RESUMEN
In vitro studies of the mode of action of cyclosporine (CsA) and tacrolimus have indicated that both drugs produce immunosuppression by a quite similar cellular and molecular mechanism to block T cell receptor emanated transcriptional activation of interleukin(IL)-2 and other cytokine genes. Herein, we show that there are distinct patterns of cytokine gene expression in rat heart allografts under equivalent effective doses ("optimal dose") of CsA and tacrolimus. The optimal doses of CsA (10 mg/kg/day) and tacrolimus (3.2 mg/kg/day), which induce similar mean graft survival time (MST), were administered in LEW recipients with ACI heart grafts from day 0 after grafting until sacrifice. Heart grafts were harvested at days 3, 5, and 7. The expression of various cell surface markers, cytokines, and cytotoxic factors was determined by immunohistology and reverse transcriptase-polymerase chain reaction (RFT-PCR). Cell populations that stained positively in the heart tissues of allograft control increased through day 7 for CD4+ and CD8+ T lymphocytes, NKR-Pla+ natural killer (NK) cells, and ED2+ macrophages. CsA and tacrolimus have comparable activity to block these cell local infiltrations. The mRNA levels of the majority of the factors were dramatically up-regulated in the allografts over time, peaking at day 5. The optimal doses of CsA and tacrolimus had similar inhibitory effects on Th1 type cytokine IL-2 and interferon [INF]-gamma), inflammatory cytokine (IL-1beta and tumor necrosis factor [TNF]-alpha), and cytotoxic factor (granzyme B and perforin) mRNA expression. However, the drugs had different effect on Th2 type cytokines (IL-4 and IL-10). Whereas IL-4 expression was not affected by tacrolimus and was enhanced by CsA, IL-10 expression was more significantly suppressed by tacrolimus than CsA. Differences in the suppression of Th2 type cytokine gene expression indicate that the in vivo molecular networks by which CsA and tacrolimus exert their full immunosuppressive activity are not necessarily the same.
Asunto(s)
Ciclosporina/farmacología , Citocinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Trasplante de Corazón/inmunología , Inmunosupresores/farmacología , Tacrolimus/farmacología , Inmunología del Trasplante/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Masculino , Ratas , Ratas Endogámicas Lew , Trasplante HomólogoRESUMEN
AIM: Hydrogen sulphide (H2S) is endogenously produced and plays an important role as a modulator of neuronal functions; however, its modulatory role in the central CO2 chemoreception is unknown. The aim of the present study was to assess the role of endogenously produced H2S in the ventilatory response to hypercapnia in adult conscious rats. METHODS: Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) inhibitors (aminooxyacetate: AOA and propargylglycine: PAG respectively) and a H2S donor (sodium sulphide: Na2S) were microinjected into the fourth ventricle (4V). Ventilation (VÌ(E)), oxygen consumption (VÌO2) and body temperature were recorded before (room air) and during a 30-min CO2 exposure (hypercapnia, 7% CO2). Endogenous H2S levels were measured in the nucleus tractus solitarius (NTS). RESULTS: Microinjection of Na2S (H2S donor), AOA (CBS inhibitor) or PAG (CSE inhibitor) did not affect baseline of the measured variables compared to control group (vehicle). In all experimental groups, hypercapnia elicited an increase in VÌ(E). However, AOA microinjection, but not PAG, attenuated the ventilatory response to hypercapnia (P < 0.05), whereas Na2S elicited a slight, not significant, enhancement. Moreover, endogenous H2S levels were found higher in the NTS after hypercapnia (P < 0.05) compared to room air (normoxia) condition. CONCLUSION: There are a few reports on the role of gaseous transmitters in the control of breathing. Importantly, the present data suggest that endogenous H2S via the CBS-H2S pathway mediates the ventilatory response to hypercapnia playing an excitatory role.
Asunto(s)
Sulfuro de Hidrógeno/farmacología , Hipercapnia/tratamiento farmacológico , Envejecimiento/metabolismo , Ácido Aminooxiacético/metabolismo , Animales , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Modelos Animales de Enfermedad , Sulfuro de Hidrógeno/metabolismo , Hipercapnia/metabolismo , Masculino , Ratas Wistar , Sulfuros/farmacologíaRESUMEN
AIM: We hypothesized that hydrogen sulphide (H2 S), acting specifically in the anteroventral preoptic region (AVPO - an important integrating site of thermal and cardiorespiratory responses to hypoxia in which H2 S synthesis has been shown to be increased under hypoxic conditions), modulates the hypoxic ventilatory response. METHODS: To test this hypothesis, we measured pulmonary ventilation (VËE) and deep body temperature of rats before and after intracerebroventricular (icv) or intra-AVPO microinjection of aminooxyacetate (AOA; CBS inhibitor) or Na2 S (H2 S donor) followed by 60 min of hypoxia exposure (7% O2 ). Furthermore, we assessed the AVPO levels of H2 S of rats exposed to hypoxia. Control rats were kept under normoxia. RESULTS: Microinjection of vehicle, AOA or Na2 S did not change VËE under normoxic conditions. Hypoxia caused an increase in ventilation, which was potentiated by microinjection of AOA because of a further augmented tidal volume. Conversely, treatment with Na2 S significantly attenuated this response. The in vivo H2 S data indicated that during hypoxia the lower the deep body temperature the smaller the degree of hyperventilation. Under hypoxia, H2 S production was found to be increased in the AVPO, indicating that its production is responsive to hypoxia. The CBS inhibitor attenuated the hypoxia-induced increase in the H2 S synthesis, suggesting an endogenous synthesis of the gas. CONCLUSION: These data provide solid evidence that AVPO H2 S production is stimulated by hypoxia, and this gaseous messenger exerts an inhibitory modulation of the hypoxic ventilatory response. It is probable that the H2 S modulation of hypoxia-induced hyperventilation is at least in part in proportion to metabolism.
