RESUMEN
Tissue accumulation and high urinary excretion of argininosuccinate (ASA) is the biochemical hallmark of argininosuccinate lyase deficiency (ASLD), a urea cycle disorder mainly characterized by neurologic abnormalities, whose pathogenesis is still unknown. Thus, in the present work, we evaluated the in vitro and in vivo effects of ASA on a large spectrum of oxidative stress parameters in brain of adolescent rats in order to test whether disruption of redox homeostasis could be involved in neurodegeneration of this disorder. ASA provoked in vitro lipid and protein oxidation, decreased reduced glutathione (GSH) concentrations, and increased reactive oxygen species generation in cerebral cortex and striatum. Furthermore, these effects were totally prevented or attenuated by the antioxidants melatonin and GSH. Similar results were obtained by intrastriatal administration of ASA, in addition to increased reactive nitrogen species generation and decreased activities of superoxide dismutase, glutathione peroxidase, and glutathione S-transferase. It was also observed that melatonin and N-acetylcysteine prevented most of ASA-induced in vivo pro-oxidant effects in striatum. Taken together, these data indicate that disturbance of redox homeostasis induced at least in part by high brain ASA concentrations per se may potentially represent an important pathomechanism of neurodegeneration in patients with ASLD and that therapeutic trials with appropriate antioxidants may be an adjuvant treatment for these patients.
Asunto(s)
Ácido Argininosuccínico/farmacología , Encéfalo/efectos de los fármacos , Depuradores de Radicales Libres/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Glutatión Peroxidasa/metabolismo , Ratas Wistar , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
The influence of angioprotector and endothelium-protector drugs pentoxifylline and unifuzol as components of supportive therapy on the efficacy of combined cytostatic treatment has been experimentally studied. It is established that pentoxifylline and unifuzol do not affect the antitumor and antimetastatic activity of doxorubicin and cyclophosphan with respect to Pliss lymphosarcoma and Walker 256 carcinosarcoma, and in some cases even potentiate the effect of cytostatic therapy.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ácido Argininosuccínico/farmacología , Carcinoma 256 de Walker/tratamiento farmacológico , Citostáticos/farmacología , Linfoma no Hodgkin/tratamiento farmacológico , Pentoxifilina/farmacología , Animales , Carcinoma 256 de Walker/patología , Proliferación Celular/efectos de los fármacos , Ciclofosfamida/farmacología , Doxorrubicina/farmacología , Sinergismo Farmacológico , Linfoma no Hodgkin/patología , Masculino , Ratas , Ratas Wistar , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Several cell types express inducible nitric oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide (LPS) or proinflammatory cytokines. For instance, muscular cells treated with LPS and interferon gamma (IFN-gamma) respond by increasing the mRNA and protein levels of NOS2, and synthesize large amounts of nitric oxide. We show here that transcriptional induction of NOS2 in muscular cells proceeds with a concomitant decrease in the levels of caveolin-1, -2 and -3. Addition of *NO-releasing compounds to C2C12 muscle cells reveals that this downregulation of the caveolin (cav) levels is due to the presence of *NO itself in the case of caveolin-3 and to the action of the LPS/IFN-gamma in the case of cav-1 and cav-2. Likewise, muscle cells obtained from NOS2(-/-) knockout mice challenged with LPS/IFN-gamma could downregulate their levels of cav-1 but not of cav-3, unlike wild-type animals, in which both cav-1 and cav-3 levels diminished in the presence of the proinflammatory insult. Laser confocal immunofluorescence analysis proves that *NO exerts autocrine and paracrine actions, hence diminishing the cav-3 levels. When the induced NOS2 was purified using an affinity resin or immunoprecipitated from muscular tissues, it appears strongly bound not only to calmodulin but also to cav-1, and marginally to cav-2 and cav-3. When the cav levels where reduced using antisense oligonucleotides, an increase in the NOS2-derived.NO levels could be measured, demonstrating the inhibitory role of the three cav isoforms. Our results show that cells expressing NOS2 diminish their cav levels when the synthesis of *NO is required.
