Stimulation by local anesthetics of the metabolism of acidic phospholipids in the rat pineal gland

The efficacy of five local anesthetics in causing stimulation of phospholipid metabolism in rat pineal gland $\text{in vitro}$ paralleled their anesthetic potency and decreased in the order: dibucaine, tetracaine, cocaine, procaine, lidocaine. When stimulation occurred, the patterns of labeling resembled that produced by propranolol, a β-adrenergic receptor blocking agent with local anesthetic activity. Isotope incorporation into phosphatidylglycerol and CDP-diglyceride was markedly enhanced and increases of labeling of phosphatidic acid and phosphatidylinositol were also seen. At concentrations of 1–10 mM, propranolol and local anesthetics inhibited labeling of phosphatidylcholine and phosphatidylethanolamine by more than 90% and incorporation of ³²P_i into other phospholipids to a smaller extent.     

Synthesis of biologically active spin-labelled radioactive cytidine diphosphodiglyceride, a novel probe for biological membranes.

A versatile synthesis of spin-labelled radioactive cytidine diphospho-sn-1,2-diacylglycerol (CDP-diglyceride) has been developed based on the combination of the enzymatic acylation of radioactive sn-glycero-3-phosphate with 12-doxyl stearic acid and the chemical conversion of the thus obtained spin-labelled radioactive phosphatidic acid with cytidine monophosphomorpholi-date into spin-labelled radioactive CDP-diglyceride. The method for the isolation and purification of the latter compound was described. This obtained CDP-[2-3H]diglyceride contained 10% of fatty acids of paramagnetic nature, presumably present as a covalently bound 12-doxyl stearic acid esters. The biological activity was tested by using the synthesized compound as a substrate in the mitochondrial biosynthesis of phosphatidylglycerol. It was found that spin-labelled CDP-[2-3H]diglyceride prepared as described can be converted in the presence of sn-[2-14C]-glycero-3-phosphate into a spin-labelled [2-3H, 2'-14C]phosphatidylglycerol with isolated rat liver mitochondria, establishing therefore that the site of its utilization is identical with the site of phosphatidylglycerol synthesis in isolated mitochondria, i.e. inner mitochondrial membrane. Results described demonstrate that the synthesized spin-labelled CDP-diglyceride can be used as a specific probe for the spin- and radioactive covalent labelling of polyglycerophosphatides of mitochondrial membranes. Some implications and further possibilities in the study of biological membranes using the spin-labelled radioactive CDP-diglyceride are discussed.     

Inhibition by gamma-hexachlorocyclohexane of acetylcholine-stimulated phosphatidylinositol synthesis in cerebral cortex slices and of phosphatidic acid-inositol transferase in cerebral cortex particulate fractions

• 1 γ-Hexachlorocyclohexane inhibits the ACh-stimulated synthesis of phosphatidylinositol in guinea pig cerebral cortex slices, as measured either by the incorporation of [2-³H]inositol or of ³²P. Phosphatidylinositol synthesis in the control slices is not inhibited.
• 2 The synthesis of phosphatidylinositol from CDP-diglyceride in cerebral cortex microsomal preparations is inhibited by γ-hexachlorocyclohexane. The incorporation of [2-³H]inositol into lipid in the absence of added cytidine nucleotide in these preparations is not inhibited.
• 3 δ-Hexachlorocyclohexane profoundly inhibits phosphatide synthesis and phosphate metabolism in cerebral cortex slices both in the presence and absence of ACh. This isomer also inhibits the exchange reaction for the incorporation of [2-³H]inositol into lipid in the microsomal preparations.
• 4 α-, and β-Hexachlorocyclohexanes do not inhibit either ACh-stimulated or control synthesis of phosphatidylinositol in cerebral cortex slices; nor do they inhibit the exchange reaction for [2-³H]inositol incorporation into lipid in the microsomal preparations.
• 5 The specific effects of γ-hexachlorocyclohexane are taken as providing evidence that ACh-stimulated phosphatidylinositol synthesis in cerebral cortex slices probably involves the CDP-diglyceride pathway. The possibility is discussed that the primary action of ACh in this system is to cause an increased activity of diglyceride kinase to provide phosphatidic acid for this pathway.