Asunto(s)
Sulfuro de Hidrógeno/farmacología , Hiperventilación/metabolismo , Hipoxia/complicaciones , Ácido Aminooxiacético/administración & dosificación , Ácido Aminooxiacético/farmacología , Animales , Sulfuro de Hidrógeno/administración & dosificación , Masculino , Área Preóptica , Ratas , Ratas WistarRESUMEN
We have documented that the locus coeruleus (LC), the main noradrenergic nucleus in the brain, is part of a thermoeffector neuronal pathway in fever induced by lipopolysaccharide (LPS). Following this pioneering study, we have investigated the role of the LC carbon monoxide (CO) and nitric oxide (NO) pathways in fever. Interestingly, despite both CO and NO are capable of activating the same intracellular target, soluble guanylate cyclase (sGC), our data have shown that LC CO is an antipyretic molecule, whereas LC NO is propyretic. Thus, aiming at further exploring the mechanisms underlying their anti- and propyretic properties, we investigated the putative interplay between the LC CO and NO pathways. Male Wistar rats were implanted with a guide cannula in the fourth ventricle (4V) and a temperature datalogger capsule in the peritoneal cavity. The animals were microinjected into the 4V with an inhibitor of heme oxygenase (HO) (ZnDPBG [zinc(II)deuteroporphyrin IX 2,4 bis ethylene glycol]), or a CO donor (CORM-2 [tricarbonyldichlororuthenium-(II)-dimer]), or an inhibitor of nitric oxide synthase (NOS) (l-NMMA [N(G)-monomethyl-L-arginine acetate]), or an NO donor (NOC12 [3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene]), and injected with LPS (100 µg/kg i.p.). Two hours later, the rats were decapitated, and the brains were frozen and cut in a cryostat. LC punches were processed to assess LC bilirubin and nitrite/nitrate (NOx) levels. Microinjection of ZnDPBG reduced LC bilirubin and increased LC NOx, whereas l-NMMA diminished LC NOx and reduced LC bilirubin. Furthermore, NOC12 caused an increase in LC bilirubin, whereas CORM-2 caused a reduction in LC NOx. These findings are consistent with the notion that in the LC during LPS fever the CO pathway downmodulates NOS activity and the NO pathway upmodulates HO activity, and, together with previous data, allow us to conjecture that LC CO blunts fever by downmodulating NOS (antipyretic property), LC NO upmodulates HO and sGC activities favoring the development of LPS fever (propyretic effect).
Asunto(s)
Monóxido de Carbono/metabolismo , Fiebre/metabolismo , Locus Coeruleus/metabolismo , Óxido Nítrico/metabolismo , Transducción de Señal/fisiología , Animales , Inhibidores Enzimáticos/farmacología , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Locus Coeruleus/efectos de los fármacos , Masculino , Microinyecciones , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacosRESUMEN
Hypoxia causes a regulated decrease in body temperature (Tb), a response that has been aptly called anapyrexia, but the mechanisms involved are not completely understood. The roles played by nitric oxide (NO) and other neurotransmitters have been documented during hypoxia-induced anapyrexia, but no information exists with respect to hydrogen sulfide (H(2)S), a gaseous molecule endogenously produced by cystathionine ß-synthase (CBS). We tested the hypothesis that H(2)S production is enhanced during hypoxia and that the gas acts in the anteroventral preoptic region (AVPO; the most important thermosensitive and thermointegrative region of the CNS) modulating hypoxia-induced anapyrexia. Thus, we assessed CBS and nitric oxide synthase (NOS) activities [by means of H(2)S and nitrite/nitrate (NO(x)) production, respectively] as well as cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) levels in the anteroventral third ventricle region (AV3V; where the AVPO is located) during normoxia and hypoxia. Furthermore, we evaluated the effects of pharmacological modifiers of the H(2)S pathway given i.c.v. or intra-AVPO. I.c.v. or intra-AVPO microinjection of CBS inhibitor caused no change in Tb under normoxia but significantly attenuated hypoxia-induced anapyrexia. During hypoxia there were concurrent increases in H(2)S production, which could be prevented by CBS inhibitor, indicating the endogenous source of the gas. cAMP concentration, but not cGMP and NO(x), correlated with CBS activity. CBS inhibition increased NOS activity, whereas H(2)S donor decreased NO(x) production. In conclusion, hypoxia activates H(2)S endogenous production through the CBS-H(2)S pathway in the AVPO, having a cryogenic effect. Moreover, the present data are consistent with the notion that the two gaseous molecules, H(2)S and NO, play a key role in mediating the drop in Tb caused by hypoxia and that a fine-balanced interplay between NOS-NO and CBS-H(2)S pathways takes place in the AVPO of rats exposed to hypoxia.