RESUMO
Nitric oxide production is increased in allograft rejection and may have both beneficial and deleterious effects on graft function and survival. In animal models, conventional immunosuppressive agents have been shown to decrease nitric oxide production. The aim of our study was to determine what effect augmentation and selective inhibition of nitric oxide production may have on graft survival by using the model of heterotopic cardiac transplantation in the rat. L-Arginine, the naturally occurring substrate for nitric oxide production, was administered subcutaneously at 200 mg/kg/day. L-NG-monomethyl-L-arginine (L-NMMA) is a selective inhibitor of nitric oxide synthase and was administered at 500 mg/kg/day to allograft recipients from the day of operation. Endogenous nitric oxide production was quantified by analysis of urinary nitrate excretion, and time to rejection was determined by graft palpation. L-Arginine did not significantly alter urinary nitrate excretion by iso- or allografts, suggesting that nitric oxide production is not a substrate-limited process in this model. Graft survival in this group was unchanged. L-NMMA produced a small increase in graft survival from 5.1 +/- 0.1 to 6.3 +/- 0.3 days compared with control allografts (P = 0.001) and abolished the rise in urinary nitrate excretion seen with control allografts. Lower doses of L-NMMA produced dose-related decrements in urinary nitrate excretion, but did not alter graft survival. We found that allograft rejection can proceed to graft loss despite complete inhibition of the increase in nitric oxide production that occurs during untreated rejection. The small increase in graft survival suggests that nitric oxide plays a minor role as a cytotoxic effector molecule in this model of acute rejection.
Assuntos
Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Coração , Óxido Nítrico/antagonistas & inibidores , Animais , Arginina/administração & dosagem , Arginina/análogos & derivados , Rejeição de Enxerto/metabolismo , Miocárdio/patologia , Nitratos/urina , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Transplante Homólogo , ômega-N-MetilargininaRESUMO
Cytokine induction of calcium-independent nitric oxide synthase is associated with production of large amounts of nitric oxide (NO). NO is a free radical that is rapidly degraded to nitrite and nitrate. Measurement of plasma and urinary nitrate is an indirect marker of NO production and previous studies have demonstrated that plasma nitrate rises with allograft rejection. The purpose of this study was to examine the temporal relationship between the rise in urinary nitrate excretion and the onset of graft rejection, and to determine the effect of conventional immunosuppression on nitrate excretion. The heterotropic model of cardiac transplantation in the rat was used, with Brown-Norway to Lewis allografts and Lewis to Lewis isograft controls. Twenty-four-hour urine specimens were collected before and after transplantation. Urinary nitrate excretion was measured by gas chromatography/mass spectrometry. Each group was treated with (1) no immunosuppression, (2) dexamethasone (3 mg/kg), or (3) CsA (10 mg/kg) on days 0, 1, and 2. Time to rejection for untreated allografts was 5.1 +/- 0.1 days, extending to 8.4 +/- 0.5 and 9.6 +/- 0.4 days with dexamethasone and CsA treatment, respectively. There was a significant rise in nitrate excretion on days 4, 7, and 9 for control, dexamethasone-treated, and CsA-treated allografts, respectively, preceding evidence of rejection. Untreated allograft rejection was associated with a peak in nitrate excretion 8 times that of basal excretion by isografts. Treatment of the allografts with dexamethasone and CsA significantly attenuated peak nitrate excretion compared with untreated allografts with a only a 2- to 3-fold rise preceding rejection. Results indicate that allograft rejection is associated with a dramatic increase in peak urinary nitrate excretion that is attenuated by standard immunosuppressive therapy. An increase in nitrate excretion precedes evidence of graft rejection, and may serve as a noninvasive marker of graft rejection.
