Abstract TP244: The Role Of Smooth Muscle Cyclic GMP-dependent Protein Kinase 2 In Ischemic-reperfusion Injury

Abstract

Background and Rationale: Cyclic GMP-dependent protein kinases (cGK1/2) act as a key mediator of the nitric oxide/cGMP signaling pathway in stroke. The cGK1/2 seems to function as a dimer under physiological conditions. Its potential mechanism in ischemia-reperfusion injury has not been investigated sufficiently. Here, we used smooth muscle cell-specific cGK1 knock-out (cGK1 KO) mice to investigate the variability of cGK1/2 in the ischemic stroke and expected dipyridamole, a non-selective phosphodiesterase inhibitor, will affect vasodilation via eNOS/cGMP/cGK1/2 signaling pathways in ischemic stroke. Methods: Mice were subjected to diverse episodes of ischemia-reperfusion injury. Laser Doppler measurements indicated that the relative cerebral blood flow values significantly decreased in cGK1 KO mice on reperfusion. Infarction volume was measured by TTC staining and relative cerebral blood flow was measured by Laser Doppler. We investigated the neurological deficit score at 23h after a 60-minute MCAO. Dipyridamole (60 mg/kg) was orally administered by gavage for 3 consecutive days. Protein levels from brain lysates in the cerebral cortex and hippocampus were studied by western blotting. To assess the time-kinetics of cGK1/2, brain samples were divided into ischemia-reperfusion periods. Neurological deficit and infarction volume were determined 24h later. Results: Dipyridamole reduced the infarction volume, improved neurological deficit score, and increased rCBF in control mice but the effect was absent in cGK1 KO mice. Basically, protein levels of both cGK1 and cGK2 in permanent MCAO brain samples were degraded during an ischemic condition time-dependent manner (1~3h). Both cGK1 and cGK2 in whole brain lysates were sustained without protein degradation during ischemia-reperfusion injury in littermate control. The protein expressions of cGK2 in both blood vessels and brains with dipyridamole were rapidly reversed on reperfusion onset in cGK1 KO mice. Conclusion: These results suggest that cGK1/2 should be balanced by eNOS/cGMP/cGK1/2 signaling pathways in the ischemic brain and the effect of both cGMP and cGK2 upregulation by dipyridamole mediating the neuroprotection in ischemic brains.