RESUMEN
Protection of ischemic myocardium is an important unmet need in reperfusion therapy of acute myocardial infarction. Myocardial ischemia and reperfusion induce necrosis and apoptosis in cardiomyocytes. Caspase processing and activation are critical steps in most receptor and nonreceptor pathways of apoptosis. Caspase inhibitors have been shown to reduce ischemia reperfusion injury in cardiac muscle. Information about dose response and time of administration are needed to optimize the design of preclinical studies. We used isolated adult rabbit cardiomyocytes subjected to metabolic inhibition (MI) and recovery to examine the role of caspases and caspase inhibitors, the dose response, and the timing of administration. In vitro inhibitory concentrations (Ki) were determined for purified caspases. Cardiomyocytes subjected to MI were treated with peptidomimetic fluoromethyl ketone inhibitors of caspases before or during MI, or at recovery. Caspase inhibitors were most effective when added before MI and included throughout recovery, but were partially protective if added after MI. The optimal concentration of the inhibitors tested was approximately 10 microM. Protection was sustained when cells were allowed to recover for 4 or 24 h. These results suggest that caspase activation is an important component of myocyte injury mediated by MI and recovery. Low doses of caspase inhibitors were identified that reduce injury in this model system, and further investigations using in vivo models are warranted.
Asunto(s)
Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/farmacología , Corazón/efectos de los fármacos , Miocardio/enzimología , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Miocardio/citología , Conejos , Transducción de SeñalRESUMEN
Hyperplasia of vascular smooth muscle cells (VSMCs) occurs during HIV infection, part of a spectrum of HIV-mediated cardiovascular and microvascular pathologies. These changes are not due to direct viral infection but may involve the receptor-mediated action of viral proteins, such as the envelope protein gp120. We sought to identify gp120 receptors which might mediate the vascular smooth muscle cell hyperplasia present in HIV infection. A homology between neuropeptide Y (NPY) and the previously identified receptor-active V2-region of gp120 defined by an octapeptide sequence (Peptide T) related to VIP was noted. Since NPY is mitogenic for VSMCs we therefore determined whether gp120 shares this activity. Rat aortic VSMCs were treated for 24 h with human (h)NPY and gp120 in the presence of 0.5% serum to measure [3H]thymidine incorporation, an index of cell proliferation. NPY increased [3H]thymidine incorporation by 80% after a 24-h treatment in a bimodal fashion, with peak effects at 10(-10) M and 10(-8) M. Gp120 was an even more potent mitogen for VSMCs with peak activity occurring at 10(-12) M. Peptide T was equipotent with gp120, and slightly less efficacious, suggesting that this domain may mediate gp120 effects on VSMCs. When combined, gp120 and NPY acted to antagonize one another, lowering DNA synthesis to basal levels. The profile of pharmacologic inhibition supports a role for NPY receptors since antagonists of Y1 and Y2 subtypes substantially or completely inhibited gp120-mediated VSMC proliferation. This is the first demonstration of the proliferative effects of HIV viral protein gp120 on VSMCs. The effect appears to be mediated via gp120 sequences related to VIP, peptide T, and NPY. These ligands may be competitive inhibitors of binding or gp120 processing. Novel treatments may emerge based upon VIP and NPY receptor antagonists if further work substantiates a role for gp120 in the vascular abnormalities of AIDS.
