Acute cardiac injury induces glial cell response and activates extracellular signaling-regulated kinase-1 and -2 in the spinal cord of Wistar rats.

We have examined the activation of glial cells and the upregulation of phosphorylated extracellular signaling-regulated kinase (ERK)-1 and -2 in upper thoracic segments of the spinal cord in rats following acute cardiac injury (ACI). ACI was established by intramyocardial injection of formalin and confirmed by hematoxylin and eosin (H&E) and terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. Following ACI, the astrocytes (determined by glial fibrillary acidic protein (GFAP) immunoreactivity (-IR)) and microglia (determined by OX-42-IR) were activated within the thoracic spinal cord. Phosphorylated (phospho-) ERK-IR was also activated in response to ACI. The upregulation of phospho-ERK was observed at 1h and became very obvious at 6h following ACI. The upregulated phospho-ERK was evidently expressed in the superficial and deep dorsal horn of the thoracic spinal cord. The activated ERK was also expressed in the intermediolateral nucleus (IML), nucleus intercalatus (IC) and the long processes projecting to the central canal, regions closely associated with autonomic outflow. Thus, the present study suggested that ACI could induce the activation of spinal ERK, which might link the nociceptive processing with the spinal sympathetic reflexes in myocardial injury in clinics.

NMDA induces NOS 1 translocation to the cell membrane in NGF-differentiated PC 12 cells.

Glutamatergic-mediated nitric oxide (NO) production occurs via the N-methyl-D-aspartic acid (NMDA) postsynaptic density protein 95 (PSD95)-neuronal nitric oxide synthase (NOS1) ternary complex. To determine whether NOS1 is targeted to the membrane subsequent to NMDA receptor activation, we examined the effect of NMDA on NOS1 subcellular localization in nerve growth factor (NGF) differentiated PC12 cells. No effect on cell viability was observed using a range of NMDA concentrations from 500 to 1000 microM. Within 3 min of stimulation with 750 microM NMDA, increased cytoplasmic NOS1 immunostaining was observed with rapid membrane staining thereafter. This was inhibited by NMDAR inhibition with MK801. This observation was confirmed using subcellular fractionation and immunoblotting. Using 4, 5-diaminofluorescein diacetate (DAF2-DA) staining and a diazotization assay, concurrent NO production was observed. When PC 12 cells were co-treated with either NMDA and N(6)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or (5R, 10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a, d] cyclohepten-5, 10-imine hydrogen maleate (MK-801), nitric oxide (NO) generation was inhibited. Stimulation in a calcium-free medium did not increase NO levels. Although no evidence of cytotoxicity was observed utilizing either the MTT assay or measures of apoptosis within the maximal interval of NOS1 translocation, cell viability was reduced following 10 h of continuous NMDA exposure. While it has been shown that NMDA triggers NOS1 activation, these results indicate that NMDAR activation also mediates NOS1 targeting to the membrane. Our data validate that NGF-differentiated PC12 cells may be employed as a useful in vitro model to further study the regulation of NOS1 subsequent to NMDAR activation.