Asunto(s)
Ácido Argininosuccínico/análogos & derivados , Caveolinas/metabolismo , Regulación hacia Abajo , Óxido Nítrico Sintasa/metabolismo , Amidinas/farmacología , Animales , Ácido Argininosuccínico/farmacología , Bencilaminas/farmacología , Caveolina 1 , Caveolina 2 , Caveolina 3 , Caveolinas/genética , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Óxido Nítrico Sintasa/deficiencia , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Oligorribonucleótidos Antisentido , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
The aim of this study was to investigate in rat gastric fundus whether L-citrulline, the co-product in the nitric oxide (NO) biosynthesis catalyzed by neuronal nitric oxide synthase (nNOS), can be converted back to the nNOS substrate L-arginine. Immunohistochemistry showed that argininosuccinate synthetase and argininosuccinate lyase, that mediate transformation of L-citrulline to L-arginine in the ureum cycle in hepatocytes, co-localize with nNOS. In longitudinal smooth muscle strips, L-arginine as well as L-citrulline (10(-3) M) was capable of completely respectively partially preventing the N(G)-nitro-L-arginine methyl ester (L-NAME) (3 x 10(-5) M)-induced inhibition of electrically induced nitrergic relaxations, whereas D-citrulline (10(-3) M) was not. The L-citrulline-mediated prevention of the L-NAME-induced inhibition was reduced by L-glutamine (3 x 10(-3) M), the putative L-citrulline uptake inhibitor, and by succinate, an argininosuccinate lyase inhibitor. The results demonstrate that the L-citrulline recycling mechanism is active in rat gastric fundus. Recycling of L-citrulline might play a role in providing sufficient amounts of nNOS substrate during long-lasting relaxations in gastric fundus after food intake.
Asunto(s)
Argininosuccinatoliasa/metabolismo , Argininosuccinato Sintasa/metabolismo , Citrulina/metabolismo , Fundus Gástrico/metabolismo , Animales , Arginasa/farmacología , Arginina/metabolismo , Arginina/farmacología , Argininosuccinatoliasa/antagonistas & inhibidores , Argininosuccinato Sintasa/antagonistas & inhibidores , Ácido Argininosuccínico/farmacología , Ácido Aspártico/farmacología , Citrulina/farmacología , Dinoprost/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Fundus Gástrico/enzimología , Ácido Glutámico/farmacología , Glutamina/farmacología , Técnicas In Vitro , Masculino , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , N-Metilaspartato/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Ratas , Ratas Wistar , Tioléster Hidrolasas/metabolismo , Ubiquitina TiolesterasaRESUMEN
Studies of the effect of nitric oxide (NO) synthesis inhibition were performed in the isometrically contracting blood-perfused canine gastrocnemius-plantaris muscle group. Muscle blood flow (Q) was controlled with a pump during continuous NO blockade produced with either 1 mM L-argininosuccinic acid (L-ArgSA) or N(G)-nitro-L-arginine methyl ester (L-NAME) during repetitive tetanic contractions (50-Hz trains, 200-ms duration, 1/s). Pump Q was set to match maximal spontaneous Q (1.3-1.4 ml. min(-1). g(-1)) measured in prior, brief (3-5 min) control contraction trials in each muscle. Active tension and oxygen uptake were 500-600 g/g and 200-230 microl. min(-1). g(-1), respectively, under these conditions. Within 3 min of L-ArgSA infusion, vascular resistance across the muscle (R(v)) increased significantly (from approximately 100 to 300 peripheral resistance units; P < 0.05), whereas R(v) increased to a lesser extent with L-NAME (from approximately 100 to 175 peripheral resistance units; P < 0.05). The increase in R(v) with L-ArgSA was unchanged by simultaneous infusion of 0.5-10 mM L-arginine but was reduced with 1-3 microg/ml sodium nitroprusside (41-54%). The increase in R(v) with L-NAME was reversed with 1 mM of L-arginine. Increased fatigue occurred with infusion of L-ArgSA; active tension and intramuscular pressure decreased by 62 and 66%, whereas passive tension and baseline intramuscular pressure increased by 80 and 30%, respectively. These data indicate a possible role for NO in the control of R(v) and contractility within the canine gastrocnemius-plantaris muscle during repetitive tetanic contractions.
