RESUMO
BACKGROUND: Triatoma infestans is the main vector of Chagas'disease in Southern Cone countries. In triatomines, symptoms suggesting neurotoxicity were observed after treatment with Jaburetox (Jbtx), the entomotoxic peptide obtained from jackbean urease. Here, we study its effect in the central nervous system (CNS) of this species. METHODS: Immunohistochemistry, Western blots, immunoprecipitation, two-dimensional electrophoresis, tandem mass spectrometry and enzymatic assays were performed. RESULTS: Anti-Jbtx antibody labeled somata of the antennal lobe only in Jbtx-treated insects. Western blot assays of nervous tissue using the same antibody reacted with a 61kDa protein band only in peptide-injected insects. Combination of immunoprecipitation, two-dimensional electrophoresis and tandem mass spectrometry identified UDP-N-acetylglucosamine pyrophosphorylase (UDP-GlcNAcP) as a molecular target for Jbtx. The activity of UDP-GlcNAcP increased significantly in the CNS of Jbtx-treated insects. The effect of Jbtx on the activity of nitric oxide synthase (NOS) and NO production was investigated as NO is a recognized messenger molecule in the CNS of T. infestans. NOS activity and NO levels decreased significantly in CNS homogenates of Jbtx-treated insects. CONCLUSIONS: UDP-GlcNAcP is a molecular target of Jbtx. Jbtx impaired the activity of T. infestans nitrergic system, which may be related with early behavioral effects. GENERAL SIGNIFICANCE: We report that the CNS of Triatoma infestans is a target for the entomotoxic peptide and propose that a specific area of the brain is involved. Besides potentially providing tools for control strategies of Chagas' disease vectors our data may be relevant in various fields of research as insect physiology, neurobiology and protein function.
Assuntos
Sistema Nervoso Central/enzimologia , Inibidores Enzimáticos/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Peptídeos/farmacologia , Proteínas de Plantas/farmacologia , Triatoma/enzimologia , Urease/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Inibidores Enzimáticos/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Nucleotidiltransferases/metabolismo , Peptídeos/química , Proteínas de Plantas/química , Urease/químicaRESUMO
We have previously shown that ovariectomy in adult Wistar rats under normal sodium (NS) intake results in an overexpression of the total Na(+)-K(+)-ATPase (NKA) α1-subunit (Di Ciano LA, Azurmendi PJ, Toledo JE, Oddo EM, Zotta E, Ochoa F, Arrizurieta EE, Ibarra FR. Clin Exp Hypertens 35: 475-483, 2013). Upon high sodium (HS) intake, ovariectomized (oVx) rats developed defective NKA phosphorylation, a decrease in sodium excretion, and an increment in mean blood pressure (MBP). Since NKA phosphorylation is modulated by dopamine (DA), the aim of this study was to compare the intracellular response of the renal DA system leading to NKA phosphorylation upon sodium challenge in intact female (IF) and oVx rats. In IF rats, HS caused an increase in urinary DA and sodium, in NKA phosphorylation state, in cytochrome P-4504A (CYP4A) expression, and in 20-HETE production, while MBP kept normal. Blockade of the D1 receptor (D1R) with the D1-like receptor antagonist SCH 23390 in IFHS rats shifted NKA into a more dephosphorylated state, decreased sodium excretion by 50%, and increased MBP. In oVxNS rats, D1R expression was reduced and D3R expression was increased, and under HS intake sodium excretion was lower and MBP higher than in IFHS rats (both P < 0.05), NKA was more dephosphorylated than in IFHS, and CYP4A expression or 20-HETE production did not change. Blockade of D1R in oVxHS rats changed neither NKA phosphorylation state nor sodium excretion or MBP. D2R and PKCα expression did not vary among groups. The alteration of the renal DA system produced by ovariectomy could account for the defective NKA phosphorylation, the inefficient excretion of sodium load, and the development of salt-sensitive hypertension.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Dopamina/metabolismo , Sódio na Dieta/farmacologia , Envelhecimento , Animais , Modelos Animais de Doenças , Hipertensão/fisiopatologia , Ovariectomia/métodos , Ratos Wistar , Receptores de Dopamina D1/metabolismo , Cloreto de Sódio na Dieta/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The androgen receptor (AR) and AR-driven genes are crucial in normal and neoplastic prostate tissue. Previous results showed a link between 20-hydroxyeicosatetraenoic acid (20-HETE) production and AR-driven prostate cancer (PCa) progression. This study aims to describe the contribution of GPR75, 20-HETE membrane receptor, in 20-HETE-mediated expression and transcriptional activity of AR in PCa. In LNCaP cells, 20-HETE increased AR expression, nuclear localization, and its transcriptional activity. Also, 20-HETE enhanced dihydrotestosterone (DHT) induced effects. All was abrogated by chemical antagonism of GPR75 (19-HEDE) or its transient knockdown. In human PCa, the expression of AR-driven genes correlated with GPR75. In LNCaP xenografts, tumors from castrated animals expressed higher levels of AR, this was impaired by inhibition of 20-HETE synthesis. These data suggest that 20-HETE, through the GPR75 receptor, regulates transcriptionally active AR in PCa cells, thus making 20-HETE/GRP75 potential targets to limit the expression of AR-driven phenotype in PCa cells.
Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Humanos , Masculino , Animais , Receptores Androgênicos/metabolismo , Próstata/metabolismo , Androgênios/farmacologia , Androgênios/metabolismo , Linhagem Celular Tumoral , Neoplasias da Próstata/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The importance of cytochrome P450 (CYP)-derived arachidonic acid (AA) metabolites, 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) as tumor growth promotors has already been described in several cancer types. The aim of this study was to evaluate the role of these compounds in the biology of pheochromocytoma/paraganglioma. These tumors originate from chromaffin cells derived from adrenal medulla (pheochromocytomas) or extra-adrenal autonomic paraganglia (paragangliomas), and they represent the most common hereditary endocrine neoplasia. According to mutations in the driver genes, these tumors are divided in two clusters: pseudo-hypoxic and kinase-signaling EETs, but not 20-HETE, exhibited a potent ability to sustain growth in a murine pheochromocytoma cell line (MPC) in vitro, EETs promoted an increase in cell proliferation and a decrease in cell apoptosis. In a mouse model of pheochromocytoma, the inhibition of CYP-mediated AA metabolism using 1-aminobenzotriazol resulted in slower tumor growth, a decreased vascularization, and a lower final volume. Also, the expression of AA-metabolizing CYP monooxygenases was detected in tumor samples from human origin, being their apparent abundance and the production of both metabolites higher in tumors from the kinase-signaling cluster. This is the first evidence of the importance of CYP- derived AA metabolites in the biology and development of pheochromocytoma/paraganglioma tumors.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Neoplasias das Glândulas Suprarrenais/induzido quimicamente , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Feocromocitoma/induzido quimicamente , Ácido 8,11,14-Eicosatrienoico/farmacologia , Adolescente , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Animais , Linhagem Celular Tumoral , Criança , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neovascularização Patológica , Feocromocitoma/patologia , Adulto JovemRESUMO
PURPOSE: Recent studies have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) is a key molecule in sustaining androgen-mediated prostate cancer cell survival. Thus, the aim of this study was to determine whether 20-HETE can affect the metastatic potential of androgen-insensitive prostate cancer cells, and the implication of the newly described 20-HETE receptor, GPR75, in mediating these effects. METHODS: The expression of GPR75, protein phosphorylation, actin polymerization and protein distribution were assessed by western blot and/or fluorescence microscopy. Additionally, in vitro assays including epithelial-mesenchymal transition (EMT), metalloproteinase-2 (MMP-2) activity, scratch wound healing, transwell invasion and soft agar colony formation were used to evaluate the effects of 20-HETE agonists/antagonists or GPR75 gene silencing on the aggressive features of PC-3 cells. RESULTS: 20-HETE (0.1â¯nM) promoted the acquisition of a mesenchymal phenotype by increasing EMT, the release of MMP-2, cell migration and invasion, actin stress fiber formation and anchorage-independent growth. Also, 20-HETE augmented the expression of HIC-5, the phosphorylation of EGFR, NF-κB, AKT and p-38 and the intracellular redistribution of p-AKT and PKCα. These effects were impaired by GPR75 antagonism and/or silencing. Accordingly, the inhibition of 20-HETE formation with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) elicited the opposite effects. CONCLUSIONS: The present results show for the first time the involvement of the 20-HETE-GPR75 receptor in the activation of intracellular signaling known to be stimulated in cell malignant transformations leading to the differentiation of PC-3 cells towards a more aggressive phenotype. Targeting the 20-HETE/GPR75 pathway is a promising and novel approach to interfere with prostate tumor cell malignant progression.
