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.     

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.     

Effect of ethanol on phospholipid turnover and calcium mobilization in chick embryos

Effects of ethanol on [3H]inositol and [14C]choline incorporation into phosphatidylinositol (PI) and phosphatidylcholine (PC), free intrasynaptosomal Ca2+ ([Ca2+]i) and synaptosomal 45Ca2+ uptake, were investigated in the brain and heart of 17-day-old chick embryos to which a 10% ethanol solution had been injected on the 3rd day of embryogenesis. In brain synaptosomes, ethanol increased the incorporation of [3H]inositol and [14C]choline into PI and PC, increased [Ca2+]i, and decreased 45Ca2+ uptake. On the other hand, in heart synaptosomal membrane, ethanol decreased the incorporation of [3H]inositol and [14C]choline into PI and PC, decreased [Ca2+]i, and increased 45Ca2+ uptake. Ethanol stimulated in vitro [3H]inositol and [14C]choline incorporation into PI and PC in the brain and heart in both the control and ethanol-treated groups. However, addition of ethanol did not affect the release of 45Ca2+ from the synaptosomal membrane of either organ in either group. Addition of ethanol inhibited 45Ca2+ uptake in a dose-dependent manner in the brain but not in the heart. In both organs, there was a relationship between phospholipid turnover and [Ca2+]i after ethanol.     

Decreased Biosynthesis of Lung Surfactant Constituent Phosphatidylcholine Due to Inhibition of Choline Transporter by Gefitinib in Lung Alveolar Cells

Purpose We investigated whether gefitinib, an anticancer agent, inhibits phosphatidylcholine (PC) biosynthesis and choline uptake by alveolar epithelial type II cells. Materials and Methods Uptake of choline and PC biosynthesis were examined in vitro, using human alveolar epithelia-derived cell line A549 and rat alveolar type (AT) II cells as models. Results Gefitinib reduced the incorporation of [³H]choline into PC in A549 and rat ATII cells. The uptake of [³H]choline by A549 and rat ATII cells was concentration-dependent, and the Km values were 15.0 and 10–100 μM, respectively. The uptake of [³H]choline by A549 and rat ATII cells was weakly Na⁺-dependent, and inhibited by hemicholinium-3. RT-PCR revealed expression of choline transporter-like protein (CTL)1 and organic cation transporter (OCT)3 mRNAs in both cells. The choline uptake by A549 and rat ATII cells was strongly inhibited by gefitinib with the IC₅₀ value of 6.77 μM and 10.5 μM, respectively. Conclusions Our results demonstrate that gefitinib reduces PC biosynthesis via inhibition of cellular choline uptake by A549 and rat ATII cells, which is mainly mediated by CTL1, resulting in abnormality of lung surfactant that can be one of mechanisms of the interstitial lung disease associated with gefitinib.     

Evidence of differential effects produced by ethanol on specific phospholipid biosynthetic pathways in rat hepatocytes.

1. The aim of the present study was to investigate the effects of ethanol in vitro on the phospholipid biosynthetic pathways in hepatocytes isolated from the rat. We have used [methyl-14C]-choline, [1-3H]-ethanolamine and L-[3-3H]-serine as exogenous precursors of the corresponding phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). 2. Incubation of hepatocytes in the presence of ethanol significantly alters the incorporation of radiolabel from [14C]-choline and [3H]-ethanolamine into the metabolic intermediates and the final products of the CDP-choline and CDP-ethanolamine pathways. Radioactivity in the metabolic intermediates of both pathways was significantly decreased and the amount of label in PE was reduced whilst that of PC was not modified. 3. In the presence of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase (ADH) activity, ethanol produces a reduction in the label of choline phosphate, ethanolamine phosphate and a significant decrease in the amount of PC and PE radiolabel. 4. On the other hand, ethanol increases the incorporation of serine into phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, although this effect is observed only in the absence of 4-methylpyrazole, indicating that this alteration is produced by some metabolite generated as a consequence of hepatic alcohol metabolism. 5. Ethanol also interferes with the methylation of phosphatidylethanolamine produced via the CDP-ethanolamine pathway but it does not alter phosphatidylethanolamine methylation when this phospholipid is produced by mitochondrial phosphatidylserine decarboxylation, suggesting the existence of different intramembrane pools of phosphatidylethanolamine, which may exhibit different sensitivity to alcohol. 6. Our results indicate that ethanol exerts two different effects on phospholipid metabolism in hepatocytes: a stimulatory effect on the incorporation of exogenous substrates into different phospholipids probably related to an alteration in the availability of lipogenic substrates as a consequence of ethanol metabolism, and another inhibitory effect produced by ethanol per se, which can be observed only when ethanol metabolism is inhibited by the presence of a specific inhibitor of alcohol dehydrogenase activity.     

