The present study provides the first evidence for long-term changes in NMDA receptor subunits composition and postsynaptic membrane protein expressions in rCCs from rats receiving esophageal acid exposure during critical periods of development. Interestingly, adult rats receiving chronic esophageal acid exposure failed to exhibit significant changes in the receptor subunit expression compare to saline-treated control. Moreover, molecular changes in rCCs from neonatal acid-treated rats were significantly higher that with neonatally handled and saline-treated, emphasizing thereby the specificity of the response. These findings indicate that the long lasting effect of acid when given early during development may be sufficient to induce discernible molecular changes in adulthood. In humans, both clinical and experimental data support early childhood as a critical time period in which trauma can induce visceral hypersensitivity and manifestation of functional disorders 22
In the brain, glutamate receptors including NMDA mediate most of the excitatory neuronal transmission and play essential role in the regulation of synaptic activity 23, 24
. The unique feature of NMDA receptors is that the receptor activation requires the binding of a co-agonist glycine in addition to glutamate 25
. Therefore, a functional NMDA receptor requires both an NR1 subunit, which has the glycine binding site, and an NR2 subunit for binding to glutamate. Among NR2 subunits, the expression patterns of NR2A and NR2B are relatively broad and both are developmentally regulated, with concurrent increase in NR2A expression and decrease in NR2B expression as neuron matures. With this rationale, in the present study we examined the expression patterns of NR1, NR2A and NR2B after infusion of acid at early stage of life as well as in the adulthood. Acute esophageal acid exposure in adult rats resulted in a transient increase in NR1 and NR2A expression in the rCC, whereas, neonatally acid expose rats receiving acute rechallenge in adulthood (P60) exhibited the highest level of NR1 and NR2A expressions indicating their enhanced susceptibility to esophageal acid exposure. Our present findings are in agreement with the previous study that reported a long-term alteration in NDMA receptor subunit mRNA from the hippocampus and cortex of rats treated with a single exposure of LPS1
We did not observe any change in NR2B following acid exposure. This result is in contrast with most of the previous studies that report upregulation of NR2B subunit in supraspinal regions under various pathological conditions 16, 26, 27
. The differential subunit expression pattern of NMDA receptor is an important factor in regulating NMDAR-dependent function and neuronal plasticity. For example, rapid calcium-mediated signaling through NR1/NR2A in contrast to slower signaling through NR1/NR2B may activate different downstream signaling and gene expression pattern. Moreover, synaptic NMDA receptor levels are not only regulated by the type of NR2 subunits, but also by lateral movement of extrasynaptic receptors in and out of the synapses 28, 29
. In this context, NR2A-subunit receptors are fairly stable in the synapse, whereas, NR2B-subunit receptors are highly mobile, 30
. Therefore, increase in cortical NR2A subunit in the present study may result in faster neuronal transmission and stable NMDA receptor expression in the synapses with a distinct downstream signaling pathway during esophageal acid- induced hypersensitivity.
NMDA receptors are reported to anchor in the postsynaptic membrane by interactions between cytoplasmic C-terminal ends of their NR2 subunits and PDZ domains of PSD-95, an abundant scaffold protein that assembles a specific set of signaling proteins around NMDA receptors 31, 32
. Recent study indicates that NMDA receptor and PSD-95 interaction may have a role in the processing of spinal nociceptive information 33
. Furthermore, PSD-95 knockdown shows a delay in the onset of mechanical and thermal hyperalgesia in chronic neuropathic pain model, indicating an involvement of this protein in NMDA receptors-mediated thermal hyperalgesia 34
. In the present study, although acute acid treatment showed an increase in PSD-95 protein expression in the rCC, the maximum expression was observed in neonatal acid and rechallenge group followed by only neonatally treated group. Therefore, the long-term effect and memory of neonatal treatment on PSD-95 expression in the rCC indicates effective surface expression and clustering of NMDA receptors at the synapses that eventually may play an important role in NMDA receptor-dependent function and neuronal plasticity.