Asunto(s)
Enfermedades Cardiovasculares/etiología , Proteína gp120 de Envoltorio del VIH/toxicidad , Infecciones por VIH/complicaciones , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Receptores de Neuropéptido Y/efectos de los fármacos , Receptores de Neuropéptido Y/fisiología , Secuencia de Aminoácidos , Animales , Enfermedades Cardiovasculares/patología , División Celular/efectos de los fármacos , Células Cultivadas , VIH/genética , VIH/patogenicidad , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Hiperplasia , Mitógenos/farmacología , Datos de Secuencia Molecular , Neuropéptido Y/genética , Neuropéptido Y/farmacología , Neuropéptido Y/fisiología , Péptido T/genética , Péptido T/toxicidad , Ratas , Homología de Secuencia de Aminoácido , Péptido Intestinal Vasoactivo/genética , Péptido Intestinal Vasoactivo/farmacología , Péptido Intestinal Vasoactivo/fisiologíaRESUMEN
We have previously reported that neuropeptide Y (NPY), a sympathetic cotransmitter and vasoconstrictor, is mitogenic for vascular smooth muscle cells (VSMCs), and now report on the mechanisms mediating these effects. In rat aortic A10 cell line, NPY's potency was greater than that of norepinephrine, and efficacy similar to that of platelet-derived growth factor, but less than that of the full serum, in stimulating cell proliferation; this effect was optimal in cell 60-80% cell density. At lower cell density and serum content, NPY stimulated DNA fragmentation/apoptosis. In rat aortic primary VSMCs (RASMCs), mitogenic effect of NPY was bimodal with the first peak at 1 pM, a decline at 1 nM, and a second peak at 10-100 nM; peptide YY had similar but less efficacious effects. The first NPY's peak was mimicked by Y2 agonists, and blocked by Y2 antagonist (T4-[NPY(33-36]4), and the second mimicked by Y1 agonist and partially blocked by Y1 antagonist, BIBP3226, suggesting a multireceptor mode of action. In A10 and in RASMCs, the expression of NPY receptors, Y1, Y2 and Y5, using RT-PCR was undetectable in quiescent cells but detected after pre-treatment with NPY. The receptor induction was NPY dose-dependent and also affected by incubation time and presence of serum. The NPY mitogenic effects were attenuated by calcium channel blockers, particularly verapamil. In primary cultures of rat coronary endothelial cells (where NPY is also mitogenic), NPY stimulated mitogen-activated protein kinase (MAPK) activity. Thus, the growth-promoting effects of NPY in vascular cells occur at concentrations lower than vasoconstrictive, and appear to be mediated by inducible Y1, Y2, and Y5 receptors, calcium entry and possibly MAPK activation.
Asunto(s)
Desarrollo de Músculos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/crecimiento & desarrollo , Neuropéptido Y/farmacología , Neuropéptido Y/fisiología , Receptores de Neuropéptido Y/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Secuencia de Bases , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Células Cultivadas , Cartilla de ADN/genética , Expresión Génica/efectos de los fármacos , Mitógenos/farmacología , Músculo Liso Vascular/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores de Neuropéptido Y/clasificación , Receptores de Neuropéptido Y/genética , Transducción de SeñalRESUMEN
Among factors underlying reperfusion injury are oxygen free radicals and Ca2+ influx via gated calcium channel or via Na+/H(+)-Na+/Ca2+ exchange which lead to calcium overload. The aim of the study was to ultrastructurally visualize the distribution of Ca2+ and to compare binding of calcium by the sarcolemma and calcium accumulation in mitochondria under therapy with an OH scavenger, dimethylthiourea (DMTU), Na+/H+ exchange inhibitor, amiloride, and calcium channel blocker, diltiazem, given alone or in combination to ischemic/reperfused hearts. Isolated working hearts subjected to 40 min ischemia and 30 min reperfusion were perfused with drugs added to the perfusate 15 min before ischemia and administered for the rest of the perfusion period. The cytochemical phosphate pyroantimonate method for localization of Ca2+ was used, and calcium distribution was analyzed with a computer image analyzer. All drugs given alone improved sarcolemmal ability to bind calcium. The best results were obtained with amiloride. All of the combined therapies gave even better results, but calcium accumulation in mitochondria diminished only with diltiazem therapy given alone or in combination with DMTU. Since the presence of Ca2+ deposits on the sarcolemma is believed to represent its normal function, and calcium sequestration by mitochondria reflects an increase in cytosolic calcium load, the lack of correlation between sarcolemmal and mitochondrial Ca2+ distribution might suggest impaired mechanisms of lowering cytoplasmic calcium or the existence of some mechanism other than Na+/Ca2+ exchange, mediated by activated Na+/H+ exchange.
Asunto(s)
Amilorida/uso terapéutico , Bloqueadores de los Canales de Calcio/uso terapéutico , Calcio/metabolismo , Diltiazem/uso terapéutico , Depuradores de Radicales Libres/uso terapéutico , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Tiourea/análogos & derivados , Animales , Quimioterapia Combinada , Corazón/efectos de los fármacos , Radical Hidroxilo/metabolismo , Técnicas In Vitro , Masculino , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Miocardio/ultraestructura , Ratas , Ratas Wistar , Sarcolema/metabolismo , Tiourea/uso terapéuticoRESUMEN
Antiarrhythmic effects and intracellular electrophysiological properties of a new antiarrhythmic compound (-)trans-4-[2-hydroxy-3(N-isopropylamino)-propoxyimino]-cis-car ane (9) were studied in several models of arrhythmia and in isolated guinea-pig myocardial preparations. Compound 9 prevented the aconitine-induced arrhythmia, reversed the ouabin-induced arrhythmia depressed the maximum rate depolarization (Vmax), and shortened the action potential duration (ADP) and the effective refractory period (ERP). The data indicate that compound 9 is an effective antiarrhythmic presumably with a class 1 B mechanism of action.
