RESUMO
Thrombin stimulates cytosolic calcium mobilization and tritiated thymidine incorporation in rat glomerular mesangial cells. This effect may be mediated by a thrombin receptor similar to the receptor found in human platelets. In order to test this possibility, a series of analogues of the thrombin receptor peptide, SFLL-RNPNDKYEPF, was evaluated for their effects on mesangial cells. Analogues of the thrombin receptor peptide containing five, six, seven and 14 amino acids were as efficacious as thrombin with respect to calcium mobilization and thymidine incorporation, although they were significantly less potent. The dissimilarity in potency between thrombin and the thrombin receptor peptides is consistent with the kinetics of the proposed mechanism of action of the enzyme, since the cleavage by thrombin of its receptor results in a tethered ligand which is at a relatively high concentration compared to the free peptides in solution. Those thrombin receptor peptide analogues which showed decreased activity in platelets were tested in mesangial cells. Removal of serine at position one, N-acetylation, or replacement of the phenylalanine at position two with alanine resulted in analogues which were inactive in stimulating mesangial cell proliferation or calcium mobilization. In addition, those analogues which had no stimulatory effects in mesangial cells were not antagonists of SFLLRN-mediated calcium mobilization and thymidine incorporation in mesangial cells.
Assuntos
Cálcio/metabolismo , Mesângio Glomerular/citologia , Receptores de Trombina/fisiologia , Trombina/fisiologia , Animais , Plaquetas/metabolismo , Divisão Celular/fisiologia , Humanos , Transporte de Íons , Ligantes , Fragmentos de Peptídeos/fisiologia , Ratos , Ratos Sprague-Dawley , Timidina/metabolismoRESUMO
8-Methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP) were assessed for their effects on cyclic AMP (cAMP) accumulation in normal human fibroblasts, epithelial cells, and mononuclear leukocytes in vitro. In the absence of ultraviolet A radiation, 8-MOP and TMP caused an increase in the concentration of cAMP, but not cyclic GMP, in all of these cells. The effect was seen within 1 min and was sustained over control levels for at least 6 h. At 15 min, the highest dose of 8-MOP or TMP increased the cAMP concentration of fibroblasts by 52% and 69%, respectively. In mononuclear leukocytes, 8-MOP led to a 142% increase in cAMP after 20 min. Several analogs of 8-MOP and TMP had no effect on the cyclic nucleotide system of the cells. The pattern of the time-dependent increase in cAMP indicated that the effect was probably due to an inhibition of phosphodiesterase, rather than an activation of adenylyl cyclase. These results demonstrate that clinically effective psoralens, in the absence of UV radiation, can alter the cAMP concentration of human fibroblasts, epithelial cells, and mononuclear leukocytes in vitro.
Assuntos
AMP Cíclico/análise , Furocumarinas/farmacologia , Pele/citologia , Relação Dose-Resposta a Droga , Fibroblastos/análise , Humanos , Raios UltravioletaRESUMO
1. The mechanism of the flushing, hypotension and tachycardia associated with i.v. administration of desGlyd(CH2)5D-Tyr(Et)VAVP (SK&F 101926; 25 micrograms kg-1) and the selective V2 antidiuretic agonist, desamino-8-D-arginine vasopressin (dDAVP; 3 micrograms kg-1) was studied in ketamine-anaesthetized rhesus monkeys. 2. The flushing associated with SK&F 101926 was reduced by pretreatment with a mast cell stabilizer and by repeated administration of peptide (within 2-4 weeks). A similar desensitization to dDAVP-associated flushing was observed on repeated administration. 3. Treatment with dDAVP also resulted in reduced SK&F 101926-associated flushing. 4. The hypotension associated with SK&F 101926 was not affected by pretreatment with a mast cell stabilizer. A similar degree of hypotension was observed with repeated administration of either SK&F 101926 or dDAVP. 5. The tachycardia associated with SK&F 101926 was reduced by pretreatment with a mast cell stabilizer or repeated administration of SK&F 101926. Repeated administration of dDAVP, however, resulted in an enhanced tachycardia. 6. Indomethacin (5 mg kg-1 i.v.) did not alter the flushing or the hypotension associated with the administration of either SK&F 101926 or dDAVP, but resulted in an enhanced tachycardia to SK&F 101926. 7. Administration of a selective V1 vasopressor antagonist did not result in flushing, hypotension or tachycardia. 8. It was concluded that the flushing response to vasopressin-like peptides in rhesus monkeys may be due to an action on mast cells, whereas the haemodynamic responses are not, but probably involve direct vasodilator actions.
