RESUMEN
BACKGROUND: ß-Resorcylidene aminoguanidine (RAG), a highly reactive derivative of aminoguanidine, possesses antithrombotic activity which involves the activation of the vascular COX-2/PGI2 pathway. This endothelium-dependent effect suggests that RAG may demonstrate vasomotor activity in arterial vessels. The aim of the present study was to investigate a possible vasoactive action of RAG in coronary arteries of rat heart. METHODS: Isolated rat hearts were perfused in the Langendorff model. To investigate the dose dependency of the effect of RAG on coronary flow, the hearts were perfused with RAG at increasing concentrations. Mechanisms of RAG-mediated vasodilation were subsequently tested using selective inhibitors of the endothelium-dependent and endothelium-independent mechanisms responsible for regulation of vascular tone. RESULTS: RAG dilated coronary arteries at concentrations above 10(-5)mol/l. Inhibition of the endothelium-dependent mechanism of vasodilation by NG-nitro-L-arginine methyl ester, indomethacin and aminobenzotriazole did not affect RAG-mediated vasodilation. Other compounds also had no impact on the vasodilating effect of RAG: the NO-dependent guanylate cyclase inhibitor - 1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one, the cAMP-dependent protein kinase inhibitor - PKAi, and the K(+) channel blockers - glibenclamide, tetraethylammonium, charybdotoxin, and apamin. CONCLUSIONS: RAG is a strong vasodilator that exerts its effect via endothelium-independent mechanisms.
Asunto(s)
Vasos Coronarios/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Guanidinas/farmacología , Vasodilatación/efectos de los fármacos , Animales , Vasos Coronarios/fisiología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiología , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Vasodilatación/fisiologíaRESUMEN
There is good evidence supporting the notion that aminoguanidine(AG)-derived compounds prevent glycation/glycooxidation-dependent processes and therefore inhibit late diabetic complications. The aim of the present work was to analyse the antithrombotic action and antiglycation activity of beta-resorcylidene aminoguanidine (RAG) in comparison with another commonly used aminoguanidine (AG)-derived compound, pyridoxal aminoguanidine (PAG). In vitro RAG and PAG prevented exhaustive glycation and glycooxidation of BSA to a similar extent. However, merely RAG showed almost complete binding to sepharose-immobilized heparin, while PAG and other AG derivatives had much poorer affinities. In the model of in vivo thrombosis in Wistar rats with extracorporeal circulation RAG (i.v. 30 mg/kg), but not PAG, produced sustained (2 h) antithrombotic effect, which was abrogated by indomethacin (5 mg/kg) and rofecoxib (1 mg/kg). The 60-day treatment of streptozotocin-diabetic animals with RAG (p.o. 4 mg/kg) significantly decreased plasma concentration of a thromboxane B(2) and reduced whole blood platelet aggregability triggered by ADP or collagen. In conclusion, although RAG and PAG displayed similar antiglycation and antioxidation activities in vitro, only RAG showed antithrombotic activity in vivo that involved activation of COX-2/PGI(2) pathway. Our results indicate that designing novel RAG derivatives with optimal antithrombotic and antiglycation activities may prove useful to treat diabetic complications.
Asunto(s)
Fibrinolíticos/uso terapéutico , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Productos Finales de Glicación Avanzada/metabolismo , Guanidinas/uso terapéutico , Trombosis/tratamiento farmacológico , Trombosis/metabolismo , Animales , Bovinos , Fibrinolíticos/farmacología , Glicosilación/efectos de los fármacos , Guanidinas/farmacología , Masculino , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Wistar , Albúmina Sérica Bovina/metabolismo , Trombosis/fisiopatologíaRESUMEN
Oxidative damage is considered to play an important role in the pathogenesis of several diseases, such as diabetes mellitus (DM), atherosclerosis, cardiovascular complications and chronic renal failure. DM is associated with the oxidative stress and formation of advanced glycation end products (AGEs). Different drugs inhibit oxidative stress and formation of advanced glycation end products. Aminoguanidine (AG) has been proposed as a drug of potential benefit in prophylaxis of the complications of DM. Recent reports show a pro-oxidant activity of AG. Therefore we examined the effect of structural analogue of AG, its Schiff base with pyridoxal-pyridoxylidene aminoguanidine (PAG) on the level of selected markers of oxidative stress. We found that PAG decreased total damage to DNA in controls as well as in diabetic group of rats. However, we also found that PAG supplementation increases susceptibility of lipoproteins to oxidation and formation of conjugated dienes in both, diabetic as well as control animals. Its administration to diabetic rats decreases antioxidant capacity of plasma. Therefore, it is necessary to search for other structural modifications of AG that would combine its higher anti-diabetic activity with less toxicity.
