RESUMO
BACKGROUND: Pravastatin, a known inducer of endothelial nitric-oxide synthase (eNOS) was demonstrated in human placenta, however the exact mechanism of it's action is not fully understood. Since placental NO (nitric oxide) synthesis is of primary importance in the regulation of placental blood flow, we aimed to clarify the effects of pravastatin on healthy (n = 6) and preeclamptic (n = 6) placentas (Caucasian participants). METHODS: The eNOS activity of human placental microsomes was determined by the conversion rate of C14 L-arginine into C14 L-citrulline with or without pravastatin and Geldanamycin. Phosphorylation of eNOS (Ser1177) was investigated by Western blot. Microsomal arginine uptake was measured by a rapid filtration method. RESULTS: Pravastatin significantly increased total eNOS activity in healthy (28%, p<0.05) and preeclamptic placentas (32%, p<0.05) using 1 mM Ca2+ promoting the dissociation of a eNOS from it's inhibitor caveolin. Pravastatin and Geldanamycin (Hsp90 inhibitor) cotreatment increased microsomal eNOS activity. Pravastatin treatment had no significant effects on Ser1177 phosphorylation of eNOS in either healthy or preeclamptic placentas. Pravastatin induced arginine uptake of placental microsomes in both healthy (38%, p < 0.05) and preeclamptic pregnancies (34%, p < 0.05). CONCLUSIONS: This study provides a novel mechanism of pravastatin action on placental NO metabolism. Pravastatin induces the placental microsomal arginine uptake leading to the rapid activation of eNOS independently of Ser1177 phosphorylation. These new findings may contribute to better understanding of preeclampsia and may also have a clinical relevance.
Assuntos
Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Placenta/metabolismo , Pravastatina/farmacologia , Pré-Eclâmpsia/tratamento farmacológico , Adulto , Arginina/metabolismo , Benzoquinonas/farmacologia , Estudos de Casos e Controles , Citrulina/metabolismo , Feminino , Humanos , Lactamas Macrocíclicas/farmacologia , Microssomos/metabolismo , Pré-Eclâmpsia/metabolismo , GravidezRESUMO
The Warburg effect occurs both in cancer cells and in inflammatory macrophages. The aim of our work was to demonstrate the role of PI3K-Akt-mTOR axis in the Warburg effect in HL-60 derived, rat peritoneal and human blood macrophages and to investigate the potential of selected inhibitors of this pathway to antagonize it. M1 polarization in HL-60-derived and human blood monocyte-derived macrophages was supported by the increased expression of NOS2 and inflammatory cytokines. All M1 polarized and inflammatory macrophages investigated expressed higher levels of HIF-1α and NOS2, which were reduced by selected kinase inhibitors, supporting the role of PI3K-Akt-mTOR axis. Using Seahorse XF plates, we found that in HL-60-derived and human blood-derived macrophages, glucose loading reduced oxygen consumption (OCR) and increased glycolysis (ECAR) in M1 polarization, which was antagonized by selected kinase inhibitors and by dichloroacetate. In rat peritoneal macrophages, the changes in oxidative and glycolytic metabolism were less marked and the NOS2 inhibitor decreased OCR and increased ECAR. Non-mitochondrial oxygen consumption and ROS production were likely due to NADPH oxidase, expressed in each macrophage type, independently of PI3K-Akt-mTOR axis. Our results suggest that inflammation changed the metabolism in each macrophage model, but a clear relationship between polarization and Warburg effect was confirmed only after glucose loading in HL-60 and human blood derived macrophages. The effect of kinase inhibitors on Warburg effect was variable in different cell types, whereas dichloroacetate caused a shift toward oxidative metabolism. Our findings suggest that these originally anti-cancer inhibitors may also be candidates for anti-inflammatory therapy.