Assuntos
Arginina Vasopressina/análogos & derivados , Desamino Arginina Vasopressina/farmacologia , Rubor/induzido quimicamente , Hemodinâmica/efeitos dos fármacos , Animais , Arginina Vasopressina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Rubor/prevenção & controle , Frequência Cardíaca/efeitos dos fármacos , Indometacina/farmacologia , Macaca mulatta , MasculinoRESUMO
The potential role of hepatocyte growth factor (HGF) in regulating extracellular matrix in mouse mesangial cells (MMC) was evaluated. Functional HGF receptors were deed in MMC by HGF-induced extracellular acidification, a response that was inhibited by the HGF inhibitor HGF/NK2, a splice variant expressing the N-terminal domain through the second kringle domain HGF also increased fibronectin and collagen alpha1 (IV) mRNA levels in these cells; the increases were associated with a concentration-dependent increase in transcriptional activity of the collagen alpha1 (IV) gene. HGF also stimulated fibronectin and collagen alpha1 (IV) mRNA levels in primary rabbit proximal tubule epithelial cells To evaluate the potential consequences of chronic elevation of HGF on renal fuction, HGF was administered continuously for 18 days to normal and diabetic C57BLKS/J lepr(db) mice. In the diabetic mice, HGF reduced creatinine clearance and increased microalbuminuria, indicating that chronic exposure to HGF impairs renal function. Thus, chronically elevated HGF may contribute to the progression of chronic renal disease in diabetes by decreasing the glomerular filtration rate and possibly promoting the accumulation of extracellular matrix.
Assuntos
Matriz Extracelular/genética , Regulação da Expressão Gênica , Mesângio Glomerular/fisiologia , Fator de Crescimento de Hepatócito/fisiologia , Animais , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Mesângio Glomerular/citologia , Humanos , Testes de Função Renal , Túbulos Renais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-met/análise , CoelhosRESUMO
Hepatocyte growth factor (HGF) induces mitogenesis, chemotaxis, and tubule formation in renal epithelial cells. This study examined the effects of wortmannin and protein kinase C (PKC) inhibitors on HGF-mediated changes in metabolic activity in glomerular mesangial cells and renal epithelial carcinoma A498 cells. The extracellular acidification rate of transformed mouse glomerular mesangial cells and A498 cells was measured as an index of metabolic activity with a microphysiometer. HGF increased the acidification rate of mesangial cells and A498 cells in a concentration-dependent fashion that was inhibited completely by the tyrosine kinase inhibitor tyrophostin-23 (100 microM). The PKC inhibitors RO-32-0432 and SKF-57048 also inhibited HGF-induced acidification. The IC50 values for SKF-57048 were 59 +/- 2 and 20 +/- 10 nM in mesangial cells and A498 cells, respectively (P < 0.05). 12-O-Tetradecanoylphorbol 13-acetate (TPA), a phorbol ester that activates PKC, increased acidification in mesangial and epithelial cells similar to HGF. Wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinase (IC50 value 1-10 nM), inhibited HGF-induced acidification with an IC50 of 93 +/- 31 and 9 +/- 1 nM in mesangial and A498 cells, respectively (P < 0.05). In contrast, there was no significant difference in the IC50 value of wortmannin for epidermal growth factor (EGF)-induced acidification between mesangial and A498 cells (23 +/- 9 vs 14 +/- 1 nM, respectively). Because the IC50 value for wortmannin in inhibiting HGF but not EGF-induced acidification was an order of magnitude higher in mesangial cells than in epithelial A498 cells, a wortmannin-sensitive PI 3-kinase pathway may not be involved in HGF-mediated acidification in mesangial cells.
