Insulin-like peptide 5 and relaxin-3 inhibit cholinergically-mediated contractions in mouse but not human isolated colon

Insulin-like peptide 5 (INSL5) is found in mouse and human colon endocrine cells. It has been implicated in control of appetite, yet its functions in the colon are unknown (1). INSL5 belongs to the relaxin family, which includes relaxin-3, structurally closest to INSL5 but with restricted expression to human and mouse brain and testis (2,3). RXFP3 and RXFP4 are receptors for relaxin-3 and INSL-5 respectively, and relaxin-3 also binds RXFP4. RXFP4 has been found in mouse and human intestine, including the myenteric plexus (1,3), whereas RXFP3 mRNA has been identified in mouse (1) but not human colon (4). This study investigated the abilities of INSL5 and relaxin-3 to modulate neuromuscular responses in mouse and human colon. The µ receptor agonist loperamide was studied as a positive control.

Loops of proximal mouse colon (3 month CD1 male; 2 mm wide) were suspended in tissue baths for electrical field stimulation of intrinsic neurons (EFS; Krebs; 5% CO2 in O2; 37°C; 1g tension) and isometric recording of circular muscle contractility. Macroscopically normal human colon was obtained from surgery for bowel cancer, following informed consent, and mucosa-free circular muscle strips prepared for EFS (5). EFS was applied at 5Hz (mouse: 0.5ms pulse width, 80V, 30s every 2 min; human: 0.5ms, 50V, 10s every 1 min) and drugs applied non-cumulatively. Loperamide was dissolved in DMSO, INSL5 in 20% DMSO 0.1% BSA and relaxin-3 in sterile PBS, before serial dilution. Data are expressed as mean ± SEM and compared to vehicle control with Student’s t-test.

In mouse colon, EFS inhibited spontaneous contractions or caused small muscle relaxation (prevented by L-NAME 300µM; n=4), consistently followed by a large amplitude ‘after-contraction’ (prevented by atropine 1µM; n=6). Tetrodotoxin 1µM prevented all responses (n=5). Loperamide 10µM reduced the after-contractions (46.1 ± 7.3 % n=3). INSL5 30nM reduced the after-contractions by 21.1 ± 2.2 % (P<0.001 compared to 0.2 ± 0.6 % inhibition with vehicle; n=3 each). A higher concentration of INSL5 (1µM) also inhibited contractions (23.8 ± 4.5 %, n=4) but the change was not statistically significant compared with vehicle (0.2% DMSO; 18.1 ± 3.4 % inhibition, n=3). Relaxin-3 0.1μM caused 27.1 ± 7.1 % inhibition (P<0.05 compared to 3.0 ± 2 % inhibition with vehicle; n=4, 3, respectively). In human colon EFS evoked contraction (prevented by atropine 1µM) or relaxation (prevented by L-NAME 300 µM), often followed by predominantly cholinergically-mediated after-contractions (5). INSL5 0.3-1 μM (n=3 each) or relaxin-3 0.1-100 nM (n=2 each) did not consistently affect responses during or after EFS.

INSL5 and relaxin-3 inhibit colon motility in mice, not in humans. For INSL5 this difference is consistent with poor RXFP4 sequence conservation between mouse and human (4). RXFP3 and relaxin-3 show better sequence conservation (2).

FS and RS contributed equally. Takeda Pharmaceuticals (FS, AP), Dunhill Medical Trust (JB), BBSRC (M-AK) and AgeUK (VWSK) are acknowledged for support.

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5. Broad J et al. (2013). Br J Pharmacol 170: 1253–1261.