Calcium-sensing receptor abrogates secretagogue- induced increases in intestinal net fluid secretion by enhancing cyclic nucleotide destruction

Abstract

The calcium-sensing receptor (CaSR) provides a fundamental mechanism for diverse cells to detect and respond to modulations in the ionic and nutrient compositions of their extracellular milieu. The roles for this receptor are largely unknown in the intestinal tract, where epithelial cells are normally exposed to large variations in extracellular solutes. Here, we show that colonic CaSR signaling stimulates the degradation of cyclic nucleotides by phosphodiesterases and describe the ability of receptor activation to reverse the fluid and electrolyte secretory actions of cAMP- and cGMP-generating secretagogues, including cholera toxin and heat stable Escherichia coli enterotoxin STa. Our results suggest a paradigm for regulation of intestinal fluid transport where fine tuning is accomplished by the counterbalancing effects of solute activation of the CaSR on neuronal and hormonal secretagogue actions. The reversal of cholera toxin- and STa endotoxin-induced fluid secretion by a small-molecule CaSR agonist suggests that these compounds may provide a unique therapy for secretory diarrheas.

Fig. 1.

Increasing extracellular Ca²⁺ abrogates secretagogue-stimulated ^netJ_V and cyclic nucleotide accumulation. (a–d) Effects of raising basolateral bath Ca²⁺ on net fluid transport (^netJ_V) in rat perfused crypts after cAMP (forskolin or cholera toxin, CTX) or cGMP (guanylin or STa endotoxin) generating secretagogues. Positive and negative values for ^netJ_V represent net absorption and secretion, respectively. (e–h) Secretagogue-stimulated increases in cyclic nucleotides are abrogated by raising extracellular Ca²⁺ to 2 mM, and this effect was absent after exposure to the phosphodiesterase inhibitor IBMX. Values in are mean ± SEM; ∗, P < 0.01 compared with 0.1 mM Ca²⁺ without secretagogue; #, P < 0.01 compared with 0.1 mM Ca²⁺ plus secretagogue. The number in parentheses is the number of crypts (a–d) or tissues (e–h) studied. Calcium (Ca) concentrations are in millimolar.

Fig. 2.

The CaSR allosteric agonist, R-568, abrogates secretagogue-stimulated fluid secretion and cyclic nucleotide accumulation in rat colonic crypts. (a) Concentration dependence of R and S enantiomers of 568 in lumen or bath perfusate on 10 μM forskolin-stimulated net fluid secretion (^netJ_V) in rat perfused colonic crypts. (b and c) R-568 dose-dependently reduced cyclic nucleotide accumulation stimulated by 500 μM guanylin or 100 nM cholera toxin, respectively. Values are mean ± SEM; ∗, P < 0.01 compared with no secretagogue; #, P < 0.01 compared with secretagogue without CaSR agonist. The number in parentheses is the number of crypts (a) or tissues (b and c) studied. Calcium (Ca) concentrations are in millimolar.

Fig. 3.

R-568 reverses the short circuit current, I_SC, induced by forskolin (FSK) or STa. Rat distal colonic strips (0.3 cm²) with the muscle layer removed were mounted in an Ussing chamber in symmetrical Hepes-buffered ringers at 37°C and gassed with 100% O₂. (a and c) Representative current traces showing the change in I_SC induced by serosal bath additions of 12.5 nM FSK (a) or 5 units/ml STa (c) and the reversal of secretagogue-induced I_SC by serosal 10 μM R-568. (b and d) Summary of the rate of reduction in negative I_SC induced by 10 μM R-568. Control rates were obtained from the steady state I_SC values 2–5 min before adding R-568. Data are mean ± SEM; ∗, P < 0.005 vs. control; #, P < 0.02 vs. control.

Fig. 4.