Assuntos
Mesângio Glomerular/fisiologia , Fator de Crescimento de Hepatócito/farmacologia , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Tirfostinas , Androstadienos/farmacologia , Animais , Catecóis/farmacologia , Células Cultivadas , Criopreservação , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Mesângio Glomerular/citologia , Mesângio Glomerular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Renais , Cinética , Camundongos , Camundongos Endogâmicos , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-met/biossíntese , RNA Mensageiro/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas , WortmaninaRESUMO
The vasopressin antidiuretic (V2) antagonist activity of the position 6 stereoisomers of four vasopressin analogs were tested for water diuretic activity in the rhesus monkey and for activity to inhibit vasopressin-stimulated adenylate cyclase activity in rhesus monkey and human renomedullary tissue in vitro. Replacement of the mercapto groups of the cysteine residues with methylene groups resulted in compounds having similar in vitro potencies to their disulfide analogs; however, these 'dicarba' compounds demonstrated more potent aquaretic activity. Position 6 D enantiomers were associated with less vasopressin antagonist activity in vitro in both species. Based upon these studies, the most potent aquaretic structure identified was the dicarba analog SK & F 105494.
Assuntos
Receptores de Superfície Celular/efeitos dos fármacos , Vasopressinas/metabolismo , Inibidores de Adenilil Ciclases , Animais , Humanos , Técnicas In Vitro , Rim/enzimologia , Macaca mulatta , Conformação Molecular , Especificidade da EspécieRESUMO
BACKGROUND: We assessed the effect of a cytokine inhibitor, compound SKF 86002 (a bicyclic imidazole), on changes in renal hemodynamics (renal blood flow and glomerular filtration rate) that occur acutely following immune injury of glomerular mesangial cells. METHODS: Injury was induced in Munich-Wistar rats by the administration of a monoclonal antibody against the mesangial cell membrane antigen Thy 1.1. An acute drop in renal blood flow (RBF) and glomerular filtration rate (GFR) occurred within one hour of injury. RESULTS: Pretreatment of animals with the cytokine inhibitor SKF 86002 prevented this drop. SKF 86002 had no effect on glomerular synthesis of vasoconstrictor eicosanoids. CONCLUSIONS: The observations indicate that in mesangial cell immune injury, cytokines mediate renal hemodynamic impairment.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Citocinas/antagonistas & inibidores , Mesângio Glomerular/imunologia , Imidazóis/farmacologia , Circulação Renal/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Anticorpos Monoclonais , Eicosanoides/biossíntese , Taxa de Filtração Glomerular/efeitos dos fármacos , Mesângio Glomerular/metabolismo , Mesângio Glomerular/fisiologia , Mediadores da Inflamação/fisiologia , Masculino , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Incubation of cultured human umbilical vein endothelial cells with factors derived from human peripheral blood mononuclear cells (MNCF) or adherent monocytes (AMF) resulted in concentration-and time-dependent increases in prostacyclin and prostaglandin E2 (PGE2) production. MNCF and AMF also stimulated prostacyclin and PGE2 biosynthesis in cultured human arterial smooth muscle cells and human dermal fibroblasts. The effect of these monokines on endothelial cells and fibroblasts was mimicked by treatment with purified human interleukin 1 (IL 1). Mononuclear cell-conditioned medium subjected to gel filtration yielded fractions (Mr 12,000 to 18,000 daltons) which simultaneously contained endothelial cell and fibroblast prostaglandin-stimulating activity and IL 1 activity. Therefore, monokines, specifically IL 1, appear to serve as chemical mediators of the interaction between monocytes and vascular cells as would occur in blood vessel injury, inflammation, and atherosclerosis.
