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. 2012 Jul;303(1):G60-70.
doi: 10.1152/ajpgi.00425.2011. Epub 2012 Apr 19.

Calcium-sensing receptor inhibits secretagogue-induced electrolyte secretion by intestine via the enteric nervous system

Sam X Cheng  1
Affiliations

Calcium-sensing receptor inhibits secretagogue-induced electrolyte secretion by intestine via the enteric nervous system

Sam X Cheng. Am J Physiol Gastrointest Liver Physiol. 2012 Jul.

Abstract

Bacterial toxins such as cholera toxin induce diarrhea by both direct epithelial cell generation of cyclic nucleotides as well as stimulation of the enteric nervous system (ENS). Agonists of the extracellular calcium-sensing receptor (CaSR) can reduce toxin-stimulated fluid secretion in ENS-absent colonic epithelial crypts by increasing phosphodiesterase-dependent cyclic-nucleotide degradation. Here we show that the CaSR is also highly expressed in tetrodotoxin (TTX)-sensitive neurons comprising the ENS, suggesting that CaSR agonists might also function through neuronal pathways. To test this hypothesis, rat colon segments containing intact ENS were isolated and mounted on Ussing chambers. Basal and cyclic nucleotide-stimulated electrolyte secretions were monitored by measuring changes in short-circuit current (I(sc)). CaSR was activated by R-568 and its effects were compared in the presence and absence of TTX. Consistent with active regulation of anion secretion by the ENS, a significant proportion of I(sc) in the proximal and distal colon was inhibited by serosal TTX, both at basal and under cyclic AMP-stimulated conditions. In the absence of TTX, activation of CaSR with R-568 significantly reduced basal I(sc) and cyclic AMP-stimulated I(sc); it also completely reversed the cAMP-stimulated secretory responses if the drug was applied after the forskolin stimulation. Such inhibitory effects of R-568 were either absent or significantly reduced when serosal TTX was present, suggesting that this agonist exerts its antisecretory effect on the intestine by inhibiting ENS. The present results suggest a new model for regulating intestinal fluid transport in which neuronal and nonneuronal secretagogue actions are modulated by the inhibitory effects of CaSR on the ENS. The ability of a CaSR agonist to reduce secretagogue-stimulated Cl(-) secretion might provide a new therapeutic approach for secretory and other ENS-mediated diarrheal conditions.

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Figures

Fig. 1.
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).
Fig. 2.
Fig. 2.
Inhibition of ENS by TTX reduces basal short-circuit current (Isc) in the colon. A: representative recording of Isc responses in a rat distal colon to addition of 2 μM TTX to serosal solution. B: quantitation of Isc responses to TTX in proximal and distal colons.
Fig. 3.
Fig. 3.
Inhibition of ENS by TTX reduces cAMP-stimulated Isc in the colon. A: representative recording of Isc responses to forskolin (FSK) and bumetanide (Bumet) in a rat distal colon segment. Following additions of 500 nM forskolin to mucosal and serosal solutions, 100 μM of bumetanide was added to serosal solution to inhibit the Na+/K+/2Cl− cotransporter, as indicated. B: quantitation of forskolin-stimulated Isc responses in proximal and distal colons. C: representative tracing of Isc responses in a rat distal colon segment to TTX under forskolin-stimulated condition. Following additions of 500 nM forskolin to mucosal and serosal solutions, 2 μM TTX was added to serosal solution, as indicated. D: quantitation of Isc responses to TTX under forskolin-stimulated condition in the proximal and distal colons.
Fig. 4.
Fig. 4.
Activation of CaSR by R-568 abolishes the effect of TTX to reduce basal Isc in the colon. A: representative recording of Isc responses in a rat distal colon segment to sequential additions of R-568, TTX, and bumetanide. All drugs were added to serosal solution with the concentrations indicated. B: quantitative changes in Isc following treatment with R-568 ± TTX ± bumetanide in the proximal and distal colons. NS, not significant. C: representative recording of Isc responses in a rat distal colon segment to sequential additions of TTX, R-568, and bumetanide. All drugs were added to serosal solution with the concentrations indicated. D: quantitative changes in Isc following treatment with TTX ± R-568 ± bumetanide in the proximal and distal colons. E summarizes the ΔIsc induced by R-568 in the absence and presence of TTX.
Fig. 5.
Fig. 5.
Activation of CaSR by R-568 attenuates the effect of TTX to reduce cAMP-stimulated Isc in the colon. A: representative recording of Isc responses to sequential additions of forskolin and R-568 in the absence of TTX in a rat distal colon segment. Following additions of 500 nM forskolin to mucosal and serosal solutions, 10 μM of R-568 was added to serosal solution to activate CaSR activity. B: quantitative changes in Isc following treatment with forskolin ± R-568 in the proximal and distal colons. C: representative recording of Isc responses to sequential additions of forskolin and R-568 in the presence of TTX in a rat distal colon segment. Following additions of 500 nM forskolin to mucosal and serosal solutions, 10 μM of R-568 was added to serosal solution to activate CaSR activity. D: quantitative changes in Isc following treatment with forskolin ± R-568 in the proximal and distal colons. E summarizes the ΔIsc induced by R-568 in the absence and presence of TTX.
Fig. 6.
Fig. 6.
R-568 produces similar inhibitory effects on Isc in the colon at 1.25 mM extracellular ionized calcium (Cao2+). Rat distal colons were treated the same way by forskolin and R-568 as in the legend of Fig. 5 except that Cao2+ of 1.25 mM was used. A: Isc responses to forskolin and R-568 in the absence of TTX. B: Isc responses to forskolin and R-568 in the presence of TTX. C summarizes the ΔIsc induced by R-568 in the absence and presence of TTX.
Fig. 7.
Fig. 7.
Prior activation of CaSR by R-568 reduces cAMP-stimulated Isc in the colon. A: representative recording of Isc response to forskolin in the absence of R-568 in a rat distal colon segment. Forskolin (500 nM) was added to mucosal and serosal solutions. B: quantitative changes in Isc following treatment with forskolin in the proximal and distal colons in the absence of R-568. C: representative recording of Isc response to forskolin in the presence of R-568 in a rat distal colon segment. 10 μM R-568 was added to serosal solution 15 min before 500 nM forskolin was added to mucosal and serosal solutions. D: quantitative changes in Isc following treatment with forskolin in the proximal and distal colons in the presence of R-568. E summarizes the ΔIsc induced by forskolin in the absence and presence of R-568.
Fig. 8.
Fig. 8.
Removal of Cl− ion from bath solutions eliminates both basal and cAMP-stimulated Isc as well as their responses to TTX and R-568. Shown are representatives of 2–6 recordings of Isc responses of distal colon to bilateral Cl− ion substitution and subsequent additions of serosal bumetanide (A), serosal TTX (B), bilateral forskolin (D), mucosal amiloride (A–D), and serosal R-568 with (D) or without (C) prior forskolin stimulation. Similar responses were noted in the proximal colon as well.
Fig. 9.
Fig. 9.
Dual-pathway model for fluid secretion in intestine and sites of antisecretory action for CaSR. Secretagogues such as cholera toxin (CTX) and forskolin induce diarrhea through direct epithelial cell generation of cyclic nucleotides and indirectly via stimulation of ENS to release secretagogues such as vasoactive peptide. The CaSR is expressed in both cell types and upon its activation can inhibit both secretory pathways.
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