THR286 PhosphorylatedaCaMKII in Anterior Cingulate Cortex Mediates Visceral Pain in Visceral Hypersensitive Rats

Hypersensitivity to visceral distension has been reported in patients with functional bowel disease such as irritable bowel syndrome (IBS). Recently, we used a viscerally hypersensitive (VH) rat mole, in which the enhanced pain responses to colorectal distention (CRD) can be observed even up to 7 weeks following the initiation of colonic anaphylaxis and appears to be independent of mucosal inflammation. Sensitization of perigenual ACC (pACC) neurons occur as a result of alteration of activity-dependent plasticity (long-term potentiation, LTP). The NR2B subunit of the NMDA receptor in the pACC plays a critical role in the modulation of ACC sensitization and pain responses. However, the cellular and molecular mechanisms responsible for this process are unknown.Given the key role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in synaptic plasticity and behavior learning and memory, and its high-affinity binding to the NR2B subunit, we therefore hypothesize that the phosphorylation of CaMKII which binds to NR2B subunits mediates the biochemical “memory” process and modulates visceral pain in VH states.To test this hypothesis, electroporation of siRNA in the pACC to silenceaCaMKII, the ACC neuronal responses and the visceral pain responses to CRD in normal and VH rats will be examined. We will perform biochemical fractionation of the cellular components of pACC and examine the changes of postsynaptic expression of CaMKII and NMDA receptors, and characterize the interaction between these two molecules.aCaMKII is autophosphorylated at Thr286, causing the enzyme to remain active independent of Ca2+/CaM binding. We will characterize the postsynaptic translocation of p-T286 CaMKII, and the enhanced binding with NR2B in the pACC of VH rats. Further, we will demonstrate that inhibition of autophosphorylation at Thr286 of CaMKII in ACC by target applying inhibitory peptide (CaMKII 281-302) reduces the translocation of phosphorylated CaMKII and its binding to NR2B subunit. Inhibitory peptide also prevents the development of visceral hypersensitivity induced by colonic anaphylaxis. In the last phase of our experiment, we will focus on genetic manipulation of rat pACC neurogenic niches to facilitate long term potentiation in the ACC neuronal circuitry, and induce visceral pain in normal rats, by substitution of the threonine autophosphorylation site with aspartate (aCaMKII T286D).Understanding the cellular and molecular mechanisms underlying the neuronal plasticity of the pACC and chronic visceral pain memory may prove vital to our understanding of the etiology of functional gastrointestinal disorders, and to identify potential therapeutic targets in the brain cortex for human functional bowel diseases.