Assuntos
Interleucina-1/fisiologia , Monócitos/fisiologia , Prostaglandinas/biossíntese , Proteínas/fisiologia , 6-Cetoprostaglandina F1 alfa/biossíntese , Células Cultivadas , Dinoprostona , Endotélio/metabolismo , Fibroblastos/metabolismo , Humanos , Monocinas , Músculo Liso Vascular/metabolismo , Prostaglandinas E/biossínteseRESUMO
Diets enriched with fish oil (FO) ameliorate kidney disease in the MRL-lpr/lpr murine model of lupus nephritis. Although the mechanisms of this effect are not known, FO is rich in the polyunsaturated fatty acid eicosapentaenoic acid (EPA) which may have profound effects on eicosanoid metabolism. In MRL-lpr/lpr mice, FO feeding reduces renal production of cyclooxygenase metabolites. However, EPA may also affect the metabolism of arachidonate by the 5-lipoxygenase (5-LO) pathway and enhanced production of 5-LO metabolites has been implicated in the pathogenesis of kidney disease in MRL-lpr/lpr mice. We therefore investigated the effects of FO feeding on production of 5-LO metabolites in 20 week old MRL-lpr/lpr mice. After 8 weeks of dietary supplementation with FO, both renal hemodynamic function and glomerular histology were improved compared to safflower oil (SO) controls. Amelioration of kidney disease was associated with alterations in the pattern of leukotriene production by macrophages and kidneys from FO fed mice. There was a significant decrease in the production of leukotriene B4 (LTB4) and tetraene peptidoleukotrienes by peritoneal macrophages isolated from mice given FO compared to control animals. Similarly, dietary supplementation with FO decreased renal production of LTB4. Reduced production of tetraene leukotrienes was accompanied by a modest increase in the production of pentaene leukotrienes by macrophages from FO fed mice. We speculate that this modulation of leukotriene production by FO feeding may have beneficial effects on renal disease in autoimmune nephritis.
Assuntos
Óleos de Peixe/farmacologia , Leucotrieno B4/biossíntese , Nefrite Lúpica/metabolismo , Administração Oral , Animais , Araquidonato 5-Lipoxigenase/biossíntese , Araquidonato 5-Lipoxigenase/metabolismo , Óleos de Peixe/administração & dosagem , Hemodinâmica , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/metabolismo , Rim/fisiopatologia , Nefrite Lúpica/enzimologia , Nefrite Lúpica/fisiopatologia , Camundongos , Prostaglandina-Endoperóxido Sintases/biossínteseRESUMO
Ureteral obstruction in rabbits is characterized by mononuclear cell invasion of the renal cortex and proliferative fibrosis that is associated with exaggerated prostaglandin synthesis in response to vasoactive and inflammatory cell agonists. In this investigation, we studied the effects of the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) and bradykinin (BK) on eicosanoid synthesis and renal vascular resistance in the ex vivo perfused hydronephrotic kidney (HNK). Administration of fMLP resulted in the dose-dependent synthesis of leukotrienes, thromboxane A2 (TXA2), prostaglandin E2 (PGE2), and prostacyclin (PGI2). Peptidoleukotriene synthesis was monitored by specific radioimmunoassay and by guinea pig ileum bioassay and it was then validated by inhibition of the ileal contractile activity with the peptidoleukotriene receptor antagonist FPL-55712. The leukotrienes produced were identified as LTB4, LTC4, LTD4, and LTE4 by comigration with authentic standards on reverse phase high-performance liquid chromatography (RP-HPLC) and by ultraviolet spectroscopy. BK administration stimulated the synthesis of TXA2, PGE2, and PGI2 but not the synthesis of leukotrienes, in contrast to the results with fMLP, suggesting the involvement of different cell types. Administration of fMLP to the HNK also resulted in a renal vasoconstriction that was partially inhibited by FPL-55712 and that was completely inhibited by the thromboxane synthase inhibitor OKY-1581. Consistent with this result, exogenous administration of LTC4 resulted in the synthesis of TXA2 and in a renal vasoconstriction that was inhibited by either FPL-55712 or OKY-1581.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hidronefrose/metabolismo , Leucotrieno B4/biossíntese , Circulação Renal , SRS-A/biossíntese , Tromboxano A2/biossíntese , Resistência Vascular , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Bradicinina/farmacologia , Hidronefrose/fisiopatologia , Rim/metabolismo , Leucotrieno B4/fisiologia , Masculino , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Coelhos , SRS-A/farmacologia , SRS-A/fisiologia , Tromboxano A2/fisiologiaRESUMO
