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).     

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.     

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.     

In vivo meal model for the evaluation of agents which affect the absorption of triglyceride and cholesterol

A meal model in which rats were trained to consume within 2 hr a high fat meal containing glycerol tri[1-¹⁴C]oleate and [³H]cholesterol was compared to the corn oil bolus model. In the meal model, dietary triglyceride was absorbed from the small intestine faster during the first 6–8 hr and more completely than intubated corn oil, as determined by analysis of intestinal contents, serum radioactivity, serum triglycerides, adipose tissue and liver lipids. The effects of Cholestyramine, Colestipol and Pluronic L-101 (1% dietary admixes) on these variables were evaluated for 5 days in the meal model. Lipid absorption during a 72-hr period was reduced by all compounds. The per cent excretion of glycerol tri[1-¹⁴C]oleate was increased significantly by Pluronic L-101 (10-fold), Cholestyramine (5.7-fold) and Colestipol (2.7-fold). The excretion of [³ H]cholesterol was enhanced significantly by Cholestyramine (1.6-fold) and Colestipol (1.3-fold). The following observations were made 4hr after the initiation of the meal. Pluronic L-101 increased significantly the retention of glycerol tri[1-¹⁴C]oleate and [³H]cholesterol in stomach (380 and 375 per cent, respectively), and of glycerol tri[1-¹⁴C]oleate in the small intestine (1100 per cent). The percent of intestinal lipid remaining as triglyce-ride from the intestinal lumen was increased significantly by Pluronic L-101 (160 per cent). Pluronic L-101 reduced significantly [¹⁴C]lipid and [³H]cholesterol in liver; Cholestyramine and Colestipol suppressed only [³H]cholesterol. In adipose tissue, Pluronic L-101 treatment reduced significantly [¹⁴C]lipid content only; Cholestyramine and Colestipol suppressed selectively [³H]cholesterol. After 5 days of treatment, only Pluronic L-101 treatment resulted in significantly reduced serum triglycerides (32 per cent), cholesterol (21 per cent) and glucose (15 per cent). These data suggest that this in vivo meal-feeding model provides a physiological technique for evaluating agents affecting lipid absorption.     

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.     

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 4-chlorobiphenyl on substrate transport and phospholipid metabolism in mouse L5178Y lymphoma cells

The toxicity of 4-chlorobiphenyl, a constituent of Aroclor 1221, was studied in mouse L5178Y cells, in vitro. 4-Chlorobiphenyl had a varied effect on the uptake of small precursor molecules. Uptake of [³H]l-leucine, [³H]l-serine, [³H]uridine and [³H]thymidine was inhibited, while that of [³H]inositol was stimulated. There was no significant effect on either [¹⁴C]ethanolamine or [¹⁴C]choline uptake. However 4-chlorobiphenyl significantly inhibited incorporation of [¹⁴C]ethanolamine into phosphatidylethanolamine and caused a 2- to 3-fold stimulation in the incorporation of [¹⁴C]choline into phosphatidylcholine. This effect on phosphatidylcholine metabolism depended on the adsorption and continued presence of 4-chlorobiphenyl on the cell plasma membrane. The stimulation of [¹⁴C]choline incorporation was reversed when treated cells were placed in fresh growth medium under conditions where 95 per cent of the 4-chlorobiphenyl was desorbed from the cell surface. The effect of 4-chlorobiphenyl on substrate uptake and phospholipid metabolism appears to depend upon the interaction of the agent with the cell membrane surface.     

Alpha 1-adrenergic stimulation of phosphatidylinositol turnover and respiration of brown fat cells

The alpha-adrenergic agonist phenylephrine (in the presence of the beta-adrenergic antagonist alprenolol) stimulated respiration and incorporation of [3H]glycerol and [32P] Pi into phosphatidylinositol of hamster brown fat cells in a concentration-dependent manner. Both responses were preferentially inhibited by prazosin as compared with yohimbine, indicating alpha 1 specificity. Uniquely, prazosin inhibition of phenylephrine-stimulated phosphatidylinositol metabolism had two components, since 30% of the response was inhibited by less than 1 nM prazosin, 10 nM gave no further inhibition, and 100 nM prazosin completely inhibited the response. The phosphatidylinositol response was still present in Ca2+-free buffer, although reduced in magnitude. The concentration relationships of the effects of agonists and antagonists were compared with those of previous results of [3H]prazosin binding and with phenylephrine potency to compete for binding. On the basis of these comparisons, it is suggested that the highly prazosin-sensitive part of the phosphatidylinositol response may be closely associated with receptor occupation.     