Assuntos
Ácidos Hidroxieicosatetraenoicos/metabolismo , Neoplasias da Próstata/patologia , Receptores Acoplados a Proteínas G/metabolismo , Amidinas/farmacologia , Androgênios/metabolismo , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Ácidos Hidroxieicosatetraenoicos/agonistas , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
20-Hydroxyeicosatetraenoic acid (20-HETE) is generated intracellularly through the ω-hydroxylation of arachidonic acid by the cytochrome P450 (in humans, CYP4A11 and CYP4F2). 20-HETE induces mitogenic responses in different cancer cells. The aim of this study was to analyze how 20-HETE impacts cell survival, proliferation, and apoptosis in prostate cancer cells. Incubation of the human androgen-sensitive cells (LNCaP) with 1-10 µM HET0016 (a selective inhibitor of 20-HETE synthesis) reduced cell viability by 49*-64%* (*p < 0.05 vs. control). This was explained by a reduction in cell proliferation (vehicle, 46 ± 3%; 1 µM, 23 ± 3%*; 10 µM, 28 ± 3%*) and by an increase in apoptosis (vehicle, 2.1 ± 0%; 1 µM, 16 ± 4%*; 10 µM, 31 ± 3%*). Furthermore, the increase in LNCaP cell viability induced by dihydrotestosterone (DHT, 0.1 nM) was abrogated by 30*-42%* by 1-10 µM HET0016. Incubation with 20-HETE (5-1000 nM) increased LNCaP cell viability up to 50%*, together with a 70%* reduction in apoptosis. PC-3 (androgen-insensitive) cell viability was not affected by either HET0016 or 20-HETE. In LNCaP cells, HET0016 (10 µM) diminished the expression of androgen receptors (AR): messenger RNA (mRNA) (40%*) and protein (50%*). DHT (10 nM) augmented CYP4F2 protein expression (1.9-fold*) and 20-HETE levels (50%*). Oppositely, enzalutamide (AR antagonist) reduced CYP4F2 mRNA and protein expressions by 30 and 25%, respectively. Thus, intracellular availability of 20-HETE is necessary to sustain LNCaP cell viability. 20-HETE may act as a signaling molecule in the pathways involved in LNCaP cell viability upon stimulation of the AR. This effect may be partially attributed to its role on securing normal AR expression levels that in turn contribute to maintain intracellular levels of 20-HETE.