Asunto(s)
Antiarrítmicos/química , Antiarrítmicos/farmacología , Propranolol/análogos & derivados , Propranolol/farmacología , Terpenos/química , Terpenos/farmacología , Animales , Arritmias Cardíacas/fisiopatología , Arritmias Cardíacas/prevención & control , Femenino , Cobayas , Técnicas In Vitro , Masculino , Propranolol/química , Ratas , Ratas WistarRESUMEN
Sympathetic nerves have long been suspected of trophic activity, but the nature of their angiogenic factor has not been determined. Neuropeptide Y (NPY), a sympathetic cotransmitter, is the most abundant peptide in the heart and the brain. It is released during nerve activation and ischemia and causes vasoconstriction and smooth muscle cell proliferation. Here we report the first evidence that NPY is angiogenic. At low physiological concentrations, in vitro, it promotes vessel sprouting and adhesion, migration, proliferation, and capillary tube formation by human endothelial cells. In vivo, in a murine angiogenic assay, NPY is angiogenic and is as potent as a basic fibroblast growth factor. The NPY action is specific and is mediated by Y1 and Y2 receptors. The expression of both receptors is upregulated during cell growth; however, Y2 appears to be the main NPY angiogenic receptor. Its upregulation parallels the NPY-induced capillary tube formation on reconstituted basement membrane (Matrigel); the Y2 agonist mimics the tube-forming activity of NPY, whereas the Y2 antagonist blocks it. Endothelium contains not only NPY receptors but also peptide itself, its mRNA, and the "NPY-converting enzyme" dipeptidyl peptidase IV (both protein and mRNA), which terminates the Y1 activity of NPY and cleaves the Tyr1-Pro2 from NPY to form an angiogenic Y2 agonist, NPY3-36. Endothelium is thus not only the site of action of NPY but also the origin of the autocrine NPY system, which, together with the sympathetic nerves, may be important in angiogenesis during tissue development and repair.
Asunto(s)
Endotelio Vascular/química , Neovascularización Fisiológica/efectos de los fármacos , Neuropéptido Y/fisiología , Sistema Nervioso Simpático/química , Animales , Aorta/efectos de los fármacos , Capilares , Colágeno/farmacología , Dipeptidil Peptidasa 4/biosíntesis , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/fisiología , Combinación de Medicamentos , Factores de Crecimiento Endotelial/farmacología , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica , Humanos , Laminina/farmacología , Linfocinas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptido Y/biosíntesis , Neuropéptido Y/genética , Neuropéptido Y/aislamiento & purificación , Neuropéptido Y/metabolismo , Neuropéptido Y/farmacología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Reacción en Cadena de la Polimerasa , Proteoglicanos/farmacología , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido Y/biosíntesis , Receptores de Neuropéptido Y/efectos de los fármacos , Receptores de Neuropéptido Y/genética , Receptores de Neuropéptido Y/fisiología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Recently, we found that vacuolar proton ATPase (VPATPase) operates in cardiomyocytes as a complementary proton-extruding mechanism. Its activity was increased by preconditioning with resultant attenuation of intracellular acidification during ischemia. In this study, we examined whether VPATPase-mediated proton efflux during metabolic inhibition/recovery may spare Na+ overload via Na+-H+ exchange, attenuate Na+-Ca2+ exchange, and decrease apoptosis. Neonatal rat cardiomyocytes were subjected to 2- to 3-hour metabolic inhibition with cyanide and 2-deoxyglucose and 24-hour recovery. The effect of VPATPase inhibition by 50 nmol/L bafilomycin A1 on apoptosis, pHi, and [Ca2+]i was studied by flow cytometry with propidium iodide, seminaphthorhodafluor (SNARF)-1-AM, and indo-1-AM staining, respectively. VPATPase inhibition increased the amount of apoptosis measured after 24 hours of recovery and abrogated the protective effect of inhibition of Na+-H+ exchange by (5-N-ethyl-N-isopropyl)amiloride (EIPA). Dual blockade of VPATPase and Na+-H+ exchange was additive in effect with EIPA on pHi during metabolic inhibition/recovery and recovery from the acid challenge with sodium propionate. VPATPase blockade increased the rate of accumulation of intracellular Ca2+ at the beginning of metabolic inhibition and abrogated the delaying effect of EIPA on intracellular Ca2+ accumulation. These results indicate that VPATPase plays an important accessory role in cardiomyocyte protection by reducing acidosis and Na+-H+ exchange-induced Ca2+ overload.
