RESUMEN
Clinical and experimental studies suggest that pharmacological postconditioning with Cyclosporin A (CsA) reduces infarct size in cardiac ischemia and reperfusion. CsA interacts with Cyclophilin D (CypD) preventing opening of the mitochondrial permeability transition pore (mPTP). Tissue kallikrein (TK) and its products kinins are involved in cardioprotection in ischemia. CypD knockout mice are resistant to the cardioprotective effects of both CsA and kinins suggesting common mechanisms of action. Using TK gene knockout mice, we investigated whether the kallikrein-kinin system is involved in the cardioprotective effect of CsA. Homozygote and heterozygote TK deficient mice (TK(-/-), TK(+/-)) and wild type littermates (TK(+/+)) were subjected to cardiac ischemia-reperfusion with and without CsA postconditioning. CsA reduced infarct size in TK(+/+) mice but had no effect in TK(+/-) and TK(-/-) mice. Cardiac mitochondria isolated from TK(-/-) mice had indistinguishable basal oxidative phosphorylation and calcium retention capacity compared to TK(+/+) mice but were resistant to CsA inhibition of mPTP opening. TK activity was documented in mouse heart and rat cardiomyoblasts mitochondria. By proximity ligation assay TK was found in close proximity to the mitochondrial membrane proteins VDAC and Tom22, and CypD. Thus, partial or total deficiency in TK induces resistance to the infarct size reducing effect of CsA in cardiac ischemia in mice, suggesting that TK level is a critical factor for cardioprotection by CsA. TK is required for the mitochondrial action of CsA and may interact with CypD. Genetic variability in TK activity has been documented in man and may influence the cardioprotective effect of CsA.
Asunto(s)
Cardiotónicos/farmacología , Ciclosporina/farmacología , Poscondicionamiento Isquémico , Isquemia Miocárdica/tratamiento farmacológico , Calicreínas de Tejido/genética , Animales , Peptidil-Prolil Isomerasa F , Ciclofilinas/genética , Ciclofilinas/metabolismo , Expresión Génica , Heterocigoto , Homocigoto , Masculino , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fosforilación Oxidativa , Ratas , Transducción de Señal , Calicreínas de Tejido/deficiencia , Canal Aniónico 1 Dependiente del Voltaje/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismoRESUMEN
PURPOSE: The feasibility of using iontophoresis to enhance the permeation rate of a model peptide was investigated in vitro using hairless mouse skin. METHODS: Angiotensin 2 (AT 2) was employed as a permeant probe, using optimum iontophoresis conditions. A number of physicochemical parameters (donor ionic strength; valence of competitive ions; pH of donor solution) were studied with the aim of exploring the mechanisms involved in the iontophoretic transport through the skin: electrokinetic transport or convective transport. For this purpose, the magnitude of the convective solvent flow was also evaluated by the permeation of (3H) H2O. The interest of pulsed currents for peptide delivery was also investigated and the effect of current density and frequency was studied. RESULTS: AT 2 transport was found to be enhanced 20-fold in comparison to passive permeation and was found to be proportional to the current density with direct currents as with pulsed currents. CONCLUSIONS: Although the flux enhancement of ions during iontophoresis is due principally to the electrical potential gradient, secondary effects such as convective solvent flow contribute also to flux enhancement of peptide delivery. This effect is dependent of physicochemical conditions of formulation.