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.     

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.     

Synergistic effect of 5-fluorouracil and N-(phosphonacetyl)-l-aspartate on cell growth and ribonucleic acid synthesis in a human mammary carcinoma

The biological effects of $\text{N-(}\text{phosphonacetyl}\text{)-}\text{l}\text{-}\text{aspartate}$ (PALA) and 5-fluorouracil (5-FU) were examined singly, and in combination, on the growth of a human mammary carcinoma (MDA) cell line in culture. All combinations of 5-FU (2.5 × 10^?7 to 1.5 × 10^?5M) and PALA (6.0 × 10^?5 to 3.6 × 10^?3 M) resulted in synergistic inhibition of cell growth as revealed by a 50 per cent isobologram.To examine the biochemical basis for the synergism, measurements of the incorporation of [³H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [¹⁴C]deoxyguanosine and [³H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10^?5M PALA and 1 × 10^?6 M 5-FU produced 65–85 per cent inhibition of cell growth after continuous treatment for 13 days. Treatment of the cells for 3 or 24 hr with the same drug regimen produced approximately a 170 per cent increase in the incorporation of 1 × 10^?6M [³H]-5-FU into poly(A)RNA in comparison to [³H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10^?5 M PALA and 1 × 10^?6 M [³H]-5-FU resulted in a 285 per cent increase in the incorporation of [³H]-5-FU into non-poly(A)RNA. The incorporation of either [¹⁴C]deoxyguanosine or [³H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [³H]deoxyuridine into DNA was elevated significantly upon 12 or 24 hr of exposure to PALA alone. PALA and 5-FU treatment resulted in a 75 per cent reduction in the concentration of UTP and no change in the concentration of 5-fluorouridine-5′triphosphate 5-FUTP) versus 5-FU treatment alone. Thus, the proportion of 5-FUTP in the total 5FUTP + UTP pool was enhanced more than 3-fold by the combination regimen. These results indicate that the synergistic effect of the combination of PALA and 5-FU on the growth of MDA cells correlates with an increased proportion of 5-FUTP in the pyrimidine nucleotide pool and, consequently, with an enhanced incorporation of 5-FU into RNA, but not with inhibition of DNA synthesis.     

Metabolism of [3H]epinephrine in rabbit fetal tissues

[³H]epinephrine was injected into rabbit fetuses through the uterine wall on day 26 of gestation and its metabolism was studied in fetal tissues. [¹⁴C]metanephrine (MN) was synthesized using a soluble rat liver preparation and it, along with [¹⁴C]epinephrine, was used as recovery markers for the quantitation of metabolites of [³H]epinephrine. The radiochemical purity of MN, the predominant metabolite isolated from the fetal liver, was achieved by obtaining a constant [³H]/[¹⁴C] after sequential thin-layer chromatography. Thirty min after the injection of labeled hormone. [³H]epinephrine comprised 25 per cent of the radioactivity in the serum and 30 per cent in the heart, while less than 5 per cent was found in the lung, liver, kidney and intestine. Unconjugated MN comprised 45 per cent of the [³H] in the serum, 63 per cent in the heart and lung and 47, 46 and 40 per cent in the liver, kidney and intestine respectively. Conjugated MN is only found in liver, kidney and intestine and accounts for 35–40 per cent of the tissue radioactivity. Enzymatic hydrolysis of conjugates with β-glucuronidase in the presence and absence of sulfatase indicates that MN is present as a glucuronide conjugate. O-methylated, deaminated metabolites accounted for 27 per cent of the radioactivity in the serum, 28 per cent in the lung and between 7 and 16 per cent in the heart, liver, kidney and intestine. Thus, in the fetal rabbit, O-methylation is the predominant route of metabolism of injected [³H]epinephrine, followed by extensive glucuronide conjugation in the fetal liver, kidney and intestine.     