Assuntos
Androgênios/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Neoplasias da Próstata/metabolismo , Androgênios/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Família 4 do Citocromo P450/antagonistas & inibidores , Família 4 do Citocromo P450/metabolismo , Di-Hidrotestosterona/metabolismo , Di-Hidrotestosterona/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Ácidos Hidroxieicosatetraenoicos/biossíntese , Masculino , Neoplasias da Próstata/genética , Receptores Androgênicos/metabolismoRESUMO
The distributions of neuropeptide Y (NPY) -like immunoreactivity (LI) and that of its Y1 receptor (Y1), as well as their coexistence with cholecystokinin (CCK) -LI, were studied in the central nervous system of Triatoma infestans by using immunohistochemistry. NPY-immunoreactive (IR) cell bodies and fibers were observed in the brain, subesophageal ganglion, and thoracic ganglia. NPY-IR somata were seen in the optic lobe and the anteromedial and caudolateral soma rinds of the protocerebrum. Immunostained cell bodies were also found in the lateral edge of the antennal lobe glomeruli as well as in the caudal part of the antennal mechanosensory and motor center. The subesophageal ganglion harbored few NPY-IR perikarya and fibers in the three neuromeres. Positive somata of the prothoracic ganglion were detected near both the cephalic and posterior connectives as well as by the root of prothoracic nerve I, whereas in the posterior ganglion, they were seen by the roots of mesothoracic and abdominal nerves. Coexpression of NPY-LI and CCK-LI was seen in cell bodies of the protocerebrum, the subesophageal and posterior ganglia. Protocerebral Y1-IR cell groups were detected in the anterolateral and posteromedial soma rinds and at the level of the lamina ganglionaris and the external optic chiasma. Numerous positive perikarya surrounded the antennal lobe glomeruli as well as the antennal mechanosensory and motor center. Other immunostained cell bodies were seen in the posterior edge of the esophageal canal and by the roots of the mandibular and the maxillary nerves. Y1-IR cell bodies of the prothoracic ganglion were found near the roots of prothoracic nerves I-II, whereas in the posterior ganglion, they were located mainly in the abdominal neuromeres. Coexpression of Y1-LI and CCK-LI were detected in several brain areas as well as in the metathoracic and abdominal neuromeres of the posterior ganglion. When assessed by immunoblotting, Y1 antibodies detected two protein bands between 34 and 46 kDa. Analysis of the distribution patterns of NPY-LI and Y1-LI suggest that peptide and receptor are mainly involved in the processing of information coming from sensory receptors.
Assuntos
Neuropeptídeo Y/análise , Receptores de Neuropeptídeo Y/análise , Triatoma/química , Animais , Sistema Nervoso Central/química , Imuno-Histoquímica , MasculinoRESUMO
Thyroidectomy augments the natriuretic response to volume expansion; however, the mechanism remains unknown. This study assessed the role of 20-hydroxyeicosatetraenoic acid (20-HETE) in the natriuretic response to an acute volume expansion in hypothyroid rats. Urine flow (1.9-fold), sodium excretion (2.4-fold), fractional sodium excretion (3.8-fold), and distal delivery of sodium (4.1-fold) increased to a greater extent in thyroidectomized rats (TX) than in sham-operated controls (SHAM) following i.v. infusion of isotonic saline (5% body weight) over 60 min. This was associated with inhibition of both proximal and distal tubular reabsorption of sodium. Administration of two mechanistic and chemical dissimilar inhibitors of the synthesis of 20-HETE, 1-aminobenzotriazole (ABT), and N-hydroxy-N'-(-4-butyl-2-methylphenyl)formamidine (HET0016) decreased the natriuretic response in TX rats. Glomerular filtration rate was lower in TX than in SHAM rats and was not altered by the CYP4A inhibitors. The expression, intrarenal distribution, and the formation of 20-HETE and expoxygenase metabolites of arachidonic acid were similar in the cortex and medulla of SHAM and TX rats. These results suggest that CYP4A-derived metabolites of arachidonic acid play an important role in the enhanced natriuretic response to volume expansion in hypothyroid rats even though TX did not alter the expression or activity of these enzymes.