Absence of effects of 2 mM Ca²⁺ or R-568 on secretagogue-stimulated net fluid secretion in CaSR null mice. (a and b) Increasing bath Ca²⁺ to 2 mM (a) or bath addition of 100 nM R-568 (b) reversed the net fluid secretion induced by 100 nM CTX (open bars; n = 8) or 500 μM guanylin (filled bars; n = 9) in perfused colonic crypts from wild-type mice, Casr^+/+::Gcm2^+/+. (c and d) The ability of bath 2 mM Ca²⁺ or bath 100 nM R-568 failed to reverse fluid secretion induced by secretagogues (n = 18) in the CaSR receptor knockout mouse, Casr^−/−:Gcm2^−/−. Values are mean ± SEM; ∗, P < 0.001 compared with no secretagogue; #, P < 0.01 compared with secretagogue without CaSR agonist. Calcium (Ca) concentrations are in millimolar.

Fig. 5.

IBMX and PLC inhibition abrogates the effects of R-568 on ^netJ_V and cyclic nucleotide accumulation in rat colonic crypts. (a) IBMX abolished the effects of 2 mM Ca²⁺ and/or 100 nM R-568 to reverse the effect of 10 μM forskolin on ^netJ_V. (b) The phospholipase C (PLC) inhibitor U73122 abolishes the ability of lumen or bath R-568 to reverse forskolin-stimulated fluid secretion. PLC inhibition blocked the ability of 100 nM R-568 to reverse cGMP or cAMP accumulation in colonic crypt cells induced by guanylin (c) or cholera toxin (CTX; d), respectively. Values are mean ± SEM; ∗, P < 0.001 compared with no secretagogue. The number in parentheses is the number of crypts (a and b) or tissues (c and d) studied. Calcium (Ca) concentrations are in millimolar.

Fig. 6.

Extracellular Ca²⁺ or R-568 reverses secretagogue-induced inhibition of fluid absorption in rat colonic crypts. (a and b) Cl⁻ entry into rat crypt cells, monitored by the rate of decrease in MQAE fluorescence, is increased by 10 μM forskolin (FSK) and 100 nM CTX, and this increase was inhibited by bumetanide, 2 mM Ca²⁺, or 100 nM R-568. (c) Bumetanide increased absorptive ^netJ_V in the absence of secretagogues, and this absorptive flux was reduced after addition of dibutyryl-cAMP (db-cAMP). (d and e) 2 mM Ca²⁺ or R-568 reversed forskolin-inhibited net fluid absorption in the presence of bath bumetanide and a luminal Cl⁻ channel inhibitor (100 μM NPPB). (f) Forskolin inhibited Na⁺-H⁺ exchanger (NHE3) activity was reversed by 2 mM Ca²⁺ or R-568. Cell acid loading was accomplished by exposure to NH₄Cl, and Na⁺-dependent cell pH recovery after removal of NH₄Cl was assessed as an index of Na⁺/H⁺ activity (51).Values are mean ± SEM; ∗, P < 0.01 compared with no secretagogue; #, P < 0.01 compared with secretagogue without inhibitor or CaSR agonist. The number in parentheses is the number of crypts studied. Calcium (Ca) concentrations are in millimolar.

Fig. 7.

Model for CaSR-mediated abrogation of secretagogue effects on fluid transport by colonic crypts. cAMP and cGMP are generated by activation of receptors coupled to adenylyl cyclase (AC) and guanylyl cyclase (GC), respectively. Stimulation of either receptor pathway [guanylin or STa toxin for cGMP; cholera toxin or forskolin (FSK) for cAMP] enhances fluid secretion and reduces fluid absorption. Activation of the CaSR by Ca²⁺ or R-568 stimulates phospholipase C (PLC) activity that generates IP₃, resulting in the release of Ca²⁺ from thapsigargin-sensitive stores. Ca²⁺-dependent activation of phosphodiesterases (PDE) in crypt cells metabolizes cyclic nucleotides and reverses the effect of secretagogues on fluid transport.