Phospholipid metabolism of stimulated lymphocytes: Comparison of the activation of acyl-CoA: Lysolecithin acyltransferase with the binding of concanavalin A to thymocytes

Calf thymocytes were isolated and incubated with concanavalin A. The effect of the mitogen on the enzyme activity of membrane-bound lysolecithin acyltransferase (acyl-CoA: $\text{1-acylglycero-3-phosphorylcholine}\text{-O-}\text{acyltransferase}$, EC 2.3.1.23) was determined as also the binding of ¹²⁵I-labelled concanavalin A to intact cells and isolated membranes.The lysolecithin acyltransferase was found to be activated three times in microsomal membranes. The activation occurred directly after binding of concanavalin A and was temperature independent, since similar activities were found in cells treated with concanavalin A at 0 and 37 °C.The acyltransferase activation using increasing concentrations of concanavalin A revealed a different behaviour, as compared to the binding of concanavalin A. While the binding of concanavalin A to intact cells expressed a normal hyperbolic saturation function the activation process of the acyltransferase described a sigmoidal relationship. Corespondingly, the interaction coefficients for both functions were different (Sips coefficient for binding = 1.0 and Hill coefficient of the enzyme activation = 1.8).These results indicate that the acyltransferase activation is due to a cooperative interaction between the ligand-receptor complex and the enzyme.     

Activation of guanylate cyclase and inhibition of adenylate cyclase by cytotoxic preparations from marine sponges of Haliclona genus.

Of seven marine sponges tested only two, $\text{Haliclona}\text{viridis}$ and $\text{Haliclona}\text{rubens}$, yielded preparations that activated rat heart microsomal guanylate cyclase and exhibited direct hemolytic activity. These two preparations also inhibited basal and fluoride-activated adenylate cyclase in rat heart microsomes and glucagon-stimulated adenylate cyclase in rat liver plasma membranes. Hemolytic activity co-purified with nucleotide cyclase-modulating activity during a standard lipid fractionation procedure. This fraction was cytotoxic to 3T3-4a Swiss mouse fibroblasts.     

Topography of phosphatidylcholine,phosphatidylethanolamine and triacylglycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of these enzymes are located on the external surface of microsomal vesicles.It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

On the mechanism of ethanol-induced accumulation of triglycerides in the liver

The possibility that ethanol or acetaldehyde has a direct effect on the activity of acyl-CoA-ligases or $\text{sn}$-glycerophosphate acyltransferases or on the biosynthesis of phosphatidic acid and triglycerides from free fatty acids was studied with subcellular preparations from rat liver. No stimulatory effect of ethanol or acetaldehyde could be observed in any case. It was further shown that the microsomal fraction of homogenate of livers of rats treated with ethanol (single peroral dose of 4.5 g of ethanol per kg body weight) did not have an increased capacity to biosynthesize phosphatidic acid. The possibility was excluded that excess cofactors necessary for formation of phosphatidic acid are responsible for the higher accumulation of triglycerides in livers of rats treated with ethanol.The results indicate that the increased formation of triglycerides in liver of rats treated with ethanol is not due to increased activity of acyl-CoA-ligase or $\text{sn}$-glycerophosphate acyltransferase or due to increased availability of $\text{sn}$-glycerophosphate, ATP or CoA-SH. It is suggested that increased availability of fatty acids is the major explanation for the increased accumulation of triglycerides in the liver after ethanol administration.     

Adenosine 3',5'-monophosphate-dependent protein kinase and amylase secretion from rat parotid gland

Tolbutamide at a concentration of 10 mM inhibited cyclic AMP-dependent protein kinase in cell-free preparations of rat parotid glands as reported in rat adipose tissues. Incubation of rat parotid slices with 10 mM tolbutamide markedly interfered with the isoproterenol stimulation of amylase secretion. A carboxy derivative of tolbutamide, 1-butyl-3-$\text{p}$-carboxyphenylsulfonylurea, had minimal inhibitory effects both on protein kinase activity and on amylase secretion. These evidences strongly suggest the participation of cyclic AMP-dependent protein kinase in amylase secretion.     

Induction,modification and inhibition of rat liver microsomal benzo(a)pyrene hydroxylase; Correlation with the S-9-mediated mutagenicity of benzo(a)pyrene

The effects of various pretreatments $\text{in vivo}$ (3MC, PB, 2 and 4FAA) and of various inhibitors $\text{in vitro}$ (7,8 BF, SKF525A and MN ^R) on the activity of rat liver microsomal BP hydroxylase were analyzed and correlated with the S-9 mediated mutagenicity of BP. 3MC is the only treatment which both induces and modifies the hydroxylase activity; it also specifically increases the enzyme mediated mutagenicity. Miconazole ^R which inhibits all the tested microsomal preparations, also reduces the mutagenicity mediated by all the S-9 preparations whereas the inhibitory effects of 7,8 BF and SKF525A are limited respectively to enzyme preparations from 3MC induced and control or PB treated rats.     

The demonstration of a cooperative action of bacterial and intestinal mucosa enzymes in the activation of mutagens.

