Studies on the mechanism of alteration by propranolol and mepacrine of the metabolism of phosphoinositides and other glycerolipids in the rabbit iris muscle

We have investigated the effects and mechanism of action of propranolol and mepacrine, two drugs with local anesthetic-like properties, on phospholipid metabolism in rabbit iris and iris microsomal and soluble fractions. In the iris, propranolol, like mepacrine [A. A. Abdel-Latif and J. P. Smith, Biochim, biophys. Acta 711, 478 (1982)], stimulated the incorporation of [14C]arachidonic acid ( [14C]AA) into phosphatidic acid (PA), CDP-diacylglycerol (CDP-DG), phosphatidylinositol (PI), the polyphosphoinositides (poly PI) and DG, and it inhibited that of phosphatidylcholine (PC), phosphatidylethanolamine (PE), triacylglycerol (TG) and the prostaglandins. Similarly, mepacrine, like propranolol [A. A. Abdel-Latif and J. P. Smith, Biochem. Pharmac. 25, 1697 (1976)], altered the incorporation of [14C]oleic acid, [3H]glycerol, 32Pi and [14C]choline into glycerolipids of the iris. Time-course studies in iris muscle prelabeled with [14C]AA showed an initial decrease in the production of DG and a corresponding increase in that of PA by the drugs, followed by an increase in accumulation of DG at longer time intervals (60-90 min). The above findings are in accord with the hypothesis that these drugs redirect glycerolipid synthesis by inhibiting PA phosphohydrolase. Propranolol and mepacrine stimulated the activities of DG kinase and phosphoinositide kinases and inhibited that of DG cholinephosphotransferase. The drugs had little effect on the activity of DG acyltransferase. It is concluded that propranolol and mepacrine redirect glycerolipid metabolism in the iris by exerting multiple effects on the enzymes involved in phospholipid biosynthesis. We suggest that these drugs could exert their local anesthetic-like effects by effecting an increase in the synthesis of the acidic phospholipids (PA, PI and the poly PI) and subsequently the binding of Ca2+- to the cell plasma membrane.     

Modifications of phospholipid metabolism induced by chlorpromazine, desmethylimipramine and propranolol in C6 glioma cells

The effects of chlorpromazine (CPZ), desmethylimipramine (DMI) and propranolol (PRO) on phospholipid metabolism in C6 glioma cells were studied by following the incorporation of 32Pi, [U-14C]glycerol, [2-3H]glycerol and [1-14C]oleate into lipids. The drugs produced a dose-dependent increase in the incorporation of 32Pi and [U-14C]glycerol, but not of [1-14C] oleate, into total phospholipids, that reached a plateau at 200 microM CPZ and 500 microM DMI and PRO. The three drugs shifted the incorporation of precursors from neutral [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)] to acidic phospholipids [phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylglycerol, phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2)] in a dose-dependent, qualitatively similar manner. The incorporation of [2-3H]glycerol into diacylglycerol was also depressed markedly by CPZ. Addition of 1 mM 1,2-dioleoylglycerol, 1-oleoyl-2-acetylglycerol or oleate only partially reversed the decrease in PC labeling caused by CPZ. 12-O-Tetradecanoylphorbol-13-acetate counteracted this effect of CPZ completely but greatly increased PC labeling even in the absence of the drug. Polyphosphoinositides rapidly incorporated 32Pi at early times reaching a plateau in about 40 min. The labeling rate of PI was not parallel to that of PIP or PIP2 and continued to increase even after the polyphosphoinositides had reached a plateau. CPZ increased PI labeling much more than that of PIP and PIP2. These data suggest that cationic amphiphilic drugs may act by inhibiting CTP:phosphocholine cytidylyltransferase, thus decreasing incorporation of precursors into PC and PE; inhibiting PA phosphohydrolase with increased formation of phosphatidyl-CMP, the intermediate for the synthesis of acidic phospholipids; and stimulating the inositol exchange reaction, forming a pool of PI that is not available for PIP and PIP2 synthesis.     

