B.V. Rama Sastry

Effects of calcium channel blocking agents on calcium and centrilobular necrosis in the liver of rats treated with hepatotoxic agents

Carbon tetrachloride, chloroform, dimethylnitrosamine, thioacetamide or acetaminophen was each administered to rats in a single hepatotoxic dose. Nifedipine, verapamil or chlorpromazine was administered in association with the hepatotoxic agents to determine if calcium channel blocking agents would prevent an increase in liver cell calcium associated with hepatotoxicity and to determine if these agents would protect against the development of centrilobular necrosis. Following a latent period different for each toxic agent, a 4- to 18-fold increase in liver cell calcium content had occurred by 24 hr. The calcium increase and the centrilobular necrosis (mean histologic score) were correlated. A relatively high calcium to necrosis ratio was obtained with dimethylnitrosamine, thioacetamide and acetaminophen. A lesser calcium to necrosis ratio was obtained with chloroform and carbon tetrachloride, the two toxic agents that destroyed the intracellular calcium sequestration activity of the liver endoplasmic reticulum. Nifedipine or chlorpromazine, administered prior to and 7 hr after the toxic agent, completely prevented the centrilobular necrosis caused by thioacetamide, carbon tetrachloride and acetaminophen; almost completely prevented necrosis with dimethylnitrosamine; and provided partial protection against chloroform toxicity. Two doses of verapamil provided partial protection against necrosis when carbon tetrachloride was the toxic agent and provided almost complete protection with dimethylnitrosamine. A reduction in liver cell calcium was associated with the protective action of the three calcium channel blocking agents. These findings are compared with earlier studies of the protective effects of calcium channel blocking agents in cardiac ischemia.

Human placental cholinergic system. Occurrence, distribution and variation with gestational age of acetylcholine in human placenta.

Abstract Although acetylcholine (ACh)-like activity was demonstrated in human placental extracts by a number of investigators, substances responsible for this activity were not identified. We found by gas Chromatographic techniques that the major component of the ACh-like activity of the term placenta was ACh (112 ± 7 nmoles/g of wet tissue). These results were confirmed by the separation of ACh from other quaternary ammonium compounds by column chromalography using Amberlite CG-50 resin. The placenta could be stored at 4 for a number of days without significant loss of ACh. Freezing and thawing of the placenta destroyed ACh. This indicates that ACh is bound within membranes. There were high concentrations of ACh in all segments of the placenta. The ACh concentrations in the concentric segments next to the periphery and the umbilical cord were lower than ACh concentrations in other segments. The concentrations of ACh in floating villi and the basal plate, which included the anchoring villi, were about 322 and 210% respectively, of that of the chorionic plate. There was variation in ACh content with gestational age: the highest concentration was found at about 22 weeks (wk) of gestation (nmoles/g at 9–12 wk, 129; 13–16 wk, 342 ± 31; 17–20 wk, 317 ± 32; 21–24 wk, 723 ± 63; 25–28 wk, 231; 29–32 wk, 249; 33–36 wk, 153 ± 15; 37–40 wk, 105 ± 7; and 41–44 wk, 88 ± 5). These observations associate ACh with syncytiotrophoblast. Choline acetyltransferase (ChA) has a similar pattern of variation with gestational age. The placental cholinergic system (as indicated by ChA ACh) was fully formed at the early fetal period of histogenesis and functional maturation, during which the fetus exhibits the fastest rate of growth.

Formation and Retention of Cotinine during Placental Transfer of Nicotine in Human Placental Cotyledon

Maternal smoking during pregnancy causes reduction of fetal breathing movements, an effect attributed to nicotine in fetal blood. Nicotine is metabolized to cotinine which has a long plasma half-life and exhibits slow clearance across membrane barriers. It is also known to activate placental phospholipase-A2-like enzymes, resulting in formation of prostaglandins. Therefore, we studied transport of nicotine in isolated perfused cotyledon of normal human term placenta. The placental cotyledon was perfused with aerated (21% O2, 5% CO2) Krebs-Ringer bicarbonate buffer (pH 7.4, 37°C) containing 2% albumin on both maternal (230 ml, 15 ml/min, 35 mm Hg) and fetal (93 ml, 1.75 ml/min, 70 mm Hg) sides in a closed recirculating system. Nicotine (2 mg) was added to the maternal perfusate; perfusate samples (1 ml) were collected from both sides at regular intervals and analyzed for nicotine and cotinine by high-pressure liquid chromatography. This study gave the following results: (1) In about 60–80 min, 18.6% of the nicotine added to the maternal perfusate was transferred to the fetal perfusate, and the maternal/fetal concentration ratio reached 1.0. These results show rapid placental transfer of nicotine, consistent with its high lipid solubility. (2) Less than 1% is metabolized to cotinine in placenta. The ratio of cotinine concentrations in maternal and fetal perfusates reached 1.0 in about 40 min. These studies were also verified using 14C-nicotine. (3) Maximal reduction in fetal breathing movements occurs at about 30 min, and recovery occurs at 90 min after tobacco smoking by the mother. These observations agree with the rate of placental transfer of nicotine. (4) When nicotine was added on the fetal side, part of it was metabolized to cotinine. However, the maximal concentration of cotinine was twice higher on fetal than on maternal side. These observations suggest that accumulation of cotinine on fetal side may activate prostaglandin formation and trigger spontaneous abortions in pregnant smokers.