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.     

Effect of ethanol on calcium-uptake and phospholipid turnover by stimulation of adrenoceptors and muscarinic receptors in mouse brain and heart synaptosomes

The effect of ethanol treatment on mouse brain and heart synaptosomal 45Ca uptake and the incorporation of [3H]inositol and [14C]choline into phosphatidylinositol (PI) and phosphatidylcholine (PC) were investigated. Ethanol in drinking water (15%) was given to mice for 3 weeks. The consumption of ethanol increased gradually during treatment but food intake was almost the same as control. The body weight of ethanol-treated mice was slightly less than that of control. The synaptosomal lipid peroxidation level of ethanol-treated mice was almost the same as control in the brain and heart. On the other hand, the synaptosomal glutathione level of ethanol-treated mice was higher than control in both brain and heart. The 45Ca uptake of brain and heart from ethanol-treated mice was 87% and 216% of control mice, respectively. Not only ethanol but also norepinephrine (NE), carbachol (Carb), or isoproterenol (IsoPro) added in vitro increased 45Ca uptake in all cases. The incorporation of [3H]inositol into PI in the brain and heart synaptosomes of ethanol-treated mice was 150% and 113% of control, respectively. The incorporation of [14C]choline into PC in the brain and heart of ethanol-treated mice was 104% and 125% of control, respectively. In vitro addition of ethanol, NE, Carb or IsoPro to brain synaptosomes increased the incorporation of [3H]inositol and [14C]choline into PI and PC, respectively, in both control and ethanol-treated mice. In the case of heart synaptosomes, NE and Carb increased the incorporation of [3H]inositol and [14C]choline into phospholipids in control mice but not ethanol-treated mice. However, IsoPro increased the incorporation by both control and ethanol-treated heart synaptosomes. These results suggest that alpha-adrenoceptors and the cholinergic system of the heart play important roles in modulating the toxic effects of ethanol.     

Choline metabolism in human term placenta— Studies on de novo synthesis and the effects of some drugs on the metabolic fate of [N-methyl-3H]choline

Fragments from human term placentae were incubated with [³H]methyl-Iabeled methionine or S-adenosyl methionine as methyl donors and ethanolamine or phosphatidyldimethylethanolamine as methyl acceptors. Analysis of acid-soluble and lipid-soluble extracts by high voltage electrophoresis or thin-layer Chromatography, respectively, gave no indication for the synthesis of products which would be expected to contain [³H]label in the choline (Ch) moiety. These findings led to the conclusion that the placenta could not perform the methylation steps required for de novo synthesis of choline. Short incubations (1, 2.5, 5 and 10 min) with [³H]Ch (5 μM, 0.8 μCi/ml) and paraoxon (10 μM) as an inhibitor of cholinesterases revealed that [³H]Ch was quite rapidly incorporated into [³HJacetylcholine (ACh) and that 18 per cent of the acid-soluble radioactivity was associated with ACh after 1 min. In the lipid-soluble fraction the major labeled product was identified as phosphatidylcholine while a minor amount of [³H]( < 5 per cent) was found in lysolecithin. Paraoxon was without effect on [³H]Ch uptake into fragments. However, its omission significantly—yet much less than expected from innervated tissues—decreased [³H] in ACh after 10 min (28 vs 44 per cent) and 20 min (37 vs 62 per cent) and increased the radioactivity remaining as free [³H]Ch. 2,4-Dinitrophenol reduced [³H]Ch uptake and inhibited preferentially the incorporation of tritium into phosphorylcholine (5 min: 2.5 vs 5.4 per cent; 10 min: 3.0 vs 9.7 per cent; 20 min: 6.5 vs 15.6 per cent; and 30 min: 7.5 vs 20.0 per cent). Removal of Na⁺ ions accelerated the uptake of [³H]Ch, but it depressed the synthesis of [³H]ACh. In Na⁺-free medium the percentage of acid-soluble radioactivity associated with ACh was always significantly lower (2.5 min: 9.8 vs 23.3 per cent; 5 min: 13.0 vs 35.9 per cent; 10 min: 19.7 vs 44.9 per cent; and 20 min: 26.8 vs 62.3 percent). Thus, although there was no indication for a high-affinity, Na³-dependent [³H]Ch uptake in placenta, ACh synthesis was markedly affected by lack of Na⁺ ions.     