While 2-aminoanthracene and 2-aminofluorene are converted to frameshift mutagens by microsomal preparations from rat livers, the microsomes from the intestinal mucosa of the same animals, under the experimental conditions used herein, either have little such activity or lack it altogether. Cell-free extracts of the colon anaerobe $\text{Bacteroides}\text{fragilis}$ may exhibit such activity to varying degrees depending upon the conditions of incubation. However mixtures consisting of cell-free extracts from $\text{B}\text{.}\text{fragilis}$ and microsomes from intestinal mucosa demonstrate significant- more than additive- activity in converting these chemicals to mutagens.     

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

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

Cytochrome P-450 and NADPH cytochrome c reductase in rat brain: formation of catechols and reactive catechol metabolites.

Microsomes isolated from whole rat brain were found to contain cytochreme P-450 (0.025 to 0.051 nmoles/mg) and NADPH cytochrome $\text{c}$ reductase activity (26.0 to 55.0 nmoles/mg/min). The oxidation of estradiol to a reactive metabolite that became covalently bound to rat brain microsomal protein was inhibited 63% by an atmosphere of CO:O₂ (9:1), indicating the involvement of a cytochrome P-450 oxygenase. In contrast, this atmosphere had no effect on the binding of either the catechol estrogen, 2-hydroxyestradiol, or several catecholamines to rat brain microsomes. An antibody prepared against NADPH cytochrome $\text{c}$ reductase was found to decrease significantly both the formation of 2-hydroxyestradiol from estradiol by rat brain microsomes and the covalent binding of the catechol estrogen and catecholamines to rat brain microsomal protein.     

Effects of anti-NADPH-cytochrome c reductase and anti-cytochrome b5 antibodies on the hepatic and pulmonary microsomal metabolism and covalent binding of the pulmonary toxin 4-ipomeanol.

An antibody prepared against purified rat liver NADPH-cytochrome $\text{c}$ reductase inhibited both the pulmonary and hepatic microsomal covalent binding of 4-ipomeanol as well as the respective NADPH-cytochrome $\text{c}$ reductase activities, findings which are consistent with previous studies which indicated the participation of cytochrome P450 in the metabolic activation of the toxin. An antibody prepared against purified rat liver cytochrome b₅, which strongly inhibited both the rat hepatic and pulmonary NADH-dependent cytochrome $\text{c}$ reductases, and was inactive against the respective NADPH-dependent cytochrome $\text{c}$ reductases, had little effect on metabolic activation of 4-ipomeanol by hepatic microsomes, but strongly inhibited both the NADH-supported and the NADPH-supported pulmonary microsomal metabolism and covalent binding of the compound. These results suggest that metabolic activation of 4-ipomeanol involves a two-electron transfer in which transfer of the second electron via cytochrome b₅ is rate-limiting in lung microsomes.     

Biosynthesis of O-acyl ethanediol phosphorylcholine in a cell-free system from rat liver.

1-$\text{0}$-Hexadecanoyl [U-¹⁴C]ethanediol can serve as substrate in the formation of 1-$\text{0}$-hexadecanoyl ethanediol 2-phosphorylcholine by particulate cell-free preparations from rat liver. Catalytic activity is largely associated with the microsomal fraction. The reaction requires CDPcholine and Mg⁺⁺. Phosphatidylcholine cannot substitute for CDPcholine, but Mn⁺⁺ is almost as effective as Mg⁺⁺. Ca⁺⁺ inhibits the reaction. The acyl ethanediol phosphorylcholine produced was identified by repeated cochromotography with authentic diol phospholipid to constant specific radioactivity, and by enzymatic and chemical degradations.     

Absence of ethanol metabolism in "acatalatic" hepatic microsomes that oxidize drugs

When liver slices of Cs^a and Cs^b mice were incubated $\text{in}\text{vitro}$, they had similar catalase activities and equal rates of ethanol metabolism. While incubated liver homogenates and microsomes from Cs^a mice oxidized ethanol and retained catalase activity, preparations from Cs^b mice did not oxidize ethanol and lost all catalase activity. Addition of beef liver catalase restored ethanol oxidation by Cs^b microsomes. The oxidations of aniline and aminopyrine proceeded at the same rate in Cs^a and Cs^b microsomes and were inhibited by ethanol. It is evident that (a) the microsomal drug-metabolizing pathway is not involved in ethanol oxidation, and (b) the postulation of a unique microsomal ethanol-oxidizing system (“MEOS”) that is independent of microsomal catalase is unwarranted.     

Danazol inhibits human adrenal 21-and 11β-hydroxylation invitro

The effects of danazol on steroidogenesis $\text{in}$$\text{vitro}$ in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant K_I = 0.8 μM) and the mitochondrial 11β-hydroxylase (K_I = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal $\text{in}$$\text{vitro}$.     