Asunto(s)
Contracción Isométrica/fisiología , Óxido Nítrico/fisiología , Resistencia Vascular/fisiología , Animales , Ácido Argininosuccínico/farmacología , Perros , Inhibidores Enzimáticos/farmacología , Femenino , Contracción Isométrica/efectos de los fármacos , Masculino , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroprusiato/farmacología , Resistencia Vascular/efectos de los fármacosRESUMEN
We evaluated in the in situ vascularly isolated canine diaphragm the role of nitric oxide (NO) in the regulation of basal vascular resistance and vascular responses to increased muscle activity (active hyperemia), brief occlusions of the phrenic artery (reactive hyperemia), and changes in arterial pressure. The vasculature of the left hemidiaphragm was either pump-perfused at a fixed flow rate or autoperfused with arterial blood from the femoral artery. Endothelial nitric oxide synthase (NOS) activity was inhibited by intraphrenic infusion of L-arginine analogues such as N(G)-nitro-L-arginine, N(G)-nitro-L-arginine methyl ester and argininosuccinic acid. Active hyperemia was produced by low (2 Hz) frequency stimulation of the left phrenic nerve. Reactive hyperemia was measured in response to 10, 20, 30, 60, and 120 sec duration occlusions of the left phrenic artery and was quantified in terms of postocclusive blood flow, vascular resistance, hyperemic duration, and hyperemic volume. Infusion of NOS inhibitors into the vasculature of the resting diaphragm increased phrenic vascular resistance significantly and to a similar extent. Reactive hyperemic volume and reactive hyperemic duration were also significantly attenuated after NOS inhibition, however, peak reactive hyperemic dilation was not influenced by NOS inhibition. It was also found that enhanced NO release contribute by about 41% to active dilation elicited by continuous 2 Hz stimulation. In addition, NOS inhibition had no effect on O2 consumption of the resting diaphragm, but significantly attenuated the rise in diaphragmatic O2 consumption during during 2 Hz stimulation. The decline in diaphragmatic O2 consumption was due to reduction in blood flow. These results indicate that NO release plays a significant role in the regulation of diaphragmatic vascular tone and O2 consumption.
Asunto(s)
Óxido Nítrico Sintasa/metabolismo , Músculos Respiratorios/irrigación sanguínea , Animales , Ácido Argininosuccínico/farmacología , Arteriopatías Oclusivas/fisiopatología , Arterias/efectos de los fármacos , Arterias/fisiología , Vasos Sanguíneos/enzimología , Perros , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Hiperemia/etiología , Hiperemia/fisiopatología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico Sintasa de Tipo III , Nitroarginina/farmacología , Consumo de Oxígeno/fisiología , Nervio Frénico/irrigación sanguínea , Nervio Frénico/fisiología , Flujo Sanguíneo Regional/efectos de los fármacos , Resistencia Vascular/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
The synthesis of guanidinosuccinic acid (GSA) increases in uremics, and GSA is implicated as a uremic toxin. The GSA synthesis increases roughly in proportion to the serum urea level that increases in patients with renal failure. Urea is a specific inhibitor of argininosuccinase, the fourth urea cycle enzyme, and might lead to the increase of argininosuccinate (ASA). We found that GSA is formed from ASA by reactive oxygen species in vitro. In this paper, we investigated GSA synthesis from ASA in isolated rat hepatocytes and the effect of reactive oxygen species on this synthesis. When isolated rat hepatocytes were incubated with 5 mmol/l ASA, GSA was formed linearly with time up to 6 h (16 nmol/g wet liver/6 h). GSA was formed depending on the ASA concentration up to 10 mmol/l. Dimethylsulfoxide, a hydroxyl radical scavenger, inhibited GSA synthesis by 65%. GSA was actively formed when the hepatocytes were incubated with 32 mmol/l urea. The GSA formation in the presence of urea was also inhibited by dimethylsulfoxide, although the inhibition was less marked. FeCl2, that increases the hydroxyl radical generation, increased GSA synthesis. These results indicate that GSA is formed from ASA in isolated hepatocytes. The results also suggest that reactive oxygen species are important for GSA synthesis in the cells.
