Activation of submucosal 5-HT(3) receptors elicits a somatostatin-dependent inhibition of ion secretion in rat colon

Abstract

Background and purpose: 5-Hydroxytryptamine (5-HT) is a key regulator of the gastrointestinal system and we have shown that submucosal neuronal 5-HT(3) receptors exerted a novel inhibitory effect on colonic ion transport. The aim of the present study was to investigate the precise mechanism(s) underlying this inhibitory effect.

Experimental approach: Mucosa/submucosa or mucosa-only preparations from rat distal colon were mounted in Ussing chambers for measurement of short-circuit current (I(sc)) as an indicator of ion secretion. Somatostatin release was determined with radioimmunoassay. Intracellular cAMP content was measured with enzyme-linked immunoadsorbent assay (elisa). Immunohistochemical techniques were used to study the expression of 5-HT(3) receptors, somatostatin and somatostatin receptors in colonic tissue.

Key results: In rat distal colonic mucosa/submucosa preparations, pretreatment with 5-HT(3) receptor antagonists enhanced 5-HT-induced increases in I(sc). However, in mucosa-only preparations without retained neural elements, pretreatment with 5-HT(3) receptor antagonists inhibited 5-HT-induced DeltaI(sc). Pretreatment with a somatostatin-2 (sst(2)) receptor antagonist in mucosa/submucosa preparations augmented 5-HT-induced DeltaI(sc). Combination of sst(2) and 5-HT(3) receptor antagonists did not cause further enhancement of 5-HT-induced DeltaI(sc). Moreover, both sst(2) and 5-HT(3) receptor antagonists enhanced 5-HT-induced increase in intracellular cAMP concentration in the mucosa/submucosa preparations. 5-HT released somatostatin from rat colonic mucosa/submucosa preparations, an effect prevented by pretreatment with 5-HT(3) receptor antagonists. Immunohistochemical staining demonstrated the presence of 5-HT(3) receptors on submucosal somatostatin neurons and of sst(2) receptors on colonic mucosa.

Conclusion and implications: Activation of neuronal 5-HT(3) receptors in the submucosal plexus of rat colon suppressed 5-HT-induced ion secretion by releasing somatostatin from submucosal neurons.

Figure 1

Effect of 5-HT₃ receptor antagonists on 5-HT-induced I_sc responses in rat distal colon mucosa/submucosa preparations. (A1) Representative I_sc recording with arrow indicating the basolateral application of 5-HT (10 µM) to the mucosa/submucosa preparations pretreated with MDL72222 (0.1 µM). (A2) Summary of the effects of MDL72222 (0.1 µM) on 5-HT-induced ΔI_sc. (B1) Representative I_sc recording showing the effect of tropanyl-3, 5-dimethylbenzoate (0.1 µM) on 5-HT-induced I_sc responses. (B2) Summary of the effects of Tropanyl-3, 5-dimethylbenzoate (0.1 µM) on 5-HT-induced ΔI_sc. Values are means ± SEM; **P < 0.01; n= 12. (C1) Effects of different concentrations of MDL72222 on 5-HT-induced I_sc responses in rat distal colonic mucosa/submucosa preparations. Numbers in the table indicate the number of tissues demonstrating the variable effect on I_sc at different doses of MDL72222. (C2) Summary of the effects of different doses of MDL72222 on 5-HT-induced ΔI_sc. Values are means ± SEM; *P < 0.05; **P < 0.01; n= 6 (0.001 µM), n= 12 (0.01 µM), n= 11 (0.1 µM), n= 14 (1.0 µM), n= 12 (10 µM). 5-HT, 5-Hydroxytryptamine; I_sc, short-circuit current.

Figure 2

Effects of 5-HT₃ receptor antagonist on 5-HT-induced I_sc responses in rat colonic mucosa/submucosa and mucosa-only preparations in the presence of TTX. (A1) Representative I_sc recording with arrow indicating the basolateral application of 5-HT (10 µM) to the mucosa/submucosa preparations pretreated with MDL72222 (0.1 µM) plus TTX (1 µM). (A2) Summary of the effects of MDL72222 (0.1 µM) plus TTX (1 µM), on 5-HT-induced ΔI_sc. Values are means ± SEM; **P < 0.01; n= 9. (B1/C1) Representative I_sc recording with arrows indicating the basolateral application of 5-HT (10 µM) to rat mucosa-only preparations pretreated with MDL72222 (0.1 µM, B1) or tropanyl-3, 5-dimethylbenzoate (0.1 µM, C1) plus TTX (1 µM) respectively. (B2/C2) Summary of the effects of MDL72222 (0.1 µM, B2) and tropanyl-3, 5-dimethylbenzoate (0.1 µM, C2) on 5-HT-induced ΔI_sc. Values are means ± SEM; *P < 0.05; **P < 0.01; n= 5 respectively. 5-HT, 5-Hydroxytryptamine; I_sc, short-circuit current; TTX, tetrodotoxin.