RESUMO
Endogenous renal dopamine is a major physiological regulator of renal ion transport; however its intracellular signaling pathways are not thoroughly understood. The present study examined the role of 20-hydroxyeicosatetraenoic acid (20-HETE), the major cytochrome P450 (CYP4A) metabolite of arachidonic acid formed in the renal cortex, on the natriuretic response to dopamine in Sprague Dawley rats. Infusion of dopamine (1.5µg/kg/min, i.v.) increased urine flow (1.9 fold over basal), sodium excretion (UNaV, 2.7 fold), fractional sodium excretion (FENa, 3.3 fold) and proximal and distal delivery of sodium by 1.5- and 2-fold respectively. Administration of two inhibitors of the synthesis of 20-HETE, 1-aminobenzotriazole (ABT) and N-hydroxy-N'-(-4-butyl-2-methylphenyl)formamidine (HET0016) reduced the response to dopamine by 65%. Induction of the renal expression of CYP4A enzymes with clofibrate did not alter the response to dopamine. The natriuretic response to dopamine was lower in Dahl salt-sensitive rats in comparison to an SS.BN5 consomic strain in which transfer of chromosome 5 from Brown Norway to Dahl salt-sensitive rats upregulates the renal expression of CYP4A protein and the production of 20-HETE. Treatment with HET0016 blocked the renal effects of dopamine in SS.BN5 rats. We also examined the influence of 20-HETE in the natriuretic response to acute volume expansion that is in part mediated via the release of endogenous dopamine. The increase in urine flow, UNaV, FENa and distal FENa following volume expansion was markedly reduced in rats treated with ABT. These results suggest that 20-HETE plays at least a permissive role in the natriuretic response to dopamine.
Assuntos
Dopamina/fisiologia , Ácidos Hidroxieicosatetraenoicos/fisiologia , Natriuréticos/fisiologia , Amidinas/farmacologia , Animais , Citocromo P-450 CYP4A/metabolismo , Taxa de Filtração Glomerular , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Córtex Renal/efeitos dos fármacos , Córtex Renal/fisiologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Masculino , Ratos , Ratos Endogâmicos Dahl , Ratos Sprague-Dawley , Triazóis/farmacologiaRESUMO
IGFs are involved in malignant transformation and growth of several tissues, including the adrenal medulla. The present study was designed to evaluate the impact of IGF-I on pheochromocytoma development. We used a murine pheochromocytoma (MPC) cell line (MPC4/30) and an animal model with a reduction of 75% in circulating IGF-I levels [liver-IGF-I-deficient (LID) mice] to perform studies in vitro and in vivo. We found that, in culture, IGF-I stimulation increases proliferation, migration, and anchorage-independent growth, whereas it inhibits apoptosis of MPC cells. When injected to control and to LID mice, MPC cells grow and form tumors with features of pheochromocytoma. Six weeks after cell inoculation, all control mice developed sc tumors. In contrast, in 73% of LID mice, tumor development was delayed to 7-12 wk, and the remaining 27% did not develop tumors up to 12 wk after inoculation. LID mice harboring MPC cells and treated with recombinant human IGF-I (LID+) developed tumors as controls. Tumors developed in control, LID, and LID+ mice had similar histology and were similarly positive for IGF-I receptor expression. The apoptotic index was higher in tumors from LID mice compared with those from control mice, whereas vascular density was decreased. In summary, our work demonstrates that IGF-I has a critical role in maintaining tumor phenotype and survival of already transformed pheochromocytoma cells and is required for the initial establishment of these tumors, providing encouragement to carry on research studies to address the IGF-I/IGF-I receptor system as a target of therapeutic strategies for pheochromocytoma treatment in the future.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Feocromocitoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Camundongos , Células PC12 , Ratos , Receptor IGF Tipo 1/metabolismoRESUMO
The inward l-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of l-dopa transport into isolated rat PTCs. Insulin (50-500 microU/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 microU/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 microM). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 microM) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 microM), but not maximal (5 microM), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 microM LY-294002) and protein kinase C (PKC; 1 microM RO-318220). Insulin-induced activation of PKC-zeta was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-zeta, and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs.