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.     

An in vitro study of hemicholinium-3 on phospholipid metabolism of Krebs II ascites cells

With [Me-¹⁴C]choline as marker and after separation of choline and phosphocholine by ion-exchange column chromatography or thin layer chromatography on alumina, it is shown that 40 μM hemicholinium-3 (HC-3) inhibits the cytosolic choline-kinase of rat liver and Krebs cells. This inhibition is competitive (Km different, Vm similar) in the first case and mixed in the second (Km and Vm different). Despite this general inhibition of the phosphocholine formation, the synthesis of phosphatidylcholine (PC) by post-nuclear supernatants of rat liver and Krebs cells is different when tested with HC-3. It is unaffected in rat liver; however, HC-3 induces a PC deficiency in Krebs cells which is time-course dependent between 15 and 120min and proportional to the drug concentrations in the interval 5–40 μM. Incorporation of AT-[γ³²P] or [2-¹⁴C]ethanolamine into phospholipids shows that the sequential methylation pathway is not detectable in Krebs cells. These results are discussed in relation to those established concerning HC-3 action on phospholipid metabolism in other tissues.     

Action of hemicholinium-3 on phospholipid metabolism in Krebs II ascites cells

Incorporation of [Me-14C]choline or/and [2-14C]ethanolamine into phospholipids of Krebs II ascites cells in toto have been tested in the presence of hemicholinium-3. With [Me-14C]choline, labelling of cell pellet, intracellular choline, phosphocholine and total lipid extract is inhibited by hemicholinium-3 in a dose-dependent way between 6.25 X 10(-6) M and 10(-3) M. These effects are caused by a diminution of the choline or/and ethanolamine transport across the cell membrane and by a choline-kinase inhibition. In Krebs cells, choline is taken up by a low affinity Na+ sensitive uptake system (KT = 46 X 10(-6) M) which is competitively inhibited by hemicholinium-3 (KTi = 161 X 10(-6) M). Krebs cells exert a counter-transport (i.e. an exchange of choline across the membrane) against a concentration gradient of 10 mM choline whereas 10 mM hemicholinium-3 has no effect. Choline-kinase is also inhibited (I50 = 57 X 10(-6) M) in Krebs cells in toto and time-course data suggest that choline transport and phosphorylation might be tightly coupled. Specific radioactivities of phosphocholine and choline-glycerophospholipids decrease owing to the effect of the drug on the uptake and phosphorylation system. With 4 X 10(-5) M hemicholinium-3 and [Me-14C]choline as a marker, labelled choline-glycerophospholipids are decreased by 22%. With [2-14C]ethanolamine, labelled ethanolamine-phospholipids are decreased by 26% and choline-glycerophospholipids remain unlabelled. With the two markers, the additional effect produces a 35% decrease. It is concluded that hemicholinium-3 might be able to induce a depression of the intracellular choline and phosphocholine pool which could provoke a serious quantitative deficiency of major phospholipids in Krebs cells.     

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.     

Effect of typical inducers of microsomal drug-metabolizing enzymes on phospholipid metabolism in rat liver.