The effect of thrombin on mesangial cell function was investigated. Thrombin caused a dose-dependent increase in [3H] thymidine incorporation (EC50 = 0.36 +/- 0.09 U/ml), intracellular calcium [(Ca++)i] mobilization (EC50 = 1.9 +/- 0.5 U/ml) and prostaglandin E2 (PGE2) production (EC50 = 0.25 +/- 0.02 U/ml) in rat glomerular mesangial cells. These effects were blocked by the thrombin inhibitor, hirudin (KB = 10.4 +/- 0.2 nM). The role of (Ca++)i mobilization and arachidonate metabolism in thrombin-stimulated proliferation was tested by the addition of the calcium channel blocker, nifedipine, and the cyclooxygenase inhibitor, indomethacin, to mesangial cell cultures. Indomethacin, at doses that completely inhibited the thrombin-mediated production of PGE2, had no significant effect on proliferation. The Ca++ channel blocker, nifedipine, inhibited both PGE2 production and [3H] thymidine incorporation in a dose-dependent fashion, but only at concentrations considered nonspecific. In addition to its effects on PGE2, thymidine incorporation and Ca++ mobilization, thrombin caused mesangial cell contraction as determined by a substrate distortion technique. This effect was not inhibited by indomethacin. These results indicate that thrombin can alter mesangial cell function in vitro.
Assuntos
Mesângio Glomerular/efeitos dos fármacos , Prostaglandinas E/biossíntese , Trombina/farmacologia , Animais , Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Mesângio Glomerular/metabolismo , Mesângio Glomerular/ultraestrutura , Hirudinas/farmacologia , Indometacina/farmacologia , Ratos , Ratos Sprague-Dawley , Timidina/metabolismoRESUMO
Clusterin, a multifunctional protein with complement blocking activity, and fibrin, a product of thrombin's enzymatic activity, are present in the kidney during acute and chronic renal failure. The role of thrombin in regulating clusterin mRNA in the kidney is not known. The effect of thrombin on clusterin mRNA expression was examined in rat glomerular mesangial and glomerular epithelial cells, and cultured human renal proximal tubular epithelial cells by northern blot. Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32-0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C.
Assuntos
Proteínas Inativadoras do Complemento/genética , Mesângio Glomerular/metabolismo , Glicoproteínas/genética , Glomérulos Renais/metabolismo , Chaperonas Moleculares , RNA Mensageiro/metabolismo , Trombina/farmacologia , Animais , Células Cultivadas , Clusterina , AMP Cíclico/fisiologia , Células Epiteliais , Epitélio/metabolismo , Mesângio Glomerular/citologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Glomérulos Renais/citologia , Proteína Quinase C/fisiologia , RatosRESUMO
The azaspirane SKF 105685 (N,N-dimethyl-8, 8-dipropyl-2-azaspiro (4.5) decane-2-propanamine dihydrochloride) has been shown to attenuate or reverse the course of immunologic disease in several animal models, possibly through the induction of nonspecific suppressor activity. To investigate its effects on immune-mediated renal disease, SKF 105685 was administered by gavage to rats with kidney allografts. Six days after transplantation, GFR (inulin clearance, 1.46 +/- 0.27 versus 0.41 +/- 0.15 mL/min per kg; P < 0.005) and RPF (p-aminohippurate clearance, 5.48 +/- 0.98 versus 1.99 +/- 0.72 mL/min per kg; P < 0.01) were significantly higher in SKF 105685-treated rats compared with vehicle-treated control rats. In addition, mononuclear inflammatory cell infiltrates were significantly reduced in SKF 105685-treated animals compared with controls. Treatment also reduced renal production of thromboxane B2 (81 +/- 22 versus 424 +/- 76 pg/min per mg of protein; P < 0.0005), prostaglandin E2 (612 +/- 165 versus 2,059 +/- 351 pg/min per mg of protein; P < 0.005), and 6-keto prostaglandin F1 alpha (217 +/- 56 versus 943 +/- 186 pg/min per mg of protein; P < 0.005), but interleukin-1 beta mRNA levels within kidney allografts were not affected by treatment. Thus, the azaspirane SKF 105685 is a novel immunosuppressive agent that substantially ameliorates renal allograft rejection in the rat. Although the mechanism of action is unknown, the beneficial effects of SKF 105685 in rejection may relate to its ability to induce suppressor activity and/or its effects on eicosanoid production.