The phosphorylation of NMDA receptor subunit regulates many cellular processes including surface expression and protein activity resulting in changes in synaptic strength underlying many forms of synaptic plasticity 35, 36
. Several kinases such as protein kinase C (PKC), calcium/calmodulin kinase II (CaMKII), protein tyrosine kinases are reported to phosphorylate various serine/threonine and tyrosine residues at the C-terminal ends of NR2 subunits of NMDA receptors 37
In order to study the effect of esophageal acid exposure on cortical NMDA receptor activation, we examined the phosphorylation pattern of C-terminal amino acids of NR2B subunits at Ser1303, Tyr1336, Tyr1472 and Tyr1252. The effect of esophageal acid exposure on NMDA receptor activation is evident in our present findings, as a significant upregulation of Ser1303 NR2B phosphorylation is observed in rCCs from rats receiving acute esophageal acid treatment at P60 and also the group with neonatal acid treatment followed by an acute exposure at P60. Interestingly, rats with only neonatal acid treatment failed to exhibit upregulation of SerP1303 NR2B, indicating this specific phosphorylation is an immediate effect of esophageal acid exposure.
We further confirmed the involvement of CaMKII in acid-induced Ser1303 NR2B phosphorylation in the rCC as microinjection of CaMKII inhibitor KN-93 in the rCC resulted in a significant reduction of SerP1303 NR2B in animals receiving acute esophageal acids exposure. KN-93 is reported to bind specifically to the CaM binding site of CAMKII and prevents its activation 38
. Recent study indicates that the phosphorylation of NMDA receptors by CaMKII enhances influx of Ca2+
through the channels 39
. CaMKII up-regulation has also been reported in the superficial laminae of the dorsal horn and DRGs after inflammation or injuries to peripheral tissues 40–42
Moreover, phosphorylation of Ser1303 NR2B by CaMKII also promotes slow dissociation of preformed CaMKII-NR2B complexes and stabilizes the receptor-enzyme in the membrane 43
Given that after Ca2+
influx through NMDA receptors, activation of CaMKII results in long-term potentiation in the hippocampus 44, 45
, our results indicate that central sensitization through cortical activation and phoshorylation of NMDA receptors could initiate a variety of intracellular processes leading to neuronal changes by activating second/third messenger systems. CaMKII being a major component of PSDs, phosphorylation and stabilization of NMDA receptor-CaMKII complex in the PSD may in turn activate multiple proteins and enzymes, such as neuronal proteins, Ca2+-
ATPase and tyrosine hydroxylate and transcription factor cAMP-responsive-element-binding protein (CREB) 46–49
We examined the distribution pattern of NMDA receptor subunits and PSD-95 in synaptic and extrasynaptic membranes in the rCCs from naïve rat. The enrichment of NMDA receptor subunits along with PSD-95 protein in the synaptic membrane preparation indicates that NMDA receptors anchoring in the cortical synapses probably is mediated by interaction between the C-terminal end of NR2 subunit with PDZ domain of PSD-95 protein of the postsynaptic membranes. We further investigated whether the acid-induced molecular changes in the rCC is mainly due to increase in NMDA receptors in the post synaptic membrane via its binding with PSD-95. The cortical membrane extracts from saline and acid-treated rats were immunoprecipitated using PSD-95 antibody. PSD-95 immunoprecipitated fractions from acid-treated rats exhibited a significantly higher expression of NR2A, and importantly SerP1303 phosphorylated NR2B subunit compared to controls. These findings clearly indicate that the NMDA receptor upregulation and phosphorylation are predominantly occurring at the postsynaptic membranes in the rCC and may be involved in synaptic transmission and increased neuronal activity in acid-induced esophageal hypersensitivity.
In conclusion, results demonstrate a long lasting change in NMDA receptors expression in neurons of the rCC following neonatal esophageal acid exposure. Further study of the complex interaction between various downstream signaling pathways may provide a better understanding for the neuronal plasticity during the development and its influence on esophageal pain mechanism of NERD and NCCP patients. We acknowledge that NMDA receptors in multifunctional brain region like CC can mediate many different functions including chronic pain. Currently, there is no quantifiable, reliable and reproducible behavioral model in experimental animals to complement the results of the present study. Therefore, in the absence of concurrent behavioral study our findings can only suggest that such molecular changes play a possible role in chronic esophageal pain.