Evidence for two methyltransferases involved in the conversion of phosphatidylethanolamine to phosphatidylcholine in the rat liver

Abstract The stepwise methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) occurs in the rat liver. The properties of two methyltransferases that are involved in this stepwise methylation of PE to PC in the rat liver are reported. Rat microsomal membranes were used as the source of the enzymes and S -adenosyl- l -[methyl- 3 H]methionine as the methyl donor. The first methyltransferase converted PE to phosphatidyl- N -methylethanolamine (PME), and the second methyltransferase converted PME to PC. The products were characterized by thin-layer chromatography and high-pressure liquid chromatography. After incubation of rat liver microsomes with the methyl donor, three peaks corresponding to PME, dimethyl-PME (PMME), and PC were found and quantified. The first methyltransferase had low K m (0.83 μ m ), pH optimum of 8, and was activated by Mg 2+ . The second methyltransferase had a high K m (~67 μ m ) and a pH optimum of 10. The proportion of the first methyltransferase in the microsomal membranes was increased by repeated washings in hypotonic medium containing EDTA. When the microsomal membranes were subjected to repeated mild sonication and centrifugation at 105,000 g a fraction of the second methyltransferase was solubilized (i.e., appeared in the nonparticulate fraction). The solubilized enzyme utilized dipalmitoyl-PME and -PMME as substrates. Both enzymes were also present in mitochondrial and nuclear membranes with highest specific activities occurring in the microsomal membranes.

The influence of cholinergic blockade on the uptake of α-aminoisobutyric acid by isolated human placental villi

Abstract Isolated villus from human term placenta contains about 167 nmol of acetylcholine (ACh) per gram of wet tissue and releases about 35 pmol of ACh/g/min when it is suspended in Krebs-Ringer bicarbonate buffer at 37°C. Chronic doses of nicotine, or smoking, which modify ACh output from human placental villus, are known to retard fetal intrauterine growth. Since one of the functions of placental villi is nutrient transport, and since it has not been possible to obtain ACh-free villi, the effects of cholinergic blockade by high concentrations of ACh (2 × 10−3 m ), phospholine (5 × 10−4 m ), nicotine (2.5 × 10−3 m ), and atropine (10−4 to 5 × 10−4 m ) on active uptake of α-aminoisobutyric acid (AIB) were studied to explain their antigrowth effects. High concentrations of ACh and nicotine decreased the rate of uptake of AIB by 34 and 41%, respectively. Atropine inhibited the AIB uptake by 29 and 61% at concentrations of 10−4 and 5 × 10−4 m , respectively. If all Ach were released from the syncytiotrophoblast, the concentration of ACh in the medium would be about 1.67 × 10−5 m . At the highest concentration of atropine used, the concentration of the active antagonist, d (−)S-hyoscyamine, in the medium is 2.5 × 10−4 m , which depressed AIB uptake by about 61%. Phospholine (an irreversible cholinesterase inhibitor) at 7 × 10−6 m increased AIB uptake by 20%, but it decreased AIB uptake at higher concentrations, with or without exogenous ACh. Mecamylamine (10−4 m ) and d-tubocurarine (10−4 m ) did not influence the AIB uptake. Among the pharmacological agents studied, nicotine increased the release of endogenous placental ACh, while atropine decreased ACh release. These observations indicate that endogenously released ACh exhibits a muscarinic effect on the placental villus and facilitates the uptake of amino acids. Blockade of the facilitating effects of ACh on amino acid uptake by placenta for long periods during pregnancy may result in a retardation of fetal growth.