Phospholipase A2 inactivation of microsomal 17β-hydroxysteroid oxidoreductase: Rates of phospholipid hydrolysis and enzyme inactivation,effects of hydrolysis products and properties of the phospholipase A2-treated enzyme

Exposure of guinea pig liver microsomes to phospholipase A₂ resulted in the nearly complete loss of 17β-hydroxy-steroid oxidoreductase (17β-HSD) activity, the time course of which correlated with phospholipid hydrolysis and lysolecithin formation. Lysolecithin and unsaturated fatty acids added to microsomes also inactivated 17β-HSD indicating that they may contribute to the inactivation by phospholipase A₂.If exposure to lysolecithin and fatty acids was minimized by including serum albumin in the reaction mixture, phospholipids were rapidly hydrolyzed; but in this case the extent of 17β-HSD inactivation was less and the rate of loss was significantly slower. The data suggest that phospholipid hydrolysis $\text{per se}$ results in a destabilization of 17β-HSD resulting in the subsequent activity loss.The inactivation of 17β-HSD by lysolecithin and fatty acids has not been reported previously and is suggestive of a possible control mechanism $\text{in vivo}$.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Studies on the immunochemical and functional similarities among iron-sulfur proteins involved in mitochondrial steroid hydroxylations

The effects of a goat anti-adrenodoxin immunoglobulin fraction on the NADPH- and NADH-dependent electron transport sequences in the mitochondria of steroidogenic tissues have been examined. The NADPH-cytochrome $\text{c}$ reductase activities of sonicated mitochondrial preparations derived from bovine adrenal cortex and rat adrenals and ovaries were inhibited in a similar manner in the presence of this antibody, while the inhibition of this activity in rat testicular mitochondrial preparations was less pronounced. The NADH-dependent reduction of cytochrome $\text{c}$ catalyzed by these mitochondrial preparations was not affected by the antibody. These results indicate that, while they may not be identical, the iron-sulfur proteins involved in mammalian mitochondrial steroid hydroxylations exhibit immunochemical and functional similarities.     

Metabolic activation of procarbazine

Procarbazine chemical degradation and rat $\text{in vitro}$ and $\text{in vivo}$ metabolism have been investigated. Procarbazine was rapidly oxidized to the azo derivative in aqueous solution in the presence of oxygen. $\text{In vitro}$ rat liver supernatany and microsomal preparations oxidize the azo function to azoxy isomers and further hydroxylate these metabolites in a manner that may by analogous to 1,2-dimethylhydrazine metabolism. The hydroxylated metabolites are activated species that chemically react to give methylating and alkylating agents. An additional metabolic pathway was observed $\text{in vivo}$. This suggests that procarbazine may be converted to free radical intermediates that decompose to give methane and N-isopropyltoluamide. Procarbazine metabolites have been separated and identified using high performance liquid chromatography and direct probe chemical ionization mass spectrometry.     

Permeability of axon membranes to local anesthetics

The permeability of the neutral form of tertiary amine local anesthetics across squid axon membranes was studied by utilizing three different experimental methods: (1) narcotic action of axon excitability was measured by monitoring the time derivative of action potential and the results were analyzed in terms of a diffusion reaction equation of local anesthetics to obtain their permeabilities; (2) the influx of local anesthetic into the axon was measured by use of the radioisotope tracer technique; and (3) the desorption rates of the neutral form of local anesthetics from lipid monolayers were measured and the desorption rate was correlated with permeability.The relative permeabilities obtained for procaine, lidocaine and tetracaine by the above three methods were comparable. The order of relative permeabilities was $\text{procaine}\text{>}\text{lidocaine}\text{>}\text{tetracaine}$, and had an inverse correlation with the partition coefficients of anesthetics at oil/water phases. Some discussion concerning the concept of permeability is made when the partition coefficient of a permeant molecule is high.     

An antiovulatory decapeptide of higher potency which has an L-amino acid (Ac-Pro) in position 1.

Ac-[Pro¹, $\text{D}\text{-}\text{Phe}{}^2$, $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH completely inhibited ovulation in cycling rats at 200μg/rat and is comparable in activity to the corresponding D-1-analogue. This Ac-Pro¹-analogue is the most potent antiovulatory peptide yet known having an $\text{L}$-amino acid residue in position 1. This result shows that for the design of potent inhibitors of ovulation, a $\text{D}$-amino acid residue is not essential in position 1. The corresponding Ac-$\text{D}$-Pro¹- and Kic¹-analogues completely inhibited ovulation at 750μg/rat, but not at 200μg/rat, and the Cpc¹-analogue was inactive at these dosages.     

Selective enhancement of bleomycin cytotoxicity by local anesthetics

The cytotoxic effect of the antitumor antibiotic bleomycin toward cultured mouse FM3A cells was greatly enhanced by exposure of the cells to local anesthetics either before or together with treatment with bleomycin. Such local anesthetics include dibucaine, tetracaine, butacaine, lidocaine and procaine. Dibucaine-induced cell sensitization to bleomycin cytotoxicity produced a decrease in cell survival that became dependent on dose and time of bleomycin treatment. This effect of local anesthetics seems to be selective to bleomycin, since dibucaine and lidocaine do not enhance the cytotoxic effect of other antitumor agents including adriamycin, mitomycin C and $\text{cis}$-diamminedichloroplatinum(II).