     

Effects of luteinizing hormone on phosphoinositide metabolism in rat granulosa cells

Rat granulosa cells isolated from mature Graafian follicles were incubated with luteinizing hormone under various conditions in order to follow the synthesis and degradation of phospholipids. During acute incubations, luteinizing hormone provoked rapid and concentration-dependent increases in the incorporation of 32PO4 into phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides. Similarly, luteinizing hormone provoked increases in labeling of phosphatidylinositol and the polyphosphoinositides when granulosa cells were incubated with myo-[2-3H]inositol. When granulosa cells were prelabeled with 32PO4 in order to label phosphatidylinositol to constant specific radioactivity (4 h), luteinizing hormone treatment significantly increased 32P-phosphatidylinositol levels (23%). Comparable increases (27%) in the cellular concentrations of phosphatidylinositol were observed in response to luteinizing hormone. In pulse-chase experiments employing 32PO4 - or [3H]inositol-prelabeled cells, luteinizing hormone did not alter phospholipid degradation. In addition, luteinizing hormone did not stimulate degradation of polyphosphoinositides. These results demonstrate that: (a) luteinizing hormone has selective effects on phospholipid metabolism in rat granulosa cells which involve phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides, (b) luteinizing hormone increases net levels of phosphatidylinositol and presumably phosphatidic acid and the polyphosphoinositides, and (c) luteinizing hormone does not increase phospholipid degradation. Our findings suggest that luteinizing hormone provokes increases in de novo synthesis of phosphatidylinositol in rat granulosa cells. These changes in phospholipid metabolism may be important for steroidogenesis and other enzymatic processes during treatment with luteinizing hormone.     

The metabolism of phosphatidylinositol in the thyroid gland of the pig

1. The metabolism of phosphatidylinositol in pig thyroid has been investigated as a basis for understanding the specific stimulation of the synthesis of this phospholipid in the gland by thyrotropin. 2. The gland contained an active Ca(2+)-dependent phosphatidylinositol-splitting enzyme with an optimum pH of 5.3-5.5. 3. The major water-soluble product (65%) formed by this catabolic enzyme was not phosphorylinositol but a related compound, which may be a cyclic phosphorylinositol. Both this and phosphorylinositol (35%) were released simultaneously from the phosphatidylinositol substrate. 4. The phosphatidylinositol-splitting enzyme was found almost exclusively in the supernatant fraction obtained by homogenization of the gland. It was not present in the acid-phosphatase-containing particulate fraction. 5. The incorporation of [2-(3)H(1)]inositol into phosphatidylinositol in the presence of either CDP-diglyceride or CTP+ATP was most active in the microsomal fraction. 6. When thyroidal microsomes were labelled with [(3)H]inositol and (32)P, and then incubated with unlabelled inositol, there was a dramatic loss of (3)H labelling from the phosphatidylinositol, which was not accompanied by an equivalent loss of (32)P from the phosphate moiety. This turnover of the inositol moiety required nucleotide coenzymes. It is postulated that the phosphatidylinositol is split into inositol and a phosphorus-containing lipid precursor of the phospholipid that remains on the microsomal membrane and is recycled. 7. Isolated thyroidal mitochondria synthesized phosphatidylinositol from [2-(3)H(1)]inositol only because of their contaminating microsomal component. 8. Some evidence has been obtained of a rapid transfer of phosphatidylinositol molecules from thyroidal microsomes to mitochondria when these were incubated together in the presence of a supernatant fraction. 9. Both phosphatidylinositol breakdown by the supernatant fraction of the gland and synthesis by the microsomes were totally inhibited by 1mm-chlorpromazine. This drug is known to suppress thyrotrophin-induced stimulation of activity in thyroid slices.     

The Influence of Divalent Cations and Substrate Concentration on the Incorporation of Myo-inositol into Phospholipids of Isolated Bovine Oligodendrocytes

The incorporation of myo-inositol into phosphatidylinositol by two routes (CTP-independent and CTP-independent) has been investigated in homogenates prepared from isolated bovine oligodendrocyte perikarya. The CTP-dependent route has the higher maximum velocity of inositol incorporation and can utilise either Mn2+ or Mg2+ as a divalent ion cofactor. This route of inositol incorporation is also strongly inhibited by Ca2+ ions at concentrations less than 1 mM. The primary site of the inhibitory action appears to be the enzyme CDP-diglyceride inositol phosphatidyl transferase (EC 2.7.8.11) though synthesis of CDP-diacylglycerol is also inhibited by endogenous Ca2+ present in the oligodendrocyte homogenate. In contrast, CTP-independent inositol incorporation into phosphatidylinositol is only stimulated by Mn2+ (Zn2+,Cu2+, Mg2+, Ca2+ and Co2+ are ineffective) and is not inhibited by Ca2+, at least up to a concentration of 1 mM.     

