RESUMO
The acute effects of ethanol (20-60 mM) on L-arginine uptake and nitric oxide (NO) formation was investigated in human placental cotyledons perfused at constant flow. Ethanol (40 mM) decreased L-[3H]arginine uptake from 27.6 +/- 2.3 to 15.8 +/- 1.3 per cent (P < 0.05) of the injected dose and significantly enhanced NO levels in the perfusate from 0.88 +/- 0.11 to 2.80 +/- 0.39 microM. Ethanol also elicited the constriction of placental vessels. The effects of ethanol (20-60 mM) on L-arginine uptake and endothelial NO synthase (eNOS) activity were also investigated in cultured human umbilical vein endothelial cells (HUVEC). After 60 min of ethanol (40 mM) exposure, basal L-[3H]arginine uptake (4.7 +/- 0.3 pmol/microg protein/min) was inhibited by 60 per cent (P < 0.05). Basal eNOS activity in HUVEC determined under "no flow" (static) conditions was significantly increased (approximately 1.8 fold) by 60 mM ethanol. These data are consistent with a stimulatory effect of ethanol on eNOS activity in both basal and flow-stimulated conditions, which may serve a protective role against its vasoconstrictive acute effect. While acute ethanol administration inhibits L-arginine uptake, the present results do not allow us to speculate on the effects of chronic ethanol exposure on NO formation in the fetoplacental unity.
Assuntos
Arginina/metabolismo , Etanol/farmacologia , Óxido Nítrico/biossíntese , Placenta/metabolismo , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Histamina/farmacologia , Humanos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Gravidez , Trítio , Veias UmbilicaisRESUMO
L-Arginine transport by the fetal side of human placenta was investigated through the characterization of L-[3H]arginine uptake in isolated perfused cotyledon. Competitive inhibition experiments suggest the presence of at least two transport systems: a Na+-independent, pH-insensitive system inhibitable by cationic amino acids, similar to system y+, and a Na+-dependent system which recognizes both cationic and neutral amino acids only in the presence of Na+, i.e. a Bo,+-like system. The kinetic analysis of L-arginine uptake in the presence of Na+ revealed that the process is mediated by saturable components: a high-affinity system (Km = 167 +/- 18.0 microM; Vmax = 0.174 +/- 0.012 micromol min-1) and a low-affinity carrier (Km = 980 +/- 112 microM; Vmax = 1.60 +/- 0.12 micromol min-1). In the absence of Na+, L-arginine uptake was fitted by one model with a Michaelis-Menten constant of 200 +/- 24.8 microM. These results suggest that the high-affinity component corresponds to the Na+-independent system y+, whilst the low-affinity system may represent the activity of the Na+-dependent Bo,+ transporter. Kinetic studies in placentae taken from aspirin-treated pregnancies showed that L-arginine is transported with a significantly higher affinity (Km = 42.5 +/- 5.7 microM), but with a lower capacity (Vmax = 0.064 +/- 0.003 micromol min-1) than in the non-treated group. The latter finding suggests that aspirin would facilitate the uptake of the NO precursor only at very low arginine concentrations.
Assuntos
Arginina/metabolismo , Aspirina/farmacologia , Placenta/metabolismo , Adulto , Aminoácidos/farmacologia , Transporte Biológico/efeitos dos fármacos , Chile , Feminino , Humanos , Concentração de Íons de Hidrogênio , Cinética , Placenta/efeitos dos fármacos , Pré-Eclâmpsia/tratamento farmacológico , Gravidez , Sódio/farmacologiaRESUMO
It has been suggested that adenosine is involved in the acute effects of ethanol in a number of tissues. The present study was undertaken to evaluate the role of adenosine on the vascular responses of perfused isolated human placental cotyledons after the acute administration of ethanol. The possibility that ethanol may effect the uptake and metabolism adenosine was also investigated. Uptake of adenosine was studied using the single-circulation paired-tracer dilution technique. Both adenosine and ethanol caused a dose-related increase in perfusion pressure of placental lobules. Pharmacologically relevant concentrations of ethanol (10-65 mM) significantly inhibited the uptake of [3H]adenosine between 25 and 50 per cent. Thin-layer chromatographic analysis of the perfusate after the administration of ethanol showed in a 17.9 +/- 0.6 per cent reduction of [3H]adenosine metabolism. These findings support the working hypothesis that placental adenosine, at least partially, mediates the placental disturbance elicited by the administration of acute ethanol, which may contribute to the pathogenesis of fetal alcohol syndrome.
