RESUMEN
UNLABELLED: There is increasing evidence that carbon monoxide (CO), a signaling molecule generated during the degradation of heme by heme oxygenase-1 (HO-1) in biological systems, has a variety of cytoprotective actions, including anti-hypoxic effects at low temperatures. However, during liver cold preservation, a direct effect needs to be established. Here, we designed a study to analyze the role of CO, delivered via a carbon monoxide-releasing molecule (CO-RM) in the maintenance of liver function, and integrity in rats during cold ischemia/reperfusion (CI/R) injury. We used an isolated normothermic perfused liver system (INPL) following a clinically relevant model of ex vivo 48 h cold ischemia stored in a modified University of Wisconsin (UW) solution, to determine the specific effects of CO in a rat model. CO was generated from 50 microM tricarbonylchloro ruthenium-glycinato (CORM-3), a water-soluble transition metal carbonyl that exerts pharmacological activities via the liberation of controlled amounts of CO in biological systems. The physiological effects of CORM-3 were confirmed by the parallel use of a specific inactive compound (iCORM-3), which does not liberate CO in the cellular environment. CORM-3 addition was found to prevent the injury caused by cold storage by improving significantly the perfusion flow during reperfusion (by almost 90%), and by decreasing the intrahepatic resistance (by 88%) when compared with livers cold preserved in UW alone. Also, CORM-3 supplementation preserved good metabolic capacity as indicated by hepatic oxygen consumption, glycogen content, and release of lactate dehydrogenase. Liver histology was also partially preserved by CORM-3 treatment. CONCLUSIONS: These findings suggest that CO-RM could be utilized as adjuvant therapeutics in UW solutions to limit the injury sustained by donor livers during cold storage prior to transplantation, as has been similarly proposed for the heart, and kidney.
Asunto(s)
Frío , Hígado , Compuestos Organometálicos/farmacología , Sustancias Protectoras/farmacología , Conservación de Tejido/métodos , Animales , Monóxido de Carbono/metabolismo , Glucógeno/metabolismo , Lactato Deshidrogenasas/metabolismo , Hígado/metabolismo , Masculino , Consumo de Oxígeno/fisiología , Ratas , Ratas WistarRESUMEN
BACKGROUND AND PURPOSE: Carbon monoxide (CO) generated by the enzyme haeme oxygenase-1 (HO-1) during the breakdown of haeme is known to mediate a number of biological effects. Here, we investigated whether CO liberated from two water soluble carbon monoxide-releasing molecules (CO-RMs) exerts inotropic effects on the myocardium. EXPERIMENTAL APPROACH: Rat isolated hearts perfused either at constant flow or constant pressure were used to test the effect of CO-RMs. KEY RESULTS: CORM-3, a fast CO releaser, produced a direct positive inotropic effect when cumulative doses (3, 10 and 30 microg min(-1)) or a single dose (5 microM) were infused at either constant coronary pressure (CCP) or constant coronary flow (CCF). The inotropic effect mediated by CORM-3 was abolished by blockade of soluble guanylate cyclase or Na(+)/H(+) exchanger, but not by inhibitors of L-type Ca(2+) channels and protein kinase C. CORM-3 also caused a slight reduction in heart rate but did not alter coronary flow. In contrast, the slow CO releaser CORM-A1 produced significant coronary vasodilatation when given at the highest concentration (30 mug min(-1)) but exerted no effect on myocardial contractility or heart rate. CONCLUSION AND IMPLICATIONS: A rapid CO release from CORM-3 exerts a direct positive inotropic effect on rat isolated perfused hearts, whereas CO slowly released by CORM-A1 had no effect on myocardial contractility but caused significant coronary vasodilatation. Both cGMP and Na(+)/H(+) exchange appear to be involved in this effect but further work is needed to determine the relative contribution of each pathway in CO-mediated inotropic effect.
Asunto(s)
Boranos/farmacología , Monóxido de Carbono/metabolismo , Carbonatos/farmacología , Cardiotónicos/farmacología , Miocardio/metabolismo , Compuestos Organometálicos/farmacología , Alcaloides/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Benzofenantridinas/farmacología , Boranos/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Carbonatos/metabolismo , Inhibidores Enzimáticos/farmacología , Corazón/efectos de los fármacos , Cinética , Masculino , Nifedipino/farmacología , Ratas , Ratas Endogámicas Lew , Transducción de Señal/efectos de los fármacosRESUMEN
Low concentrations of carbon monoxide (CO) can protect tissues against ischemia-reperfusion (I-R) injury. We have recently identified a novel class of compounds, CO-releasing molecules (CO-RMs), which exert important pharmacological activities by carrying and delivering CO to biological systems. Here, we examined the possible beneficial effects of CO liberated from CO-RMs on the damage inflicted by cold storage and I-R in isolated perfused kidneys. Hemodynamic and biochemical parameters as well as mitochondrial respiration were measured in isolated perfused rabbit kidneys that were previously flushed with CO-RMs and stored at 4 degrees C for 24 h. Two water-soluble CO-RMs were tested: (1) sodium boranocarbonate (CORM-A1), a boron-containing carbonate that releases CO at a slow rate, and (2) tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), a transition metal carbonyl that liberates CO very rapidly in solution. Kidneys flushed with Celsior solution supplemented with CO-RMs (50 microM) and stored at 4 degrees C for 24 h displayed at reperfusion a significantly higher perfusion flow rate (PFR), glomerular filtration rate, and sodium and glucose reabsorption rates compared to control kidneys flushed with Celsior solution alone. Addition of 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor, prevented the increase in PFR mediated by CO-RMs. The respiratory control index from kidney mitochondria treated with CO-RMs was also markedly increased. Notably, renal protection was lost when kidneys were flushed with Celsior containing an inactive compound (iCO-RM), which had been deliberately depleted of CO. CO-RMs are effective therapeutic agents that deliver CO during kidney cold preservation and can be used to ameliorate vascular activity, energy metabolism and renal function at reperfusion.