Assuntos
Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Transplante de Rim , Compostos de Espiro/uso terapêutico , Animais , Artrite Experimental/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Eicosanoides/biossíntese , Inflamação , Interleucina-1/biossíntese , Transplante de Rim/imunologia , Transplante de Rim/patologia , Masculino , Ratos , Ratos Endogâmicos ACI/imunologia , Ratos Endogâmicos Lew/imunologia , Ratos Endogâmicos/imunologia , Circulação Renal , Transplante HomólogoRESUMO
Three hours following injection of anti-GBM (glomerular basement membrane) antibody (10 mg/kg, i.v.) into rats, glomerular production of LTB4 was significantly increased as compared to untreated rats (497 +/- 26 vs. 244 +/- 18 pg of LTB4/mg protein). Twenty-four hours following administration of anti-GBM antibody, renal function (blood urea nitrogen, BUN; plasma creatinine, PCr; creatinine clearance, CCr; fractional excretion of sodium, FENa; fractional excretion of urea, FEUrea) was determined to be impaired proportionally to the amount of injected antibody (5 to 30 mg/kg, i.v.). In a second series of experiments, a selective 5-lipoxygenase (5-LO) inhibitor, SK&F 107649, was used to investigate the involvement of 5-LO products in the pathophysiology of anti-GBM antibody-induced glomerulonephritis. In preliminary experiments. SK&F 107649 (50 to 200 mg/kg, p.o.) inhibited ionophore (A23187)-stimulated whole blood leukotriene (LT) B4 production in a dose-dependent fashion (P < 0.05 at doses > or = 100 mg/kg). The anti-GBM antibody-mediated decrease in glomerular filtration rate (GFR) and increase in BUN and PCr were dose-dependently attenuated by SK&F 107649 (50 to 200 mg/kg, p.o. BID). These data confirm that glomerular LTB4 production is stimulated in anti-GBM antibody-induced glomerulonephritis, and demonstrate that inhibition of 5-LO by SK&F 107649 normalizes BUN and PCr and attenuate the decrease in GFR following anti-GBM antibody treatment. These results provide additional evidence for a role of 5-LO products in immune-mediated renal disease, and suggest that pharmacologic inhibition of 5-LO may be of therapeutic value.