Assuntos
Adenosina/metabolismo , Etanol/farmacologia , Placenta/metabolismo , Transporte Biológico/efeitos dos fármacos , Feminino , Humanos , Troca Materno-Fetal/efeitos dos fármacos , GravidezRESUMO
Uptake and metabolism of hypoxanthine by human placenta were studied using the single-circulation paired-tracer technique. In isolated cotyledons perfused through the fetal (basal) circulation, at mean pressures of 31.7 +/- 4.0 mmHg and mean flow rates maintained at 5.5 +/- 0.15 ml/min, the [3H]hypoxanthine uptake was 36 +/- 2.4 per cent (16.5 +/- 1.1 pmol/g wet weight). Hypoxanthine uptake was significantly inhibited by unlabelled (mM) hypoxanthine (0.5), adenine (0.5), guanine (0.5) and papaverine (15.0), but was unaffected by nitrobenzylthioinosine (0.01). Adenosine failed to inhibit hypoxanthine uptake. The kinetic analysis of hypoxanthine uptake showed it to be partially mediated by a saturable (apparent K(m) = 12.1 +/- 1.85 microns; Jmax = 7.1 +/- 0.52 nmol/min) and Na(+)-dependent mechanism. A greater fraction of hypoxanthine influx proceeded through a non-saturable process. Thin layer chromatographic analysis of venous perfusate after the intra-arterial injection of [3H]hypoxanthine showed a negligible degradation of nucleobase. These overall results show that hypoxanthine uptake at the fetal side of human placenta occurs by a saturable plus a non-saturable process. The carrier showed specificity for nucleobases and high affinity-low capacity for hypoxanthine. Since the fetal blood concentration of hypoxanthine is normally low, its uptake would be mediated by the high affinity transport system. Because the non-saturable mechanism can be operative at high concentrations of hypoxanthine, it may have primary importance to clear the nucleobase coming from the fetus during intrauterine hypoxia.
Assuntos
Hipoxantina/metabolismo , Placenta/metabolismo , Adenina/farmacologia , Adenosina/farmacologia , Transporte Biológico , Velocidade do Fluxo Sanguíneo , Feminino , Feto/metabolismo , Guanina/farmacologia , Humanos , Hipoxantina/farmacologia , Papaverina/farmacologia , Placenta/irrigação sanguínea , Gravidez , Sódio/farmacologia , TrítioRESUMO
The purpose of this study was to clarify the role of endogenous nitric oxide and prostanoids in ethanol-induced perturbation of microcirculation in perfused human placenta. Infusion of ethanol into chorionic plate vessels at 10-65 mM increases perfusion pressure in a concentration-dependent fashion, and is an indicator of fetal-placental vasoconstriction. Simultaneous infusion of N(omega)-nitro-L-arginine, methylene blue and endothelial cell removal significantly enhances the ethanol-induced increase in perfusion pressure. In contrast, sodium nitroprusside attenuates this effect. Indomethacin did not significantly modify the ethanol-induced response. In conclusion, inhibition of the action of endogenous nitric oxide is associated with an increase in fetal-placental vasoconstriction. These results suggest that endogenous nitric oxide acts as a vasodilator that reduces ethanol-induced vasoconstriction, thus improving microcirculation, and leads to decreased placental damage.
