Increased formation of phosphatidic acid induced with vasopressin or Ca2+ ionophore A23187 in rat hepatocytes

The effects of vasopressin and Ca2+ ionophore A23187 on phospholipid metabolism were investigated in rat hepatocytes. Vasopressin stimulated the incorporation of [32P]Pi into phosphatidic acid within 2 min but then it returned to control level after 10 min. On the other hand, the stimulation of the incorporation of [32P]Pi into phosphatidylinositol continued with incubation times up to 20 min. The Ca2+ ionophore A23187 also increased the 32P-labeling in phosphatidic acid, although it had no effect on [32P]Pi incorporation into phosphatidylinositol. Concerning the incorporation of [3H]glycerol, vasopressin did not enhance its incorporation into phosphatidic acid and phosphatidylinositol. The Ca2+ ionophore A23187 increased the incorporation into phosphatidic acid without significant effects on that into phosphatidylinositol. In the hepatocytes prelabeled with [3H]arachidonic acid, stimulated degradation of phosphatidylinositol with the addition of vasopressin and resultant formation of phosphatidic acid were observed within 5 min. The transient accumulation of diacylglycerol, the product of phosphatidylinositol hydrolysis, also occurred within 5 min with vasopressin. On the other hand, with the Ca2+ inophore A23187, stimulated degradation of triacylglycerol to diacylglycerol and the consequent formation of phosphatidic acid were observed. The Ca2+ ionophore A23187 caused a significant release of free [3H]arachidonic acid, although vasopressin had no effect.     

Cationic amphiphilic drugs as a potential tool for modifying phospholipids of tumor cells. An in vitro study of chlorpromazine effects on Krebs II ascites cells

[³²P]orthophosphate and [U-¹⁴C]glycerol incorporation into Krebs ascites cell lipids was studied in vitro in the presence of chlorpromazine (CPZ)¹. At concentrations not exceeding 0.1 mM, the drug produced no cell damage within 3 hr incubation. Under these conditions, CPZ inhibited the incorporation of [³²P]orthophosphate into phosphatidylcholine and phosphatidylethanolamine and of [U-¹⁴C]glycerol into phosphatidylcholine and triglycerides, in a dose-dependent manner. On the other hand, synthesis of phosphatidic acid and phosphatidylglycerol was greatly enhanced, whereas phosphatidylinositol showed no major change. These results are compatible with an inhibition of phosphatidate phosphohydrolase, redirecting phospholipid biosynthesis towards the anionic classes. In vitro treatment of cells for 3 hr induced profound changes of phospholipid composition, which displayed a relative increase of phosphatidylglycerol and phosphatidic acid at the expense of phosphatidylcholine and phosphatidylethanolamine. The use of amphipathic cationic drugs can thus offer an interesting model for studying the relationship between cell proliferation and membrane phospholipid biosynthesis.     

Alpha1-adrenergic stimulation of phosphatidylinositol-phosphatidic acid turnover in rat parotid cells

The regulation of phosphatidylinositol turnover by alpha-adrenergic agonists in rat parotid acinar cell aggregates was examined with respect to kinetics and agonist-antagonist interactions. Phosphatidylinositol turnover was followed by the changes in the specific activities of [32P]phosphatidic acid and [32P]phosphatidylinositol. The specific activity of phosphatidic acid increased rapidly (within 1 min) after addition of epinephrine (10(-5) M), reached a maximal level within 12-16 min, and then decreased. Incorporation of 32P into phosphatidylinositol exhibited a lag phase of about 5 min and then increased continuously for an additional 40 min. The absolute amounts of phosphatidic acid and phosphatidylinositol did not change. The concentrations of epinephrine needed to stimulate 32P incorporation into phosphatidic acid and phosphatidylinositol, measured at 15 and 30 min, respectively, were similar; Ka values of 2.05 +/- 0.46 X 10(-6) M for phosphatidic acid and 2.98 +/- 0.30 X 10(-6) M for phosphatidylinositol were found. The effects of agonists on 32P labeling of phosphatidylinositol, in order of potency, were epinephrine greater than or equal to norepinephrine greater than phenylephrine much greater than normetanephrine. When various adrenergic antagonists were evaluated for their ability to inhibit 10(-5) M epinephrine-stimulated 32P incorporation into both phosphatidic acid and phosphatidylinositol, the order of antagonist potency was prazosin greater than or equal to phenoxybenzamine greater than phentolamine greater than or equal to yohimbine greater than much greater than propranolol. These findings indicate that phosphatidylinositol-phosphatidic acid turnover in the rat parotid gland is mediated by the alpha 1-adrenergic receptor system.     