Asunto(s)
Apoptosis/fisiología , Calcio/metabolismo , Macrólidos , Miocardio/enzimología , ATPasas de Translocación de Protón/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Animales Recién Nacidos , Antiarrítmicos/farmacología , Antibacterianos/farmacología , Apoptosis/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/enzimología , ATPasas de Translocación de Protón/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Intercambiador de Sodio-Calcio/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vacuolas/enzimologíaRESUMEN
The physiological role of neuropeptide Y (NPY), a sympathetic cotransmitter and vasoconstrictor, has not been determined yet. We used a specific nonpeptide antagonist to the NPY Y1 receptor [BIBP-3226; (R)-N2-(diphenacetyl)-N-[(4-hydroxyphenyl) methyl]-D-arginineamide] to study the involvement of NPY in stress-induced vasoconstriction in the mesenteric bed. In rats subjected to cold water stress (COLD), plasma NPY immunoreactivity levels increased progressively from 0.15 +/- 0.01 to 0.32 +/- 0.05 pmol/ml and remained elevated during recovery. Administration of BIBP-3226 (3 mg.kg-1.h-1 infusion) tended to decrease the stress-induced pressor response and significantly attenuated the post-COLD elevation of blood pressure. The COLD-induced fall in the superior mesenteric artery blood flow and the increase of up to 300% in the mesenteric vascular resistance were either reduced or eliminated by BIBP-3226. Conversely, the Y1 antagonist had no effect on the COLD-induced tachycardia. This study provides the first evidence of the physiological role of NPY. The peptide is released during stress and increases mesenteric vascular resistance via activation of its Y1 receptors. Specific Y1-receptor antagonists may therefore be of potential benefit in prevention or treatment of stress-induced vasospasm.
Asunto(s)
Arterias Mesentéricas/fisiología , Receptores de Neuropéptido Y/fisiología , Vasoconstricción/fisiología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Presión Sanguínea/efectos de los fármacos , Frío , Hemodinámica , Masculino , Ratas , Ratas Wistar , Receptores de Neuropéptido Y/antagonistas & inhibidores , Flujo Sanguíneo Regional/efectos de los fármacos , Estrés Mecánico , Resistencia Vascular/efectos de los fármacosAsunto(s)
Daño por Reperfusión Miocárdica/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Endotelio/citología , Endotelio/metabolismo , Depuradores de Radicales Libres/farmacología , Glicocálix/efectos de los fármacos , Glicocálix/metabolismo , Corazón/efectos de los fármacos , Masculino , Molsidomina/análogos & derivados , Molsidomina/metabolismo , Molsidomina/farmacología , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocardio/citología , Miocardio/metabolismo , Ratas , Ratas Wistar , Tiopronina/farmacología , Vasodilatadores/metabolismo , Vasodilatadores/farmacologíaRESUMEN
OBJECTIVES: The importance of NO-induced vasodilator tone in maintaining adequate coronary flow to sustain hemodynamic function in aerobically perfused heart and the role of NO in the injury development in ischaemic/reperfused heart was studied. METHODS: Effect of NO synthesis inhibitor (N omega-nitro-L-arginine, L-NOARG) on isolated working rat hearts subjected to either 90 min of aerobic perfusion or to a global ischaemia (27.5 to 42.5 min) followed by 40 min reperfusion was studied. To overcome coronary flow reducing effect of L-NOARG either perfusion pressure was raised from 75 to 120 cm H2O or adenosine (400 nM) was administered. RESULTS: In the hearts perfused at coronary pressure of 75 and 120 cm H2O, L-NOARG (10 microM) reduced coronary flow by 30% and 17%, respectively, while cardiac output was not affected. Only a transient increase in adenosine and lactate outflow occurred in L-NOARG-treated hearts. The post-ischaemic recovery of functions was impaired in L-NOARG-treated hearts, an effect not correlating with L-NOARG-induced reduction in coronary flow. Although the pre-ischaemic coronary flow was similar in the untreated hearts perfused at 75 cm H2O and in L-NOARG-treated hearts perfused at 120 cm H2O, the post-ischaemic recovery in the latter group was still impaired as compared to that in the untreated hearts. Likewise, coronary flow was similar in the untreated hearts and in those treated with L-NOARG plus adenosine, nevertheless, the post-ischaemic recovery in the latter group was impaired as compared to that in the untreated hearts. CONCLUSIONS: While the inhibition of NO synthesis resulted in coronary flow reduction it did not induce a state of permanent ischaemia in isolated rat heart. L-NOARG-induced augmentation of the ischaemia/reperfusion injury was related to the deficit of NO, itself, rather than to the reduction in myocardial perfusion.
