Calcitonin gene-related peptide (CGRP) is a potent neuropeptide important for gut motility, vasodilation, and immune responses. There are two forms of CGRP, a and b, that differ by 3 amino acids and are encoded by distinct genes. aCGRP, encoded by Calca, predominates in most of the nervous system and has been linked to many biological functions, including migraine and neurogenic inflammation. bCGRP, encoded by Calcb, predominates in the enteric nervous system (ENS), but its functional significance is unclear. To determine how bCGRP regulates gut motility in vivo, we studied Calcb^GFP/GFP (Calcb-null) mice. Total gastrointestinal transit time and fecal pellet output were completely normal in these Calcb-null mice despite loss of the major ENS form of CGRP. Even more surprisingly, intestinal CGRP levels measured by ELISA were the same in Calcb-null mice and wildtype littermates, suggesting that either the ENS makes a minimal contribution to gut CGRP or that aCGRP is upregulated to compensate for bCGRP loss. To distinguish these possibilities, we generated Calcb^CreER/+; ROSA26^DTA/+ mice in which bCGRP-expressing neurons, most of which are intrinsic primary afferent neurons (IPANs), could be genetically ablated. Depletion of bCGRP neurons in adult mice caused a dramatic loss of intestinal CGRP. In contrast, chemical denervation of aCGRP-expressing spinal afferent neurons in dorsal root ganglia (DRG), including those that innervate the gut, did not have a similar effect. These data indicate that the ENS is the major source of intestinal CGRP and can compensate for bCGRP loss. Consistently, Calca transcript levels in the small intestine were low at baseline and upregulated by Calcb loss in a gene dose-dependent manner. Conversely, Calcb levels were high in the ENS at baseline and unchanged by Calca loss. This reciprocal regulation was not observed in DRG where Calca was highly expressed at baseline with no induction of Calcb expression upon Calca loss. To identify the cells that upregulate Calca when Calcbis lost, we used fluorescent in situ hybridization to label Calca transcripts in the intestines of Calcb^GFP/GFP (Calcb-null) mice. Calca expression was evident within GFP⁺ cells that were immunoreactive for CGRP, suggesting that IPANs sense and respond to intestinal CGRP deficiency by upregulating Calca. IPANs express the CGRP receptor complex and pharmacological blockade of CGRP was sufficient to provoke upregulation of Calca within the gut. Taken together, these data indicate that IPANs in the ENS can respond to intestinal CGRP levels through autocrine signaling. This novel mechanism of CGRP regulation is unique to the gut and has implications for understanding the adverse GI effects of the many drugs targeting CGRP signaling for migraine relief.

Kathryn Grabowski

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