Asunto(s)
Diabetes Mellitus/metabolismo , Guanidinas/farmacología , Estrés Oxidativo/efectos de los fármacos , Piridoxal/farmacología , Aldehídos/metabolismo , Animales , Antioxidantes/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Daño del ADN , Diabetes Mellitus/sangre , Diabetes Mellitus/inducido químicamente , Diabetes Mellitus/enzimología , Guanidinas/administración & dosificación , Guanidinas/química , Lipoproteínas/metabolismo , Masculino , Malondialdehído/metabolismo , Oxidación-Reducción , Piridoxal/administración & dosificación , Piridoxal/química , Ratas , Ratas Wistar , Solubilidad , Agua/químicaRESUMEN
In the present investigation, we attempted to study possible mechanisms of the interactions of resorcylidene aminoguanidine (RAG), the agent with a recognized anti-glycation and antioxidative activity, with rat liver mitochondria. We hypothesized that RAG affects organization of the lipid bilayer in mitochondrial membranes and thus impairs transmembrane Ca(2+) redistribution, transmembrane potential, and respiration capacity. Isolated mitochondria were exposed to RAG (50-200 microM) and several parameters of their function monitored employing spectrofluorimetric, cytometric, and respirometric techniques. Mitochondrial membrane potential and membrane fluidity were tracked using the staining with rhodamine 123 (Rh123) and 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. Mitochondrial respiration and oxidative phosphorylation was monitored with a high-resolution respirometry, and mobilization of Ca(2+) was detected using spectrofluorimetry with Calcium Green 5-N. RAG depolarized and fluidized mitochondrial membrane, as deduced from reduced fluorescence of intramitochondrial Rh123 and decreased DPH fluorescence anisotropy. The slight inhibitory effect of 100-200 microM RAG on mitochondrial respiratory capacity was observed merely when monitored in the presence of ADP. The reduced sensitivity of mitochondria to calcium-induced depolarization was significant only at higher RAG concentrations (100-200 microM). Moreover, RAG induced pronounced conformational changes in two model proteins: bovine serum albumin and cytochrome c. These findings indicate that regardless of its depolarizing and fluidizing properties, RAG does not largely affect the mitochondrial respiration, although it may significantly lower oxidative phosphorylation when used at higher concentrations.
Asunto(s)
Guanidinas/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Animales , Calcio/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Membrana Dobles de Lípidos/metabolismo , Masculino , Mitocondrias Hepáticas/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Oxígeno/metabolismo , Ratas , Ratas Wistar , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Triptófano/químicaRESUMEN
BACKGROUND: Pyridoxylidene aminoguanidine is an appropriate inhibitor of protein glycation, respectively formation of advanced glycation products, which are connected with mechanism of pathogenesis in chronic diabetic complications. Moreover, it was found that in comparison with aminoguanidine, pyridoxylidene aminoguanidine does not influence the level of vitamin B6 in liver and kidneys in vivo. The aim of this study was to test cytotoxic effect of pyridoxylidene aminoguanidine in vitro, in regard to its potential use as inhibitor of advance protein glycation in diabetic patients. METHODS: The potential genotoxic activity of pyridoxylidene aminoguanidine in vitro was assessed by the micronucleus test and the karyological analysis. The direct contact method using diploid human cell line B-HEF-2 was performed to evaluate cytotoxicity. The concentrations of 5 x 10(-3), 2.5 x 10(-3) and 1 x 10(-3) ml/l were used in all tests. RESULTS: Microscopic analysis did not proved any changes in morphology of exposed fibroblasts. The inhibitive effect of pyridoxylidene aminoguanidine was increased with rising concentration. The proliferative activity of exposed cells to concentrations of 1 x 10(-3), 2.5 x 10(-3), 5 x 10(-3) mol/l was inhibited approximately by 30%, 60% and 80%, respectively. The frequency of micronuclei and rate of numerical or structural aberrations was not increased. CONCLUSION: Obtained results confirmed that pyridoxylidene aminoguanidine in selected concentrations has an inhibitive effect on the proliferation activity of exposed cells, but did not develop any cytotoxic effect on B-HEF-2 cells.