Asunto(s)
Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Óxido Nítrico/fisiología , Adenosina/metabolismo , Adenosina/farmacología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Circulación Coronaria/efectos de los fármacos , Corazón/fisiopatología , Lactatos/metabolismo , Ácido Láctico , Masculino , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/fisiopatología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina , Perfusión , Ratas , Ratas WistarRESUMEN
To determine the effect of post-ischaemic reperfusion on the ultrastructure of the endothelial glycocalyx and the role of oxygen free radicals, isolated working rat hearts were subjected to 20 min ischaemia followed by 3 or 30 min of reperfusion. Ruthenium red and lanthanum chloride were used to delineate the endothelial glycocalyx, and histochemical manganese/diaminobenzidine (Mn+2/DAB) or iron/diaminobenzidine (Fe+2/DAB) techniques were applied to visualize superoxide and hydrogen peroxide in myocardial capillaries. We found that ischaemia alone led to only a slightly flocculent appearance of the glycocalyx and its disruption was not observed until the onset of reperfusion. Prolongation of reperfusion to 30 min had no further effect on the ultrastructure of the glycocalyx. The ultrastructure of endothelial cells was normal. The disruption of the glycocalyx correlated in time and place with the appearance of Mn+2/DAB and Fe+2/DAB reaction products on the luminal surface of endothelial cells. Treatment with 5 mM N-(2-mercaptopropionyl)-glycine (MPG), an .OH radical scavenger, starting before ischaemia prevented the disruption of the glycocalyx, while 100 mM 3-morpholinosydnonimine (SIN-1), capable of generating both NO and -O2 simultaneously when applied at the time of reperfusion, increased the mean density of capillaries positively stained with Mn+2/DAB and Fe+2/DAB, and caused substantial disruption of the glycocalyx and damage to endothelial cells, which was not prevented by MPG. Our results suggest that the onset of reperfusion is critical for injury to the endothelial glycocalyx. Most probably the hydroxyl radical derived from the Fenton reaction is responsible for this injury. Peroxynitrite and/or nitric dioxide, if present upon reperfusion, may also account for damage of the endothelial glycocalyx.
Asunto(s)
Endotelio Vascular/metabolismo , Glicoproteínas/metabolismo , Peróxido de Hidrógeno/metabolismo , Miocardio/metabolismo , Polisacáridos/metabolismo , Superóxidos/metabolismo , Animales , Endotelio Vascular/ultraestructura , Técnicas In Vitro , Masculino , Microscopía Electrónica , Miocardio/ultraestructura , Ratas , Ratas Wistar , ReperfusiónRESUMEN
OBJECTIVES: The relative contribution of oxygen free radicals, disturbances in calcium homeostasis and Na+/H+ exchange in the development of injury in the ischemic/reperfused heart was investigated. The study was designed to assess whether these factors initiate independent mechanisms of injury or, alternatively, they share a common mechanism of toxicity. METHODS: Isolated working rat hearts were subjected to different periods (30-55 min) of global ischemia and then were reperfused for 30 min. We compared the effects of oxygen radical scavengers (10 mM dimethylthiourea, DMTU and 0.6 mM desferrioxamine), inhibitors of Na+/H+ exchange (0.15 mM amiloride and 15 microM dimethylamiloride, DMA) and of 0.1 microM diltiazem, which was used to limit calcium overload, given alone or in combination, on the rate of myocardial injury development (recovery of hemodynamic function, LDH release, incidence of severe arrhythmias and structural integrity of cardiomyocytes were estimated at reperfusion following different periods of ischemia). RESULTS: All interventions studied, when given alone, provided nearly equivalent cardioprotection. DMTU or desferrioxamine when applied in combination with diltiazem provided additive cardioprotection, relatively limited, however, as compared to the remarkable cardioprotection achieved by DMTU or desferrioxamine in combination with amiloride. CONCLUSIONS: All mechanisms studied may contribute in an equal manner to the rate of injury development in the ischemic/reperfused heart. The oxygen free radicals-induced myocardial injury may be partially attributed to some disturbance in intracellular calcium homeostasis, possibly calcium overload, whereas the damaging effect of the Na+/H+ exchange activated upon reperfusion is probably largely related to some other mechanism.