Kinetic mechanisms of human placental choline acetyltransferase

Abstract Choline acetyltransferase (ChA) from human placenta was assayed by the formation of [acetyl- 14 C]choline (ACh) from choline (Ch) and [acetyl- 14 C]CoA (ACoA). The bisubstrate kinetics of this enzyme (0.15 nmoles ACh min mg protein; ACoA, 10 −5 M; Ch, 5 × 10 −4 ; pH 7.4, 37°) were studied by the following experiments: (1) variation of the initial velocity as a function of Ch concentration (6 × 10 −5 to 2.5 × 10 −4 M) at fixed ACoA concentrations (6 × 10 −6 to 2.5 × 10 −5 M); (2) variation of the initial velocity as a function of ACoA concentration (6 × 10 −6 to 2.5 × 10 −5 M) at fixed Ch concentrations (6 × 10 −5 to 2.5 × 10 −4 M); (3) enzyme inhibition by ACh (10 −2 M) and CoA (2.5 × 10 −5 , 10 −4 M) with Ch as the variable substrate and ACoA as the fixed substrate; and (4) enzyme inhibition by ACh and CoA with ACoA as the variable substrate and Ch as the fixed substrate. In experiments (1) and (2), double reciprocal plots intersecting in the third quadrant were obtained. In experiment (3), ACh is a competitive inhibitor and CoA is a noncompetitive inhibitor. In experiment (4), CoA is a competitive inhibitor and ACh a noncompetitive inhibitor. These results suggest that the steady state concentrations of ternary complexes (ACoA. ChA. Ch, CoA. ChA. ACh) are very low. CoA was about 100 times stronger as a product inhibitor than ACh. Therefore, an ordered Theorell-Chance mechanism was tentatively postulated with ACoA as the leading substrate and CoA as the obligatory product. A ping-pong mechanism has been proposed for the human placental ChA in the published literature which was based upon the parallel double reciprocal plots in experiments (1) and (2) at higher substrate concentrations for ACoA and choline than those used by us. At these high substrate concentrations, the experimental points fall within the region of mixed first-order and zero-order kinetics instead of first-order kinetics on the initial velocity-substrate concentration curves. Therefore, the intersecting double reciprocal plots in experiments (1) and (2) were altered into plots which appeared approximately parallel. At high substrate concentrations of ACoA, a significant degree of substrate inhibition and product inhibition due to CoA was observed.

Occurence of choline acetyltransferase and acetylcholine and other quaternary ammonium compounds in mammalian spermatozoa

Abstract These investigations were undertaken to show the presence of acetylcholine (ACh) and other quaternary ammonium compounds and choline acetyltransferase (ChA) activity in mammalian spermatozoa. The spermatozoa from the fresh ejaculates of the bull and man were washed with calcium-free Krebs-Ringer phosphate solution and extracted with acetonitrile. The quarternary ammonium compounds from the extracts were subjected to pyrolysis gas chromatography. In the gas chromatogram of the quaternary ammonium compounds of bull spermatozoa, two peaks were found which represented 2-dimethylaminoethyl acetate and 2-dimethylaminoethyl propionate. In the gas chromatogram of the quaternary ammonium compounds of the human spermatozoa, a peak for 2-dimethylaminoethyl acetate was found. These observations suggest that ACh and propionylcholine (PCh) occur in bull spermatozoa and that ACh occurs in human spermatozoa. Bull spermatozoa contained 4.27 ± 1.41 pmoles (mean ± S.E.M.) ACh/10 6 cells and 1.47 ± 0.48 pmoles PCh/10 6 cells, and human spermatozoa contained 28.57 ± 3.38 pmoles ACh/10 6 cells. ChA activity was determined in the washed spermatozoa by a radiometric assay. Incubation of the homogenates of the sperm cells with [ 14 C]acetylcoenzyme A and choline resulted in the synthesis of [ 14 C]ACh, which was separated, using a column of an anion exchange resin, and assayed. ChA activity in bull spermatozoa was 200.19 ± 32.08 pmoles ACh synthesized/10 6 cells/10 min, and ChA activity in human spermatozoa was 130.90 ± 14.19 pmoles ACh synthesized/10 6 cells/10 min. The specific activity of ChA in the tails of bull spermatozoa was about five times higher than in the heads or midpieces.

PROSTAGLANDIN E2 IN HUMAN PLACENTA : ITS VASCULAR EFFECTS AND ACTIVATION OF PROSTAGLANDIN E2 FORMATION BY NICOTINE AND COTININE