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

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 microM 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 microM)-stimulated DAG accumulation was found to be related to the breakdown of [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([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-microM)-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 [32P]PIP and of [32P]PIP2 and that the second peak is predominantly associated with [32P]PIP2 breakdown and phosphatidylcholine hydrolysis.     

Effects of chlorpromazine and trifluoperazine on choline metabolism and phosphatidylcholine biosynthesis in cultured chick heart cells under normoxic and anoxic conditions

The effects of chlorpromazine and trifluoperazine on phosphatidylcholine biosynthesis in the heart were investigated in isolated cardiac cells under normoxic and anoxic conditions. The cells were obtained from 7-day-old chick embryos and were maintained in culture. After 96 hr, cells were maintained either in an incubator with oxygen at room air concentration (normoxia) or in an incubator containing 95% nitrogen and 5% CO2 (anoxia). Pulse chase experiments with [methyl-3H]choline were conducted using a 2-hr incubation with choline. Chlorpromazine and trifluoperazine at 10(-5) M produced a significant (P less than 0.05) increase in the incorporation of choline into both phosphocholine and phospholipid. High concentrations of chlorpromazine or trifluoperazine i.e. 10(-4) M, damaged myocardial cells as reflected in a significant (P less than 0.05) reduction in cellular protein and a further reduction in labelled choline in phosphocholine or phospholipid after adjusting for the lower protein concentrations. Anoxia altered choline metabolism but 6 hr of anoxia was the minimum time needed for the effect to be observable. Anoxia, for 24 hr, produced a significant (P less than 0.05) reduction in labelled choline in phosphocholine without a significant change in incorporation of label in phospholipid or cellular protein. Both chlorpromazine and trifluoperazine at 10(-5) M prevented anoxic-induced changes in phosphocholine metabolism. Thus, chlorpromazine and trifluoperazine affect phospholipid biosynthesis in cardiac cells and prevent anoxia-induced changes in phosphatidylcholine biosynthesis.     

Stimulation of phosphatidic acid synthesis in bovine aortic endothelial cells in response to activation of P2-purinergic receptors.

In this study we used the bovine thoracic aorta endothelial cell line AG 4762 and primary bovine aortic endothelial cells to investigate the formation of phosphatidic acid (PA) in response to activation of P2-purinergic receptors. 2-Methylthio ATP (2MeSATP) stimulated the formation of [32P]-PA in bovine aortic endothelial cells labelled with 32P(i) for 2.5 hr. A comparison of the response to other ATP analogues suggests that this was mediated via a P2Y-purinergic receptor. Using various agonists at 30 microM there was a correlation between the formation of [32P]PA and of total inositol phosphates in the presence of lithium. The 2MeSATP-stimulated accumulation of [32P]PA showed an initial high rate, followed by a more sustained slower rate. The initial response was independent of extracellular calcium while the later response was dependent on calcium influx. The protein kinase C stimulator phorbol myristate acetate (PMA) produced only a very small enhancement of [32P]PA accumulation compared to 2MeSATP. The 2MeSATP stimulation of both inositol phosphates and [32P]PA was almost eliminated by the presence of PMA. Using cells prelabelled with [3H]methylcholine 2MeSATP produced only a small non-significant enhancement of [3H]choline formation; PMA by contrast formed a much larger amount of [3H]choline. There was no evidence of a change in [3H]phosphocholine. The dissociation between phospholipase D (PLD) activation and [32P]PA accumulation and the correlation between stimulation of [32P]PA accumulation and phospholipase C (PLC) activation all suggest that, using this protocol for labelling cells, the principle route of the stimulation of formation of [32P]PA is via the activation of PLC followed by metabolism of diacylglycerol (DAG) by DAG kinase. These results show that activation of P2Y-purinergic receptors on aortic endothelial cells leads to the formation of phosphatidic acid and that both PLD and PLC pathways are likely to contribute to this response.     

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.     

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.     

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.     

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.     