Effects of dl-propranolol on the synthesis of glycerolipids by rabbit iris muscle

Propranolol (0.03?0.3 mM), an amphiphilic cationic drug which is used therapeutically as a β-blocker, was found to alter significantly the incorporation of [¹⁴C]glucose, [¹⁴C]glycerol, [¹⁴C]acetate, ³²Pi, [³H]cytidine, [³H]inositol, [¹⁴C]choline, [¹⁴C]ethanolamine and [¹⁴C]serine into phospholipids of the iris muscle. Furthermore, it was found to exert a stimulatory effect on the [¹⁴C]serine incorporation into phosphatidylserine of the muscle and microsomes. In contrast, sotalol, another β-blocker-but lacking the hydrophobicity of propranolol-exerted no effect on lipid metabolism. Whereas norepinephrine stimulated only the turnover of the phosphate moiety of phosphatidic acid and phosphatidylinositol, in general propranolol caused the following changes: (a) it stimulated by 2- to 6-fold the labelling of phosphatidic acid and phosphatidylinositol from [¹⁴C]glucose, [¹⁴C]glycerol, [¹⁴C]acetate, ³²Pi and [³H]inositol, (b) it increased by 5- and 38-fold the incorporation of ³²Pi and [³H]cytidine, respectively into CDP-diglyceride, (c) it inhibited appreciably the incorporation of [¹⁴C]glucose, [¹⁴C]glycerol, [¹⁴C]acetate and ³²Pi into phosphatidylcholine and phosphatidylethanoalmine. However, while it inhibited significantly the [¹⁴C]choline incorporation into the former, it stimulated by 60 per cent the ethanolamine incorporation into the latter phospholipid. These results indicate that propranolol probably redirects phospholipid synthesis de novo, by inhibiting phosphatidate phosphohydrolase, such that the increase obtained in the biosynthesis of phosphatidylinositol is accompanied by a corresponding decrease in the synthesis of phosphatidylcholine and phosphatidylethanolamine.Propranolol also caused a 250 per cent increase in the [¹⁴C]serine incorporation into phosphatidylserine of the iris muscle and 28 per cent increase in that of microsomes. The drug appears to stimulate the Ca²⁺ -uptake by muscle and microsomes, which in turn could act to stimulate the Ca²⁺-catalyzed base-exchange reaction.In addition the metabolic pathways involved in the biosynthesis of the major phospholipids of the iris, a smooth muscle, are reported for the first time. These pathways were found to be essentially similar to those reported for other tissues.     

Action of neomycin on the metabolism of polyphosphoinositides in the guinea pig kidney.

The effect of neomycin on the metabolism of phospholipids was investigated in the guinea pig kidney both in vivo and in vitro. In vivo, after an intraperitoneal injection of [³²P]orthophosphate, the polyphosphoinositides (phosphatidylinositol phosphate and phosphatidylinositol diphosphate) were the most rapidly labeled phospholipids reaching maximal radioactivity within 40 min. The labeling of these lipids paralleled that of the nucleotide phosphate pool. After chronic treatment of animals with neomycin (100 mg/kg body weight daily for 6 days), phosphate incorporation into the polyphosphoinositides was decreased whereas incorporation into other lipids remained unaffected. Also, the amount of phosphatidylinositol diphosphate was reduced from 270 to 170 nmoles/g of kidney. No significant changes in phospholipid labeling were found in the liver. Experiments in vitro with kidney homogenates demonstrated inhibition of labeling of phosphatidylinositol diphosphate from [γ-³²]ATP and inhibition of the enzymatic hydrolysis of previously labeled [³²P]phosphatidylinositol phosphate and diphosphate in the presence of neomycin. A direct antibiotic-lipid interaction is discussed as a mechanism for the observed effects of neomycin on renal polyphosphoinositide metabolism.     