Further studies on the formation of cardiolipin and phosphatidylglycerol in rat liver mitochondria. Effect of divalent cations and the fatty acid composition of CDP-diglyceride.

The divalent cation requirement for mitochondrial cardiolipin biosynthesis has been further investigated. The relative order of divalent cation activity was Co-2+ greater than Mn-2+ greater than Mg-2+. Cardiolipin was not formed in the incubations with Zn-2+, Fe-2+, Cu-2+, Hg-2+, and Ca-2+. Cardiolipin synthesis in the presence of optimal cincentration of Co-2+ was inhibited by Ca-2+. A series of CDP-diglycerides was synthesized having differences in fatty acid chain lenth and degree of unsaturation. These compounds were tested in mitochondrial cardiolipin and phosphatidylglycerol synthesis. Although there were some minor differences between phosphatidylglycerol and cardiolipin synthesis, in general, saturated shorter chain CDP-diglycerides (dilauroyl and dimyristoyl) were better substrates than the longer chain dipalmitoyl and distearoyl homologues. Introduction of double bonds into distearoyl CDP-diglyceride resulted in more rapid rates of synthesis (e.g. dioleoyl and dilinoleoyl CDP-diglyceride). Significance of the results is dicussed with regard to possible mechanisms of linoleic acid incorporation into rat liver cardiolipin.     

Insulin-stimulated phosphoinositide metabolism in isolated fat cells

Treatment of isolated fat cells with insulin produced increases of up to 4.8-fold in the incorporation of [3H]inositol into phosphatidylinositol. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 30 microunits/ml of insulin. Insulin increased the labeling of phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but not phosphatidylinositol 4-monophosphate in cells which had been preincubated with [3H]inositol for 90 min. Incubation of the cells in a Ca2+-free buffer increased the basal level of phosphatidylinositol labeling and enhanced the effect of insulin. Glucagon and isoprenaline, both of which stimulate lipolysis, had no effect on phosphatidylinositol labeling but did potentiate insulin-stimulated incorporation of [3H]inositol into phosphatidylinositol. Phosphoinositide breakdown was measured by the accumulation of inositol phosphates. Insulin did not increase the level of the inositol phosphates at all concentrations of the hormone tested. By comparison, phenylephrine and vasopressin were able to stimulate phosphoinositide breakdown. Pretreatment of the cells with insulin enhanced the effect of phenylephrine on inositol phosphates' accumulation, suggesting that insulin may potentiate phenylephrine-mediated phosphoinositide turnover. From these data we conclude that insulin stimulates the de novo synthesis of phosphatidylinositol and phosphatidylinositol 4,5-biphosphate, but has no effect on phosphoinositide breakdown.     

Phospholipid metabolism and lysosomal enzyme secretion by leukocytes. Effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP.

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

Affinity of phosphatidylglycerophosphate synthetase for the liponucleotides in Bacillus subtilis

Abstract In Bacillus subtilis , the synthesis of phosphatidylglycerol, the more reactive phospholipid, was investigated in vitro. The phosphatidylglycerophosphate synthetase was exclusively localized in the membrane fraction. Three phospholipids (phosphatidylglycerophosphate, phosphatidylglycerol and diphosphatidylglycerol) were synthesized by this fraction. Exogenous CDP-diglyceride and dCDP-diglyceride were substrates with the same K _m (0.11 mM). In coupled system with CDP-diglyceride synthetase, endogenous dCDP-diglyceride was a less effective substrate than CDP-diglyceride.     

Phosphatidylinositol synthesis in castor bean endosperm: cytidine diphosphate diglyceride:inositol transferase

CDP-diglyceride:inositol transferase in endoplasmic reticulum fractions from castor bean (Ricinus communis) endosperm was partially characterized. The enzyme had a pH optimum of 8.5 and required Mn²⁺ for activity. Maximal activity was at 1.5 millimolar MnCl₂. A K_m of 0.30 mM was calculated for myo-inositol and 1.35 millimolar was estimated for CDP-dipalmitoylglyceride. Concentrations of CDP-dipalmitoylglyceride above 1.2 millimolar inhibited the enzyme. A deoxycholate concentration of 0.1% (w/v) stimulated the reaction slightly while Triton X-100 inhibited at all concentrations tested. Some incorporation of myo-inositol into phosphatidylinositol occurred in the absence of CDP-diglyceride.     