Assuntos
Hipoglicemiantes/metabolismo , Insulina/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Levodopa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Inibidores Enzimáticos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Hipoglicemiantes/farmacologia , Imuno-Histoquímica , Imunoprecipitação , Insulina/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Fosforilação , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
Previous studies propose 20-hydroxyeicosatetraenoic acid (20-HETE), a major arachidonic acid metabolite of cytochrome P-450 (CYP), as a possible mediator of Na(+)-K(+)-ATPase inhibition by dopamine (DA). The aim of this study was to investigate the intracellular mechanisms involved in this effect and to elucidate the DA receptor associated with the 20-HETE pathway in the rat kidney. DA (10(-5) M) inhibited Na(+)-K(+)-ATPase activity in microdissected tubular segments to 59.4 +/- 3.8% of control activity. This response was suppressed by the CYP4A inhibitor 17-octadecynoic acid (10(-6) M), which had no effect per se, thus confirming the participation of CYP arachidonic acid metabolites in DA-induced Na(+)-K(+)-ATPase inhibition. We next examined whether 20-HETE is involved in the signaling pathways triggered by either D(1) or D(2) receptors. Neither fenoldopam nor quinpirole (D(1) and D(2) agonists, respectively, both 10(-5) M) modified Na(+)-K(+)-ATPase activity when tried alone. However, coincubation of a threshold concentration of 20-HETE (10(-9) M) with fenoldopam resulted in a synergistic inhibition of Na(+)-K(+)-ATPase activity (66 +/- 2% of control activity), while 20-HETE plus quinpirole had no effect. Furthermore, 20-HETE (10(-9) M) synergized with forskolin (10(-5) M) and with the diacylglycerol analog 1-oleoyl-2-acetoyl-sn-glycerol (OAG; 10(-11) M; 62.0 +/- 5.3 and 69.9 +/- 2.0% of control activity, respectively), indicating a cooperative role of 20-HETE with the D(1)-triggered pathways. In line with these results, no additive effect was observed when OAG and 20-HETE were combined at concentrations which per se produced maximal inhibition (10(-6) M). These results demonstrate that the inhibition of Na(+)-K(+)-ATPase activity by DA in the proximal tubule may be the result of the synergism between 20-HETE and the D(1) signaling pathway.
Assuntos
Ácidos Hidroxieicosatetraenoicos/fisiologia , Túbulos Renais Proximais/metabolismo , Receptores de Dopamina D1/fisiologia , Receptores de Dopamina D2/fisiologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Colforsina/farmacologia , Diglicerídeos/farmacologia , Dopamina/farmacologia , Agonistas de Dopamina/farmacologia , Sinergismo Farmacológico , Ácidos Graxos Insaturados/farmacologia , Fenoldopam/farmacologia , Ácidos Hidroxieicosatetraenoicos/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/enzimologia , Masculino , Proteína Quinase C-alfa/metabolismo , Ratos , Ratos Wistar , Receptores de Dopamina D1/metabolismo , Transdução de Sinais/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Frações Subcelulares/enzimologia , Distribuição TecidualRESUMO
The biochemical characterization of nitric oxide synthase (NOS) and its distribution in the central nervous system (CNS) were studied in the heteropteran bug Triatoma infestans. NOS-like immunoreactivity was found in the brain, subesophageal ganglion, and thoracic ganglia by using immunocytochemistry. In the protocerebrum, NOS-immunoreactive (IR) somata were detected in the anterior, lateral, and posterior soma rinds. In the optic lobe, numerous immunostained somata were observed at the level of the first optic chiasma, around the lobula, and in the proximal optic lobe. In the deutocerebrum, NOS-IR perikarya were mainly observed in the lateral soma rind, surrounding the sensory glomeruli, and a few cell bodies were seen in association with the antennal mechanosensory and motor neuropil. No immunostaining could be detected in the antennal nerve. The subesophageal and prothoracic ganglia contained scattered immunostained cell bodies. NOS-IR somata were present in all the neuromeres of the posterior ganglion. Western blotting showed that a universal NOS antiserum recognized a band at 134 kDa, in agreement with the expected molecular weight of the protein. Analysis of the kinetics of nitric oxide production revealed a fully active enzyme in tissue samples of the CNS of T. infestans.