Some factors regulating [1-14C] acetate incorporation into aflatoxins by spheroplasts and spheroplast lysates from Aspergillus parasiticus

Factors regulating aflatoxin biosynthesis were studied in spheroplasts and lysates of Aspergillus parasiticusNRRL 3240. Over a 2 hr period, ribose, sucrose and maltose stimulated the incorporation of [1-¹⁴C] acetate into aflatoxins. Of a number of inorganic salts studied, zinc sulphate, magnesium sulphate, cobaltous chloride, cadmium acetate and strontium chloride stimulated the incorporation of labeled acetate into aflatoxins. Vanadyl sulphate and ferrous sulphate inhibited de novo aflatoxin formation in spheroplast lysates. Strontium chloride at concentrations of 0·01–0·5 mM stimulated [1-¹⁴C] acetate incorporation. Silver nitrate, copper sulphate and chlorides of manganese, calcium and barium markedly stimulated the incorporation of radioactivity into aflatoxins. An inverse correlation between the incorporation of [1-¹⁴C] acetate into aflatoxins and lipids was noted. In lysates, addition of pyruvic acid markedly increased de novo aflatoxin synthesis. Acetoacetate and bicarbonate enhanced incorporation of radioactivity at lower concentrations. Citric acid at 1–10 mM concentrations was stimulatory to incorporation of label into aflatoxins while malonic acid was inhibitory. In lysates, EDTA enhanced acetate incorporation into aflatoxins and inhibited incorporation of label in intact spheroplasts. This effect was only partially reversed by the addition of ZnSO₄ or MgSO₄.     

Ethanol-induced stimulation of phosphoinositide turnover and calcium influx in isolated hepatocytes

Ethanol has been shown to mobilize intracellular calcium in isolated rat hepatocytes by activation of phosphoinositide-specific phospholipase C. However, addition of ethanol to 32P-labeled hepatocytes resulted in a rapid increase in the level of [32P]phosphatidylinositol 4-phosphate over a period of 2 min, concomitant with a small decrease in [32P]phosphatidylinositol 4,5-bisphosphate and an increase in [32P]phosphatidic acid levels. These results indicate that polyphosphatidylinositol metabolism was stimulated by ethanol simultaneously with the activation of phospholipase C. Ethanol also caused a transient increase in the influx of extracellular calcium into quin 2-loaded hepatocytes over a similar period of time. The results demonstrate that ethanol, in common with calcium-mobilizing hormones, directly or indirectly stimulated polyphosphoinositide regeneration and allowed for increased movement of calcium across the hepatocyte plasma membrane.     

Effect of the tridecamer of compound 48/80, a Ca2+-dependent histamine releaser,on phospholipid metabolism during the early stage of histamine release from rat mast cells