In vivo metabolic intermediates of phospholipid biosynthesis in Rhodopseudomonas sphaeroides

The in vivo metabolic pathways of phospholipid biosynthesis in Rhodopseudomonas sphaeroides have been investigated. Rapid pulse-chase-labeling studies indicated that phosphatidylethanolamine and phosphatidylglycerol were synthesized as in other eubacteria. The labeling pattern observed for N-acylphosphatidylserine (NAPS) was inconsistent with the synthesis of this phospholipid occurring by direct acylation of phosphatidylserine (PS). Rather, NAPS appeared to be kinetically derived from an earlier intermediate such as phosphatidic acid or more likely CDP-diglyceride. Tris-induced NAPS accumulation specifically reduced the synthesis of PS. Treatment of cells with a bacteriostatic concentration of hydroxylamine (10 mM) greatly reduced total cellular phospholipid synthesis, resulted in accumulation of PS, and stimulated the phosphatidylglycerol branch of phospholipid metabolism relative to the PS branch of the pathway. When the cells were treated with a lower hydroxylamine dosage (50 microM), total phospholipid synthesis lagged as PS accumulated, however, phospholipid synthesis resumed coincident with a reversal of PS accumulation. Hydroxylamine alone was not sufficient to promote NAPS accumulation but this compound allowed continued NAPS accumulation when cells were grown in medium containing Tris. The significance of these observations is discussed in terms of NAPS biosynthesis being representative of a previously undescribed branch of the phospholipid biosynthetic sequence.     

Phosphatidyl glycerolphosphate serves as glycerolphosphate donor in polymer synthesis

Phosphatidyl glycerolphosphate was found to serve as the glycerolphosphate donor for polymer synthesis. When CDP-diglyceride and radiolabeled glycerolphosphate were incubated with the membrane enzyme prepared from Streptococcus sanguis, active syntheses of radiolabeled lipids and polymers were observed. The synthesis of polymer was not inhibited by low concentration of unlabeled phosphatidylglycerol. When [3H, 32P]glycerolphosphate was used, the polymer synthesized contained both 3H and 32P. The lipids formed were characterized as phosphatidylglycerol and phosphatidyl glycerolphosphate. The polymers formed from the latter were characterized as lipoteichoic acid like compounds by sodium dodecylsulfate-polyacrylamide gel electrophoresis.     

Differential effect of progesterone on the labeling of phosphatidylinositol with [3H]inositol and [32P]phosphate in the uterus of the estrogen-treated ovariectomized rat

In rat uterine mince incubated in vitro [3H]inositol was found to be incorporated into phosphatidylinositol (PI) predominantly via a pathway which could be markedly and dose dependently activated with Mn2+ (0.1-10 mM) and inhibited by Ca2+ (1-10 mM). These ions had no effect on the incorporation of [32P]phosphate (32P) into PI indicating a distinct inositol-exchange mechanism for the labeling of PI with [3H]inositol. Treatment of ovariectomized rats for 5 days with 2 micrograms estradiol dipropionate (EDP) increased about 3-fold (when measured in the presence of 1 mM Mn2+) and 4-5-fold (when measured in the presence of 1 mM Ca2+) the inositol-exchange activity in the rat uterus, and these effects were suppressed by 40 and 30% respectively by the concomitant administration of 2 mg progesterone (P). EDP alone or in combination with P increased to the same extent (by a factor of 2-3) the rate of labeling with 32P of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and plasmenylethanolamine (PmE). The labeling rate of PI was increased 1.5-1.7-fold by treatment with EDP and this increase was selectively augmented further to about 2.5-fold by the simultaneous administration of P. Treatment with P alone had no significant effect on the incorporation of either labeled precursor. Steroid hormone treatments had no effect on the amount of these phospholipids in 100 mg uterine tissue, but they increased about 1.7-fold the rate of labeling of ATP with 32P. We conclude that P, when administered together with estradiol, regulates differentially the turnover of the inositol and phosphate moieties of PI with possible physiological consequences.     

Intracellular distribution of enzymes of phospholipid metabolism in several gram-negative bacteria.

