Endocrine Abstracts

The oxytocin (OT) treatment protects heart against ischemia. Here we investigated heart-derived H9c2 cells in simulated ischemia-reperfusion (I-R) experiments in order to examine the mechanism of OT-induced cardioprotection.

Results: I-R was induced in an anoxic chamber for 2 h and followed by 2 h of reperfusion. In comparison to normoxia, I-R resulted in decrease of formazan production by H9c2 cells to 63.5±1.7% (MTT assay) and enhanced apoptosis from 1.7±0.3% to 2.8±0.4% (Tunel test). Using these assays we found that treatment with OT (1–500 nM) exerted dose-dependent protection during I-R, especially when OT was added at the time of reperfusion. This involved OT receptor (OTR) because the cells exposed to I-R and treated with specific OTR siRNA responded to OT by enhanced apoptosis. The OT stimulation causes intracellular signaling involving Pi3K, Akt and eNOS, known as the canonical factors of signalosomes. Confocal microscopy demonstrated in OT-treated cells, the phosphorylated Akt co-localized with the mitochondrial marker Cox IV. In addition, the NOS dissociation from caveolin-3 and eNOS phosphorylation was accompanied by increased production of NO. The increased cell viability induced by OT was abolished by the co-treatment of the cells with a PKG inhibitor, KT-5823. Furthermore, stimulation with OT resulted in enhanced release of atrial natriuretic peptide. Using the CM-H₂DCFDA probe we have also observed the paradox that OT treatment stimulates moderate levels of reactive oxygen species (ROS) production in cells whereas inhibits excess of ROS produced as a consequence of ischemia evoked by I-R.

Conclusion: The OTR protected H9c2 cells against I-R, especially if activated at the onset of reperfusion. The OTR-transduced signals include Akt and PKG. These kinases translocate to the mitochondria, where they act in a localized signalosome involving activation of ROS in a positive feedback loop.