When the tridecamer component of compound 48/80 (Fraction D, Fr. D), a Ca²⁺-dependent histamine releaser, was incubated with rat mast cells that had been prelabeled with [³²P]phosphate, [³H]inositol or [³H]glycerol, it induced a rapid decrease in [³²P]phosphatidylinositol-4,5-bisphosphate (PIP₂) followed by increases of [³H]inositol-1,4,5-triphosphate (Ins P₃) and [³H]diacylglycerol during the 10 sec prior to detectable histamine release. Fr.D-induced changes of the metabolism of these compounds occurred even in the absence of Ca²⁺, but to a lesser extent than in the presence of Ca²⁺. In contrast, the accumulation of [³H]arachidonic acid into phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidic acid (PA) in [³H]arachidonic acid-prelabeled mast cells was Ca²⁺-dependently stimulated by Fr.D with a concomitant decrease in [³H]phophatidylethanolamine (PE). These Ca²⁺-dependent changes in PC and PE were not observed in mast cells preloaded with [³²P]phosphate, while [³²P]PI and [³²P]PA increased Ca²⁺ independently. Fr.D also increased ⁴⁵Ca²⁺ uptake by mast cells within 5 sec after the stimulation. These results indicate that Fr.D binding to mast cell Ca²⁺ independently induces rapid changes of PI cycle-related metabolism of plasma membrane components, while it also induces Ca²⁺-dependent accumulation of arachidonic acid into PC, PI and PA in association with the decrease of PE, which may be important during the latent period prior to the Ca²⁺-dependent release of histamine from Fr.D-stimulated mast cells.     

Effect of pentobarbital on regional brain phospholipid synthesis

Rats were given 45 mg/kg i.p. sodium pentobarbital 15 min prior to the intraventricular injection of 200 μCi [³²P]phosphoric acid and 50 μCi [³H]glycerol. The animals were sacrificed 1 hr later, subcellular fractions were prepared from four subcortical brain regions and phospholipids were extracted. Pentobarbital significantly increased the ratio of [³H]- and [³²P]-triphosphatidylinositol (TPI) to diphosphatidylinositol (DPI) in the microsomal but not synaptosomal fractions. The possible relationship of this change to nicotinic receptor activity is discussed. Pentobarbital specifically decreased ³²Pi but not [³H]glycerol incorporation into synaptosomal phosphatidylinositol (PI). Thus, pentobarbital induced the opposite of the “neurotransmitter effect” on PI turnover. Pentobarbital either decreased or had no effect on the incorporation of ³²Pi and [³H]glycerol into phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE).     

Muscarinic receptor stimulated phosphoinositide turnover in cardiac atrial tissue

The properties of muscarinic agonist stimulated phosphoinositide turnover in canine atrial slices were investigated. In slices prelabeled with 32Pi, carbachol stimulated a 20-30% decrease of 32P-labeled phosphatidylinositol 4'-phosphate (PIP) and phosphatidylinositol 4',5'-bisphosphate (PIP2) content within 10-15 sec. This was followed by a resynthesis of these lipids to control levels after 30 sec. Carbachol-stimulated PIP and PIP2 turnover was followed by a relatively slower increase in 32P incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) which was maximal after 5-10 min. Carbachol increased 32P-labeling of PA and PI in most regions of right and left atria with equal effectiveness. Muscarinic receptor stimulated increases in PA and PI labeling showed high specificity for certain muscarinic agonists and, unlike most tissues, this muscarinic receptor mediated phospholipid effect was dependent on extracellular calcium. Carbachol did not increase 32P incorporation into PA and PI if Mn2+, Co2+, Mg2+, or La3+ was substituted for extracellular Ca2+. Unlike other muscarinic agonists, acetylcholine increased 32P incorporation into phosphatidylcholine in addition to PA and PI. Low concentrations of calcium channel blockers, verapamil, nifedipine or diltiazem, did not block carbachol-stimulated changes in PA and PI labeling in the presence of Ca2+; however, higher concentrations (greater than or equal to 10 microM) of verapamil increased PA and PI labeling. Ouabain enhanced carbachol-stimulated 32P incorporation into PA but attenuated incorporation into PI. The mechanisms associated with the actions of these agents on phospholipid metabolism and their possible physiological significance are discussed.     