Desensitization of platelets to iloprost. Loss of specific binding sites and heterologous desensitization of adenylate cyclase

The binding characteristics of [3H]prostacyclin and [3H]iloprost ([3H]5-[(E)-(1S,5S,6R,7R)-7-hydroxy-6-[(E)-(3S,4RS) -3-hydroxy-4-methyl-1-octen-6-inyl]-bicyclo[3.3.0]octan-3-yl idene] -pentanoic acid) and platelet adenylate cyclase activities were investigated in platelet-rich plasma preincubated with iloprost. The exposure of platelets to 0.1 microM iloprost (12 h, 20 degrees C) caused a significant loss of iloprost binding sites (P less than 0.01) without causing changes in binding affinity. This loss of specific [3H]iloprost binding was time- and dose-dependent. The reduction of iloprost receptor density was accompanied by an impaired responsiveness of platelet adenylate cyclase to iloprost, prostaglandin D2 and forskolin. In contrast, basal adenylate cyclase activity was not affected by iloprost pretreatment. The diminished response of the enzyme to GTP and NaF pointed to an involvement of the stimulatory guanyl nucleotide-binding protein (Gs) in iloprost-induced heterologous desensitization. Consequently, [32P]NAD+ and cholera toxin were used for the direct labelling of Gs. Platelet membranes desensitized to iloprost incorporated less label into the 45 kD subunit of Gs. These data suggest that the site of action of iloprost for heterologous desensitization of human platelet adenylate cyclase is located on Gs.     

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).     

Stimulatory effect of calmodulin antagonists on phospholipid base-exchange reactions in rabbit platelet membranes

The properties of Ca2+-dependent incorporation of [3H]serine, [3H]ethanolamine and [3H]choline into the corresponding phospholipids mediated by base-exchange enzymes in rabbit platelet membranes were studied in the presence or absence of the calmodulin antagonists chlorpromazine, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), all of which markedly activate three base-exchange reactions. The base-exchange activities were dependent on Ca2+ both in the presence and absence of the drugs. Other metal ions tested did not stimulate the base-exchange reactions, even in the presence of the drugs. Apparent Km values for serine, ethanolamine and choline were not affected significantly by the concentration of Ca2+, with or without the drugs. [3H]Serine incorporation into phospholipid was competitively inhibited by ethanolamine and choline, [3H]choline incorporation was competitively inhibited by serine and ethanolamine, whereas [3H]ethanolamine incorporation was competitively inhibited by serine and noncompetitively by choline. These competitive and noncompetitive relations between each base were also not affected by the drugs. The amount of 45Ca2+ binding to platelet membranes was decreased by the drugs dose dependently. A weaker calmodulin antagonist, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), only slightly stimulated the base-exchange reactions, but did clearly inhibit 45Ca2+ binding to the membranes, in the same manner as that of the other calmodulin antagonists used. The concentration of chlorpromazine, trifluoperazine, W-7 and W-5, required to produce half-maximal inhibition of Ca2+ binding, was approximately 30 microM. These results suggest that the calmodulin antagonists used activate the base-exchange reactions only in the presence of Ca2+ without changing the affinity of each free base to base-exchange enzymes. The activation of the base-exchange reactions was not due to the increase in free Ca2+ caused by the drug-induced inhibition of Ca2+ binding to platelet membranes.     

Chlorimipramine--but not imipramine--rapidly reduces [3H]imipramine binding in human platelet membranes

A single dose of 50 mg chlorimipramine was followed by a rapid and pronounced decrease in [3H]imipramine binding to platelet membranes. Incubation of human platelets or platelet membranes with 25 nM chlorimipramine similarly reduced [3H]imipramine binding. Imipramine, desmethylchlorimipramine, chlorpromazine and some serotonin uptake inhibitors did not have this effect. The effect was not due to chlorimipramine remaining in the membranes during the binding analysis.     

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.     