Assuntos
Etanol/farmacologia , Óxido Nítrico/fisiologia , Placenta/irrigação sanguínea , Solventes/farmacologia , Vasoconstrição/efeitos dos fármacos , Antídotos/farmacologia , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Técnicas In Vitro , Indometacina/farmacologia , Azul de Metileno/farmacologia , Microcirculação/efeitos dos fármacos , Nitroarginina/farmacologia , Nitroprussiato/farmacologia , Perfusão , Placenta/efeitos dos fármacos , Gravidez , Prostaglandinas/farmacologia , Tocolíticos/farmacologia , Vasodilatadores/farmacologiaRESUMO
ATP-diphosphohydrolase (apyrase. EC 3.6.1.5) has both ATPase and ADPase activity that are stimulated by bivalent metals, with Ca2+ being the most effective. The possible physiological function of this enzyme, associated with placental and renal microvilli, is related to the extracellular metabolism of nucleotides. A comparison of the biochemical properties of human placenta and rat kidney apyrase is presented, showing similarities in Mr. bivalent metal stimulation, nucleotide nonspecificity, insensitivity towards specific ATPase inhibitors, and lack of essential sulfhydryl and aliphatic hydroxyl groups. We describe the treatment of membrane preparations from both tissues with different detergents and the isoelectric focusing of the solubilized proteins to partially purify apyrase. An ectoenzyme localization is assigned both in microvillus membranes and in the vasculature on the basis of organ perfusion experiments with nucleotides in the presence of antibodies. Placental and kidney microvillus membranes inhibited ADP-induced platelet aggregation, in agreement with an extracellular role. Initial studies on enzyme regulation suggested the existence of at least two types of modulatory proteins: an activating protein in the cytosol of both tissues, and an inhibitory protein associated with placental microsomes. Possible hormonal regulation was investigated in kidneys using in vivo estradiol treatment, but only slight changes in total apyrase activity were observed.
Assuntos
Apirase/metabolismo , Rim/enzimologia , Placenta/enzimologia , Animais , Apirase/química , Estradiol/farmacologia , Humanos , Agregação Plaquetária/efeitos dos fármacos , RatosRESUMO
ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has both ATPase and ADPase activity that are stimulated by bivalent metais, with Ca2+ being the most effective. The possible physiological function of this enzyme, associated with placental and renal microvilli, is related to the extracellular metabolism of nucleotides. A comparison of the biochemical properties of human placenta and rat kidney apyrase is presented, showing similaiities in Mr, bivalent metal stimulation, nucleotide nonspecificity, insensitivity towards specifjc ATPase inhibitors, and lack of essential sulfhydryl and aliphatic hydroxyl groups. We describe the treatment of membrane preparations from both tissues with different detergents and the isoelectric focusing of the solubilized proteins to partially purify apyrase. An ectoenzyme localization is assigned both in microvillus membranes and in the vasculature on the basis of organ perfusion experiments with nucleotides in the presence of antibodies. Placental and kidney microvillus membranes inhibited ADP-induced platelet aggregation, in agreement with an extracellular role. Initial studies on enzyme regulation suggested the existence of at least two types of modulatory proteins: an activating protein in the cytosol of both tissues, and an inhibitory protein associated with placental microsomes. Possible hormonal regulation was investigated in kidneys using in vivo estradiol treatment, but only slight changes in total apyrase activity were observed.
Assuntos
Humanos , Animais , Ratos , Apirase/metabolismo , Rim/enzimologia , Placenta/enzimologia , Agregação Plaquetária , Apirase/química , Estradiol/farmacologiaRESUMO
Uptake and metabolism of adenosine by human placenta were studied using the single-circulation paired-tracer technique. When isolated cotyledons were perfused through the fetal (basal) circulation at mean pressures of 36 +/- 3.3 mmHg and mean flow rates of 6.6 +/- 0.3 ml/min the maximal [3H]adenosine uptake was 51.3 +/- 3.9 per cent. The uptake was not changed when the vascular resistance was pharmacologically increased. Adenosine uptake was significantly inhibited by adenosine, inosine and nitrobenzylthioinosine (NBMPR), but was unaffected by hypoxanthine. The kinetic analysis of adenosine transport showed it to be a saturable and, Na(+)-independent process, with a Km of 60.8 microM and a Jmax of 0.148 mumol/min. Thin layer chromatographic analysis showed that about 65 per cent of [3H]adenosine was metabolized (10-30 sec) in a single passage through the fetoplacental circulation. [3H]hypoxanthine and [3H]adenine were the major products recovered in the venous perfusate. In the presence of NBMPR the fractional recovery of [3H]adenine and [3H]phosphorylated derivatives was reduced while that of [3H]hypoxanthine was increased. These overall results show that the uptake of adenosine is a Na(+)-independent, NBMPR-sensitive, carrier-mediated process, which appears to be specific for nucleosides, and suggests that metabolization of adenosine proceeds both intra- and extracellularly.
