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Calcimimetic acts on enteric neuronal CaSR to reverse cholera toxin-induced intestinal electrolyte secretion - PubMed

  • ️Mon Jan 01 2018

Calcimimetic acts on enteric neuronal CaSR to reverse cholera toxin-induced intestinal electrolyte secretion

Lieqi Tang et al. Sci Rep. 2018.

Abstract

Treatment of acute secretory diarrheal illnesses remains a global challenge. Enterotoxins produce secretion through direct epithelial action and indirectly by activating enteric nervous system (ENS). Using a microperfused colonic crypt technique, we have previously shown that R568, a calcimimetic that activates the calcium-sensing receptor (CaSR), can act on intestinal epithelium and reverse cholera toxin-induced fluid secretion. In the present study, using the Ussing chamber technique in conjunction with a tissue-specific knockout approach, we show that the effects of cholera toxin and CaSR agonists on electrolyte secretion by the intestine can also be attributed to opposing actions of the toxin and CaSR on the activity of the ENS. Our results suggest that targeting intestinal CaSR might represent a previously undescribed new approach for treating secretory diarrheal diseases and other conditions with ENS over-activation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

R568 anti-secretory effects under basal conditions. Summarized are steady-state Isc changes in response to the sequential serosal additions of R568 (10 µM) and lidocaine (Lido, 1.6 mM)/TTX (2 µM). The R568 anti-secretory effect is present in colons of wild-type mice and mice expressing neuronal CaSR (villinCre/Casrflox/flox or villin mice) but is absent in mice lacking neuronal CaSR (nestinCre/Casrflox/flox or nestin mice). NS, no significance.

Figure 2
Figure 2

R568 anti-secretory effects under cholera toxin-stimulated conditions. Mouse intestines were pretreated with cholera toxin, in vivo, in live animals to induce hypersecretion (see Methods), followed by examining Isc changes of pre-treated intestines, ex vivo, in Ussing chambers. Summarized are steady-state Isc changes of these pretreated intestines in response to the sequential serosal additions of R568 (10 µM) and lidocaine (Lido, 1.6 mM)/TTX (2 µM). The R568 anti-secretory effect is present in colons of wild-type mice and mice expressing neuronal CaSR (villinCre/Casrflox/flox or villin mice) but is absent in mice lacking neuronal CaSR (nestinCre/Casrflox/flox or nestin mice). NS, no significance.

Figure 3
Figure 3

R568 anti-secretory effects under forskolin-stimulated conditions. Mouse intestines were pretreated with forskolin (FSK, 500 nM, added to the serosal bath), ex vivo, in Ussing chambers to elevate cyclic AMP and excite neurons to induce hypersecretion. Following this pre-treatment, R568 (10 µM) and lidocaine (Lido, 1.6 mM)/TTX (2 µM) were subsequently added to the serosal solution of Ussing chambers with the sequence of their additions indicated. Summarized are steady-state Isc changes of these pretreated intestines and effects of R568 and lidocaine/TTX. R568 abolishes the forskolin’s excitatory effect in colons of wild-type mice and mice expressing neuronal CaSR (villinCre/Casrflox/flox or villin mice) but not in mice lacking neuronal CaSR (nestinCre/Casrflox/flox or nestin mice). NS, no significance.

Figure 4
Figure 4

Proposed dual-pathway model for CaSR inhibition of cholera toxin-induced hypersecretion in the intestine. Vibrio cholerae generates cholera toxin (CTX) to evoke hypersecretion using two pathways and CaSR agonists block both pathways. (1) CTX induces a non-neuronal fluid secretory response due to binding of toxin directly to enterocytes, leading to the generation of cyclic AMP and activation of CFTR and NKCC1. This direct secretory response is blocked by activation of CaSR on enterocytes, either apically or basolaterally, through enhancing cyclic nucleotide destruction. (2) CTX induces a neurally mediated secretory response by stimulation of secretomotor reflex pathways in the ENS. This neurally mediated indirect secretory response is blocked by activation of CaSR on neurons (cell bodies, indicated in filled red circles, and nerve terminals, indicated in black lines) possibly by modulations of cyclic nucleotide metabolism and ion channel properties, leading to inhibition of neuronal cell excitability and/or synaptic excitation (see text for more discussions). Besides enterocytes and neurons, the cholera toxin-induced hypersecretion requires the release of 5-hydroxytryptamine (5-HT, indicated by blue dots) from enterochromaffin (EC, indicated in red) cells of the mucosa, which also express CaSR. It remains to be addressed whether cholera toxin-evoked release of 5-HT from EC cells is blocked by CaSR on EC cells.

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