Asunto(s)
Ácido Argininosuccínico/metabolismo , Guanidinas/metabolismo , Hígado/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Succinatos/metabolismo , Animales , Ácido Argininosuccínico/farmacología , Células Cultivadas , Dimetilsulfóxido/metabolismo , Dimetilsulfóxido/farmacología , Relación Dosis-Respuesta a Droga , Compuestos Ferrosos/metabolismo , Compuestos Ferrosos/farmacología , Depuradores de Radicales Libres/metabolismo , Depuradores de Radicales Libres/farmacología , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Factores de Tiempo , Urea/metabolismo , Urea/farmacologíaAsunto(s)
Hipercapnia/fisiopatología , Hipoxia/fisiopatología , Óxido Nítrico/fisiología , Circulación Pulmonar/fisiología , Animales , Ácido Argininosuccínico/farmacología , GMP Cíclico/metabolismo , Técnicas In Vitro , Masculino , Óxido Nítrico/antagonistas & inhibidores , Perfusión , Circulación Pulmonar/efectos de los fármacos , Conejos , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiologíaRESUMEN
1. The role played by nitric oxide (NO) in the regulation of blood flow to the canine isolated hemidiaphragm was evaluated by determining (a) the effects of the L-arginine analogues NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-L-arginine (L-NOARG), and argininosuccinic acid (ArgSA) on baseline vascular resistance and of the latter two agents on endothelium-dependent (acetylcholine, ACh) and endothelium independent (sodium nitroprusside, SNP) vasodilatation; (b) the effects of L- and D-arginine on baseline vascular resistance; and (c) the effects of L-glutamine, an inhibitor of intracellular recycling of L-citrulline to L-arginine, on baseline resistance and on the response to ACh and SNP. 2. L-NAME, L-NOARG and ArgSA (6 x 10(-4) M final concentration) increased baseline diaphragmatic vascular resistance to a similar extent (28.6 +/- 4.2%, 26.7 +/- 4.3% and 32.8 +/- 4.6% respectively). L-NOARG and ArgSA reversed the vasodilator effect of ACh but not of SNP. 3. L- and D-arginine had no effect on vascular resistance. 4. L-Glutamine (10(-3) M) increased baseline vascular resistance by 10 +/- 1.9% (P < 0.05) but did not alter responses to either ACh or SNP. 5. Basal NO release plays a role in the regulation of baseline diaphragmatic vascular resistance. L-Arginine analogues tested potently and specifically inhibited this process. Moreover, extracellular L-arginine appears to have no effect on baseline diaphragmatic vascular resistance.
Asunto(s)
Diafragma/irrigación sanguínea , Óxido Nítrico/fisiología , Resistencia Vascular/efectos de los fármacos , Acetilcolina/farmacología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Ácido Argininosuccínico/farmacología , Diafragma/efectos de los fármacos , Diafragma/metabolismo , Perros , Técnicas In Vitro , Tono Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , NG-Nitroarginina Metil Éster , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Nitroarginina , Nitroprusiato/farmacología , Flujo Sanguíneo Regional/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
The control of phosphatidylcholine biosynthesis in the hamster liver was examined. Livers of hamsters fasted for 24 and 48 h were perfused with labelled choline. Under both fasting conditions, the incorporation of labelled choline into phosphatidylcholine was reduced. After 48 h of fasting, the 52% reduction in phosphatidylcholine biosynthesis was caused by changes in several factors including a diminishing rate of choline uptake and severe reductions in the pool sizes of ATP and CTP (to 33-37% control values) which resulted in a decrease in the pools of choline-containing metabolites. The activation of cytidylyltransferase after 48 h of fasting might be regarded as a compensatory mechanism for the maintenance of phosphatidylcholine biosynthesis. After 24 h of fasting, a 25% reduction in phosphatidylcholine biosynthesis was observed. The ATP and CTP levels were decreased but the reduction was not severe enough to affect the choline uptake or the labelling of the phosphocholine fraction. The activities of the cytidylyltransferase remained unchanged but an accumulation of labelled CDP-choline was detected. Although choline-phosphotransferase activity was not changed in the microsomes, the enzyme activity was attenuated in the postmitochondrial fraction. Further analysis revealed that cholinephosphotransferase in the liver was inhibited by an endogenous inhibitor in the cytosol which was later identified as argininosuccinate. The level of argininosuccinate was elevated during fasting and the change quantitatively accounted for the attenuation of cholinephosphotransferase activity. The inhibition of choline-phosphotransferase by argininosuccinate, coupled with a substantial decrease in the diacylglycerol level, would provide the hamster liver with an immediate mechanism for the transient modulation of phosphatidylcholine biosynthesis during short-term fasting.