Asunto(s)
Amilorida/uso terapéutico , Diltiazem/uso terapéutico , Daño por Reperfusión Miocárdica/prevención & control , Tiourea/análogos & derivados , Amilorida/análogos & derivados , Animales , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Endocardio/patología , Técnicas In Vitro , Masculino , Isquemia Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/patología , Ratas , Ratas Wistar , Tiourea/uso terapéuticoRESUMEN
The effects of perfusate calcium reduction, allopurinol and dimethylthiourea on reperfusion-induced arrhythmias and purine wash-out in isolated rabbit and rat hearts were compared. The overall incidence of reperfusion-induced ventricular tachycardia (VT) was 88% and 94% and that of ventricular fibrillation (VF) was 44% and 88% in the control rabbit and rat hearts, respectively. VF was reduced to 10% and 0% in rat and rabbit hearts subjected to perfusate calcium reduction (0.4 mM for 1 min before ischemia and for 1 min before and throughout reperfusion), respectively. In allopurinol, 1 mM, perfused rat hearts the overall incidence of VF was not changed and only the incidence of a sustained VF (that lasting for at least 10 min) was reduced. VT and VF were prevented in allopurinol-perfused rabbit hearts. Dimethylthiourea, 10 mM, reduced the incidence of VF in rat hearts to 16% and did not significantly affect VT and VF in rabbit hearts. In untreated rat hearts, the major purine compounds washed out upon reperfusion were inosine, hypoxanthine, xanthine and urate. Allopurinol augmented the wash-out of adenosine and abolished that of xanthine and urate. In untreated rabbit hearts, the major purine washed out were inosine, adenosine and hypoxanthine. Allopurinol did not cause further increase in adenosine wash-out in rabbit hearts. We speculate that: (1) calcium mediated arrhythmogenic mechanism is operating both in reperfused rat and rabbit heart; (2) free radical mediated mechanism is of an importance only in rat heart; (3) neither a decreased free radical production secondary to xanthine oxidase inhibition nor the augmentation of adenosine wash-out is a likely explanation for the antiarrhythmic effect of allopurinol in reperfused hearts; and (4) high level of myocardial adenosine accumulation during ischemia, probably secondary to low xanthine oxidase activity, may play a role of a natural defence mechanism in ischemic/reperfused rabbit heart.
Asunto(s)
Alopurinol/farmacología , Arritmias Cardíacas/fisiopatología , Calcio/metabolismo , Corazón/fisiología , Miocardio/metabolismo , Purinas/metabolismo , Daño por Reperfusión/fisiopatología , Tiourea/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacología , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/metabolismo , Calcio/análisis , Calcio/fisiología , Femenino , Radicales Libres/metabolismo , Corazón/efectos de los fármacos , Incidencia , Masculino , Miocardio/química , Miocardio/ultraestructura , Oxidación-Reducción , Conejos , Ratas , Ratas Wistar , Receptores Purinérgicos P1/análisis , Receptores Purinérgicos P1/fisiología , Daño por Reperfusión/complicaciones , Daño por Reperfusión/metabolismo , Taquicardia Ventricular/epidemiología , Taquicardia Ventricular/etiología , Taquicardia Ventricular/prevención & control , Tiourea/farmacología , Xantina Oxidasa/fisiologíaRESUMEN
OBJECTIVE: A proposal that injury in ischaemic/reperfused rat heart is critically dependent on the availability of free iron rather than on the efficiency of O2-. and H2O2 production was examined. METHODS: Isolated working rat hearts from 152 male Wistar rats (200-250 g weight), subjected to 20-40 min of global ischaemia and reperfused for 30 min, were perfused with 10 mumol.litre-1 Fe[III] or Fe[II] and/or 0.6 mmol.litre-1 desferrioxamine, 10 mmol.litre-1 dimethylthiourea, and 1 mmol.litre-1 allopurinol. Curves relating the recoveries of haemodynamic functions and the reperfusion lactate dehydrogenase release to the duration of the preceding ischaemic period were constructed. Morphological examination was also performed. RESULTS: In the untreated hearts, the duration of ischaemia resulting in 50% loss of cardiac output was 29 min. This time was decreased to 24 min and 20 min by Fe[III] and Fe[II], respectively, and was increased to 36 min and 37 min by desferrioxamine and dimethylthiourea, respectively. Desferrioxamine prevented the effect of Fe[III] but not that of Fe[II], whereas dimethylthiourea prevented the effect of Fe[II]. Neither the effect of Fe[III] nor that of Fe[II] was prevented by allopurinol which, however, proved to be beneficial in the untreated hearts. CONCLUSIONS: The beneficial effect of desferrioxamine and dimethylthiourea suggest that it is intensification of the Fenton reaction by iron which accounts for iron induced aggravation of the reperfusion injury. Thus we speculate that the availability of free iron, rather than O2-. and H2O2, is a limiting factor in the development of injury in an ischaemic/reperfused rat heart. What remains unclear is why allopurinol is unable to prevent iron induced changes.