The effect of administration of phenobarbital (PB), polychlorinated biphenyls(PCBs) or 3-methyl-cholanthrene (3-MC) on the metabolism of phospholipids in rat liver was studied by the i.p. injection of [³²P]orthophosphate or [Me-¹⁴C]choline chloride. The inducers were given to animals for 2 successive days PB had no significant effect on the incorporation rate of ³²Pi into liver microsomal phospholipid classes 48 hr after the first administration. The rate of incorporation of [¹⁴C]choline into phosphatidylcholine (PC) in both subcellular components of the liver and blood plasma decreased slightly at this experimental period. In addition, the ratio of [¹⁴C] sp. act. of phosphorylcholinc to that of microsomal phosphulipid was considerably higher on PB-trcated rats as compared with the ratio in control rats. These and previous findings strongly suggest that proliferation of liver endoplasmic reticulum (ER) membranes induced by PB would be accompanied by the stimulation of phospholipid synthesis at the early process of induction and subsequently followed by the decrease in turnover rate of microsomal phospholipids. The administration of PCBs, on the other hand, caused strong inhibition of both ³²Pi and ¹⁴C incorporation into PC in liver subcellular fractions. The secretion of PC from liver cells to blood plasma was also strongly depressed. In addition, phospholipid catabolizing activity was found to be depressed in the liver. These results indicate that hypertrophy of ER membranes in the liver after PCBs administration could be due to a depression of both secretion of lipoprotein from liver cells to plasma and catabolic activity toward membranous phospholipids in liver cells. The decrease in ³²Pi incorporation into liver microsomal PC was also observed in rats treated with 3-MC. There occurred a considerable accumulation of ¹⁴C activity in phosphorylcholine in the liver of rats treated with either PCBs or 3-MC, suggesting strongly that these drugs caused an inhibition of PC synthesis at the site of CDP-choline formation, namely the inhibition of the reaction catalyzed by cholinephosphate cytidylyltransferase.     

Effect of morphine in vivo on uptake of [3H]choline and release of [3H]acetylcholine from rat striatal synaptosomes

The effect of morphine on the rat striatal cholinergic system was investigated in vitro by measuring the rates of [³H]choline uptake and [³H]acetylcholine release in striatal synaptosomes after in vivo injections of morphine sulfate. Morphine caused a 50 per cent increase in the V_max of [³H]choline uptake. Although a concomitant increase was also measured in the amount of [³H] acetylcholine released, it could be explained by the previous increase in uptake. It is suggested that morphine had an overall stimulatory effect on the striatal cholinergic system which may be a transynaptic phenomenon rather than a direct effect on the cholinergic cell.     

Alterations in lipid metabolism and cellular morphology in the liver of female rats treated with ethmozine

Alterations in lipid metabolism and cellular morphology in the liver were examined in female rats treated with 100 mg Ethmozine/kg/day for 7 days. The incorporation of either [3H]acetate with [methyl-14C]choline, or [methyl-14C]methionine was used to monitor the effect of the drug on neutral and phospholipid syntheses. Ethmozine (ETH) reduced the incorporation of choline into phosphatidylcholine (PC) by 50%, but the transmethylation of phosphatidylethanolamine to form PC was unaffected. The formation of lyso-PC was reduced by one-half irrespective of the donor radiolabel. An accumulation of both micro- and macrovesicles (fat) was found in the centri- and midlobular zones of the liver, which is likely the result of increased synthesis and decreased secretion of triacylglycerol (TAG). Incorporation of acetate into TAG was increased fivefold by ETH treatment, and to a lesser degree into cholesterol and cholesterylester/squalene.     

Altered lipid metabolism in 2,5-hexanedione-induced testicular atrophy and peripheral neuropathy in the rat

2,5-Hexanedione (HD) induces testicular atrophy and peripheral neuropathy in rats. Since altered lipid metabolism is frequently associated with these disease states, lipid metabolism was investigated in vitro in testes and sciatic nerves of rats fed 1% HD in the drinking water for 6 weeks. Testes from HD-treated rats were 30–60% smaller and weighed threefold less than testes from pair-fed control (PFC) rats. Compared to testes from PFC rats, testes from HD rats exhibited increased incorporation of [¹⁴C]acetate into phospholipids (344%), triacylglycerols (281%), and cholesteryl esters + hydrocarbons (246%) but decreased incorporation into free fatty acids (25%) and sterols + diacylglycerols (65%). The increased incorporation of [¹⁴C]acetate into phospholipids induced by HD reflected an approximate 300% increase into phosphatidyl choline, lysophosphatidyl choline, phosphatidyl serine + phosphatidyl inositol, and phosphatidyl ethanolamine and a disproportionate 800% increase into sphingomyelin. HD rats exhibited clinical signs of peripheral neuropathy, including everted and flat foot placement and hindlimb weakness; similar changes were not observed in PFC rats. In sciatic nerves, the incorporation of [¹⁴C]leucine was decreased into sterols + diacylglycerols (47%), digitoninprecipitable sterols (45%), and cholesteryl esters + hydrocarbons (40%) in HD compared to PFC rats; incorporation of [¹⁴C]leucine into free fatty acids, triacylglycerols, and phospholipids was similar in HD and PFC rats. In contrast to the testis and nerve, lipid metabolism in the liver was similar in HD and PFC rats. The concentrations of 2,5-hexanedione and 2,5-dimethylfuran, respectively, were 0.6 ± 0.3 and 6.5 ± 0.9 μg/g wet weight in the testes and 3.1 ± 0.4 and 3.0 ± 0.4 μg/g wet weight in the livers of HD rats. The data indicate that altered metabolism is associated with HD-induced testicular atrophy and distal axonopathy.     