Inhibition of sterologenesis but not glycolysis in 2,5-hexanedione-induced distal axonopathy in the rat

If glycolysis is inhibited in distal axonopathy, there should be a concomitant inhibition of lipogenesis from glucose. To investigate this possibility, lipogenesis from [¹⁴C]glucose and [³H]acetate was studied in sciatic nerves incubated with iodoacetate, a known inhibitor of glycolysis, in sciatic nerves incubated with 2,5-hexanedione, a putative inhibitor of glycolysis, and in sciatic nerves from rats exhibiting clinical signs of peripheral neuropathy induced by 2,5-hexanedione. Nerves incubated with 1.0 mm iodoacetate, in comparison with untreated nerves, exhibited decreased incorporation of [¹⁴C]glucose into sterols + diacylglycerols (33-fold), free fatty acids (14-fold), triacylglycerols (27-fold), and phospholipids (21-fold). In addition, these nerves exhibited decreased incorporation of [³H]acetate into sterols + diacylglycerols (30-fold), free fatty acids (2-fold), triacylglycerols (23-fold), and phospholipids (12-fold). In contrast, the incorporation of [¹⁴C]glucose into sterols + diacylglycerols, free fatty acids, and triacylglycerols was not affected by 1.0 mm 2,5-hexanedione. Compared to untreated nerves, nerves incubated with 1.0 mm 2,5-hexanedione exhibited a small decrease (15%) in the incorporation of [¹⁴C]glucose into phospholipids. Nerves from rats given 1% 2,5-hexanedione in the drinking water for 6 weeks, in comparison with those from pair-fed control rats, exhibited decreased (45%) incorporation of [¹⁴C]glucose and [³H]acetate into digitonin-precipitable sterols. Nerves from 2,5-hexanedione-treated and pair-fed control rats exhibited similar incorporation of [¹⁴C]glucose and [³C]acetate into free fatty acids, triacylglycerols, and phospholipids. The data indicate that while sterologenesis is inhibited in distal axonopathy, glycolysis is not.     

Effects of pentobarbital and veratridine on phosphatidylinositol and phosphatidate metabolism in rat parotid acinar cells

The metabolism of phosphatidylinositol and phosphatidate was studied in isolated rat parotid cells, incubated in a physiological buffer containing [32P]phosphate or [3H]glycerol. Carbamylcholine and epinephrine stimulated 32P incorporation into both of these phospholipids, causing their half-maximal effects at 2 and 0.8 microM respectively. The former concentration is much lower than that anticipated from binding studies. The Hill coefficients for carbamylcholine activation of 32P incorporation were 0.61 +/- 0.05 for phosphatidate and 0.64 +/- 0.05 for phosphatidylinositol. Pentobarbital (0.58 mM) inhibited the increased 32P incorporation caused by 5 microM carbamylcholine but not 100 microM carbamylcholine. Pentobarbital inhibited the incorporation of 3H equally in the presence and absence of epinephrine, indicating that the effect of pentobarbital on 32P incorporation is on turnover and not on de novo synthesis. Veratridine (200 microM) had no effect on phospholipid metabolism in the presence and absence of either carbamylcholine or epinephrine, which contrasts with our previous findings in synaptosomes [J.C. Miller and I. Leung, Biochem. J. 178, 9 (1979)].     

Pelanserin inhibition of serotonin-induced phosphatidylinositol turnover and contraction in rabbit aorta

Stimulation of rabbit aortic rings with serotonin or quipazine increased the incorporation of [³²P]Pi into phosphatidylinositol (Pl) and induced a dose-related contraction. The effects of serotonin and quipazine were blocked by 5-HT₂ antagonists with the following order of potency: pelanserin = ketanserin ? methysergide. Indorenate, a 5-HT₁ agonist, failed to modify phosphatidylinositol labeling in rabbit aorta but elicited a very weak contraction at high concentrations. Pelanserin blocked epinephrine-stimulated phosphatidylinositol labeling, being two orders of magnitude less potent than prazosin. The results demonstrate that pelanserin is a potent antagonist of the stimulation of Pl labeling and vasoconstrictor effects of 5-hydroxytryptamine and suggest that 5-HT₂ blockade is involved as its major pharmacologic action.     