Assuntos
Rejeição de Enxerto/metabolismo , Transplante de Coração , Nitratos/urina , Óxido Nítrico/urina , Animais , Biomarcadores/urina , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos , Transplante HomólogoRESUMO
1. We confirmed that endothelium-independent contraction of the rabbit pulmonary artery (RPA) is mediated through both an endothelin A (ET(A)R) and endothelin B (ET(B2)R) receptor. 2. The response of endothelium-denuded RPA rings to endothelin-1 (ET-1, pD2 = 7.84 +/- 0.03) was only partially inhibited by BQ123 (10 microM), an ET(A)R antagonist. 3. Pretreatment with 1 nM sarafotoxin S6c (S6c), an ET(B)R agonist, desensitized the ET(B2)R and significantly attenuated the response to ET-3 (pD2 = 7.40 +/- 0.02 before, <6.50 after S6c). 4. Pretreatment with S6c had little effect on the response to ET-1, but BQ123 (10 microM) caused a parallel shift to the right of the residual ETAR-mediated response to ET-1 (pD2 = 7.84 +/- 0.03 before S6c, 7.93 +/- 0.03 after S6c, 6.81 +/- 0.05 after BQ123). 5. Binding of radiolabelled ET-1 to early passage cultures of RPA vascular smooth muscle cells (VSMC) displayed two patterns of competitive displacement characteristic of the ET(A)R (BQ123 pIC50 = 8.73 +/- 0.05) or ET(B2)R (S6c pIC50 = 10.15). 6. Competitive displacement experiments using membranes from late passage VSMC confirmed only the presence of the ET(A)R (ET-1 pIC50 = 9.3, BQ123 pIC50 = 8.0, S6c pIC50 < 6.0). 7. The ET(A)R was functionally active and coupled to rises in intracellular calcium which exhibited prolonged homologous desensitization. 8. Using a reverse transcriptase polymerase chain reaction for the rabbit ET(B2)R, we demonstrated the absence of mRNA expression in phenotypically modified VSMC. 9. We conclude that the ET(B2)R expressed by VSMC which mediates contraction of RPA is rapidly down-regulated at the transcriptional level during phenotypic modulation in vitro.
Assuntos
Artéria Pulmonar/química , Receptores de Endotelina/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/biossíntese , Proteínas de Ligação a Calmodulina/química , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo , Antagonistas dos Receptores de Endotelina , Endotelina-1/farmacologia , Endotelina-3/farmacologia , Feminino , Regulação da Expressão Gênica , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Peso Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Peptídeos Cíclicos/farmacologia , Fenótipo , Artéria Pulmonar/fisiologia , RNA Mensageiro/metabolismo , Coelhos , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/biossíntese , Receptores de Endotelina/genética , Receptores de Endotelina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
1. The mouse is the animal of choice for studies involving genetic manipulation and transgenic and knockout mice are valuable tools for physiological studies. We have studied adrenocorticotrophin (ACTH)- and steroid-induced hypertension in both rat and humans. The aim of the present study was to develop a model of ACTH-induced hypertension in the mouse and to assess a chronically implanted telemetric device for measurement of blood pressure (BP). 2. Male Swiss Outbred and Quackenbush Swiss (QS) mice (35-45 g) were implanted with TA11PA-C20 BP devices (Data Sciences International, St Paul, MN, USA) under isoflurane anaesthesia. Seven to 10 days later, mice were monitored telemetrically for baseline BP for 4 days. Mice were then randomly allocated to: (i) sham treatment with normal saline s.c.; or (ii) ACTH at 500 microg/kg per day, s.c. Mice were monitored 24 h/day for 10 days. 3. Sham treatment (n = 7) did not affect BP (114 +/- 2/84 +/- 1 to 115 +/- 2/84 +/- 1 mmHg; P = NS). Adrenocorticotrophin treatment (n = 5) raised BP from 112 +/- 7/82 +/- 4 to 138 +/- 3/104 +/- 4 mmHg, which was significantly different from sham treatment (P = 0.0021 for systolic BP; P < 0.0001 diastolic BP). The increase in BP with ACTH was comparable with that seen in previous studies in humans, sheep and rat. Sham and ACTH-treated animals each lost 3% bodyweight. 4. Administration of ACTH (500 microg/kg per day) raises BP in two strains of mice, measured using a telemetry system. This model will allow the selective use of transgenic and/or knockout mice to further elucidate the mechanism of ACTH- and steroid-induced hypertension.
Assuntos
Hormônio Adrenocorticotrópico/administração & dosagem , Hipertensão/fisiopatologia , Telemetria/métodos , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Hipertensão/induzido quimicamente , Masculino , Camundongos , Fatores de TempoRESUMO
1. Adrenocorticotrophic hormone (ACTH) raises blood pressure in humans, sheep, rat and mouse. In rat and humans, but not sheep, the hypertension can be explained by glucocorticoid excess. 2. In both rat and humans, the hypertension is associated with a rise in cardiac output and renal vascular resistance. 3. In both rat and humans, the nitric oxide system is implicated in glucocorticoid hypertension. 4. In both rat and humans, hypertension due to naturally occurring glucocorticoids is not prevented by drugs that block classical glucocorticoid or mineralocorticoid receptors. 5. Abnormalities in glucocorticoid metabolism may contribute to some forms of 'essential' hypertension.
