CGRP and its receptor, CLR•RAMP1, have been previously localized in the central and peripheral nervous systems, and are involved in the regulation of diverse physiologic effects, including nociception, secretion, and smooth muscle relaxation [10
]. CGRP has a prominent role in the enteric nervous system as well, and animal studies have suggested a role for the peptide in modulation of intestinal neurotransmission, motility and secretion [12
]. Given that the distribution of CLR and RAMP1 in human enteric tissue had yet to be characterized, the primary objectives of this study was to confirm the presence of CLR and RAMP1 within the human GI tract and determine its subcellular and tissue distribution together with its ligands, CGRP and IMD.
We show that CLR and RAMP1 are widely expressed in normal human GI tissue, with mRNA transcripts amplified from stomach, ileum, and colonic tissue by RT-PCR (). Immunohistochemical analysis of human GI tissues using immunofluorescence and confocal microscopy showed that CLR is expressed within the human stomach, ileum, and colon. In agreement with our observation, an earlier study reported presence of CLR in human stomach and also in other peripheral tissues such as the heart, lung, pancreas, and testis [13
]. Within the stomach, prominent CLR-IR was observed in the cytoplasm of the fundic chief cells, and sporadic CLR-IR was found within the epithelium and myenteric plexus of enteric tissue [13
]. However, no study has localized RAMP1 or co-localized CLR and RAMP1 in human gut. In this study, we found prominent CLR- and RAMP1- IR within nerve bundles in the myenteric plexus, as well as nerve fibers throughout the circular and longitudinal muscle in the stomach ( and ). The differences observed in CLR-IR in the stomach between our study and that previously reported [13
], may be due to the different antigenic sequences used to generate the antibodies. Hagner et al [13
] raised an antibody to the C-terminus of human CLR (455
), whereas our antibody was raised to the C-terminus of rat CLR (455
]. These are 6 substitutions between these sequences, which may account for the discrepancies observed in the staining. Another explanation could be the differences in the immunohistochemical staining procedures.
In the ileum and proximal colon, CLR is found in the myenteric plexus and surrounding submucosal neurons (). Similarly, RAMP1-IR is found in neurons in the myenteric plexus (). The localization of CLR and RAMP1 in the myenteric plexus demonstrates that the two subunits of the receptor are in proximity and can potentially heterodimerize to form a functional CGRP receptor where it may respond to locally secreted CGRP in the human GI tract.
When we localized human tissues for CLR and the potential agonists, CGRP and IMD, we observed no co-localization of CLR with CGRP or IMD. However, we did observe CLR-IR and CGRP- and IMD-IR in very close proximity in nerve fibers in the circular and longitudinal muscle layers of the stomach, ileum, and colon (). The results suggest that CGRP and IMD are appropriately localized with CLR to function as ligands for CLR•RAMP complexes and to exert biologic effects under appropriate stimulation. However, there is no evidence that CLR•RAMP may be autoreceptors for CGRP and IMD in the tissues we examined. It is also possible that other RAMP proteins, namely RAMP2 and 3 may also be present in the human gut and may interact with CLR to form a higher affinity receptor for IMD or indeed adrenomedullin. In support of this, mRNA transcripts for RAMP2 and RAMP3 were amplified from rat duodenum, jejunum, ileum, and proximal and distal colon [6
]. Further, a recent study has shown that when co-expressed with RAMP3, CLR interacts with IMD and adrenomedullin with similar affinities, that are up to two orders of magnitude greater than CGRP [15
]. Thus, agonists may be secreted in an autocrine manner to act on their receptors to mediate their effects in the gut.
Previous studies have demonstrated the effects of CGRP and IMD on gastrointestinal secretion and motility. Enteric neurons containing CGRP have long been implicated in mediating gut vasodilatation [18
] and motility [4
]. Rodent studies have identified CGRP secretion after mechanical stimulation of the mucosa, and coordinated ascending contraction and descending relaxation is suppressed after application of CLR-RAMP1 antagonists [10
]. Likewise, rodent studies with IMD demonstrated its role in suppressing gastric emptying and food intake [28
]. Together, these data corroborate the notion that CGRP and IMD play a prominent role in normal human gut physiology. Functional studies in human tissue are a logical progression of these earlier findings, which is now facilitated by the localization of CLR•RAMP1, CGRP, and IMD within the human gastrointestinal tract. The co-localization of the subunits of the CGRP receptor suggests a functional role in tissue that is punctuated by the receptor’s close tissue (cellular) association with the agonists CGRP and IMD. Future studies will attempt to characterize receptor activity and function in human cells and tissue to ascertain the role of CLR•RAMP1, CGRP and IMD in human enteric physiology and disease.