Asunto(s)
Monóxido de Carbono/farmacología , Riñón/efectos de los fármacos , Preservación de Órganos , Daño por Reperfusión/prevención & control , Animales , Monóxido de Carbono/metabolismo , Frío , Riñón/irrigación sanguínea , Riñón/fisiología , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxadiazoles/farmacología , Consumo de Oxígeno/efectos de los fármacos , Quinoxalinas/farmacología , ConejosRESUMEN
The inducible stress protein heme oxygenase-1 (HO-1) has been linked to tissue and organ protection against the deleterious actions of many pathological conditions, including endotoxin challenge. Similar protection can be achieved by the main products of heme oxygenase activity, namely bilirubin and carbon monoxide (CO). Since the identification of novel chemical compounds that liberate CO in biological systems (CO-releasing molecules or CO-RMs), our group and others have had access to a convenient and simple pharmacological tool that enables to study the role of CO in physiological functions. This article will review the scientific literature published to date on CO-RMs, with emphasis on the in vitro, ex vivo and in vivo experimental models employed to determine the contribution of CO to cellular mechanisms. In addition, we will report on the effect of heme oxygenase-related substances, such as bilirubin, CORM-3 and hemin, in a model of endotoxin-induced hypotension. Among the three different approaches examined, CORM-3 proved the most effective agent in reducing the fall in blood pressure caused by endotoxin. Furthermore, heme oxygenase-related substances affected the endotoxin-stimulated induction and distribution of hepatic HO-1 and inducible nitric oxide synthase (iNOS). Thus, it emerges that CO-RMs could exert important biological actions in the context of endotoxic-mediated dysfunction.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Monóxido de Carbono/metabolismo , Endotoxinas/farmacología , Hipotensión/inducido químicamente , Hipotensión/fisiopatología , Animales , Regulación Enzimológica de la Expresión Génica/genética , Corazón/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Miocardio/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Mensajero/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Emerging evidence reveals that heme oxygenase-1 (HO-1) and its product carbon monoxide (CO) can exert diverse biological and cytoprotective effects. Our group has recently identified a new class of compounds (CO-releasing molecules or CO-RMs) that can carry and deliver CO to biological systems and can be used to examine the physiological properties of CO. Here, we evaluated the influence of endogenously-generated CO (via HO-1 induction by hemin) and CO liberated from exogenously supplied CO-RMs on mitochondrial function. Renal mitochondria were isolated either from rats with increased HO-1 or from untreated animals, the latter being exposed to different concentrations of CO-RMs (10-100 microM). We found that mitochondrial oxygen uptake was significantly reduced in kidneys after HO-1 induction and, in a similar fashion, CO-RMs inhibited mitochondrial function in a concentration-dependent manner. Specifically, a marked depression of state 3 was observed resulting in a significant decrease in respiratory control index (RCI) values. When mitochondria were incubated with the inactive forms of CO-RMs, which are devoid of CO, the respiratory parameters remained unchanged. In summary, the results indicate that HO-1 induction and enhanced CO decrease renal oxygen consumption and alter mitochondrial function suggesting that CO could be a physiological regulator of mitochondrial oxidative phosphorylation.
Asunto(s)
Monóxido de Carbono/metabolismo , Respiración de la Célula/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Animales , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemina/farmacología , Masculino , Estructura Molecular , Ratas , Ratas Sprague-DawleyRESUMEN
Various ring- and nitrogen-substituted benzenesulphonamides have been prepared and tested as potential inhibitors of carbonic anhydrase. N-Methoxysulphonamides showed no inhibitory activity, as predicted by the classic work of Krebs on N-substituted inhibitors. By contrast, N-hydroxysulphonamides proved to be very effective inhibitors of carbonic anhydrase. Using 111Cd-NMR it has been possible to analyse the molecular interaction of 4-fluoro-N-hydroxybenzenesulphon[15N]amide, with 111Cd-substituted bovine carbonic anhydrase. A large cadmium-111:nitrogen-15 spin-coupling shows that this inhibitor is directly bound to the metal via its nitrogen rather than through an oxygen atom. The mode of this binding is similar to that for the unsubstituted sulphonamide inhibitor, 4-fluorobenzenesulphon[15N]amide. The 111Cd-chemical shift of the signal for the inhibited enzyme shows that the N-hydroxysulphonamide is bound as its anion. From the relative intensities of free and complexed enzyme signals it can be deduced that the cadmium enzyme complex with the N-hydroxysulphonamide has a longer life-time than that formed with the unsubstituted sulphonamide. By contrast, native zinc-containing bovine carbonic anhydrase shows similar I50 values with both of these sulphonamides. Attempts to monitor the binding using 15N-NMR were unsuccessful, possibly due to a very long relaxation time for the nitrogen nucleus in the N-hydroxysulphonamide when bound to the enzyme leading to loss of the 15N signal.