Effects of protein deprivation and re-feeding on P2X_2 receptors in enteric neurons

AIM:To investigate the effects of malnutrition and refeeding on the P2X2 receptor,nitric oxide synthase(NOS),calretinin,calbindin and choline acetyltransferase(ChAT) in neurons of the rat ileum.METHODS:We analyzed the co-localization,numbers and sizes of P2X2-expressing neurons in relation to NOS-immunoreactive(IR),calbindin-IR,ChAT-IR,and calretinin-IR neurons of the myenteric and submucosal plexus.The experimental groups consisted of:(1) rats maintained on normal feed throughout pregnancy until 42 d post-parturition(N);(2) rats deprived of protein throughout pregnancy and 42 d post-parturition(D);and(3) rats undernourished for 21 d post-parturition and then given a protein diet from days 22 to 42(DR).The myenteric and submucosal plexuses were evaluated by double labeling by immunohistochemical methods for P2X2 receptor,NOS,ChAT,calbindin and calretinin.RESULTS:We found similar P2X2 receptor immunoreactivity in the cytoplasm and surface membranes of myenteric and submucosal neurons from the N,D and DR groups.Double labeling of the myenteric plexus demonstrated that approximately 100% of NOS-IR,calbindin-IR,calretinin-IR and ChAT-IR neurons in all groups also expressed the P2X2 receptor.In the submucosal plexus,the calretinin-IR,ChAT-IR and calbindin-IR neurons were nearly all immunoreactive for the P2X2 receptor.In the myenteric plexus,there was a 19% increase in numbers per cm2 for P2X2 receptor-IR neurons,64% for NOS-IR,84% for calretinin-IR and 26% for ChAT-IR neurons in the D group.The spatial density of calbindin-IR neurons,however,did not differ among the three groups.The submucosal neuronal density increased for calbindin-IR,calretinin-IR and ChAT-IR neurons.The average size of neurons in the myenteric plexus neurons in the D group was less than that in the controls and,in the re-fed rats;there was a 34% reduction in size only for the calretinin-IR neurons.CONCLUSION:This work demonstrates that expression of the P2X2 receptor is present in inhibitory,intrinsic primary afferent,cholinergic secretomotor and vasomotor neurons.Undernutrition affected P2X2 receptor expression in the submucosal plexus,and neuronal and size.These changes were rescued in the re-fed rats.