Fig. 1.

Calcium-sensing receptor (CaSR) is highly expressed in the enteric nervous system (ENS) of the colon. Shown are representative images of single (A–F) or double (G–L) labeling immunofluorescence studies. Rat distal colon tissues were stained with anti-CaSR (red, A–D), anti-class III β-tubulin (green, E and F), or both (G–L). Note that intense CaSR staining is evident in the nerve fibers and neurite projections (white arrowheads) and somas (white arrows) of the submucosal Meissner's plexus (A) and the myenteric Auerbach's plexus (B), the pattern similar to that of class III β-tubulin (green, E and F). Consistent with our previous study (11), labeling on apical and basolateral membranes of the crypt epithelium was also noted (C). No signal similar to the CaSR was seen when the CaSR primary antibody was preabsorbed with an excess amount (1 μg/ml) of the immunizing peptide (D). Note that the large red fluorescent structures noted in the mucosa layer (orange arrows, D; also seen in C) were interstitial cells that exhibit autofluorescence; these latter were also seen in F (orange arrows), in which the CaSR primary and secondary antibodies were absent. G–L: representative immunofluorescence images with double labeling. Rat distal colon tissues were probed with both anti-CaSR and anti-class III β-tubulin as described in materials and methods. G and J: staining of nerve fibers and somas of the ENS was observed with anti-CaSR (red). H and K: staining against class III β-tubulin to identify neural tissues (green). I and L: merged images with labeling against CaSR (red) and the class III β-tubulin (green) demonstrating expression of both proteins in the same neutral tissues (yellow), including the nerve terminals innervating the crypt epithelium (L, white arrowheads). Note that, although labeling overlap is seen in most neuronal tissues, structures labeled by anti-class III β-tubulin alone but not by anti-CaSR are also noted (I and L, green arrowheads), suggesting that CaSR is present in most, but not all, neuronal tissues of the ENS. A similar labeling pattern of CaSR was noted in proximal colon (not shown).