Asunto(s)
Compuestos Férricos/farmacocinética , Compuestos Ferrosos/farmacocinética , Radicales Libres/efectos adversos , Miocardio/metabolismo , Daño por Reperfusión/metabolismo , Alopurinol/farmacología , Animales , Disponibilidad Biológica , Deferoxamina/farmacología , Corazón/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Masculino , Microscopía Electrónica , Miocardio/ultraestructura , Ratas , Ratas Endogámicas , Daño por Reperfusión/patología , Tiourea/análogos & derivados , Tiourea/farmacología , Factores de TiempoRESUMEN
1. The release of three endothelial mediators, namely, endothelial-derived relaxing factor (EDRF), prostacyclin (PGI2) and endothelin, and of two sympathetic neurotransmitters, noradrenaline and neuropeptide Y (NPY), from resting or sympathetically stimulated rabbit Langendorff hearts was investigated at normal or elevated coronary flow. The sympathetic nerves to the hearts were stimulated at 5 Hz for 30 s and the cardiac effluent was analysed for nitrite (metabolite of EDRF) with electron paramagnetic resonance spectrometry, for 6-keto-PGF1 alpha (metabolite of PGI2) with gas chromatography/mass spectrometry, for endothelin- and NPY-like immunoreactivity with radioimmunoassay, and for noradrenaline and purines with liquid chromatography. 2. During perfusion of the hearts at normal flow (35 +/- 1.4 ml min-1) the effluent concentration of nitrite was 0.15 +/- 0.02 microM, that of 6-keto-PGF1 alpha 0.74 +/- 0.08 nM, and that of endothelin-like immunoreactivity 0.18 +/- 0.01 pM. Nerve stimulation augmented the release of 6-keto-PGF1 alpha from 76 +/- 8 to 99 +/- 10 pmol (3 min)-1 (P less than 0.05), but did not affect the release of nitrite or endothelin-like immunoreactivity. Nerve stimulation also facilitated the outflow of noradrenaline and of NPY-like immunoreactivity by 52 +/- 11 pmol (3 min)-1 and 19 +/- 7 fmol (3 min)-1, respectively. 3. Elevation of the coronary flow to 79 +/- 3.2 ml min-1 did not affect the effluent concentrations of nitrite, 6-keto-PGF1 alpha and endothelin-like immunoreactivity, implying that their outflows were augmented. Sympathetic stimulation at elevated coronary flow did not further augment the outflow of endothelial mediators or of NPY-like immunoreactivity, but increased the outflow of noradrenaline by 62 +/- 12%, in comparison to stimulation at normal flow. Perfusion of the heart with the noradrenaline uptake blocker desipramine (5 microM) completely abolished the promoting effecting of elevated coronary flow on noradrenaline outflow during sympathetic stimulation. 4. These data indicate that an increase in coronary flow in perfused rabbit hearts is paralleled by a corresponding facilitation of the formation of the endothelial mediators, EDRF, prostacyclin and endothelin. Such an elevation of mediator formation does not affect nerve stimulation-induced release of sympathetic transmitters in the heart.
Asunto(s)
Circulación Coronaria/fisiología , Endotelinas/metabolismo , Epoprostenol/metabolismo , Proteínas Musculares/metabolismo , Neurotransmisores/metabolismo , Animales , Velocidad del Flujo Sanguíneo/fisiología , Desipramina/farmacología , Endotelio Vascular/metabolismo , Corazón/efectos de los fármacos , Neuropéptido Y/metabolismo , Óxido Nítrico , Norepinefrina/metabolismo , Técnicas de Cultivo de Órganos , ConejosRESUMEN
Calcium channel blockers (nifedipine, verapamil, diltiazem), calmodulin antagonists (trifluoperazine, calmidazolium, compound 48/80) and anti-free radical agents (allopurinol, desferrioxamine, mannitol, L-methionine) were tested for their potency to stabilize human erythrocytes against hypotonic hemolysis. The anti-free radical agents and compound 48/80 did not confer the membrane stabilization. Nifedipine, verapamil, diltiazem, calmidazolium and trifluoperazine at low concentrations, protected the cells from the hypotonic hemolysis while at higher concentrations they caused lysis. Similar biphasic changes were produced by the detergents sodium dodecyl sulphate (SDS) and Triton X-100. The drug concentration-dependency of the biphasic changes in the erythrocytes osmotic fragility produced by calcium channel blockers and calmodulin antagonists was not affected by low concentrations of SDS and Triton X-100. On the other hand, these drugs did not prevent the hemolysis produced by high concentrations of the detergents. The above as well as the observation that the membrane stabilization is conferred only by relatively high concentrations of calcium channel blockers and calmodulin antagonists suggest that membrane stabilization is not responsible for anti-ischemic effects of these agents reported in the literature.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calmodulina/antagonistas & inhibidores , Membrana Eritrocítica/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Masculino , Fragilidad Osmótica/efectos de los fármacosRESUMEN
The vascular endothelium is not merely a passive physical barrier between the blood and the tissue surrounding the blood vessel, but may actively participate in key processes of metabolic, secretory, and vasoregulatory character. In addition, the endothelium plays an important role in the control of platelet activation. Under certain conditions endothelial cells have been shown to produce powerful vasodilators, like endothelium-derived relaxing factor (EDRF) and prostacyclin (GPI2), and vasoconstrictors like endothelium-derived constricting factor (EDCF) and endothelin (ET) (Griffith et al., 1988; Vanhoutte & Katusic, 1988). In contrast to the extensive studies performed to characterize the actions and nature of EDRF, recently identified chemically as nitric oxide (Moncada et al., 1988), relatively little is known about EDCF(s). This paper reviews recent data on EDCF, with special emphasis on the newly discovered vasoconstrictor peptide, endothelin (ET).
