RESUMEN
Phospholipase A2 (PLA2) and phospholipid methylases (PLM) play significant roles in transmitter release and membrane signal transduction, respectively. Previous studies have indicated that PLMs occur in the rat brain synaptosomal and retinal membranes, and they are activated under halothane anesthesia. The influence of halothane on PLA2 is not known. Therefore, we have investigated the effect of halothane on retinal PLA2 activity. Rat retinal sonicates were assayed for PLA2 activity using 1-palmitoyl-2[1-14C]arachidonyl-phosphatidylethanolamine (PE, 2.2 nmol) in Tris buffer (10 mM, pH 7.4) at 37 degrees C with and without halothane (0.25-2.0 mM) in the assay medium. These studies gave the following results: (1) Rat retinal sonicates contained PLA2 activity of 4.2+/-0.8 pmol PE hydrolyzed/100 ng protein/hr; (2) Halothane (0.25-2.0 mM) increased PLA2 activity by 20 to 150% depending upon concentration; (3) The lower concentration of halothane (0.25 mM) exhibited high activation of PLA2 (150%); (4) High concentrations of halothane (1.0-2.0 mM) caused a low degree of activation of PLA2 (20%); and (5) During phospholipid methylation of retinal membranes with S-adenosyl-L-methionine in the presence of halothane, increased amounts of fatty acid methyl esters (FAME) were formed. This increase in FAME (45%) was possibly due to the hydrolysis of phospholipids by activated PLA2, liberating fatty acids which were methylated. This increase in FAME (45%) was inhibited by mepacrine (quinacrine) (10 microM), an inhibitor of PLA2. These observations suggest that the release of retinal transmitters (dopamine, acetylcholine and others) is affected during halothane anesthesia, due to activation of PLA2 and enhanced fusogenic activity of vesicular membranes with plasma membrane and depletion of vesicles.
Asunto(s)
Anestésicos por Inhalación/farmacología , Halotano/farmacología , Fosfolipasas A/efectos de los fármacos , Retina/efectos de los fármacos , Animales , Calcio/metabolismo , Dopamina/metabolismo , Metilación , Fosfolipasas A2 , Fosfolípidos/metabolismo , Ratas , Ratas Endogámicas F344 , Retina/metabolismoRESUMEN
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 microg/ml) enhanced this effect at all concentrations of PGE2. Nicotine (2 microg/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.
Asunto(s)
Cotinina/farmacología , Dinoprostona/fisiología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Placenta/efectos de los fármacos , Adulto , Dinoprostona/biosíntesis , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Técnicas In Vitro , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Placenta/fisiología , Embarazo , Vasoconstricción/efectos de los fármacosRESUMEN
The macula is a constituent of the sensory retina that is necessary for sharp contrast and color vision. A significant relationship has been found between tobacco smoking and age-related macular degeneration. Opsin, rhodopsin and phospholipase A2 (PLA2) are located in excitable membranes of retina which are enriched with polyunsaturated fatty acids (PUFA). A question may arise as to whether nicotine and its major metabolite cotinine influence PLA2 so that arachidonic acid (AA) and proinflammatory prostaglandins (PG) are produced in the retina. Therefore, the effects of nicotine and cotinine on the retinal PLA2 were studied. PLA2 activity of rat retinal sonicates was assayed using 1-palmitoyl-2[1-14C]arachidonyl-Phosphatidylethanolamine (PE, 2.2 nmol) as a substrate in Tris buffer (10 mM, pH 7.4) at 37 degrees C with and without nicotine or cotinine in the assay medium. These studies gave the following results: (1) Rat retinal PLA2 activity was 4.2+/-0.8 pmol PE hydrolyzed/100 ng protein/hr. (2) Nicotine in low concentrations (1-150 nM) activated PLA2 (EC50 5 nM). (3) Cotinine also activated PLA2 (EC50 300 nM). (4) Only high concentrations of nicotine (> 1.0 microM) and cotinine (> 25 microM) exhibit inhibition of PLA2. (5) All three known PLA2 inhibitors, mepacrine, 4-bromophenacyl bromide and bromoacetylcholine bromide (IC50: 0.5mM, 88 microM, 30 mM, respectively) inhibited retinal PLA2 activity. These observations suggest that polyunsaturated fatty acids are cleaved, and arachidonic acid, the precursor for prostaglandins and related pro-inflammatory mediators, is liberated by nicotine and cotinine. Oxidative stress (reduced levels of antioxidants), vascular insufficiency, as well as activation of PLA2 by nicotine and cotinine may contribute for retinal degeneration in smokers during aging.
Asunto(s)
Cotinina/farmacología , Nicotina/farmacología , Fosfolipasas A/metabolismo , Retina/efectos de los fármacos , Acetofenonas/farmacología , Acetilcolina/análogos & derivados , Acetilcolina/farmacología , Animales , Inhibidores Enzimáticos/farmacología , Degeneración Macular/etiología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Quinacrina/farmacología , Ratas , Ratas Endogámicas F344 , Retina/enzimologíaRESUMEN
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 degrees 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.
Asunto(s)
Cotinina/metabolismo , Intercambio Materno-Fetal , Nicotina/metabolismo , Placenta/metabolismo , Adulto , Antipirina/análisis , Antipirina/metabolismo , Cotinina/análisis , Femenino , Humanos , Técnicas In Vitro , Nicotina/análisis , Perfusión/métodos , EmbarazoRESUMEN
There is periparturitional increase of prostaglandin E2 (PGE2) in the plasma and amniotic fluid of humans. PGE2 increases uterine contractions and also increases uterine blood flow to sustain the contractions. A question arises as to what role PGE2 plays in human placental circulation. It may regulate feto-placental blood flow and closure of placental resistance vessels at parturition. Therefore, we have investigated (a) the release of PGE2 into fetal and maternal circulations, and (b) the influence of PGE2 on the feto-placental pressure in the 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 degrees C) containing 2% albumin on both maternal (230 ml, 12 ml/min., 0.6" Hg) and fetal (93 ml, 1.75 ml/min., 1.75" Hg) sides in a closed recirculating system. In one group of cotyledons, perfusion samples (2 ml) were collected at regular intervals from both perfusates for 3 hrs. and PGE2 was determined in aliquots (0.5 ml) of samples by a specific radioimmunoassay. In a second set of cotyledons, exogenous PGE2 was administered into fetal perfusate, and pressure was monitored as a function of time. These experiments gave the following results: 1) During the initial 20 min., a constant level of PGE2 (2.3-4.4 pg/ml) was maintained in both perfusates. At 3 hrs., the concentrations increased to about 110 ng/ml on the fetal side and 30 ng/ml on the maternal side. The total amount of PGE2 accumulated in the fetal and maternal reservoirs reached to 10.16 and 7.03 ng, respectively. 2) PGE2 (10-150 ng/ml) increased the feto-placental perfusion pressure in a concentration dependent manner. At 150 ng/ml, the pressure increased to 125-240% of control pressure observed at the beginning of the experiment. These studies suggest that a) placental trophoblast has the capacity for the synthesis and release of PGE2 into fetal and maternal circulations; b) PGE2 exhibits differential effects in the placental and uterine blood vessels, vaso-constriction in placental vessels and vasodilation in uterine blood vessels, and (c) PGE2 exhibits dual effects on blood vessels possibly by activating two different subtypes of PG-receptors.