Figure 3

Effect of the sst₂ receptor antagonist CYN 154806 on 5-HT-induced I_sc responses in rat distal colon mucosa/submucosa preparations. (A1) Representative I_sc recording with arrow indicating the basolateral application of 5-HT (10 µM) to the mucosa/submucosa preparations pretreated with CYN 154806 (0.5 µM). (A2) Summary of the effect of CYN 154806 (0.5 µM) on 5-HT-induced ΔI_sc. Values are means ± SEM; **P < 0.01; n= 7. (B1) Representative I_sc recording with arrow indicating the basolateral application of 5-HT (10 µM) to the mucosa/submucosa preparations pretreated with CYN 154806 (0.5 µM) and MDL72222 (0.1 µM). (B2) Summary of the effect of CYN 154806 (0.5 µM) and MDL72222 (0.1 µM) on 5-HT-induced ΔI_sc. Values are means ± SEM; **P < 0.01; n= 4. 5-HT, 5-Hydroxytryptamine; I_sc, short-circuit current; sst₂, somatostatin receptor-2.

Figure 4

Intracellular cAMP measurement. Summary of the effects of pretreatment with the sst₂ receptor antagonist, CYN 154806 (0.5 µM), 5-HT₃ receptor antagonists, MDL72222 (0.1 µM) and tropanyl-3, 5-dimethylbenzoate (0.1 µM), and 5-HT₄ receptor antagonists, GR113808 (0.5 µM) on 5-HT (10 µM)-stimulated intracellular cAMP production in the rat colonic mucosa/submucosa preparations. **P < 0.01; n= 6 respectively. 5-HT, 5-Hydroxytryptamine; sst₂, somatostatin receptor-2.

Figure 5

Expression of 5-HT₃ receptor in somatostatin neurons of the rat colonic submucosal plexus. (A–C) Immunoreactivity of 5-HT₃ receptor (A) and somatostatin (B) in the frozen sections of the rat distal colon. (D–F) Immunoreactivity of 5-HT₃ receptor in whole-mount preparations of the rat distal colon. 5-HT₃ receptor (green) was expressed in two neurons of a submucosal ganglion and nerve fibres. Neuronal phenotype was confirmed using a pan-neuronal marker HuC/D (red). (G–I) 5-HT₃ receptor (green) and somatostatin (red) was colocalized in neurons in whole-mount preparations of the submucosal plexus. Scale bars, 20 µm. 5-HT, 5-Hydroxytryptamine.

Figure 6

Expression of sst₂ receptors in the epithelium of the rat distal colonic mucosa. sst₂ receptor immunoreactivity was found to be abundantly expressed in the crypts of the rat distal colonic mucosa (A. lower magnification; B. higher magnification). (C) Omission of the primary antibody of sst₂ receptors resulted in no immunostaining. sst₂, somatostatin receptor-2.

Figure 7

Radioimmunoassay of somatostatin release in rat colonic mucosa/submucosa preparations. A summary of the effects of pretreatment with different concentrations of 5-HT₃ antagonist, MDL72222 (0.1 µM, n= 6) and tropanyl-3, 5-dimethylbenzoate (0.1 µM, n= 5), as well as the sst₂ receptor antagonist, CYN 154806 (0.5 µM, n= 5) on the 5-HT (10 µM, n= 6)-stimulated somatostatin release. n= 7. *P < 0.05 compared with 5-HT treatment alone. 5-HT, 5-Hydroxytryptamine; sst₂, somatostatin receptor-2.

Figure 8

An illustration of the working model of 5-HT₃ and 5-HT₄ receptors in rat colonic submucosal plexus and mucosa. (A) Submucosal 5-HT₃ receptor-mediated somatostatin-dependent inhibitory neural pathway. Activation of 5-HT₃ receptors in the submucosal plexus leads to the release of somatostatin. Somatostatin binds with to the sst₂ receptors on the colonic epithelium and reduces the intracellular cAMP concentration, which leads to a decrease of ion secretion in the rat colon. Somatostatin also inhibits secretomotor neuron activity, which leads to a decrease of ion secretion. (B) Epithelial non-neuronal pathway mediated by 5-HT₃ and 5-HT₄ receptors. In this pathway, 5-HT binds to 5-HT₃ and 5-HT₄ receptors in the rat colonic epithelium and stimulates ion secretion in rat colon. 5-HT, 5-Hydroxytryptamine; sst₂, somatostatin receptor-2.