Immunohistochemical Localization of the Melanocortin-4 Receptor (MC4R) in the Colon Myenteric Plexus

Abstract

Alpha-melanocyte stimulating hormone (α-MSH) is a tricdecaneuropeptide that can bind four melanocortin receptors (MC1R, MC3R-MC5R) to exert various receptor-dependent effects. MC4R regulates several functions which include energy homeostasis, enhancement of sexual function, blood pressure, and heart rate, and suppression of inflammation. Although MC4R is predominantly expressed in the central nervous system (CNS), peripheral expression was also observed in the enteric nervous system (ENS) of the stomach and duodenum. α-MSH exerts anti-inflammatory effects via MC4R in the brain and attenuates experimental-induced inflammatory bowel disease (IBD) in the colon by an unknown mechanism. We asked if ENS MC4R expression extends to the colon and if treatment with Melanotan II (MTII), an α-MSH analog, or leptin, stimulates increased MC4R expression. We used reverse-transcriptase polymerase chain reaction (RT-PCR) to detect MC4R mRNA in the rat colon and immunohistochemistry (IHC) to determine cell types that express MC4R. We focused on enteric glial cells and myenteric neurons using floating sections of longitudinal muscle- myenteric plexus (LMMP) and formalin-fixed, paraffin-embedded rat colon cross-sections. RT- PCR showed MC4R mRNA expression in the colon and IHC localized MC4R to the myenteric plexus. Likewise, a co-localization assay showed that the MC4R-positive cells were also positive for neuron-specific beta III tubulin. In enteric glial cells, MC4R was co-localized with glial fibrillary acidic protein (GFAP), a glial cell marker. There was no significant difference in MC4R expression among MTII, leptin, or vehicle treatment groups.