Asunto(s)
Ácido Argininosuccínico/farmacología , Diacilglicerol Colinafosfotransferasa/metabolismo , Privación de Alimentos/fisiología , Hígado/enzimología , Fosfatidilcolinas/metabolismo , Animales , Ácido Argininosuccínico/aislamiento & purificación , Compartimento Celular , Colina/metabolismo , Colina Quinasa/análisis , Citidililtransferasa de Colina-Fosfato , Cricetinae , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Diacilglicerol Colinafosfotransferasa/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Masculino , Mesocricetus , Nucleotidiltransferasas/análisis , Fracciones Subcelulares/enzimologíaRESUMEN
The role of the endothelium in the hyporesponsiveness of alpha-adrenoceptor-mediated contractions of the rat aorta was investigated. The norepinephrine-induced maximal contraction was diminished after repeated addition of the agonist. The hyporesponsiveness of the maximal contraction was endothelium dependent, being prevented by NG-monomethyl-L-arginine (0.5 mM), L-argininosuccinic acid (0.5 mM), puromycin (IC50 = 100 microM), actinomycin D (IC50 = 80 nM) but not by indomethacin, which suggests that nitric oxide (NO) synthase is induced. The sensitivity of the rings to NO-induced relaxation remained unchanged. The above-mentioned hyporesponsiveness of norepinephrine-induced maximal contractions of aorta rings was also observed after a 5-h incubation without norepinephrine. The agonist-independent hyporesponsiveness was also prevented by NG-monomethyl-L-arginine, puromycin and actinomycin D, which suggests that NO synthase is induced. Moreover, the norepinephrine-independent hyporesponsiveness was prevented by polymyxin B (10 micrograms/ml), which suggests that bacterial lipopolysaccharide (LPS) might be involved. The concentration of contaminating LPS was 89 +/- 11 ng/ml. When the concentration of contaminating LPS was reduced to 40-70 pg/ml, the hyporesponsiveness of the maximal contraction did not occur after repeated addition of norepinephrine or alter a 5-h incubation without the agonist. An addition of 30 or 100 ng/ml of E. coli lipopolysaccharide to the organ bath reproduced the hyporesponsiveness of the maximal contraction. After a 5-h incubation of aortic rings with 30 ng/ml LPS, only the endothelium-intact ring showed a reduced contraction. However, a 24-h incubation reduced the contraction even in the absence of endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Endotelio Vascular/fisiología , Lipopolisacáridos/farmacología , Norepinefrina/farmacología , Vasoconstricción/efectos de los fármacos , Aminoácido Oxidorreductasas/metabolismo , Animales , Aorta , Arginina/análogos & derivados , Arginina/farmacología , Ácido Argininosuccínico/farmacología , Dactinomicina/farmacología , Escherichia coli/química , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico Sintasa , Polimixina B/farmacología , Puromicina/farmacología , Conejos , Ratas , Ratas Sprague-Dawley , omega-N-MetilargininaRESUMEN
To assess the effect of endothelium-derived relaxing factor (EDRF) on diaphragmatic vascular resistance at rest and during contractions, we studied an in situ isolated diaphragm preparation in anesthetized and mechanically ventilated dogs. The arterial supply of the left diaphragm (phrenic artery) was catheterized and perfused with arterial blood at a fixed flow rate. Drugs were infused through a side port of the arterial catheter at 1/100th of the phrenic arterial flow. The inferior phrenic vein was catheterized to complete the isolation from the systemic circulation. Three sets of experiments were performed. In set 1 (n = 3), we infused endothelium-dependent (acetylcholine, ACh) and endothelium-independent (sodium nitroprusside, SNP) dilators at increasing concentrations. ACh and SNP infusion elicited a dose-dependent