CaMKII-Mediated Upregulation of SerP1303 NR2B Phosphorylation in Rostral Cingulated Cortex in Response to Acute Esophageal Acid Exposure in Rats

Abstract

Phosphorylation of NMDA receptors regulates many cellular processes including surface expression and protein activity resulting in changes in synaptic strength underlying many forms of synaptic plasticity. Several kinases such as protein kinase C(PKC), calcium/calmodulin kinase II (CaMKII),and protein tyrosine kinases phosphorylate various serine/threonine and tyrosine residues at the C-terminal ends of NR2 subunits of NMDA receptor. AIMS: To examine the effect of esophageal acid stimulation on NMDA receptor subunit phosphorylation in rat rostral cingulate cortex (rCC). Methods: Twelve Sprague Dawley rats were anesthetized (Nembutal, 30 mg/Kg, IP) and their mid esophagus were perfused with either 0.1N HCl (n=6) or saline (n=6) for 20 min at 0.1 ml/min. Four hours later the rat`s brains were removed and sliced sagitally at 2 mm from midline. The cingulate cortex obtained from the first slice on either side of the midline was transected rostrocaudally and the rCCwas analyzed usingWestern Blots. To investigate the involvement of CaMKII inNR2B phosphorylation, KN93, a specific inhibitor of CaMKII was microinjected into rCC 30 min before esophageal acid or saline infusion. The expression of CaMKII and activated phospho CaMKII (pCaMKII) were also examined in the rCC from acid and saline-treated animals. The intensity of expression was normalized against β-actin and expressed as mean ± SD. RESULTS: Following acute esophageal acid treatment a significant increase in SerP1303 NR2B was observed in rCC compared to saline-treated animals (p<0.001). Tyrosine phosphorylated NR2B subunits (TyrP1252, Tyr1336 and Tyr1472) failed to demonstrate significant differences in expression between acid and saline-treated animals. KN-93 treatment resulted in significant downregulation of SerP1303 NR2B in acid treated group (p<0.05 vs vehicles). A significant increase in pCaMKII, but not total CaMKII was observed in rCC from acid-treated animals (p<0.05 vs saline controls). Conclusions: our results indicate the involvement of CaMKII-mediated NR2B phosphorylation in esophageal acid-induced neuronal plasticity in the rostral cingulate cortex (rCC). This effect is blocked by specific CaMKII inhibitor KN-93. These findings may have ramifications in acid related symptom control.