Tobacco smoking by pregnant women increases the frequency of spontaneous abortions and preterm births. Human labor is associated with enhanced intrauterine phospholipid metabolism and production of prostaglandin E2 (PGE2) which induces labor, initiates uterine contractions and maintains the homeostasis of placental blood flow. Therefore, we studied: (a) the influence of nicotine and cotinine on the effects of PGE2 on placental vasculature in perfused human placental cotyledon, and (b) the activation of placental phospholipase A2 (PLA2) by nicotine and cotinine using 1-palmitoyl-2-[1-14C]arachidonyl-phosphatidylethanolamine (PE, 2.2 nmol) as substrate. These studies revealed that: (1) increasing concentrations of PGE2 (10– 150 ng/ml) increased umbilical perfusion pressure by 170 ± 10% (n = 6) of control (100%). Cotinine (2 µg/ml) enhanced this effect at all concentrations of PGE2. Nicotine (2 µg/ml) prevented the effect of PGE2; (2) both cotinine (EC50 470–500 fmol/l) and nicotine (EC50 18–32 pmol/l) activated PLA2 in human placental tissues. These observations indicated that cotinine was more potent than in nicotine activating PLA2 and potentiating the vasoconstrictive effects of PGE2 on fetal placental circulation. Nicotine activates nicotinic receptors and releases placental acetylcholine, a vasodilator of placental arteries. Acetylcholine stimulates muscarinic receptors of endothelial cells resulting in the release of endothelium-derived relaxing factor (EDRF), and possibly nitric oxide. Therefore, nicotine prevents or abolishes the vasoconstrictive effects of PGE2 through the release of EDRF. Cotinine is inactive at nicotinic and muscarinic receptors. Therefore, accumulation of cotinine, the major metabolite of nicotine, in fetal circulation may contribute to production of PGE2 and induction of preterm labor and spontaneous abortions.

Depression of Amino Acid Uptake in Human Placental Villus by Cocaine, Morphine and Nicotine

There are several drugs which are known to cause intrauterine growth retardation in experimental animals and man (Tuchmann-Duplessis, 1975). This retarded growth may predispose neonates to perinatal morbidity and mortality. Morphine is known to be associated with retarded intrauterine growth, the extent of which is apparently related to the amount of exposure (Stone et al., 1971). Nicotine and smoking are also implicated in intrauterine fetal growth retardation (Tuchmann-Duplessis, 1975; Hasselmeyer et al., 1979). Prior studies in our laboratory have demonstrated dose-related inhibition of the uptake of the amino acid, a-aminoisobutyric acid (AIB), by components of tobacco smoke (Barnwell and Sastry, 1982). Intrauterine fetal growth retardation among cocaine addicts has not been well documented. However, its use as an euphoric drug has recently become more popular, and greater numbers of pregnant females may be exposing their offspring to the drug (Ellinwood and Kilbey, 1975). Amino acids for fetal protein synthesis and metabolic energy have to be transported from maternal blood to the fetal circulation through the placenta. There is, however, little information on the effects of the above drugs on amino acid transport in human placenta.

Maternal tobacco smoking and changes in amino acid uptake by human placental villi: Induction of uptake systems, gammaglutamyltranspeptidase and membrane fluidity

Abstract Maternal smoking depressed the active uptake of amino acids by human placentae and lowered their levels in the placenta and umbilical vein. During starvation of cells for amino acids, more amino acid carriers are induced and incorporated into the plasma membrane. A question arises whether there could be similar changes due to maternal smoking in the placental amino acid uptake carrier systems. Therefore, the characteristics of (a) the uptake of 2-amino[1- 14 C]-isobutyric acid (AIB) by isolated placental villi, (b) gammaglutamyltranspeptidase (GGTP), a critical enzyme of the gammaglutamyl cycle (GGC) for the uptake of amino acids in human placenta, and (c) lipid structural parameters (reciprocal of fluidity), by steady state fluorescence polarization of plasma membrane vesicles of microvilli (MV) and microsomal membranes (MM) of umbilical and chorionic plate arteries of placentae of smoking and non-smoking mothers were investigated. The above investigations gave the following results: 1. (a) Washed placental villi of smokers exhibited higher capacity for AIB uptake than those of non-smokers. The higher uptake capacity was mainly due to increase in V max for AIB uptake in smokers. K m increased for placental AIB uptake in smokers. 2. (b) Maternal smoking lowered GGTP activity of MV by decreasing its V max . Therefore, maternal smoking decreases the formation of gammaglutamyl-amino acid (GGAA) on the surface of trophoblast which are absorbed by the trophoblast. The degree of absorption of GGAA is considered as an inverse environmental signal for the cell to regulate amino acid transport systems. Maternal smoking seems to decrease the formation and absorption of GGAA and thereby induce the formation of new carriers for AIB uptake. 3. (c) Maternal smoking increased the values for lipid structural order parameters and microviscosity of MV and induced tolerance against fluidization by ethyl alcohol in MM of umbilical and chorionic arteries. The alterations could increase K m for AIB uptake system and decrease the sensitivity of umbilical and chorionic arteries to vasoconstrictive substances like 5-hydroxytryptamine and catecholamine which are released by nicotine. All these changes tend to overcome the deficits produced in placental amino acid transport and satisfy the demands of the growing fetus for amino acids.