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.     

Deoxyribonucleic acid binding of 3-hydroxy- and 9-hydroxybenzo[a]pyrene following further metabolism by mouse liver microsomal cytochrome P1-450.

In the presence of NADPH and microsomes from 3-methylcholanthrene-treated C57BL/6N mice, [³H]3-OH-benzo[a]pyrene is metabolized to reactive intermediates which covalently bind to deproteinized salmon sperm DNA in vitro. Enzymatically digested DNA, containing bound [³H]3-OH-benzolajpyrene derivatives, generates an elution profile from Sephadex LH20 chromatography which resembles similar chromatograms with [³H]benzo[a]pyrene. All peaks resulting from [³H]benzo[a]pyrene activation appear to be prominently represented in [³H]3-OH-benzo[a]pyrene activation, except that several peaks which emerge near the end of the eluting gradient of methanol and water are much reduced. Notably, a peak designated E, and attributed to benzo[a]pyrene-7, 8-diol-9, 10-oxide binding in [³H]benzo[a]pyrene incubations, is also prominently represented in incubations with [³H]3-OH-benzo[a]pyrene. Radioactivity in all of these peaks is inhibited effectively if one-seventh the concentration of 1-OH-benzo[a]pyrene is included in the incubation with [³H]3-OH-benzo[a]pyrene. Microsomes from 3-methylcholanthrene-treated DBA/ 2N mice cause insignificant binding. UDP-glucuronic acid markedly reduces all peaks except E, and 1, 2-epoxy-3, 3, 3-trichloropropane reduces all peaks except C and E. 9-Hydroxybenzo[a]pyrene is further metabolized to DNA binding species by microsomes from either 3-methylcholanthrene-treated DBA/2N or C57BL/6N mice. UDP-glucuronic acid prevents about 50 per cent of the binding with microsomes from DBA/2N mice but not with microsomes from C57BL/6N. In contrast, UDP-glucuronic acid does prevent binding in some of these same peaks when [³H]benzotaipyrene is the starting substrate with microsomes from C57BL/6N mice. UDP-glucuronic acid does not prevent binding in peak E in incubations with [³H]benzo[a]pyrene or [³H]3-hydroxybenzo[a]pyrene.     

Radioisotope studies of purine metabolism during administration of guanine and allopurinol in the pig

In pigs pre-fed guanine, some 33 per cent of [8-¹⁴C]guanine administered orally appeared in the urine in 24 hr, principally in the form of allantoin. Little incorporation (less than 1 per cent) of radioactivity into body tissues occurred and only 5 per cent of the radioactivity could be found in the faeces.When allopurinol was added to the guanine diet the pattern of excretion of [8-¹⁴C]-guanine changed considerably. Only 11 per cent of the radioactivity was recovered from the urine in 24 hr while 83 per cent appeared in the faeces in 3 days. Again, less than 1 per cent of the radioactivity was found in the tissues at slaughter.Intravenous administration of [8-¹⁴C]guanine to a pig on the above mixture resulted in the rapid incorporation of approximately 50 per cent of the radioactivity into body tissues with a slow subsequent daily excretion of approximately 2 per cent of this radioactivity in faeces and urine. The finding of 13 per cent of the radioactivity in the faeces is considered evidence of purine excretion into the gut. The recovery of urinary radioactivity (34 per cent of dose) principally in xanthine, but also in hypoxanthine, showed the existence of a rapid additional route of guanine catabolism via hypoxanthine. Experimental evidence is also presented to demonstrate the existence of a reciprocal relationship between urinary [¹⁴C]hypoxanthine and allopurinol riboside excretion suggesting competitive inhibition of allopurinol riboside formation by hypoxanthine in vivo.In the allopurinol treated pig, orally administered [6-¹⁴C]allopurinol was rapidly absorbed and almost totally excreted in the urine in 24 hr (90 per cent). The remainder of the radioactivity (approximately 7 per cent) was excreted in the faeces in 3 days and no radioactivity could be detected in tissue nucleic acids or in tissues to any extent (less than 0.01 per cent of the dose).The significance of these results in relation to the metabolic studies is discussed.