Cell-free extracts of Salmonella typhimurium, Serratia marcescens, Enterobacter aerogenes, and Micrococcus cerificans contained the following enzymatic activities related to phospholipid metabolism: cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride):l-serine O-phosphatidyltransferase (phosphatidylserine synthase), phosphatidylserine decarboxylase, CDP-diglyceride:sn-glycero-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase), phosphatidylglycerophosphate phosphatase, and CDP-diglyceride hydrolase. The intracellular distribution of these enzymatic activities as determined by sucrose density gradient centrifugation of cell-free extracts was shown to be similar in each species investigated. The phosphatidylserine decarboxylase, phosphatidylglycerophosphate synthase, and CDP-diglyceride hydrolase activities were all associated with the cell envelope fraction, whereas the phosphatidylserine synthase activity was associated mainly with the ribosomal fraction. These enzymatic activities are comparable and have an intracellular distribution similar to those found in Escherichia coli cell-free extracts. Therefore, the pathways established for phospholipid biosynthesis in E. coli can also account for the synthesis of the major phospholipids (phosphatidylethanolamine and phosphatidylglycerol) in several other gram-negative organisms. In addition, the unusual ribosomal association of the phosphatidylserine synthase from E. coli (Raetz and Kennedy, J. Biol. Chem. 247:2008-2014, 1972) appears to be a general property for this activity in several other bacterial species.     

Influence of potassium and calcium ions on the phosphatidylinositol and phosphatidylcholine metabolism in rat fibroblasts after growth stimulation by calf serum.

In secondary cultures of embryonic rat fibroblasts which were arrested in G1 (G0) by serum depletion and subsequently triggered into the cell cycle by readdition of growth factors isolated from fetal calf serum the influence of the potassium and calcium concentrations in the medium on phosphatidylinositol and phosphatidylcholine metabolism was investigated. The incorporation of inorganic [32P]phosphate into phosphatidylinositol is dependent on the potassium content of the culture medium. The specific activity of 32P in phosphatidylinositol is increased at K+ concentrations between 0.1 and 1 mM. Also calcium (between 0.01 and 2 mM) slightly stimulates phosphatidylinositol metabolism. Also the incorporation of myo-[3H]inositol is increased at potassium concentrations between 0.2 and 1 mM, whereas calcium is slightly inhibitory. The labelling of phosphatidylcholine with either [32P]phosphate or [3H]choline is not dependent on the potassium and calcium concentrations of the culture medium. Moreover, the phospholipid metabolism of permanently growing epithelioid and fibroblastoid cells lines, which were investigated, is considerably less dependent on the K+ and Ca2+ ions.     

Effect of unsaturated fatty acids and Ca2+ on phosphatidylinositol synthesis and breakdown

CDP-diglyceride : inositol transferase was inhibited by unsaturated fatty acids. The inhibitory activity decreased in the following order: arachidonic acid greater than linolenic acid greater than linoleic acid greater than oleic acid greater than or equal to palmitoleic acid. Saturated fatty acids such as myristic acid, palmitic acid, and stearic acid had no effect. Calcium ion also inhibited the activity of CDP-diglyceride : inositol transferase. In rat hepatocytes, arachidonic acid inhibited 32P incorporation into phosphatidylinositol and phosphatidic acid without any significant effect on 32P incorporation into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Ca2+ ionophore A23187 also inhibited 32P incorporation into phosphatidylinositol. However, 32P incorporation into phosphatidic acid was stimulated with Ca2+ ionophore A23187. Phosphatidylinositol-specific phospholipase C was activated by unsaturated fatty acids. Polyunsaturated fatty acids such as arachidonic acid and linolenic acid had a stronger effect than di- and monounsaturated fatty acids. Saturated fatty acids had no effect on the phospholipase C activity. The phospholipase C required Ca2+ for activity. Arachidonic acid and Ca2+ had synergistic effects. These results suggest the reciprocal regulation of phosphatidylinositol synthesis and breakdown by unsaturated fatty acids and Ca2+.     

Enzymes of phospholipid metabolism in rat pancreatic islets: subcellular distribution and the effect of glucose and calcium

The effect of glucose and calcium on the activities of the phosphatidylinositol cycle enzymes, CDP-diglyceride inositol transferase, diacylglycerokinase, and lysophosphatidylcholine 2-acyltransferase in rat pancreatic islets was studied. Calcium inhibited the activity of CDP-diglyceride inositol transferase but had no effect on lysophosphatidylcholine 2-acyltransferase and diacylglycerokinase activities. Upon preincubation of islets in a concentration of glucose known to stimulate insulin release, the activity of lysophosphatidylcholine 2-acyltransferase, but not that of diacylglycerokinase or the CDP-diglyceride inositol transferase, was stimulated. Subcellular fractionation of pancreatic islets showed that secretory granule membranes were enriched in CDP-diglyceride inositol transferase, whereas lysophosphatidylcholine 2-acyltransferase activity was highest in the microsomal membranes. The activation of 2-acyltransferase by incubating islets in insulinotropic glucose, and the calcium sensitivity of CDP-diglyceride inositol transferase, suggest that these enzymes may have roles in regulation of insulin secretion.     