RESUMO
The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) pathway plays a pivotal role in macrophage polarization, but other signaling routes may also be involved. The aim of this study was to reveal the relationship of activation between rat peritoneal macrophages and their polarization, to detect the signaling routes involved, and find selective protein kinase inhibitors decreasing the production of inflammatory proteins in activated peritoneal macrophages. Rat macrophages were elicited with i.p. casein injection. CD80 and CD206 markers, NOS2 (Nitric oxide synthase 2), arginase, cytokines and phagocytosis were investigated by ELISA (Enzyme Linked Immunosorbent Assay), Western Blot, fluorescent microscopic and flow cytometry. Statistical methods were ANOVA (Analysis Of Variance) and Student t-tests. Resident and elicited cells expressed both CD80 and CD206 polarization markers. The involvement of MAPK (mitogen-activated protein kinases) and JAK/STAT pathways in the polarization was evidenced by a phosphorylation array, supported by Western blotting, by cytokine markers and by the inhibitory effects of kinase inhibitors. The expression of NOS2 and inflammatory cytokines was higher in elicited cells suggesting their M1 polarization. This effect was reduced by the inhibitors of MAPK and JAK/STAT pathways. Phagocytosis was also higher in elicited macrophages and decreased by these inhibitors. Nevertheless, they cannot change macrophage polarization unambiguously, as levels of CD80 and CD206 markers were not changed. For comparison, human blood macrophages were also studied. Similar effects and several differences were observed between the two types of macrophages, suggesting the role of the previous differentiation in defining their characteristics. Selected anti-cancer protein kinase inhibitors of p38, MAPK and JAK/STAT pathways are possible candidates for the therapy of inflammatory diseases.
Assuntos
Citocinas , Macrófagos Peritoneais , Óxido Nítrico Sintase Tipo II , Inibidores de Proteínas Quinases , Animais , Humanos , Ratos , Citocinas/metabolismo , Janus Quinases , Macrófagos Peritoneais/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fagocitose , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Statins are used to treat hypercholesterolemia, with several pleiotropic effects. Alongside their positive effects (for example, decreasing blood pressure), they can also bring about negative effects/symptoms (such as myopathy). Their main mechanism of action is inducing apoptosis, the key step being the release of cytochrome c from the mitochondria. This can be facilitated by oxidative stress, through which glutathione is oxidized. In this research, glutathione was used as a respiratory substrate to measure the mitochondrial oxygen consumption of rat liver with an O2 electrode. The reduction of cytochrome c was monitored photometrically. Hydrophilic (pravastatin) and lipophilic (simvastatin) statins were used for the measurements. Pravastatin reduces the reduction of cytochrome c and the oxygen consumption of the mitochondria, while simvastatin, on the other hand, increases the reduction of cytochrome c and the mitochondrial oxygen consumption. The results make it seem probable that statins influence the mitochondrial oxygen consumption through cytochrome c. Simvastatin could enhance the oxidizing capacity of free cytochrome c, thereby increasing oxidative stress and thus facilitating apoptosis. The observed effects could further the understanding of the mechanism of action of statins and thereby aid in constructing optimal statin therapy for every patient.
RESUMO
JAK/STAT pathway plays a well-known role in macrophage polarization, but other signaling routes may also be involved. The aim of this study was to identify new signaling pathways and repolarize macrophages by selected protein kinase inhibitors. HL-60 derived macrophages were chosen as model cells and human blood macrophages were used for comparison. M1 and M2 polarization of HL60 derived and human blood macrophages was promoted by LPS + IFNγ (LIF) and IL-4 treatments, respectively. In HL-60 derived macrophages, M1 polarization was mediated by Erk1/2 and p38 phosphorylation, while HSP27 phosphorylation was involved in M2 polarization. The inhibition of both MAPK and JAK/STAT pathways reduced the expression of NOS2, IP-10 and TNFα, IL-8 production was decreased by the inhibition of AMPK and PKD, the upstream kinase of HSP27. HSP27 phosphorylation was inhibited by NB 142, a PKD inhibitor. The expression of CD80 (M1 marker) was reduced by MAPK and JAK/STAT inhibitors, without increasing CD206 (M2 marker). On the other hand, CD206 was reduced by PKD and AMPK inhibitors, without increasing CD80 marker. Phagocytic capacity of HL-60 derived macrophages was higher in M1 macrophages and decreased by trametinib and a p38 inhibitor, while in human blood macrophages, where AT 9283, a JAK/STAT inhibitor also caused a significant decrease in M1 polarized macrophages, no difference was observed between M1 and M2 macrophages. Our results suggest that the repolarization of macrophages cannot be achieved by inhibiting their signaling pathways; nevertheless, the expression of certain polarization markers was decreased, therefore a "depolarization" could be observed both in M1 and M2 polarized cells. Selected protein kinase inhibitors of M1 polarization, decreasing NOS 2 and inflammatory cytokines may be potential candidates for therapeutical trials against inflammatory diseases.