Assuntos
Glomerulonefrite/prevenção & controle , Inibidores de Lipoxigenase/farmacologia , Tetra-Hidronaftalenos/farmacologia , Ureia/análogos & derivados , Animais , Araquidonato 5-Lipoxigenase/fisiologia , Membrana Basal/imunologia , Relação Dose-Resposta a Droga , Imunofluorescência , Taxa de Filtração Glomerular , Glomerulonefrite/patologia , Glomerulonefrite/fisiopatologia , Imunoglobulina G/administração & dosagem , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Leucotrieno B4/antagonistas & inibidores , Leucotrieno B4/biossíntese , Masculino , Ratos , Ratos Sprague-Dawley , Ovinos , Ureia/farmacologiaRESUMO
Thrombin-mediated down-regulation of endothelin (ET) receptors was studied in rat glomerular mesangial cells. Overnight incubation of mesangial cells with thrombin (10 nM) resulted in a significant decrease (67%) in the number of ET receptors, with no change in affinity. Northern analysis of the mRNA from these cells showed a corresponding decrease in the ETA receptor message. Such a decrease in ET receptors could result from an increase in ET levels caused by an increase in synthesis and/or a decrease in degradation. It has been previously reported that thrombin stimulates ET production in endothelial and mesangial cells. Because ET is known to be degraded by neutral endopeptidase (NEP), which is present at high levels in the kidney, the potential effects of thrombin on NEP activity were evaluated. There was a decrease of NEP activity in mesangial cells at 16 and 24 hr after treatment with 10 nM thrombin. This effect was specific for thrombin, because NEP activity was not altered after treatment with thrombin in the presence of hirudin, an inhibitor of thrombin activity. The thrombin-mediated decrease in NEP activity correlated with a decrease in NEP protein and mRNA levels, as determined by Western and Northern analyses, respectively. To determine whether the thrombin-mediated decrease in ET receptors had a functional corollary, ET-1-stimulated intracellular calcium mobilization was measured. Overnight incubation with 10 nM thrombin resulted in a significant inhibition of ET-stimulated intracellular calcium mobilization. This effect was specific for ET, because thrombin pretreatment did not affect vasopressin-stimulated intracellular calcium mobilization in mesangial cells. These results indicate that the thrombin-mediated down-regulation of ET receptors is due, in part, to a thrombin-stimulated increase in ET resulting from the down-regulation of NEP and the reported increase in ET synthesis. In addition, pretreatment of mesangial cells with ET-1 caused a significant decrease (85%) in ET receptor number and ET-1-mediated intracellular calcium release (84%), without affecting vasopressin- or thrombin-mediated responses.
Assuntos
Regulação para Baixo/fisiologia , Neprilisina/metabolismo , Receptores de Endotelina/fisiologia , Trombina/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Endotelinas/metabolismo , Mesângio Glomerular/citologia , Mesângio Glomerular/enzimologia , Mesângio Glomerular/metabolismo , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Receptores de Endotelina/metabolismoRESUMO
Azaspiranes are novel immunomodulators which are effective in a variety of autoimmune diseases. One azaspirane analog, SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine dihydrochloride), caused a decrease in total serum cholesterol in dogs after oral administration. To determine whether an effect on cholesterol was common to this class of compounds, the immunomodulatory activity was compared with the cholesterol-lowering activity of six azaspirane analogs. The compounds were given to beagles at a dose of 1 mg/kg p.o. for 28 days, and the effect on serum cholesterol was determined. The results from this study showed a clear dissociation between the immunomodulatory and hypocholesterolemic activities of these compounds. Studies performed to determine the mechanism of the decrease in serum cholesterol caused by SK&F 105685 indicated that it was not due to inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase or acyl-CoA:cholesterol acyltransferase activities, or to a potentiation of cholesterol-7 alpha-hydroxylase activity. In addition, analysis by gas chromatography of the nonsaponifiable sterol fraction in dog plasma after treatment with SK&F 105685 or SK&F 106333 showed a decrease in cholesterol and an accumulation of lathosterol and an unknown sterol, indicating that the conversion of these sterols is inhibited and cholesterol synthesis is blocked at these steps. SK&F 105685 affected the sterol profile in human hepatoblastoma cells (Hep G2) in a similar way. Characterization of the unknown sterol by gas chromatography and mass spectrometry indicated that the unknown sterol is very similar to cholesterol and lathosterol, but its identity has yet to be established. These results show that the hypocholesterolemic effects of azaspiranes are related to inhibition of one or more of the final steps in the biosynthetic pathway of cholesterol.