The role of protease-activated receptor-2 (PAR2) in the modulation of beating of the mouse isolated ureter: lack of involvement of mast cells or sensory nerves

Abstract

1 The localization of protease-activated receptor-2 (PAR2) and the effects of PAR2 activators were investigated in the mouse isolated ureter in order to test the hypothesis that PAR2 activation may initiate neuropeptide release from sensory nerve fibres and hence contribute to inflammation. 2 PAR2 was localized by fluorescence immunohistochemistry to both the smooth muscle and epithelium of the ureter. Macrophage-like cells in the adventitia of the ureter were also PAR2-immunoreactive. PAR2-immunoreactivity was not observed in mast cells or nerve fibres. 3 In circular muscle preparations of the ureter in which continuous rhythmic beating was induced by KCl (20 mM) and the thromboxane A2 mimetic U46619 (0.3 microM), trypsin (0.3 U ml-1) reduced beat frequency to 84.6+/-2.0% of control rates. The PAR2-selective peptide agonist SLIGRL-NH2 concentration-dependently (0.1-3.0 microM) slowed beat frequency to a maximum of 72.7+/-2.0%. 4 Histamine (1-300 microM) was more efficacious than SLIGRL-NH2 in inhibiting ureter beat frequency in a concentration-dependent manner to a maximum (at 300 microM) of 7.9+/-2.5% of the control rate. 5 Pretreatment of preparations with capsaicin (10 microM for 30 min) markedly attenuated the inhibitory effect of histamine, but not that of SLIGRL-NH2, indicating a role for sensory nerves in the inhibitory effect of histamine only. 6 The inhibitory effect of SLIGRL-NH2 on ureter beat frequency was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM) or the cyclo-oxygenase inhibitor, indomethacin (3 microM). 7 In conclusion, PAR2 activation causes inhibition of beating in the mouse ureter that is not mediated by axon reflex release of inhibitory neuropeptides. This inhibitory effect of PAR2 appears to be mediated directly on smooth muscle cells, although the contribution of non-NO, non-prostanoid epithelium-derived factors cannot be ruled out.

Figure 1

Immunohistochemical localization of PAR2 in the mouse ureter. (a) Transverse section (3 μm) of a ureter. Epithelial cells (arrows) are intensely stained, while smooth muscle cells contain less widespread, punctate fluorescence (arrowheads). Scale bar represents 10 μm. (b) Whole mount preparation of a ureter showing intense PAR2-immunoreactivity of macrophages (arrows) in the adventia. Asterisks indicate adipocytes. Scale bar represents 50 μm.

Figure 2

Effect of PAR2 activators and histamine on induced beating in the mouse isolated ureter. (a, b, c) Representative original traces of the inhibitory effects of trypsin, SLIGRL-NH₂ and histamine respectively on beat frequency. (d) Cumulative concentration-effect curve to SLIGRL-NH₂. (n=9), (e) Cumulative concentration-effect curve to histamine (n=5). Horizontal scale bars indicate 1 min; vertical scale bars indicate 0.5 mN.

Figure 3

(a) Representative traces of two preparations of a ureter from the same animal. In the control preparation (left panel), addition of capsaicin (3 μM; at dot) abolishes beating, whilst in the capsaicin pretreated preparation (right panel), addition of capsaicin has no effect. Horizontal scale bars indicate 1 min; vertical scale bars indicate 0.5 mN. (b) Group data illustrating the effect of capsaicin pretreatment on responses to SLIGRL-NH₂ (3 μM) and histamine (300 μM). Asterisk indicates P<0.05 (n=5; unpaired t-test).

Figure 4

Lack of effect of L-NOARG (100 μM) and indomethacin (3 μM) on the inhibition of beating in the mouse ureter induced by SLIGRL-NH₂ (3 μM). Data from five preparations.