RESUMO
OBJECTIVE AND DESIGN: The role of NO and adipocytokines in childhood obesity was studied, supposing that obesity provokes inflammation. Children were admitted to the pediatric clinic for a regular check up because of obesity. SUBJECTS: Obese (n = 79) and healthy (n = 12) children were selected and divided into subgroups according to their age, gender, glucose tolerance and nitric oxide synthase (NOS II) positivity. METHODS: Urine and blood nitrite plus nitrate, the expression of NOS II in white blood cells, serum adipocytokines and clinical characteristics were analyzed in each group. Significance was tested by unpaired two-tailed t test and by ANOVA. RESULTS: NOS II was only detected in the white blood cells of a subgroup (17/79) of obese children. Serum leptin and resistin concentrations were significantly higher, adiponectin was lower compared to healthy children. Significant correlations were observed between serum adiponectin and resistin levels (reciprocal, R (2) = 0.4), and between body mass index and serum leptin levels. CONCLUSIONS: NOS II expression in white blood cells was observed in a minority of patients. Low-grade inflammation in obese children was suggested by the increased resistin levels, particularly in NOS II-positive patients. Correlation between different adipocytokines was restricted for a few subgroups.
Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico/metabolismo , Obesidade/metabolismo , Adipócitos/metabolismo , Adipocinas/metabolismo , Adiponectina/biossíntese , Adolescente , Índice de Massa Corporal , Proteína C-Reativa/biossíntese , Criança , Pré-Escolar , Feminino , Humanos , Inflamação , Interleucina-6/biossíntese , Leptina/biossíntese , Leucócitos/metabolismo , Masculino , Óxido Nítrico Sintase Tipo II/biossíntese , Resistina/biossínteseRESUMO
BACKGROUND: Weak androgens have an antioxidant effect in vitro which is represented as a beneficial change in the antioxidant status. OBJECTIVE: Our aim was to clarify whether dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulphate (DHEAS) oral administration results in beneficial antioxidant changes in Sprague-Dawley adult male rats in vivo. METHODS: Groups of experimental animals were fed a high-fat or a normal-fat diet and treated with DHEA or DHEAS in the drinking fluid. The control group was fed a high-fat diet together with untreated drinking fluid. Total scavenger capacity (TSC) was measured before and after 4 weeks of treatment in blood samples using a chemiluminometric assay. Fat content, superoxide dismutase (SOD), catalase and glutathione S-transferase (GST) activity in the liver were determined by Sudan staining and spectrophotometric assessments, respectively, from the fresh frozen tissue. RESULTS: DHEA and the DHEAS treatment showed significantly increased TSC in the groups fed a high-fat diet. The control group and the DHEA- or DHEAS-treated groups on normal diets showed no significant changes in TSC. The total score of liver fat content in the high-fat diet groups showed a marked positivity with Sudan staining, and the groups treated with DHEA or DHEAS had a markedly decreased amount of fat in the liver slides compared to the untreated group on the high-fat diet. Liver SOD activity was decreased in all high-fat diet groups and elevated only in the groups on a normal diet with DHEA or DHEAS treatment. Liver catalase and GST activities were decreased in the groups where TSC was significantly increased. CONCLUSION: Our results support the hypothesis that DHEA and DHEAS supplementation can improve the antioxidant status in lipid-rich dietary habits.