Asunto(s)
Guanidinas/toxicidad , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Aberraciones Cromosómicas , Diploidia , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Humanos , Pruebas de Micronúcleos , Fosfato de Piridoxal/toxicidadRESUMEN
The hyperglycaemia and oxidative stress, that occur in diabetes mellitus, cause impairment of membrane functions in cardiomyocytes. Also reduced sensitivity to Ca-overload was reported in diabetic hearts (D). This enhanced calcium resistance is based on remodelling of the sarcolemmal membranes (SL) with down-regulated, but from the point of view of kinetics relatively well preserved Na,K-ATPase and abnormal Mg- and Ca-ATPase (Mg/Ca-ATPase) activities. It was hypothesised that in these changes may also participate the non-enzymatic glycation of proteins (NEG) and the related free radical formation (FRF), that decrease the membrane fluidity (SLMF), which is in reversal relationship to the fluorescence anisotropy (D 0.235 +/- 0.022; controls (C) 0.185 +/- 0.009; p < 0.001). In order to check the true role of SLMF in hearts of the diabetic rats (streptozotocin, single dose, 45 mg/kg i.v.) animals were treated in a special regimen with resorcylidene aminoguanidine (RAG 4 mg/kg i.m.). The treatment with RAG eliminated completely the diabetes-induced decrease in the SLMF (C 0.185 +/- 0.009; D + RAG 0.167 +/- 0.013; p < 0.001) as well as in NEG (fructosamine microg x mg(-1) of protein: C 2.68 +/- 0.14; D 4.48 +/- 0.85; D + RAG 2.57 +/- 0.14; p < 0.001), and FRF in the SL (malondialdehyde: C 5.3 +/- 0.3; D 8.63 +/- 0.2; D + RAG 5.61 +/- 0.53 micromol x g(-1); p < 0.05). Nevertheless, the SL ATPase activity in diabetic animals was not considerably influenced by RAG (increase in D + RAG vs. D 3.3%, p > 0.05). On the other hand, RAG increased considerably the vulnerability of the diabetic heart to overload with external Ca2+ (C 100% of hearts failed, D 83.3%, D + RAG 46.7% of hearts survived). So we may conclude, that: (i) The NEG and FRF caused alterations in SLMF, that accompanied the diabetes-induced remodelling of SL, also seem to participate in the protection of diabetic heart against Ca2+-overload; (ii) Although, the changes in SLMF were shown to influence considerably the ATPase activities in cells of diverse tissues, they seem to be little responsible for changes in ATPases-mediated processes in the SL of chronic diabetic hearts.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Guanidinas/farmacología , Corazón/fisiopatología , Fluidez de la Membrana/fisiología , Sarcolema/metabolismo , Animales , ATPasas Transportadoras de Calcio/metabolismo , Células Cultivadas , Miocardio/patología , Ratas , Ratas Wistar , Sarcolema/efectos de los fármacos , Sarcolema/patología , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
We estimated in vitro membrane fluidity gradient in erythrocytes (RBC) from diabetic patients, using a fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH). The rate constant of DPH incorporation (k) into the membranes was determined by fitting experimental data to an exponential equation. Four important findings were made. First, membrane fluidity in the hydrocarbon region of RBC from diabetic patients is decreased compared with control cells (P<0.01). Second, the rate constant k of DPH incorporation into the membranes of RBC from diabetic patients was lower (P<0.01), which indicates an altered fluidity gradient in the membranes. Third, resorcylidene aminoguanidine (RAG) decreased significantly (P<0.001) the anisotropy values in RBC membranes from diabetic patients, which means that it apparently acted as a fluidizing agent. Lastly, no significant differences in the rate constants k were found between the control membranes (from RAG untreated RBC) and the membranes isolated from RAG pretreated blood from diabetic patients, as well as between the control membranes and those from RAG pretreated control blood. In conclusion, RAG affects lipid-protein interactions in RBC membranes, which results in membrane lipid bilayer fluidization and leads to the restoration of natural physiological membrane dynamic parameters in RBC from diabetic patients.