Selective modifications in the de novo biosynthesis of retinal phospholipids and glycerides by propranolol or phentolamine

The effects of propranolol or phentolamine on the metabolism of phospholipids, diacylglycerol, and triacylglycerol were studied in the bovine retina in vitro. Lipid labeling was followed during short-term incubation of intact bovine retinas with [U-₁₄C]glycerol and [1-¹⁴C]palmitic acid. Each of these precursors was recovered in the appropriate lipid moiety. Most of the [¹⁴C]glycerol appeared progressively in triacylglycerol (TG) through the sequence from phosphatidic acid (PA) to diacylglycerol (DG). Labeled palmitate appeared in much lower quantities than labeled glycerol in all glycerolipids except phosphatidylcholine (PC). Propranolol and phentolamine greatly enhanced the [¹⁴'C]glycerol specific activities of PA, phosphatidylinositol (PI), and phosphatidylserine (PS), whereas labeling in other glycerolipids was much lower than in controls. The labeling in TG with both precursors was found to be less than 50% of the control values; however, a late increase in DG labeling was observed. The effects of these drugs on broken cell preparations were also described, although lipid synthesis from labeled glycerol in these preparations was only 9% that of intact retinas. It appeared that an amphiphilic cationic structure was necessary to produce these drug effects; propranolol glycol, the hydrophobic moiety of propranolol, did not elicit the same effects. It is suggested that, among other changes, the drugs inhibited phosphatidate phosphohydrolase and redirected the flux predominantly toward PI. Support for the proposed multiple lipid effects elicited by these drugs was provided by the dual changes found in the labeling of DG.     

Mechanism of depression of brain phospholipid levels by an epileptogenic drug

Treatment of the rat with U18666A [3 beta-(2-diethylaminoethoxy) androst-5-en-17-one HCl] resulted in development of a chronic seizure state and 20-40% reductions in the concentration of all major phospholipid in whole brain. The mechanism of the phospholipid changes was explored in the present study. Incorporation of intracerebrally injected [1,3-3H]glycerol and [32P]orthophosphate into glycerolipids was decreased by 30-40% in treated rats. U18666A added in vitro to brain slices totally blocked glycerolipid synthesis at a high drug level (10(-3) M) but stimulated incorporation into diacylglycerol, phosphatidic acid and phosphatidylinositol at a lower level (10(-4) M). When added in vitro to cell fractions from liver or brain, U18666A readily inhibited phosphatidate phosphohydrolase and the acyltransferase enzymes which convert glycerolphosphate to phosphatidic acid and which convert diacylglycerol to triacylglycerol. Fifty percent inhibition of all three enzymes occurred at drug concentrations of between 0.4 and 1.0 mM. Phosphatidate cytidylyltransferase, an enzyme important to formation of phosphatidylinositol, was comparatively resistant to inhibition. Taken together, the results indicate that the marked reduction in the concentration of brain phospholipids caused by treatment of the young rat with U18666A is likely due to decreased phospholipid synthesis secondary to inhibition of several key enzymes in glycerolipid synthesis and, particularly, to inhibition of glycerolphosphate acyltransferase and phosphatidate phosphohydrolase.     

Comparison of the effect of trihalomethanes on lipid metabolism in rat liver slice

The effect of a series of trihalomethanes, CHCl3, CHBrCl2, CHBr2Cl, and CHBr3, on in vitro lipid metabolism was compared using rat liver slices. The incorporation of 32Pi and [3H]glycerol into phospholipid of liver slices was inhibited in the presence of trihalomethanes. The inhibitory effect followed the number of bromine atoms in the trihalomethane molecule in the initial period of incubation. CHBr3 markedly inhibited the incorporation of [3H]glycerol into triacylglycerol (TG); CHBr2Cl was less effective and the other two trihalomethanes were without effect. The activities of glycerophosphate acyltransferase, phosphatidate phosphatase and diacylglycerol acyltransferase were changed by the exposure to trihalomethanes. The effects of CHBr2Cl and CHBr3 were much more severe as compared to those of CHBrCl2 and CHCl3. The change in the enzyme activities could explain the alteration in the incorporation of [3H]glycerol into TG.     

The binding of haloperidol to human blood platelets and interactions with 5-hydroxytryptamine and dopamine.