Assuntos
Antioxidantes/metabolismo , Desidroepiandrosterona/farmacologia , Gorduras na Dieta/administração & dosagem , Fígado Gorduroso/prevenção & controle , Glutationa Transferase/metabolismo , Superóxido Dismutase/metabolismo , Administração Oral , Animais , Antioxidantes/análise , Catalase/efeitos dos fármacos , Catalase/metabolismo , Sulfato de Desidroepiandrosterona/farmacologia , Modelos Animais de Doenças , Fígado Gorduroso/patologia , Glutationa Transferase/efeitos dos fármacos , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Valores de Referência , Espectrofotometria , Superóxido Dismutase/efeitos dos fármacosRESUMO
The uptake of L-arginine into mouse peritoneal macrophages can be inhibited by numerous amino acids and derivatives. Kinetic studies showed an almost entirely competitive inhibition for both cationic and neutral amino acids and derivatives suggesting that the comparison of their binding specificity by using a quantitative structure-activity relationship (QSAR) study is reasonable. The properties of the most efficient inhibitors were the following: the length of the aliphatic side chain, a general structural similarity to L-arginine (>0.79), cationic character, L-configuration, the presence of an alpha-amino group (with a mean pK(a) of 9.41), the van der Waals volume (mean 225 A(3)) and a low logP value (mean: -2.99). The significance of four other descriptors (neutral character, presence and the pK(a) of an alpha-carboxyl group, and the presence of a modified guanidino group) is much lower. Similar results were obtained for the hCAT-1 cell line, but the significance of the descriptors was slightly different. The L-configuration, van der Waals volume, the low logP value and the length of aliphatic side chain were the most significant, while the pK(a) value of the side chain (mean pK(a)=11.6) was found to be more important than that of the alpha-amino group. In addition, the general similarity to L-arginine, the presence of an amino group in the terminal position of the side chain (Orn, Lys) and the basic character were significant descriptors, while the significance of the acidity is negligibly low. As a final conclusion, the following descriptors were found to be important generally for the cationic transporters: the van der Waals volume, hydrophobicity (log P); L-configuration; the size of the side chain; the general similarity to L-arginine; the presence of an alpha-amino group; the general basicity of the molecule; the pK(a) values of the alpha-amino group (in macrophages) or that of the side chain (in CAT-1 cells). These descriptors can be regarded as the general structurally important binding characteristics of the cationic amino transporters.
Assuntos
Arginina/metabolismo , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Macrófagos Peritoneais/metabolismo , Animais , Arginina/química , Sítios de Ligação/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Transportador 1 de Aminoácidos Catiônicos/antagonistas & inibidores , Transportador 1 de Aminoácidos Catiônicos/química , Linhagem Celular Tumoral , Humanos , Cinética , CamundongosRESUMO
The aim of our study was to investigate the adaptation of the hypothalamic circulation to chronic nitric oxide (NO) deficiency in rats. Hypothalamic blood flow (HBF) remained unaltered during chronic oral administration of the NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 1 mg/ml drinking water) although acute NOS blockade by intravenous l-NAME injection (50 mg/kg) induced a dramatic HBF decrease. In chronically NOS blocked animals, however, acute l-NAME administration failed to influence the HBF. Reversal of chronic NOS blockade by intravenous l-arginine infusion evoked significant hypothalamic hyperemia suggesting the appearance of a compensatory vasodilator mechanism in the absence of NO. In order to clarify the potential involvement of vasodilator prostanoids in this adaptation, cyclooxygenase (COX) mRNA and protein levels were determined in the hypothalamus, but none of the known isoenzymes (COX-1, COX-2, COX-3) showed upregulation after chronic NOS blockade. Furthermore, levels of vasodilator prostanoid (PGI(2), PGE(2) and PGD(2)) metabolites were also not elevated. Interestingly, however, hypothalamic levels of vasoconstrictor prostanoids (TXA(2) and PGF(2alpha)) decreased after chronic NOS blockade. COX inhibition by indomethacin but not by diclofenac decreased the HBF in control animals. However, neither indomethacin nor diclofenac induced an altered HBF-response after chronic l-NAME treatment. Although urinary excretion of PGI(2) and PGE(2) metabolites markedly increased during chronic NOS blockade, indicating COX activation in the systemic circulation, we conclude that the adaptation of the hypothalamic circulation to the reduction of NO synthesis is independent of vasodilator prostanoids. Reduced release of vasoconstrictor prostanoids, however, may contribute to the normalization of HBF after chronic loss of NO.