Effect of clofibrate on the metabolism of oleate in the perfused rat liver

The effect of clofibrate on the metabolism of [1-¹⁴C]- and [U-¹⁴C]oleate was examined in the perfused rat liver. Clofibrate feeding severely reduced hepatic triglyceride secretion and enhanced ketone body production. The increase in the rate of incorporation of labeled tracers into perfusate oxidation products and ketone bodies due to the clofibrate treatment was demonstrated only with [U-¹⁴C]oleate. Clofibrate strongly reduced the rate of incorporation of oleate into perfusate triglyceride, whereas that into the phospholipid fraction of the post-perfused liver doubled. In consequence, the sum of the radioactivities in esterified lipids in the perfusate and the post-perfused liver was not altered by clofibrate. A clofibrate-dependent increase in phospholipid synthesis may restrict the amount of exogenous fatty acid which is available for the formation of triglyceride-rich lipoproteins.     

Effect of 2,5-Hexanedione on lipid biosynthesis in sciatic nerve and brain of the rat

Distal axonopathy induced by n-hexane and methyl n-butyl ketone has been attributed to a common metabolite, 2,5-hexanedione. Since altered lipid metabolism is frequently associated with neuropathy, the effects of 2,5-hexanedione on lipid biosynthesis from [1-¹⁴C]acetate in sciatic nerve and brain of rats given 1% 2,5-hexanedione in drinking water have been studied, in vitro. Clinical signs of neuropathy appeared after 6 weeks. Loss of body weight induced by 2,5-hexanedione was similar to that observed in pair-fed control rats. Compared to nerves from pair-fed controls, nerves from rats fed 2,5-hexanedione exhibited decreased incorporation of [1-¹⁴C]acetate into triacylglycerols (32%), total sterols + diacylglycerols (54%), digitonin-precipitable sterols (55%), squalene (55%), and ubiquinone (43%). Incorporation of [1-¹⁴C]acetate into phospholipids, fatty acids, and cholesteryl esters was similar in nerves of 2,5-hexanedione-treated rats and pair-fed controls. In brain, incorporation of [1-¹⁴C]acetate into lipids was similar in 2,5-hexanedione-treated and pair-fed control rats, except into the fatty acid fraction which was significantly decreased by 11%. The data support the hypothesis that lipid metabolism, in particular sterol metabolism, is altered in hexacarbon-induced distal axonopathy.     

The phosphorylation of rat liver ribosomes following administration of dimethylnitrosamine

Following a single dose of dimethylnitrosamine (DMNA) the phosphorylation of rat liver ribosomes was strongly enhanced. The incorporation of [³²P]phosphate into the 40 S subunit was stimulated 5- and 9-fold 2 and 5 hr, respectively, after administration of the drug. The phosphorylation of the large ribosomal subunit was not affected. The enhanced phosphorylation of the small subunit was due to a 20-fold stimulated incorporation of phosphate into ribosomal protein S6, the only one affected by DMNA.     

The effect of the methionine antagonist l-2-amino-4-methoxy-trans-3-butenoic acid on the growth and metabolism of walker carcinosarcoma in vitro

l-2-Amino-4-methoxy-trans-3-butenoic acid (Ro07–7957) is a structural analogue ot methionine with a potent tumour growth inhibitory activity in vitro. This agent is transported by the methionine carrier system in Walker carcinoma and causes an initial dose-related depression of the acid-soluble pool of methionine. The depression of the incorporation of l-[methyl-³H]methionine into acid-insoluble material in the presence of Ro07–7957 is greater than that of l-[2-³H]methionine and l-[4,5(n)-³H]lysine, suggesting an inhibition of the methylation of macromolecules as well as an inhibition of protein synthesis. There is no effect of the drug on the incorporation of [³H]thymidine into acid-insoluble material during the first 24 hr after treatment, while the incorporation of [5-³H]uridine is stimulated 40 per cent. The ratio of incorporation of l-[2-³H]methionine to l-[methyl-¹⁴C]methionine into proteins increases with increasing drug concentration, suggesting an inhibition of protein methylation. This effect is more prevalent at 24 hr than after 8 hr of treatment. The specific activity of tRNA methylase using E. coli MRE 600 tRNA as substrate is elevated more than two-fold within 24 hr after treatment, as is also the intracellular level of S-adenosyl-l-methionine (SAM). The effects of this agent on macromolecular metabolism is in some respects similar to that observed with the carcinogen ethionine, and suggests the initial formation of an inhibitor of methylation, which is followed by a later attempt by the cells to maintain homeostasis by production of increased amounts of tRNA methylating enzymes.     

Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylgglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 μM carbachol was biphasic: the first peak occurred at 5 min and the second one at 20 min. It was also demonstrated that the second peak was regulated in part by a calmodulin/protein kinase C-dependent mechanism. Based on the kinetic analysis of DAG formation and [³²P]phosphoinositide breakdown, the first peak of carbachol (1 μM)-stimulated DAG accumulation was found to be related to the breakdown of [³²P]phosphatidylinositol 4-monophosphate ([³²P]PIP) and [³²P]phosphatidylinositol 4,5-biphosphate ([³²P]PIP₂). The second peak was found to be related to [³²P]PIP₂ breakdown. Carbachol stimulated the release of [³H]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of [³H]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of [³H]ethanolamine metabolites. A calmodulin/protein kinase C-dependent mechanism was also studied and was found to be involved in carbachol-stimulated phosphatidylcholine hydrolysis; W-7, a calmodulin inhibitor and staurosporine, a protein kinase C inhibitor, inhibited the carbachol (1-μM)-induced release of [³H]choline metabolites at 20 min in a dose-dependent manner, but did not have inhibitory effects at 5 min. These results suggest that the first peak of DAG accumulation induced by carbachol is predominantly associated with the breakdown of [³²P]PIP and [³²P]PIP₂ and that the second peak is predominantly associated with [³²P]PIP₂ breakdown and phosphatidylcholine hydrolysis.     

Palmitic acid-1-14C incorporation and turnover in lung phospholipids of rats treated with chlorphentermine,RMI 10.393 and Ro 4-4318

The effects of 3 lipidosis-inducing drugs on the incorporation and turnover of palmitic acid-1-¹⁴C in lung phospholipids was studied.In rats treated with 1 dose of chlorphentermine or RMI 10.393, the incorporation of palmitate-1-¹⁴C into most lung phospholipid fractions was moderately decreased, but markedly lowered after 1 dose of Ro 4-4318.Eight doses of chlorphentermine and RMI 10.393 strongly inhibited the incorporation of palmitate-1-¹⁴C into lung phospholipids, whereas with 8 doses of Ro 4-4318 the incorporation was highly increased.Thirty hours after the last of 3 injections of the labeled palmitic acid the turnover of most lung phospholipids was considerably lower in chlorphentermine and RMI 10.393-treated rats than in controls. Ro 4-4318, however, induced a highly increased turnover of most phospholipids. After 54 h, this effect had practically disappeared.Our studies showed that phospholipid storage after treatment with chlorphentermine and RMI 10.393 is mainly due to decreased degradation of phospholipids, whereas increased synthesis accounts for the effect of Ro 4-4318.     

The cholecystokinin analogues JMV-180 and CCK-8 stimulate phospholipase C through the same binding site of CCK(A) receptor in rat pancreatic acini

