Effects Of Adenosine Al Receptor On The Opening Of Mitochondrial Permeability Transition Pore In Hypoxic Cardiocytes And Its Mechanisms

Objectives:To investigate whether adenosine A1 receptor (A1R) activation could affect the opening of mitochondria permeability transition pore (MPTP) in hypoxic cardiocytes and its mechanisms.Methods:1. Neonatal rat cardiocytes cultured in the mixed gas (94%N2,5%CO2,1% O2) were employed as the hypoxic model.2. Neonatal rat cardiocytes in primary culture were randomized as group A, the normoxic group; group B, cells cultured under hypoxia; group C, cells under hypoxia treated with A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 500nmol/L); group D, cells under hypoxia treated with CCPA(500nmol/L) and mitoKATP inhibitor 5-hydroxydecanoate (5-HD, 500μmol/L), and group E, cells under hypoxia treated with CCPA(500nmol/L) and PKC inhibitor chelerythine(CHE, 2μmol/L).3. The opening of MPTP was observed by coloading with Calcein-AM and cobalt chloride; the mitochondrial membrane potential was detected by TMRE; the changes of ROS and the viability of cardiocytes were determined by using fluorescent probe DCFH-DA and CCK-8, separately.4. The expression of PKC were determined by immunofluorescence and western blotting (WB).Results:1. The expression of PKC was detected in a small amount and mainly existed in cytoplasm in both normoxic group and hypoxic group. However, the expressions of PKC in total protein increased significantly, especially obvious in cytomembrane in groups hypoxia+CCPA and hypoxia+CCPA+ 5-HD.2. The viability of cardiocytes decreased remarkably in hypoxic group, while this change did not arise when cells were treated with hypoxia and CCPA together. However, both PKC and mitoKATP inhibitors abolished this protective role of CCPA in hypoxic cardiocytes.3. Compared with normoxic group, the fluorescence intensity decreased significantly in hypoxic group, which reflects obvious opening of MPTP. The A1R agonist CCPA decreased the opening of MPTP induced by hypoxia notably. But both PKC and mitoKATP inhibitors abolished this protective role of CCPA in hypoxic cardiocytes.4. Compared with normoxic group, concentration of ROS increased and mitochondrial membrane potential declined in hypoxic cardiocytes. But the mitochondrial membrane potential and concentration of ROS in cells treated with hypoxia and CCPA did not change. However, both PKC and mitoKATP inhibitors abolished this role of CCPA.Discussion and Conclusions1. The A1R agonist CCPA up-regulation of PKC expression in total protein. PKC activation might not depend on being translocated to cellular membrane, and possibly it might rely on being translocated to membranes of some organells.2. In CCPA signal transduction of cardiocytes, mitoKATP inhibitor could not block the influence of CCPA on PKC, suggesting that PKC might be in the upstream of mitoKATP in the signal transduction process.3. CCPA might reduce the opening of MPTP induced by hypoxia, and this effect might be controlled by PKC—mitoKATP signal pathway.4. The mechanism that mitoKATP influenced the opening of MPTP might be through maintaining mitochondrial membrane potential and reducing the augment of ROS after hypoxia.5. A1R agonist (CCPA) could decrease the opening of MPTP in hypoxic cardiocytes, the mechanism of this protection role was from PKC—mitoKATP to MPTP further. And it provided us a new way to prevention and treatment of post-burn"Shock Heart".6. The results of the present study demonstrated that CCPA may reduce the opening of MPTP induced by hypoxia. The mechanism for CCPA to modulate opening of MPTP in hypoxic cardiocytes might be: CCPA activates PKC through combination with A1 receptor on the membrane, and the activated PKC promotes the opening of mitoKATP, resulting in maintaining the mitochondrial membrane potential and reducing ROS production, and further reducing the the opening of MPTP in hypoxic cardiocytes.