Assuntos
Adaptação Fisiológica/fisiologia , Artérias Cerebrais/fisiologia , Circulação Cerebrovascular/fisiologia , Hipotálamo/metabolismo , Óxido Nítrico/deficiência , Prostaglandinas/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Animais , Arginina/farmacologia , Artérias Cerebrais/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Círculo Arterial do Cérebro/efeitos dos fármacos , Círculo Arterial do Cérebro/fisiologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/metabolismo , Inibidores Enzimáticos/farmacologia , Epoprostenol/metabolismo , Hiperemia/induzido quimicamente , Hipotálamo/irrigação sanguínea , Hipotálamo/efeitos dos fármacos , Masculino , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Prostaglandina D2/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Wistar , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/metabolismo , Vasodilatadores/farmacologiaRESUMO
Certain receptor tyrosine kinase (RTK) inhibitors reduced the phagocytic capacity of rat macrophages, without influencing the binding of bacteria to macrophage surface. The NO production of elicited rat macrophages was also decreased due to the inhibition of the expression of NOS II. The most potent inhibitory compound was PD166326 (6-(2,6-dichloro-phenyl)-2-(4-hydroxy-phenylamino)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one) belonging to a family of RTK inhibitors of broad spectra. These impairing effects could be explained by the apoptosis inducing property of the inhibitor, evidenced by the destroyed mitochondrial membrane potential. The MTT cell viability test indicated a slight, but significant injury of macrophages. In addition to this compound, two other tested RTK inhibitors caused less marked impairment of macrophage functions, while four compounds were not efficient on macrophages at all. Nevertheless, these damaging effects of the inhibitors did not reduce the anti-tumor effect of the RTK inhibitors on COS 7 cells as evidenced by MTT test and apoptosis study. However, these side effects may be important when RTK inhibitors are selected against tumor growth, indicating that certain inhibitors may impair the immune defense during therapeutical application.
Assuntos
Fagocitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Apoptose , Células COS , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/imunologia , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos WistarRESUMO
A new family of G-protein-coupled receptors (GPRC6A) has recently been described and characterized with unknown physiological role. Three isoforms of GPRC6A are expressed from the same gene by alternative splicing. Agonists for GPRC6A are basic amino acids, particularly the analogues and derivatives of L-arginine and L-ornithine. These compounds are known ligands of nitric oxide synthase and arginase isoenzymes, but amino-acid sequences of these enzymes have not shown significant homologies. Various hypotheses for physiological roles of these receptors have been proposed but not yet substantiated. In order to define the most potent agonists, and elucidate further the biological significance of the receptor family, a detailed ligand-based quantitative structure-activity relationship study may be helpful.
Assuntos
Arginase/antagonistas & inibidores , Arginina/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Animais , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Isoformas de Proteínas , Receptores Acoplados a Proteínas G/genéticaRESUMO
Rat peritoneal macrophages were induced to produce high amounts of nitric oxide (NO) when rats were challenged by MTH68/H, (a live attenuated oncolytic Newcastle disease virus strain). The increase in NO production was observed to be viral particle dose dependent. The higher NO production measured could be due to the enhanced expression of NO synthase II enzyme. In addition, viral administration caused a higher macrophage cell count in the peritoneal cavity of treated rats. Interleukin-1 and granulocyte-monocyte colony stimulating factors were also produced by the induced macrophages. COS 7, a transformed cell line was killed by both NO donors and activated macrophages; the latter effect was markedly decreased in the presence of the inhibitors of NO production. Cytotoxic effect of NO was evidenced by the decrease of cell viability and proliferation of COS 7 cells. Excessive NO production may also be cytotoxic for macrophages themselves as proved by the addition of exogenous NO donors. These results strongly suggested the participation of induced NO synthesis of macrophages in the anti-tumor effect of MTH-68/H vaccine treatment.
Assuntos
Macrófagos Peritoneais/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/biossíntese , Vírus Oncolíticos/imunologia , Vacinas Virais/toxicidade , Animais , Arginase/metabolismo , Células COS/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Hidrazinas/farmacologia , Técnicas In Vitro , Interleucina-1/metabolismo , Macrófagos Peritoneais/metabolismo , Masculino , Óxido Nítrico/farmacologia , Nitritos/metabolismo , Ratos , Ratos WistarRESUMO
The relationship between diabetes mellitus Type 1 and nitric oxide (NO) synthesis was studied in multiple low-dose streptozotocin (STZ)-treated rats and diabetic children. The aim of our experimental work was to test the effect of hyperglycemic state on the level of urinary stable NO end products and on the expression of inducible nitric oxide synthase (NOS II) in white blood cells (WBC). It was also studied whether the measurements of these parameters were suitable to predict the presence of early diabetes before its onset. The occurrence of insulitis in streptozotocin-treated rats could not be clearly demonstrated. Urinary nitrite plus nitrate level significantly increased both in diabetic rats and in children compared to controls. However, the increase of the activity and the expression of inducible NOS II were only observed in rat white blood cells and this effect was prevented by insulin treatment. In human samples, less than 25% of children showed elevated NOS II expression in white blood cells without any correlation to the level of urinary NO end products and glycated hemoglobin in blood. Correlation was found only between the activity and expression of NOS II in white blood cells of patients whose white blood cells were positive for the presence of NOS II. Measurement of urinary nitrite plus nitrate content as well as the determination of NOS II expression of white blood cells in an early phase of diabetes are not suitable predictors in humans probably due to the basic differences in the mechanism of streptozotocin-induced rat and spontaneous human Type 1 diabetes.
