Subepithelial trypsin induces enteric nerve-mediated anion secretion by activating proteinase-activated receptor 1 in the mouse cecum

Abstract

Serine proteases are versatile signaling molecules and often exert this function by activating the proteinase-activated receptors (PAR(1)-PAR(4)). Our previous study on the mouse cecum has shown that the PAR(1)-activating peptide (AP) and PAR(2)-AP both induced electrogenic anion secretion. This secretion mediated by PAR(1) probably occurred by activating the receptor on the submucosal secretomotor neurons, while PAR(2)-mediated anion secretion probably occurred by activating the receptor on the epithelial cells. This present study was aimed at using trypsin to further elucidate the roles of serine proteases and PARs in regulating intestinal anion secretion. A mucosal-submucosal sheet of the mouse cecum was mounted in Ussing chambers, and the short-circuit current (I(sc)) was measured. Trypsin added to the serosal side increased I(sc) with an ED(50) value of approximately 100 nM. This I(sc) increase was suppressed by removing Cl(-) from the bathing solution. The I(sc) increase induced by 100 nM trypsin was substantially suppressed by tetrodotoxin, and partially inhibited by an NK(1) receptor antagonist, by a muscarinic Ach-receptor antagonist, and by 5-hydroxytryptamine-3 (5-HT(3)) and 5-HT(4) receptor antagonists. The I(sc) increase induced by trypsin was partially suppressed when the tissue had been pretreated with PAR(1)-AP, but not by a pretreatment with PAR(2)-AP. These results suggest that the serine protease, trypsin, induced anion secretion by activating the enteric secretomotor nerves. This response was initiated in part by activating PAR(1) on the enteric nerves. Serine proteases and PARs are likely to be responsible for the diarrhea occurring under intestinal inflammatory conditions.

Fig. 1

Changes to I _sc and G _t induced by serosal trypsin. a Time-course characteristics for the changes in I _sc and G _t induced by 1 nM–100 μM trypsin added to the serosal side at the arrowed time (n = 4–8). Mean values are presented. b Concentration dependence of the serosal trypsin-induced increase in I _sc (ΔI _sc) and G _t (ΔG _t). Values were obtained when the peak I _sc increases were reached. Each data value is presented the mean ± SE, the number of animals used being 4–8

Fig. 2

Ionic basis for the increase in I _sc induced by 100 nM serosal trypsin. Bumetanide (100 μM, serosal) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 μM, mucosal) were added, or Cl ⁻ was removed (from both the mucosal and serosal bathing solutions) 20 min before adding 100 nM trypsin to the serosal side. Peak values for the increases in I _sc (ΔI _sc) induced by trypsin were determined and are expressed as a percentage of the control response obtained in the adjacent tissue. Each data value is presented as the mean ± SE, with n = 4 for bumetanide and n = 5 for the other compounds. *0.01 < P < 0.05 and ***P < 0.001, compared with the control response that had been determined in adjacent tissue by a paired t test

Fig. 3

Changes in G _t (ΔG _t) induced by 100 nM serosal trypsin in the presence of various inhibitors. The values were obtained when the peak I _sc increases were reached. Each data value is presented as the mean ± SE, with n = 3–6. *0.01 < P < 0.05, **0.001 < P < 0.01, and ***P < 0.001, compared with the control response that had been determined in the adjacent tissue by a paired t test

Fig. 4

Role of enteric submucosal neurons in serosal 100 nM trypsin-induced Cl⁻ secretion. Tetrodotoxin (TTX, 300 nM), 3-tropanyl-3,5-dichlorobenzoate (20 μM), SB-204070 (10 μM), atropine (10 μM), hexamethonium (10 μM), L-703,606 (10 μM) and pyrilamine (10 μM) were each added to the serosal side, and procaine (50 μM) was added either to the mucosal or serosal side 20 min before adding trypsin (100 nM) to the serosal side. The peak values for the increases in I _sc (ΔI _sc) induced by trypsin were determined, and are expressed as a percentage of the control response obtained in the adjacent tissue. Each data value is presented as the mean ± SE, with n = 3 for 3-tropanyl-3,5-dichlorobenzoate + SB-204070 (10 μM), n = 6 for mucosal procaine and pyrilamine, and n = 5 for the other compounds. *0.01 < P < 0.05, **0.001 < P < 0.01, and ***P < 0.001, compared with the control response by a paired t test

Fig. 5

Effect of inhibitors of arachidonic acid metabolism on I _sc induced by 100 nM serosal trypsin. Indomethacin (10 μM) was added to both the mucosal and serosal sides, and NDGA (50 μM) and SKF-525A (30 μM) were each added to the serosal side 20 min before adding trypsin (100 nM) to the serosal side. Each data value is presented as the mean ± SE, with n = 5. ***P < 0.001, compared with the control response that had been determined in the adjacent tissue by a paired t test

Fig. 6

Effect of the protease inhibitor cocktail (P8340; Sigma) on the I _sc increase induced by trypsin. The protease inhibitor cocktail was added to the serosal solution (diluted 1,000-fold) 20 min before adding 100 nM trypsin to the serosal solution. Peak values for the increases in I _sc (ΔI _sc) induced by trypsin were determined and are expressed as a percentage of the control response obtained in the adjacent tissue. Each data value is presented as the mean ± SE, with n = 4. ***P < 0.001, compared with the control response that had been determined in the adjacent tissue by a paired t test

Fig. 7

Involvement of proteinase-activated receptor type 1 (PAR₁) in the serosal trypsin-induced I _sc increase. a–c Attenuation of the ΔI _sc induced by trypsin after pre-exposure to SFFLRN-NH ₂, a PAR₁-activating peptide (AP). a The control ΔI _sc induced by serosal trypsin (100 nM). Mean values are presented, n = 4. b ΔI _sc induced by trypsin was obtained in the tissue that had been pretreated with serosal SFFLRN-NH₂. Mean values are presented, n = 4. c Peak values for ΔI _sc induced by serosal trypsin (100 nM) in the absence (control; shown in a) and presence (SFFLRN-NH ₂; shown in b) of pre-exposure to PAR₁-AP. Each data value is presented as the mean ± SE. *P < 0.05, paired t test. d–e Attenuation of ΔI _sc induced by the serosal PAR₁-AP (30 μM), after pre-exposure to serosal trypsin (100 nM). d The control ΔI _sc induced by SFFLRN-NH₂ was obtained in tissue that had been pretreated with trypsin, but only after the soybean trypsin inhibitor (0.59 mg/ml, serosal side) had been added. Mean values are presented, n = 4. e The soybean trypsin inhibitor was added after trypsin had worked for 20 min, and ΔI _sc induced by SFFLRN-NH₂ was finally obtained. Mean values are presented, n = 4. f The peak values for the increases in I _sc (ΔI _sc) induced by SFFLRN-NH₂ after pre-exposure to trypsin (trypsin treated; shown in e) are expressed as a percentage of the control response (control; shown in d) obtained in the adjacent tissue. Each data value is presented as the mean ± SE. *P < 0.05, paired t test

Fig. 8

Effects of pretreating by SLIGRL-NH ₂, a PAR₂-AP, on the trypsin-induced increase in I _sc (ΔI _sc). a The control ΔI _sc value induced by serosal trypsin (100 nM). Mean values are presented, n = 14. b ΔI _sc induced by trypsin was obtained for tissue that had been treated with serosal SLGRL-NH₂. Mean values are presented, n = 7. c ΔI _sc values induced by serosal trypsin are presented in the tissue that had been pre-exposed to SLGRL-NH₂ or not. Each data value is presented as the mean ± SE. *P < 0.05, unpaired t test