Assuntos
Sistema Nervoso/enzimologia , Óxido Nítrico Sintase/metabolismo , Triatoma/enzimologia , Animais , Western Blotting , Encéfalo/enzimologia , Gânglios dos Invertebrados/enzimologia , Cinética , Masculino , Fibras Nervosas/enzimologia , Sistema Nervoso/citologia , Óxido Nítrico/biossíntese , Transporte ProteicoRESUMO
The purpose of this study was to determine the basal levels of dopamine (DA) and to examine the enzymes involved in DA metabolism in different microdissected nephron segments from rat kidneys. Segments were incubated with DA (50 nM) or DA plus monoamine oxidase (MAO) or catechol-O-methyl transferase (COMT) inhibitors. Basal DA levels were higher in the proximal convoluted tubule (PCT, 10.8+/-3.7 pg/mm) and in the medullary collecting duct (MCD, 10.9+/-4.0 pg/mm) than in the medullary thick ascending limb of Henle's loop (MTAL, 4.9+/-0.9 pg/mm) (P<0.05). The percentage of exogenously added DA that was not metabolised was similar in both PCT (67+/-13%) and MCD (65+/-5%) and lower in MTAL (35+/-7%), suggesting that MTAL is a major site of DA metabolism. Inhibition of MAO (pargyline 1 mM) significantly increased the basal content of DA and the percentage of the added non-metabolised DA (to 95+/-10%) in PCT but had no effect on MTAL or MCD. Conversely, inhibition of COMT (nitecapone or Ro-41-0960, both 1 mM) slightly increased the basal levels of DA only in MTAL, whereas the percentage of added DA not metabolised rose to 97+/-10% in MTAL and to 91+/-15% in MCD. COMT inhibition had no effect in PCT. In conscious rats pargyline (50 mg/kg) increased urinary DA from 680+/-34 to 1,128+/-158 ng/d/100 g BW (P<0.01) while nitecapone (40 mg/kg) produced a slight non-significant increment. Our results show that DA is present all along the rat nephron and that renal DA is metabolised continuously and predominantly by MAO in proximal segments, and by COMT in the more distal ones.
Assuntos
Dopamina/metabolismo , Néfrons/enzimologia , Animais , Benzofenonas/farmacologia , Catecol O-Metiltransferase/metabolismo , Inibidores de Catecol O-Metiltransferase , Catecóis/farmacologia , Córtex Renal/enzimologia , Medula Renal/enzimologia , Túbulos Renais Coletores/enzimologia , Túbulos Renais Proximais/enzimologia , Alça do Néfron/enzimologia , Masculino , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Natriurese/efeitos dos fármacos , Pargilina/farmacologia , Pentanonas/farmacologia , Ratos , Ratos WistarRESUMO
A stimulatory role for insulin in the uptake of neutral amino acids has been reported for a variety of tissues. Here we examine the effect of insulin on L-dopa uptake by proximal tubule cells (PT cells) isolated from control and fructose-fed rats (FR-rats, 10% w/v fructose solution in tap water), a model of insulin resistance. Insulin (200 microU/ml) increased L-dopa uptake into PT cells by about 50% (705+/-186 vs.1117+/-140 pmol L-dopa/mg protein per minute) (p<0.05). The higher uptake correlated with a 40% increase in the number of high-affinity L-dopa transport sites (L-dopa 0.2 microM) (0.59+/-0.05 vs. 0.82+/-0.09 pmol L-dopa/mg protein per minute), without changing their affinity. The effect of insulin was not modified by ouabain (1 mM), nocodazole (1-10 microM) or colchicine (50-100 microM), whereas it was abolished by cytochalasin D or latrunculin B (both 1 microM). This suggests that the process is independent of Na(+),K(+)-ATPase activity or the microtubule network but that it requires the integrity of the actin cytoskeleton. L-dopa transport by the low-affinity transport sites (L-dopa 5 microM) was not affected by insulin, neither was the effect of insulin observed in PT cells isolated from FR-rats. In line with this, FR-rats showed lower renal L-dopa reabsorption as compared to control animals (81+/-4 vs. 51+/-9%). Taken together, our results support the involvement of insulin in the multifactorial regulation of renal L-dopa reabsorption.