Assuntos
Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 1/sangue , Óxido Nítrico/sangue , Animais , Criança , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Humanos , Leucócitos/metabolismo , Nitratos/sangue , Óxido Nítrico Sintase Tipo II/sangue , Nitritos/metabolismo , RatosRESUMO
Nitric oxide (NO) production was increased in macrophages during inflammation. Casein-elicitation of rodents causing a peritoneal inflammation offered a good model to study alterations in the metabolism of L-arginine, the precursor of NO synthesis. The utilization of L-arginine for NO production, arginase pathway and protein synthesis were studied by radioactive labeling and chromatographic separation. The expression of NO synthase and arginase was studied by Western blotting.Rat macrophages utilized more arginine than mouse macrophages (228+/-27 versus 71+/-12.8pmol per 10(6) macrophages). Arginine incorporation into proteins was low in both species (<15% of labeling). When NO synthesis was blocked, arginine was utilized at a lower general rate, but L-ornithine formation did not increase. The expression of enzymes utilizing arginine increased. NO production was raised mainly in rats (1162+/-84pmol citrulline per 10(6) cells) while in mice both arginase and NO synthase were active in elicited macrophages (677+/-85pmol ornithine and 456+/-48pmol citrulline per 10(6) cells). We concluded, that inflammation induced enhanced L-arginine utilization in rodent macrophages. The expressions and the activities of arginase and NO synthase as well as NO formation were increased in elicited macrophages. Specific blocking of NO synthesis did not result in the enhanced effectivity of the arginase pathway, rather was manifested in a general lower rate of arginine utilization. Different rodent species reacted differently to inflammation: in rats, high NO increase was found exclusively, while in mice the activation of the arginase pathway was also important.
Assuntos
Arginina/metabolismo , Inflamação/metabolismo , Macrófagos Peritoneais/metabolismo , Óxido Nítrico/metabolismo , Animais , Arginase/metabolismo , Caseínas/toxicidade , Fracionamento Celular , Células Cultivadas , Inflamação/induzido quimicamente , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ratos , Ratos WistarRESUMO
The cerebrovascular effects of the heme oxygenase-carbon monoxide pathway were studied in the rat hypothalamus. Intraperitoneal administration of the heme oxygenase inhibitor zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG, 45 micro mol/kg) had no significant effect on the resting cerebral blood flow, but increased hypothalamic nitric oxide synthase activity by 67% without changing the CSF cyclic GMP concentration. After pharmacologic inhibition of nitric oxide synthase, the diminished cerebral blood flow was further reduced by 22% after administration of ZnDPBG, and the effect showed direct correlation with the baseline perfusion level. Therefore, endogenous carbon monoxide may significantly contribute to the cerebral vasoregulation under resting conditions and in pathophysiologic states associated with diminished nitric oxide synthesis.
Assuntos
Circulação Cerebrovascular/fisiologia , Heme Oxigenase (Desciclizante)/metabolismo , Hipotálamo/irrigação sanguínea , Hipotálamo/enzimologia , Óxido Nítrico Sintase/metabolismo , Equilíbrio Ácido-Base/fisiologia , Animais , Monóxido de Carbono/metabolismo , Circulação Cerebrovascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Heme Oxigenase-1 , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Ratos , Ratos Wistar , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
The heme oxygenase (HO)-carbon monoxide pathway was earlier shown to increase hypothalamic blood flow after inhibition of nitric oxide synthesis in rats. We hypothesized that this effect is mediated by prostaglandin E2 (PGE2). Inhibition of constitutive HO activity decreased cerebral PGE2 production and simultaneously increased hypothalamic nitric oxide synthase (NOS) activity without changing hypothalamic blood flow. Furthermore, HO blockade induced cyclooxygenase-dependent decrease and NOS-mediated increase of the hypothalamic blood flow after inhibition of NOS and cyclooxygenase, respectively. Therefore, constitutive carbon monoxide release seems to have two indirect effects on the hypothalamic circulation: vasodilation mediated by PGE2 and vasoconstriction as a result of NOS inhibition.