1. This study was designed to address the controversy related to the involvement of phospholipase C in the signalling pathway linked to CCK(A) receptor stimulation by the cholecystokinin analogue JMV-180, a full agonist for amylase release, in rat pancreatic acini. 2. JMV-180 was shown to stimulate phospholipase C by measuring the incorporation of [(32)P]-orthophosphoric acid ([(32)P]-Pi) into phosphatidic acid (PtdOH) and phosphatidylinositol (PtdIns). Both responses elicited by JMV-180 were time and concentration dependent. Maximal effects elicited by JMV-180 were 39.08+/-0.72 and 8.02+/-0.40% for the labelling of [(32)P]-PtdIns and [(32)P]-PtdOH, respectively, as compared to the maximal effects of CCK-8, a full agonist of the CCK(A) receptor. 3. [(32)P]-Pi incorporation into PtdOH and PtdIns was sensitive to lithium, demonstrating that both responses are a consequence of phospholipase C activation. However, since lithium blocks the phosphoinositide cycle by an uncompetitive mechanism, its effect was only apparent at high concentrations of CCK-8 (>10 pM), which elicited stimuli above 20 and 60% of the maximal [(32)P]-PtdOH and [(32)P]-PtdIns labelling, respectively. 4. JMV-180 inhibited the incorporation of [(32)P]-Pi into PtdOH and PtdIns as stimulated by CCK-8, down to its own maximal effect. The estimated IC(50) values for the inhibition curves were not significantly different from those calculated assuming the same single binding site for both agonists. These results indicated that the well established role of JMV-180 as a partial agonist for CCK(A) receptor-linked signalling responses, also applies for the stimulation of phospholipase C. 5. The comparison of CCK-8 and JMV-180 dose-response curves of amylase release to those of PtdIns and PtdOH labelling with [(32)P]-Pi showed the existence of an amplification mechanism between phospholipase C and amylase release for both agonists. 6. In conclusion, we show that JMV-180, as well as CCK-8, stimulate phospholipase C upon interaction with the same binding site at the CCK(A) receptor in rat pancreatic acini.     

Effect of polychlorinated biphenyls (PCBs) administration on phospholipid biosynthesis in rat liver

The effect of PCBs or phenobarbital on the biosynthesis of phospholipids in hepatic endoplasmic reticulum of rats was studied by the intraperitoneal injection of [³²P]orthophosphate, [Me?¹⁴ C]choline or [2?³H]glycerol. Significant increases in liver microsomal phospholipid content after the administration of either PCBs or phenobarbital indicated the actual proliferation of endoplasmic reticulum membranes. The rate of both [³²P] and [¹⁴C] incorporations into microsomal choline-containing phospholipids, such as phosphatidylcholine, sphingomyelin and lysophosphatidylcholine, was reduced to one fifth by PCBs administration compared with control animals. The incorporation of [³²P]orthophosphate into phosphatidylethanolamine or other phospholipid classes was less or not affected, respectively, by PCBs administration. The specific inhibitory effect of PCBs on the incorporation into cholinecontaining phospholipids was not observed when [2?³-H]glycerol was used as a precursor. Phenobarbital administration, however, increased significantly the rate of [³²P] incorporation into liver phospholipids, especially phosphatidylcholine. It is suggested that the increase in microsomal phospholipid content by PCBs administration is not due to the stimulation of synthesis but to the inhibition of the catabolism of membrane phospholipids and that the increase in content caused by phenobarbital is due at least in part, to the stimulation of synthesis. The possible site(s) of PCBs-induced inhibition of phospholipid biosynthesis in rat liver is discussed.     

Adrenergic and cholinergic stimulation of 32P-labeling of phospholipids in rabbit iris muscle.