decline in phrenic vascular resistance (Rphr) at concentrations greater than 10(-8) M and 0.50 micrograms/ml, respectively. In set 2 (n = 15), we infused an inhibitor of EDRF synthesis and release, L-argininosuccinic acid (ArgSA), at increasing concentrations (10(-4), 3 x 10(-4), and 6 x 10(-4) M). ArgSA produced a dose-dependent increase in Rphr. Infusion of another EDRF inhibitor (NG-nitro-L-arginine, LNA, 6 x 10(-4) M) elicited increase in Rphr similar to that induced by ArgSA. In set 3 (n = 25), we infused ArgSA or LNA (6 x 10(-4) M) simultaneously with ACh and SNP and during sustained (2-Hz) contractions of the diaphragm. Both ArgSA and LNA completely reversed ACh vasodilation, whereas SNP vasodilation was reversed by 26 and 11%, respectively. ArgSA or LNA infusion during contractions reversed vasodilation by 48 and 52%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Diafragma/irrigación sanguínea , Hiperemia/etiología , Óxido Nítrico/fisiología , Acetilcolina/farmacología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Ácido Argininosuccínico/farmacología , Diafragma/efectos de los fármacos , Diafragma/fisiología , Perros , Hiperemia/fisiopatología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Óxido Nítrico/antagonistas & inhibidores , Nitroarginina , Nitroprusiato/farmacología , Resistencia Vascular/efectos de los fármacos , Resistencia Vascular/fisiología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
1. Argininosuccinic acid (ASA), a naturally occurring NG derivative of arginine, and the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) were compared for their ability to reduce responses to nitric oxide (NO) derived from endothelial cells (aorta) and nitrergic nerves (anococcygeus muscle). 2. In isolated rings of rat aorta, endothelium-dependent relaxation responses to acetylcholine were abolished by L-NAME (0.1 mmol/L) and were reduced by ASA (0.1 and 0.3 mmol/L). Relaxations induced by sodium nitroprusside (SNP) were not affected by L-NAME but were reduced by ASA. 3. In rat isolated anococcygeus muscles, relaxations elicited by nitrergic nerve stimulation at 1 Hz were abolished by L-NAME (0.1 mmol/L) but were only slightly reduced by ASA (1 mmol/L). The effect of ASA was not sustained. L-Arginine (1 mmol/L) prevented the effect of L-NAME but not that of ASA. Neither ASA or L-NAME inhibited SNP-induced relaxation in the anococcygeus muscle. 4. The results suggest that ASA inhibits NOS but this does not totally account for its effects in reducing NO-mediated relaxations produced by the endothelium-dependent vasodilator acetylcholine in rat aortic rings and stimulation of nitrergic nerves in the rat anococcygeus muscle.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Ácido Argininosuccínico/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculos/efectos de los fármacos , Óxido Nítrico/farmacología , Animales , Aorta Torácica/fisiología , Arginina/análogos & derivados , Arginina/antagonistas & inhibidores , Arginina/farmacología , Estimulación Eléctrica , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Músculo Liso Vascular/fisiología , Músculos/inervación , NG-Nitroarginina Metil Éster , Ratas , Ratas Endogámicas , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Fura-2 microfluorometry revealed that as little as 10(-13) M L-argininosuccinate, an intermediate of the L-arginine synthesizing pathway, is sufficient to reduce the increase in intracellular free calcium produced by L-glutamate in the acutely isolated cerebellar neurons of immature rats. In these neurons L-argininosuccinate also reduced the response to quisqualate, but not the response to kainate or N-methyl-D-aspartate. The results suggest that L-argininosuccinate may function as a transmitter or modulator in the brain.