Effects of changes in calcium concentration on basal and stimulated 32P incorporation into phospholipids in rat pineal cells

Abstract: The Ca²⁺ requirement for α-agonist stimulation of ³²P incorporation into acidic phospholipids (the phosphatidylinositol effect) of dispersed pineal cells was evaluated by means of several different compounds that interfere with Ca²⁺ disposition. Simple omission of Ca²⁺ led to slight increases in basal and norepinephrine-stimulated phosphatidyl-CMP (CDP-diacylglycerol) and phosphatidylglycerol labeling without affecting phosphatidylinositol labeling. In the absence of Ca²⁺, EGTA (200 μM) or the ionophore for divalent cations A23187 (10 μM) elicited large increases in phosphatidic acid, phosphatidyl-CMP, and phosphatidylglycerol labeling while strongly inhibiting the phosphatidylinositol effect. The Ca²⁺ translocation inhibitor LaCI₃ also reduced the magnitude of this effect. The phosphatidylinositol effect is, however, not induced by increased Ca²⁺ entry into the cytosol, since A23187 did not mimic the effect of norepinephrine. Under conditions where membrane Ca²⁺ was lowered, the addition of 1 mM-inositol greatly reduced phosphatidic acid, phosphatidylglycerol, and phosphatidyl-CMP labeling with concomitant increases in basal and norepinephrine-stimulated phosphatidylinositol labeling approaching that observed in the presence of norepinephrine and 2.5 mM-Ca²⁺. In the presence of 2.5 mM-Ca²⁺, inositol had negligible effects on phosphatidylinositol labeling. It was concluded that changes in membrane Ca²⁺ availability and/or disposition alter phospholipid metabolism and concurrently reduce the magnitude of the phosphatidylinositol effect, perhaps by making the pool of readily available inositol in pinealocytes rate-limiting.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli. In vitro and in vivo incorporation into phospholipids

sn-Glycerol 3-phosphorothioate, a bacteriocidal analog of sn-glycerol 3-phosphate in strains of Escherichia coli with a functioning glycerol phosphate transport system, was investigated for its ability to be incorporated into phospholipid under in vitro and in vivo conditions. A cell-free particulate fraction from E. coli strain 8 catalyzes the transfer of sn-[3H]glycerol 3-phosphoro[35S]thioate to chloroform-soluble material in the presence of either CDP-diglyceride or palmitoyl coenzyme A. With CDP-diglyceride as the co-substrate, the product of the reaction was tentatively identified as phosphatidylglycerol phosphorothioate. No formation of phosphatidylglycerol was observed, suggesting that the specific phosphatase required for the synthesis of phosphatidylglycerol does not catalyze, or else at a greatly reduced rate, the hydrolysis of the phosphorothioate monoester linkage. The kinetics of incorporation of sn-[3H]glycerol 3-phosphate and phosphorothioate into chloroform-soluble material in the presence of CDP-diglyceride are almost identical. In the presence of palmitoyl coenzyme A, sn-[3H]glycerol 3-phosphoro[35S]thioate was converted to the phosphorothioate analog of phosphatidic acid. Kinetic analysis showed that the apparent Km values for the incorporation of the phosphate and the phosphorothioate derivatives into phospholipid were 0.4 and 0.8 mM, respectively. The Vmax for the phosphorothioate analog was approximately half that for the phosphate derivative. Chemically synthesized thiophosphatidic acid was not a substrate for CTP:phosphatidic acid cytidylyltransferase. sn-[3H]Glycerol 3-phosphoro[35S]thioate was incorporated into phospholipid by cultures of E. coli strain 8. The major phosphorothioate-containing phospholipid synthesized in vivo was identified as 1,2-diacyl-sn-[3H]glycerol 3-phosphoro[35S]thioate. The phosphorothioate analog of phosphatidylglycerol phosphate was not observed despite our observations that this analog can be synthesized in vitro. Our results indicate that the phosphorothioate analog is an effective sn-glycerol 3-phosphate surrogate and suggest that a major reason for its toxicity toward E. coli strain 8 may be due to a total blockade of endogenous phospholipid biosynthesis.