Assuntos
Glucocorticoides/metabolismo , Hipertensão/fisiopatologia , Hormônio Adrenocorticotrópico/metabolismo , Animais , Modelos Animais de Doenças , Hemodinâmica , Humanos , Hipertensão/metabolismo , Sistema Nervoso/fisiopatologia , Óxido Nítrico/metabolismo , Receptores de Esteroides/metabolismoRESUMO
The role of nitric oxide in heart failure is unknown. The high-capacity inducible isoform of nitric oxide synthase is present in the myocardium of patients with idiopathic dilated cardiomyopathy. Plasma nitrate, the stable end-product of nitric oxide production, was significantly increased in patients with heart failure compared with normal controls (means 51.3 and 24.6 mumol/L). Vasodilation caused by increased nitric oxide may compensate for the vasoconstrictor effect of neurohumoral adaptions to heart failure. Alternatively, excess production may be detrimental to the heart by a direct negative inotropic effect.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Insuficiência Cardíaca/metabolismo , Nitratos/sangue , Óxido Nítrico/biossíntese , Idoso , Cardiomiopatia Dilatada/sangue , Feminino , Insuficiência Cardíaca/sangue , Testes de Função Cardíaca , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Adrenocorticotropic hormone (ACTH)-induced hypertension in the rat is characterized by nitric oxide deficiency. Tetrahydrobiopterin (BH4) is an essential cofactor for the enzyme nitric oxide synthase and glucocorticoids have been reported to reduce cytokine-induced BH4 production. Accordingly we hypothesized that ACTH-induced hypertension would be reversed by BH4 supplementation. Male Sprague-Dawley rats (n = 33) were treated with BH4 in vehicle (10 mg/kg/day i.p.) or vehicle alone (5 mg/kg/day i.p. of ascorbic acid in 4 mM HCl) for 10 days. ACTH (0.2 mg/kg s.c.) or saline daily injection was started 2 days after BH4 or vehicle treatment and continued for 8 days. Systolic blood pressure (SBP) was measured on alternate days using the tail cuff method. Treatment with HCl, ascorbic acid or BH4 alone had no effect on SBP. In saline treated rats, neither BH4 nor its vehicle modified SBP. In ACTH treated rats, SBP was increased in both BH4 (from 128 +/- 6 to 142 +/- 4 mmHg, T0 to T10, P < 0.0005, one way ANOVA) and vehicle groups (from 127 +/- 3 to 158 +/- 7 mmHg, T0 to T10, P < 0.001, one way ANOVA). There was no significant difference in SBP between BH4 + ACTH treated and vehicle + ACTH treated rats. Thus, daily injection of BH4 (10 mg/kg i.p.) failed to prevent the development of ACTH-induced hypertension in rat.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Antioxidantes/farmacologia , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Hipertensão/fisiopatologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Hipertensão/induzido quimicamente , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
1. The purpose of the present study was to examine the effects of papaverine-HCl, administered into the lumen of the human internal mammary artery (IMA) during harvesting of this vessel, on vascular reactivity in vitro and to specifically test the hypothesis that intraluminal administration of papaverine-HCl impairs endothelium-dependent vasodilation. 2. The present study measured in vitro dilator and constrictor responses of terminal segments of human IMA. Internal mammary artery segments were obtained either prior to routine administration of intraluminal papaverine (pre-P) or after papaverine administration (post-P) in patients undergoing coronary artery bypass grafting. In addition, the viability of cultured human saphenous vein endothelial cells exposed to papaverine-HCl was examined. 3. Cumulative concentrations of U46619, 5-hydroxytryptamine and phenylephrine (PE) produced active contractions in post-P IMA rings. Contractile responses to low concentrations of endothelin-1 were significantly enhanced in post-PIMA compared with pre-P IMA segments. 4. Maximal endothelium-dependent vasodilator responses of pre-P IMA segments to cumulative concentrations of acetylcholine (ACh) and the calcium ionophore A23187 were 49 +/- 7 and 66 +/- 4%, respectively, of the initial active tension induced by PE (1 mumol/L). 5. Maximal endothelium-dependent vasodilator responses were markedly attenuated in post-P IMA (6 +/- 6 and 11 +/- 10% for ACh and A23187, respectively; P < 0.0001 for both vasodilators compared with pre-P). Post-P IMA relaxed completely to the endothelium-independent vasodilator sodium nitroprusside. 6. Exposure of cultured human saphenous vein endothelial cells to papaverine-HCl (1.2 and 12.0 mg/mL) for 1 h resulted in the reduced viability of these cells. 7. The loss of endothelium-dependent relaxation could dangerously predispose the IMA graft to vasospasm in the postoperative period.