Asunto(s)
Ácido Argininosuccínico/farmacología , Cerebelo/metabolismo , Glutamatos/farmacología , Neuronas/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Fura-2 , Ácido Glutámico , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Ácido Quiscuálico/farmacología , Ratas , Ratas Endogámicas , Espectrometría de FluorescenciaRESUMEN
The roles of tumor necrosis factor (TNF alpha) and reactive nitrogen intermediates (RNI) as effectors of macrophage-mediated tumor cell killing were investigated in a variety of tumor cell lines. Three TNF alpha-sensitive tumor targets were also susceptible to resting bone-marrow-derived mononuclear phagocytes (BMMP). This macrophage lytic activity was markedly diminished or even abolished by anti-TNF alpha, indicating that TNF alpha is the major effector of macrophage-mediated killing of these targets. The other 21 tumor cell lines examined were resistant to TNF alpha but, in their large majority, were more or less susceptible to killing by interferon gamma (IFN gamma)- and Corynebacterium parvum (CP)-activated BMMP. Among the various analogues of L-arginine used to assess the role of L-arginine-derived RNI as mediators of macrophage tumoricidal activity, NG-monomethyl-L-arginine (NMMA) was most efficient in suppressing RNI secretion by activated macrophages. In some macrophage tumor-cell combinations, NMMA inhibited both the generation of RNI and the expression of tumoricidal activity in a dose-dependent manner, suggesting a central role for RNI as effectors. In other combinations, NMMA in concentrations that abolished secretion of RNI either affected tumor-cell killing only after its induction by IFN gamma, or not at all. The findings not only support the thesis that macrophages posses various means of coping with tumor cells but also suggest that the mechanism becoming operative is determined predominantly by the pathway of macrophage activation and the properties of the tumor-cell type.
Asunto(s)
Arginina/análogos & derivados , Supervivencia Celular , Macrófagos/inmunología , Neoplasias/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Arginina/fisiología , Ácido Argininosuccínico/farmacología , Línea Celular , Humanos , Inmunidad Celular , Interferón gamma/fisiología , Activación de Macrófagos , Macrófagos/metabolismo , Masculino , Ratones , Nitroarginina , Propionibacterium acnes/patogenicidad , Ratas , omega-N-MetilargininaRESUMEN
This study examined the actions of L-arginine, a putative precursor of endothelium-derived nitric oxide, and arginine analogs on endothelium-dependent relaxation of isolated rings of bovine pulmonary artery. L-Arginine did not consistently relax arterial rings unless rings were first rendered refractory to endothelium-dependent relaxation by pretreatment with 1 microM A23187 for 45 min. L-Arginine-elicited relaxation was endothelium-dependent, antagonized by oxyhemoglobin or methylene blue, and unaffected by indomethacin. L-Argininosuccinic acid caused endothelium-dependent contractions and irreversible inhibition of endothelium-dependent but not nitroglycerin-elicited relaxation, which was not overcome by addition of L-arginine. Inhibition of endothelium-dependent relaxation by L-NG-monomethyl arginine, however, was reversible and overcome by L-arginine. Therefore, endothelium-dependent relaxants may cause arginine depletion in endothelial cells and endogenous argininosuccinic acid may modulate the biosynthesis of endothelium-derived nitric oxide from arginine.
Asunto(s)
Arginina/análogos & derivados , Arginina/farmacología , Ácido Argininosuccínico/farmacología , Endotelio Vascular/fisiología , Músculo Liso Vascular/efectos de los fármacos , Acetilcolina/farmacología , Animales , Calcimicina/farmacología , Bovinos , Técnicas In Vitro , Relajación Muscular/efectos de los fármacos , Nitroglicerina/farmacologíaRESUMEN
The nitro analogs of aspartate and argininosuccinate were synthesized and tested as substrates and inhibitors of argininosuccinate synthetase and argininosuccinate lyase, respectively. The Vmax for 3-nitro-2-aminopropionic acid was found to be 60% of the maximal rate of aspartate utilization in the reaction catalyzed by argininosuccinate synthetase. Only the nitronate form of this substrate, in which the C-3 hydrogen is ionized, was substrate active, indicating a requirement for a negatively charged group at the beta carbon. The V/K of the nitro analog of aspartate was 85% of the value of aspartate after correcting for the percentage of the active nitronate species. The nitro analog of argininosuccinate, N3-(L-1-carboxy-2-nitroethyl)-L-arginine, was a strong competitive inhibitor of argininosuccinate lyase but was not a substrate. The pH dependence of the observed pKi was consistent with the ionized carbon acid (pK = 8.2) in the nitronate configuration as the inhibitory material. The pH-independent pKi of 2.7 microM is 20 times smaller than the Km of argininosuccinate at pH 7.5. These results suggest that the tighter binding of the nitro analog relative to the substrate is due to the similarity in structure to a carbanionic intermediate in the reaction pathway.