1 The binding of [3H]-5-hydroxytryptamine ([3H]-5-HT), [3H]-haloperidol and [3H]-dopamine to human blood platelets was investigated and the effects of unlabelled haloperidol on [3H]-5-HT binding and (+)- and (-)-butaclamol on [3H]-haloperidol were studied. 2 Scatchard analysis did not show any specific binding of [3H]-haloperidol or [3H]-dopamine to platelets, but two binding sites were identified for binding of [3H]-5-HT. 3 Unlabelled haloperidol reduced the binding of 5-HT in concentrations comparable to those inhibigint 5-HT-induced platelet aggregation; (+)- and (-)-butaclamol did not affect the binding of [3H]-haloperidol. 4 It is concluded that haloperidol binding represents saturation of the platelet membrane, and that the platelet is not a suitable model for investigation of dopamine-drug interactions.     

The effect of ethanol on phospholipid metabolism in rat pancreas

The phospholipid effect involves agonist-induced breakdown of phosphatidyl inositol (or polyinositides) generating second messengers followed by increased incorporation of 32P during the resynthetic phase of the cycle. Ethanol, an aetiological factor in pancreatitis, has been shown to have various effects on pancreatic secretion. In this study ethanol decreased the incorporation of 32P into phosphatidyl inositol but had no effect on the stimulated breakdown of prelabelled phosphatidyl inositol. However, in addition to recycling of phosphatidyl inositol stimulation of pancreatic tissue results in increased incorporation of precursors into other phospholipids. Cholecystokinin increased the incorporation of both [U-14C] glucose and 32P into phosphatidyl ethanolamine 3-fold but had no effect on 32P incorporation into phosphatidyl choline. As well as increased incorporation of 32P into phosphatidyl inositol (8-fold) cholecystokinin also increased the incorporation of [U-14C] glucose into phosphatidyl inositol (4-5-fold) implying significant de novo synthesis of 1,2 diacyl glycerol in addition to the currently accepted recycling of the 1,2 diacyl glycerol back to phosphatidyl inositol. Ethanol caused an inhibition of 32P incorporation into total phospholipid of rat pancreas during basal and stimulated conditions. When individual phospholipids were separated ethanol was found to decrease the incorporation of 32P into phosphatidyl choline under basal conditions and into all phospholipids during cholecystokinin stimulation. With [U-14C] glucose as the precursor, ethanol inhibited its incorporation into phosphatidyl choline only. Ethanol did not alter the total 32P radioactivity in the aqueous phase of the pancreatic extract nor the percent incorporated into nucleotides. This excluded decreased uptake of 32P and incorporation into nucleotides as a mechanism for the differential inhibition of 32P versus [U-14C] glucose incorporation into phospholipids other than phosphatidyl choline under stimulated conditions.     

Mechanism of the enrichment of phosphatidylcholine in liver accompanying enzyme induction by phenobarbital

Summary The mechanism of the increase of phosphatidylcholine in liver, accompanying enzyme induction by phenobarbital, has been studied in rats. Using radioactively labeled precursors, the two main pathways of phosphatidylcholine biosynthesis-the CDP-choline pathway and the methylation of phosphatidylethanolamine-were analyzed after pretreatment with 4 doses of phenobarbital (80 mg/kg) on 3 consecutive days.After i.v. injection of choline [Me-³H], choline [Me-¹⁴C] or NaH₂[³²P]O₄ the specific radioactivity (sp. act.) of phosphatidylcholine (dpm/nmol) was decreased by 60%, and after methionine [Me-³H] or ethanolamine [1.2-¹⁴C] by 40% compared to control rats.These changes are partly due to the increased concentration of phosphatidylcholine and phosphatidyl-ethanolamine, causing the incorporated precursors to dilute, and partly to a secondary effect which leads to a reduction of the sp. act. of free choline in pretreated animals.The concentration of glycerylphosphorylcholine, one of the metabolites of phosphatidylcholine catabolism, was also diminished by almost 50%.From these results it may be concluded that the increase of phosphatidylcholine is due to a retardation of its breakdown rather than to an increase of its synthesis.