The phospholipid and cholesterol composition of the iris muscle of the rabbit was determined, and the incorporation of ³²Pi into the individual phospholipids of irises incubated in Krebs Ringer bicarbonate buffer that contained glucose in the presence or absence of adrenergic or cholinergic neurotransmitters and their agonists and antagonists was investigated. The results of studies on the characteristics and the effects of norepinephrine and other neuropharmacological agents on the ³²P-la-beling of phosphatidic acid (PhA). phosphatidylinositol (PhI), phosphatidylcholine (PhC). phosphatidyl-ethanolamine (PhE) and phosphatidylserine (PhS) in the iris muscle are reported. Addition of l-nore-pinephrine. l-epinephrine, dopamine or acetylcholine (0.003 to 0.3 mM) to irises which were preincubated for 20 min in ³²P-Krebs-Ringer without cold phosphate stimulated significantly the isotope incorporation into PhA and Phi. but not PhC. In contrast histamine, noremetanephrine. metanephnnc. adrenochrome. 6-hydroxydopamine and eserine (0.003 to 0.3 mM) had a negligible effect on the isotope incorporation. At higher concentrations of norepinephrine (10mM), labeling of PhA, PhI and PhC was elevated to 989. 630 and 185 per cent of that of the control respectively. It was concluded that in the iris muscle α-receptors and not β-receptors are involved in the stimulatory action of norepinephrine on ³²Pi incorporation into phospholipids. This conclusion is based on the following findings. (a) Only α-stimulators. such as norepinephrine and phenylephrine. increased significantly the labeling of PhA and PhI. This effect was blocked by the α-blockers. phenoxybenzamine and phentolamine. (b) Isoproterenol, a β-stimulator, had no effect on the labeling of PhA and PhI. Sotalol and propranolol, both β-blockers, did not block the norepinephrine stimulation of ³²Pi incorporation, (c) Cyclic AMP (or dibutyryl cyclic AMP), which has been suggested as a β-receptor mediator, exerted no influence on the phospholipid effect. Atropine blocked completely the acetylcholine-stimulated ³²P-labeling of PhA and PhI. At 0.3 mM, phentolamine and propranolol increased by several-fold the isotope labeling of PhA, PhI and, unexpectedly, of CDP-diglyceride and inhibited that of PhC. The properties of the norepinephrine stimulation of ³²P-labeling in the iris muscle can be summarized as follows, (a) It is concentration dependent; but this dependence varies with the condition of incubation, (b) It is temperature dependent, but the effect is lost upon freezing and thawing, (c) It can be demonstrated during a period of 3 days of aging at 4. (d) Glucose is required for maximal stimulation, (e) The phospholipid effect is not specific to a particular subcellular fraction, (f) Addition of norepinephrine brought about a 25 per cent decrease in the level of PhI and a 63 per cent increase in the level of PhA. (g) Time-course studies on the ³²P-labeling of phospholipids in the presence and absence of norepinephrine revealed that, in contrast to PhI and PhC, the specific radioactivity of PhA increased with time of incubation (0.90 min) only when the neurotransmitter was added, (h) Direct activation of the enzyme diglyceride kinase by norepinephrine does not appear to be the molecular mechanism underlying the phospholipid effect. Our data suggest that in the iris muscle norepinephrine increases the turnover of PhA and PhI phosphorus by stimulating the hydrolysis of endogenous PhA or PhI. or both, to form more membraneous diglyceride. The latter is then rephosphorylated by diglyceride kinase to form more labeled PhA.     

Chlorpromazine and human platelet glycerolipid metabolism: precursor specificity and significance of drug-platelet interaction time

Chlorpromazine is known to have a number of effects on glycerolipid metabolism in a variety of cell types, and in some cases reports are contradictory. To investigate the basis for some of these discrepancies, we reinvestigated the effects of chlorpromazine on some aspects of platelet glycerolipid metabolism. Time-courses conducted with [3H]glycerol or [3H]palmitic acid showed that the effects of chlorpromazine on the labelling of phosphatidylcholine, diacylglycerol, and triacyglycerol were highly dependent upon platelet-drug interaction time. The time-dependent changes in labelling patterns were independent of the presence of radiolabel during incubation, and were not the results of time-dependent changes in the platelets per se. The effects of chlorpromazine on the labelling of platelet glycerolipids by [3H]glycerol, [3H]palmitic acid, [32P]P(i) ([32P]phosphatase), and [14C]choline were compared. Dose-response curves conducted at 30-min incubation time showed that chlorpromazine potently inhibited labelling of diacylglycerol and diacyglycerol-derived lipids (triacyglycerol and phosphatidylcholine) by the 3H-labelled precursors. Labelling of phosphatidylcholine by [32P]P(i) or [14C]choline was, however, not affected at all by the drug. We conclude that the effects of chlorpromazine on platelets are highly time-dependent, and that the prolonged effects are most likely to be of biological significance. Furthermore, in platelets the effects of the drug on the labelling of phosphatidylcholine by isotope-labelled precursors are highly dependent on the route of incorporation of the specific precursor chosen.