalamic hormones, growth hormone releasing factor (GRF) and somatostatin. In addition, the insulin-like growth factors (IGF or somatomedins) which are themselves growth hormone dependent, inhibit growth hormone release in vitro, therefore acting to close the negative feedback loop. The studies reported here examine some of the differences between inhibition of growth hormone secretion by somatostatin and IGF-I in vitro. The major finding is that cycloheximide, a protein synthesis inhibitor, blocks inhibition of GRF-stimulated growth hormone release caused by IGF-I, without changing the inhibition caused by somatostatin. The experiments were done by exposing mixed rat adenohypophysial cells to secretagogues with or without cycloheximide for 24 h in a short term culture. Somatostatin (0.6 nM) totally blocked rat GRF (1 nM) stimulated growth hormone release to values 48% of control (nonstimulated values), while IGF-I (27 nM) only reduced the GRF-stimulated growth hormone release by 27 +/- 3% (N = 5). Cycloheximide (15 micrograms/mL) totally blocked the effect of IGF-I but not somatostatin. A low concentration (0.12 nM) of somatostatin, which only partly inhibited growth hormone release, was also unaffected by cycloheximide. In purified rat somatotrophs, somatostatin (0.1 nM) inhibited GRF-stimulated cAMP levels slightly and reduced growth hormone release while IGF-I (40 nM) had no effect. We suggest that IGF-I inhibits only the secretion of newly synthesized growth hormone, while somatostatin inhibits both stored and newly synthesized growth hormone pools.     

Ghrelin stimulates insulin-induced glucose uptake in adipocytes

The gastric and hypothalamic hormone ghrelin is the endogenous agonist of the growth hormone secretagogue receptor GHS-R1(a). Ghrelin stimulates growth hormone release and appetite via the hypothalamus. However, putative direct peripheral effects of ghrelin remain poorly understood. Rat adipose tissue expresses GHS-R1(a) mRNA, suggesting ghrelin may directly influence adipocyte function. We have investigated the effects of ghrelin on insulin-stimulated glucose uptake in isolated white adipocytes in vitro. RT-PCR confirmed the expression of GHS-R1(a) mRNA in epididymal adipose tissue. However, GHS-R1(a) expression was not detected in the peri-renal fat pads. Ghrelin increased insulin-stimulated deoxyglucose uptake in isolated white adipocytes extracted from the epididymal fat pads of male Wistar rats. Ghrelin 1000 nM significantly increased deoxyglucose uptake by 55% in the presence of 0.1 nM insulin. However, ghrelin administration in the absence of insulin had no effect on adipocyte deoxyglucose uptake, suggesting that ghrelin acts synergistically with insulin. Des-acyl ghrelin, a major circulating non-octanylated form of ghrelin, had no effect on insulin-stimulated glucose uptake. Furthermore, acylated ghrelin had no effect on deoxyglucose uptake in adipocytes from peri-renal fat pads suggesting that ghrelin may influence glucose uptake via the GHS-R1(a). Ghrelin therefore appears to directly potentiate adipocyte insulin-stimulated glucose uptake in selective adipocyte populations. Ghrelin may play a role in adipocyte regulation of glucose homeostasis.     

Prolactin-like hormone in the nematode Trichinella spiralis larvae

Expression of prolactin (PRL) or prolactin-like hormone has been reported in invertebrates. We investigated the larval phase of Trichinella spiralis: (a) to express 23 kDa PRL, (b) to define its localization and (c) to test its possible biological activity. Immunostaining in isolated larvae demonstrated positive material to 23 kDa PRL by all along the stichosome, specifically in the stichocytes. Homogenized immunoblot larvae showed a 23 kDa protein band. To assess PRL release and its biological activity, larvae were incubated in culture medium and the excretory/secretory products were analyzed by the Nb2 cells bioassay. A cellular growth equivalent until 10 nM PRL and using antibody against 23 kDa PRL, the growth was blocked. In conclusion our result provides evidence that PRL-like hormone is expressed and secreted by the larvae of T. spiralis.     

Rat ghrelin stimulates growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

Recently, ghrelin (Ghr), a new peptide which specifically stimulates growth hormone (GH) release from the pituitary, was identified in the rat and human stomach. Ghrelin has been shown to stimulate GH release by acting through a growth hormone secretagogue (GHS) receptor in the rat. The present study describes the in vitro effect of rat Ghr on the release of GH and two forms of prolactin (PRL(177) and PRL(188)) in the tilapia, Oreochromis mossambicus. Rat Ghr stimulated the release of GH in a dose-related manner after 8 and 24 hr of incubation. Rat Ghr also significantly stimulated the release of PRL(177) and PRL(188) in a dose-related manner after 24 hr. Rat Ghr had no effect on the pituitary content of GH or PRL(188), but significantly increased PRL(177) content. These results show for the first time that rat Ghr significantly stimulates GH and PRL release in teleosts, and suggest that Ghr and a GHS receptor are present in fish.     

Alkaline phosphatase activity in cultured urinary bladder cancer cells.

Established cell lines derived from human urinary bladder carcinomas produce heat-stable alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] which resembles the oncofetal enzyme of HeLa S3. Rat bladder cancer cell lines derived from chemically induced tumors produce heat-labile alkaline phosphatase. Corticosteroids and/or hyperosmolality do not influence the enzyme of rodent cells, but induce increased levels of activity in human cells. The increase is most pronounced when human cells multiply in hyperosmolar medium containing prednisolone. Under these conditions a rise of over 100-fold in specific activity is noted. This synergistic effect, not seen with other cultured heteroploid cells, may represent a specific characteristic of cells derived from human bladder tumors.     

Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol

Assays for two distinct phosphatidate phosphohydrolase activities were established based upon a differential inhibition by N-ethylmaleimide (NEM). The activity that is insensitive to this reagent in rat liver is predominantly in the plasma membrane fraction, whereas the NEM-sensitive activity is in the cytosolic and microsomal fractions. The NEM-insensitive activity is further distinguished from the NEM-sensitive phosphohydrolase by: (a) being relatively stable to heat; (b) not being inhibited by phenylglyoxal, butane-2,3-dione, cyclohexane-1,2-dione, 2,4-dinitrofluorobenzene, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and diethyl pyrocarbonate; (c) being inhibited by NaF and phosphatidylcholine; and (d) not being stimulated by Mg2+. The NEM-insensitive activity was specific for phosphatidate. Both phosphohydrolase activities could be inhibited by chlorpromazine, propranolol, sphingosine, and spermine. The NEM-sensitive phosphatidate phosphohydrolase activity was increased by incubating hepatocytes for 12 h with glucagon and dexamethasone, and this effect was antagonized by insulin. The NEM-sensitive phosphohydrolase is concluded to be involved in glycerolipid synthesis. The activity of the NEM-insensitive phosphohydrolase was not altered by preincubation of rat hepatocytes in the short or long term with vasopressin, glucagon, insulin, triiodothyronine, or dexamethasone, but it might be modulated indirectly by sphingosine. The NEM-insensitive enzyme of the plasma membranes could be involved in signal transduction via the agonist-stimulated degradation of phosphatidylcholine through the phospholipase D pathway.     

Pulmonary surfactant synthesis. A highly active microsomal phosphatidate phosphohydrolase in the lung.

Lung cell-free homogenate, which contains about twice the units of phosphatidate phosphohydrolase per mg of protein compared to liver, was fractionated by differential centrifugation and the fractions were assayed for phosphatidate phosphohydrolase and marker enzymes of endoplasmic reticulum, mitochondria, and lysosomes. Over 60% of the lung phosphatidate phosphohydrolase was associated with the endoplasmic reticulum, compared to 50% of the total liver enzyme. Thus a major portion of the more active lung enzyme is potentially involved in lipid biosynthesis by the endoplasmic reticulum. Less than 0.2% of the total lung enzyme was found in a lamellar body fraction, consistent with previous findings. The lung microsomal phosphohydrolase was specific for lipid substrates, showing equal activity towards phosphatidic acid or lysophosphatidic acid and relatively low activities towards glycerophosphates. It had a neutral pH optimum, similar to the liver enzyme, but differed somewhat in its relative activity at extremes of pH. Stability at 65 degrees C was greater for the lung enzyme. Fluroide inhibited lung (or liver) microsomal phosphatidate phosphohydrolase, while tartrate, MgCl2, or EDTA had no effect. The presence of a high activity of phosphatidate phosphohydrolase in lung endoplasmic reticulum is consistent with the rapid synthesis of pulmonary surfactant phosphatidylcholine.     

Studies on the interactions between phospholipids and membrane-bound enzymes in microsomes. Effects of phospholipases C on the glucose-6-phosphatase system of rat liver microsomes

The role of phospholipids in the glucose-6-phosphatase system, including glucose-6-P phosphohydrolase and glucose-6-P translocase, was studied in rat liver microsomes by using phospholipases C and detergents. In the time course experiments on detergent exposure, the maximal activation of glucose-6-P phosphohydrolase varied according to the nature of the detergent used. On treatment of microsomes with phospholipase C of C. perfringens, the activity of glucose-6-P phosphohydrolase without detergent (i.e. without rupture of translocase activity) was gradually decreased with the progressive hydrolysis of phosphatidylcholine and phosphatidylethanolamine on the microsomal membrane, and was restored by incubation of these microsomes with egg yolk phospholipids. The extent of decrease in this phosphohydrolase activity in the detergent-exposed microsomes (with rupture of translocase activity) also varied depending on the detergent used (Triton X-114 or taurocholate). When 66% of the phosphatidylinositol on the membrane was hydrolyzed by phosphatidylinositol-specific phospholipase C of B. thuringiensis, the inhibition of glucose-6-P phosphohydrolase activity without detergent was very small. Although the inhibition of enzyme activity with detergent was apparently greater than that without detergent, the enzyme activity was stimulated by the breakdown of phosphatidylinositol when the enzyme activity was measured at lower concentration (0.5 mM) of substrate, glucose-6-P. The latency of mannose-6-P phosphohydrolase, a plausible index of microsomal integrity, remained above 70% after the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol. The results show that the glucose-6-phosphatase system requires microsomal phospholipids for its integrity, suggesting that there exists a close relation between phosphatidylinositol and glucose-6-P translocase.     

Oleic acid promotes the activation and translocation of phosphatidate phosphohydrolase from the cytosol to particulate fractions of isolated rat hepatocytes

The incubation of hepatocytes with 1-4mM-oleate increased the total activity of phosphatidate phosphohydrolase that was measured in the presence of Mg2+ to about 2-fold. This was accompanied by an increase in the proportion of the enzyme that was isolated with the particulate fractions. Conversely, the addition of up to 4mM-oleate decreased the recovery of phosphatidate phosphohydrolase in the cytosolic fraction from about 70% to 3% when hepatocytes were lysed with digitonin. Most of the increase in the membrane-associated phosphohydrolase activity was isolated after cell fractionation in the microsomal fraction that was enriched with the endoplasmic-reticulum marker arylesterase. It is proposed that the translocation of phosphatidate phosphohydrolase facilitates the increased synthesis of triacylglycerols in the liver when it is presented with an increased supply of fatty acids.