Assuntos
Adenosina/metabolismo , Placenta/metabolismo , Transporte Biológico/fisiologia , Feminino , Humanos , Hipoxantina , Hipoxantinas/farmacologia , Técnicas In Vitro , Perfusão , Gravidez , Nucleosídeos de Purina/farmacologia , Sódio/farmacologia , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Vasoconstrição/fisiologiaRESUMO
The main neurotransmitter of the mouse urinary bladder is ATP, which is hydrolyzed to AMP and adenosine; the latter compound, in contrast to ATP, relaxes the smooth muscle. Diazepam also relaxes the urinary bladder and, since some peripheral and central effects of the benzodiazepines are thought to be induced by inhibition of adenosine uptake or by inhibiting calcium channels, the effects of diazepam, adenosine, R-phenylisopropyladenosine, cyclohexyladenosine, and of the calcium channel antagonists, diltiazem, verapamil and nifedipine, were studied on the contractile responses of the mouse isolated urinary bladder. The contractile responses of the bladder's smooth muscle were elicited by transmural stimulation and by application of ATP or acetylcholine. All drugs mentioned decreased the contractile responses of the bladder. The inhibitory effect of diazepam was similar to that induced by adenosine, R-phenylisopropyladenosine, cyclohexyladenosine, and nifedipine. 8-Phenyltheophylline, an adenosine receptor antagonist, did not decrease the relaxatory response of diazepam, which might exclude a P1 purinoceptor-mediated mechanism in the response studied. Diazepam did not significantly change the inhibitory effects of diltiazem and nifedipine on the contractile response to acetylcholine. The similar patterns of relaxant effects, exerted by diazepam, adenosine analogues and calcium channel antagonists, suggest the interference of benzodiazepine, and adenosine and its analogues on calcium channels of the urinary bladder smooth muscle. The inability of diazepam to further increase the effects of diltiazem and nifedipine on the responses to acetylcholine, reinforces the hypothesis that diazepam is acting through a common mechanism with calcium antagonists.
Assuntos
Adenosina/análogos & derivados , Adenosina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Diazepam/farmacologia , Relaxantes Musculares Centrais/farmacologia , Músculo Liso/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Vasodilatadores/farmacologia , Acetilcolina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Interações Medicamentosas , Estimulação Elétrica , Técnicas In Vitro , Masculino , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , Potássio/farmacologia , Bexiga Urinária/fisiologiaRESUMO
1. The nerve-evoked contractions elicited by transmural electrical stimulation of mouse urinary bladders superfused in modified Krebs Ringer buffer containing 1 microM atropine plus 3.4 microM guanethidine were inhibited by adenosine (ADO) and related nucleoside analogues with the following rank order of potency: R-phenylisopropyladenosine (R-PIA) greater than cyclohexyladenosine (CHA) greater than 5'N-ethylcarboxamido adenosine (NECA) greater than ADO greater than S-phenylisopropyladenosine (S-PIA). Tissue preincubation with 8-phenyltheophylline (8-PT) displaced to the right, in a parallel fashion, the NECA concentration-response curve. 2. The contractions elicited by application of exogenous adenosine 5'-triphosphate (ATP) were also inhibited by ADO and related structural analogues. The rank order of potency to reduce the motor response to ATP was: NECA greater than 2-chloroadenosine (CADO) greater than R-PIA greater than ADO greater than CHA greater than S-PIA. 3. The ADO-induced ATP antagonism was of a non-competitive nature and was not specific. Tissue incubation with 10 microM NECA not only reduced the motor responses elicited by ATP, but also 5-hydroxytryptamine, acetylcholine and prostaglandin F2 alpha. The action of NECA was antagonized following tissue preincubation with 8-PT. The inhibitory action of NECA was not mimicked by 10 microM CHA. 4. The maximal bladder ATP contractile response was significantly increased by tissue preincubation with 5-30 microM 8-PT. 5. The 0.15 Hz evoked muscular twitch was significantly increased by 8-PT while dipyridamole consistently reduced the magnitude of the twitch response. These results are consonant with the hypothesis that an endogenous ADO tone modulates the bladder neurotransmission. 6. A working model is proposed suggesting the presence of ADO-Al and A2 receptors in the mouse urinary bladder. The A1 receptor subpopulation is probably of presynaptic origin whereas the smooth muscle membranes contain a population of the A2 receptor subtype.