The regulatory action of acetylcholine and its receptors on B4 and C4 leukotriene formation in the porcine endometrium after experimental inflammogenic Escherichia coli infection

Abstract

Introduction: Endometritis is a very common pathology in animals which changes endometrial leukotriene (LT) formation and muscarinic 2 and 3 receptor subtypes (M2R/M3R) and α-7 nicotinic acetylcholine (ACh) receptor (α-7 nAChR) expression patterns. With the relationship between ACh, its receptors and LT production remaining unclear, the role of M2R, M3R and α-7 nAChR in action of ACh on the 5-lipoxygenase (5-LO), LTA4 hydrolase (LTAH) and LTC4 synthase (LTCS) protein abundances in the inflamed porcine endometrium and on the tissue secretion of LTB4 and LTC4 were studied.

Material and methods: On day three of the oestrous cycle in gilts aged 7-8 months, 50 mL of either saline solution (control group, n = 5) or an E. coli suspension at 10⁹ colony-forming units/mL (E. coli group, n = 5), was injected into each uterine horn. Endometrial explants obtained eight days later, were incubated with ACh alone, antagonists of M2R, M3R and α-7 nAChR alone, or with ACh together with particular antagonists for 16 h. Enzyme abundances in endometrial tissue were estimated by Western blotting, and LT concentrations in medium by ELISA.

Results: Severe acute endometritis developed in the E. coli group. In the endometrial explants from both groups, ACh elevated 5-LO, LTAH and LTCS protein abundances and LTB4 and LTC4 release. In the E. coli group, ACh-induced 5-LO and LTCS abundances and LTB4 release were increased versus the control group. In both groups, the M3R antagonist with ACh reduced all ACh-stimulated enzyme abundances and LT release in comparison to the abundances and release mediated by ACh alone. This effect on LTCS protein abundance and LTB4 release was also produced by the M2R antagonist with ACh in the E. coli group. Compared to the effect of ACh alone, exposure of the E. coli group endometrium to the α-7 nAChR antagonist with ACh led to a rise in LTAH and LTCS protein abundances and LTB4 and LTC4 secretion.

Conclusion: In the inflamed pig endometrium, ACh increased 5-LO, LTAH and LTCS protein abundances and LTB4 and LTC4 release by M3R, and LTCS protein abundance and LTB4 release also by M2R. By interaction with α-7 nAChR, ACh reduced LTAH and LTCS protein abundances and the release of these LTs. Thus, in an indirect manner, ACh can affect LT-controlled processes.

Keywords: B4 and C4 leukotriene production; acetylcholine; endometritis; pig.

Fig. 1.

Western blot results for the effects of acetylcholine (ACh) alone or with antagonists of muscarinic 2 and 3 receptor subtypes (M2R/M3R) and α-7 nicotinic ACh receptor (α-7 nAChR) on 5-lipoxygenase (5-LO; A), leukotriene (LT) A4 hydrolase (LTAH; B) and LTC4 synthase (LTCS; C) protein abundances in the endometrium of gilts from the control (grey bars, n = 5) and E. coli (black bars, n = 5) groups. Blots with representative bands for each enzyme in particular groups are depicted in Supplementary Figure 2. a, b, c – different letters for any pair of values within a group indicate significant difference (P-value < 0.05–0.001) between the control values (CV), action of ACh and actions of particular antagonists with ACh; *** – statistical difference between groups for the same type of treatment (P < 0.001)

Fig. 2.

ELISA results for the effects of acetylcholine (ACh) alone or with antagonists of muscarinic 2 and 3 receptor subtypes (M2R/M3R) and α-7 nicotinic ACh receptor (α-7 nAChR) on the leukotriene (LT)B4 (A) and LTC4 (B) secretion from the endometrium of gilts from the CON (grey bars, n = 5) and E. coli (black bars, n = 5) groups. a, b, c – different letters for any pair of values within a group indicate significant difference (P-value < 0.05–0.001) between the control values (CV), action of ACh and actions of particular antagonists with ACh; * P < 0.05, ** P < 0.01, *** P < 0.001 – statistical difference between groups for the same type treatment