Asunto(s)
Endotelio Vascular/análisis , Péptidos/análisis , Secuencia de Aminoácidos , Animales , Endotelinas , Humanos , Datos de Secuencia Molecular , Péptidos/fisiologíaRESUMEN
Endothelin is a 21-residue peptide formed during incubation of isolated porcine endothelial cells. Due to its pronounced vasoconstrictor activity, endothelin has been proposed to play a role in the regulation of vascular tone. We studied the effects of synthetic endothelin (0.1-10 nM) on coronary flow, mechanical performance, myocardial oxygen uptake, and formation of purines, and outflow of 6-ketoprostaglandin F1 alpha (metabolite of prostacyclin) in rabbit hearts perfused with saline by the Langendorff method. Endothelin dose-dependently decreased the coronary flow, at 10 nM, by about 75%. Heart rate, ventricular contractility, myocardial oxygen uptake, and purine release were affected by endothelin no more than by a corresponding mechanical reduction of the coronary flow. In contrast, the diastolic relaxation appeared to be directly diminished by endothelin. The concentration of 6-ketoprostaglandin F1 alpha in the cardiac effluent was dose-dependently elevated by about 14 times by endothelin (10 nM) (p less than 0.001). A corresponding mechanical restriction of the coronary flow insignificantly affected the effluent concentration of 6-ketoprostaglandin F1 alpha. The calcium channel blocker nifedipine (1 microM) completely abolished the decrease in diastolic relaxation induced by endothelin and markedly counteracted the peptide-induced increase in effluent concentration of 6-ketoprostaglandin F1 alpha, but did not affect the vasoconstrictor activity. These data demonstrate that endothelin induces vasoconstriction and facilitates the outflow of prostacyclin in the rabbit heart. In addition, the peptide appears to affect diastolic relaxation in this organ.
Asunto(s)
6-Cetoprostaglandina F1 alfa/análisis , Circulación Coronaria/efectos de los fármacos , Endotelio Vascular , Corazón/efectos de los fármacos , Miocardio/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Péptidos/farmacología , Purinas/análisis , Animales , Cromatografía de Gases , Cromatografía Liquida , Endotelinas , Femenino , Corazón/fisiología , Frecuencia Cardíaca/efectos de los fármacos , Técnicas In Vitro , Masculino , Espectrometría de Masas , ConejosRESUMEN
Prostacyclin and purine efflux rates from the isolated rabbit heart in response to variations of flow rate or perfusion pressure were investigated. Increases in coronary flow by 25, 50, and 100% augmented the effluxes of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) and purines equally, by up to four times. Increases in coronary pressure by 25, 50, and 100% augmented the outflow of 6-keto-PGF1 alpha by up to 20 times, whereas the outflow of purines increased no more than 6.5 times. Neither reduction of perfusate Ca2+ by 50% nor administration of quinacrine (1 microM) affected the basal efflux of 6-keto-PGF1 alpha or its response to an increase in coronary pressure. Both interventions did, however, reduce the pressure-induced purine efflux by approximately 50%. Pulsatile flow did not affect either the outflow of 6-keto-PGF1 alpha or that of purines, in comparison to steady flow at the same rate. The data demonstrate that an increase in coronary pressure activates a specific mechanism for prostacyclin production that appears independent of extracellular Ca2+ and of phospholipase activity.