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.     

Uptake and metabolism of [3H]choline by the rat phrenic nerve-hemidiaphragm preparation

Summary A whole nerve-muscle preparation (about 160 mg) or an end-plate preparation (about 25 mg) of the rat phrenic nerve-hemidiaphragm were incubated with [³H]choline, to investigate choline uptake and choline metabolism. Choline uptake was measured from the disappearance of choline from the incubation medium during the loading period and from the retention of tritium in the tissue after the loading and washout period.Based on the results obtained with both methods the end-plate preparation takes up three times as much choline than the whole nerve-muscle preparation or a small muscle strip that was cut outside the end-plate region and had a similar size as the end-plate preparation. Choline uptake was not markedly affected by the degree of nerve activity or by a chronic denervation. However, hemicholinium-3 significantly reduced (50%) the choline uptake by the end-plate preparation.Most of the choline (70–88%) taken up was metabolized and incorporated into membrane structures. Phosphatidylcholine was the predominant metabolite in both preparations. The ratio of phosphatidylcholine/lysophosphatidylcholine in the end-plate preparation (16) was significantly lower than in the whole nerve-muscle preparation (31). This might indicate a higher metabolism of phosphatidylcholine in the end-plate preparation.It is suggested that choline uptake by the rat phrenic nerve-hemidiaphragm occurs mainly by the muscle fibres. The innervated part of the muscle fibres can accumulate more choline than the peripheral part outside the end-plate region, probably because of a very active choline phospholipid metabolism within the end-plate region. Send offprint requests to I. Wessler at the above address     

METABOLISM AND PRESYNAPTIC INHIBITORY EFFECTS OF ADENOSINE IN RAT VAS DEFERENS

• 1 In the rat vas deferens, adenosine caused rapid presynaptic inhibition of the twitch response stimulated at 0.2 Hz, this effect being maximal within 30 sec. The metabolism of [¹⁴C]adenosine was examined under these conditions. For 2–40juM adenosine and for incubations up to 120 sec, deamination of adenosine was quantitatively greater than incorporation into adenine ribonucleotides.
• 2 Exposure of tissues to 2juM deoxycoformycin reduced deamination of 10juM [¹⁴C]adenosine by about 90%, but did not alter the presynaptic inhibitory action of adenosine. Exposure of tissues to 1juM hydroxy-nitrobenzylthioguanosine, an adenosine uptake inhibitor, reduced the incorporation of 2–40juM [¹⁴C]-adenosine into adenine ribonucleotides by 25–39% and potentiated the presynaptic inhibitory action of adenosine by 46–105%.
• 3 Postganglionic sympathetic denervation of the tissue did not alter the metabolism of [¹⁴C]adenosine, indicating that the metabolism observed occurred almost exclusively in smooth muscle.
• 4 It is concluded that the concentration of adenosine in the immediate vicinity of the presynaptic adenosine receptor is not greatly affected by deamination but is influenced by adenosine uptake. It is also possible that hydroxynitrobenzylthioguanosine may modify the affinity or intrinsic activity of the presynaptic adenosine receptor.

     

The effect of isoproterenol on the metabolism of phosphatidylinositol by rat heart in vitro

The effect of epinephrine and isoproterenol (10^?4 M) on the incorporation of ³²P orthophosphate and [1-¹⁴C] palmitic acid into rat heart slices was investigated in pulse and chase experiments. Epinephrine stimulated the uptake of ³²P into phosphatidic acid and phosphatidyl-inositol. Isoproterenol stimulated the labelling of phosphatidic acid but had no effect on phosphatidyl-inositol metabolism in the first 60 min of the incubation. Propranolol stimulated the incorporation of ³²P and [1-¹⁴C] palmitic acid into phospholipids in the first 20–30 min of the incubation but counteracted isoproterenol thereafter. These results indicate that the main effect of isoproterenol on phospholipid metabolism in rat heart is to increase the turnover of phosphatidic acid and to slow down the conversion of phosphatidic acid into phosphatidylinositol.