Asunto(s)
Alanina/análogos & derivados , Arginina/análogos & derivados , Argininosuccinatoliasa/metabolismo , Argininosuccinato Sintasa/metabolismo , Ácido Argininosuccínico/análogos & derivados , Ligasas/metabolismo , Liasas/metabolismo , Alanina/síntesis química , Alanina/metabolismo , Alanina/farmacología , Argininosuccinatoliasa/antagonistas & inhibidores , Argininosuccinato Sintasa/antagonistas & inhibidores , Ácido Argininosuccínico/síntesis química , Ácido Argininosuccínico/metabolismo , Ácido Argininosuccínico/farmacología , Concentración de Iones de Hidrógeno , CinéticaRESUMEN
The behaviour of arginyl-tRNA synthetase (EC 6.1.1.19) in the presence of the arginine biosynthetic precursors, argininosuccinate, ornithine and citrulline, was studied in several Escherichia coli K12 strains and in E. coli W. The results of kinetic measurements with partially purified extracts indicate that the arginyl-tRNA synthetase of E. coli is not inhibited by the arginine precursors. The apparent affinity constant Km for arginine of the K12 enzyme is about 3.4 muM in the absence and in the presence of these precursors, whereas the W enzyme an apparently slightly lowered Km and a decreased [14C]arginyl-tRNA equilibrium level in the presence of argininosuccinate. This however was shown to be due to isotopic dilution of [14C]arginine by non-radioactive amino acid formed from argininosuccinate by argininosuccinate lyase (EC 4.3.2.1) contaminating the synthetase preparation. This finding emphasizes the necessity of using pure arginyl-tRNA synthetase in order to study the possible regulatory involvement of this enzyme in the control of the arginine regulon in vitro.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Arginino-ARNt Ligasa/metabolismo , Arginina/análogos & derivados , Ácido Argininosuccínico/farmacología , Citrulina/farmacología , Escherichia coli/enzimología , Ornitina/farmacología , Arginina/metabolismo , Argininosuccinatoliasa/metabolismo , Escherichia coli/efectos de los fármacos , CinéticaRESUMEN
As determined by equilibrium dialysis, bovine liver argininosuccinase of molecular weight 202,000 binds 4 mol of argininosuccinate or arginine/mol of enzyme. Negative homotropic interactions occur in the binding of both ligands at 0.15 ionic strength in the presence of phosphate. Argininosuccinate binds to two sites (Kdiss 1.6 times 10(-5) M) and four sites (Kdiss 2.9 times 10(-4) M) at low and high substrate concentration. Similarly, arginine binds to two sites (Kdiss 4.9 times 10(-4) M), and four sites (Kdiss 1.6 times 10(-3) M). At 0.05 ionic strength in Tris-HCl buffer, the four enzyme sites bind argininosuccinate independently and arginine binding remains negatively cooperative. Kinetic analysis gave double reciprocal plots that showed negative cooperatively also. The changes in Km were analogous to changes in Kdiss, thus indicating that the substrate binding sites correspond to catalytic sites. Since the catalytically active enzyme is a tetramer composed of four identical or closely similar subunits (Lusty, C.J., and Ratner, S. (1972) J. Biol. Chem. 247, 7010-7022), the present results show that each subunit contains one catalytic site. Ionic strength, phosphate ions, and GTP have each been found to influence negative cooperatively through a change in the affinity for argininosuccinate. The significance of the negative homotropic interactions and of the specific stimulation of activity by GTP is discussed with respect to different conformational forms of the enzyme and the in vivo regulation of argininosuccinase activity.