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

All study procedures were approved by the Local Ethics Committee for Experiments on Animals (University of Warmia and Mazury in Olsztyn, Poland, Consent No. 65/2015). The guidelines in EU Directive 2010/63/EU for animal studies were also followed. The experiment was carried out on sexually mature Large White × Landrace gilts aged 7–8 months and weighing 107.3 ± 1.8 kg/mean ± standard error of the mean (SEM). Behavioural oestrus was detected by a tester boar. There was no vaginal discharge from the experimental pigs and their second oestrous cycles were regular. For acclimatisation, the gilts were transported from a commercial farm (Agro-Wronie Sp. z o.o., Wronie, Wąbrzeźno, Poland) to the animal house at the University of Warmia and Mazury (Olsztyn, Poland) three days prior to the beginning of the study. Each gilt was housed in an individual pen measuring approximately 5 m² and kept at a temperature of 18 ± 2°C, in 14.5 ± 1.5 h of natural daylight and 9.5 ± 1.5 h of darkness. The gilts were given a commercial diet and had access to water.

At the end of the acclimatisation period (day 3 of the second oestrous cycle and day 0 of the experiment), the gilts were randomly assigned to two groups: the control group (n = 5), in which the gilts received saline injections into their uterine horns and the E. coli group (n = 5), in which the gilts received E. coli injections into their uterine horns. The study procedures were reported earlier (11). Briefly, the animals were premedicated with atropine (Atropinum sulfuricum WZF; Warszawskie Zakłady Farmaceutyczne Polfa S.A., Warsaw, Poland), azaperone (Stresnil; Janssen Pharmaceutica, Beerse, Belgium) and ketamine hydrochloride (Ketamina; Biowet, Puławy, Poland). General anaesthesia was induced by ketamine hydrochloride. After median laparotomies had been performed, one injection of 10 mL was given into each of five equidistant sites of each uterine horn in the E. coli group and in the control group. The experimental group received a suspension of the E. coli serotype O25:K23/a/:H1 at 10⁹ colony forming units/mL concentration, which was provided by the Department of Microbiology at the National Veterinary Research Institute (Puławy, Poland). The quantity of injected bacteria was the same as was used in a previous experiment which revealed that injections into pig uterine horns of the same strain of E. coli caused severe (mostly) or moderate acute endometritis after eight days (15). The animals from both groups were untreated in the period from surgery until euthanasia, which was performed by an overdose of sodium pentobarbital on day 8 of the study (the expected day 11 of the oestrous cycle). The collected uteri were transported on ice to the laboratory within 20 min.

The uterine horns were washed twice using sterile phosphate-buffered saline. Next, a fragment of the wall was harvested from the middle part of each uterine horn. The wall was divided into endometrial and myometrial layers with the help of a scalpel blade. The endometrium fragments were cut into slices weighing 60–70 mg and then rinsed in Medium 199 with 4-(2- hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) modification and Earle’s salts, L-glutamine and 2 mM HEPES and without sodium bicarbonate (catalogue No. M2520; Sigma-Aldrich, St. Louis, MO, USA). Single endometrial explants were put into glass vials with 2 mL of Medium 199 and 0.1% bovine serum albumin (BSA; catalogue No. A2058, Sigma-Aldrich), gentamicin at 500 μL/500 mL (catalogue No. G1272) and neomycin at 100 μL/500 mL (catalogue No. N1142 (both from Sigma-Aldrich). Endometrium slices underwent preincubation for 1.5 h and incubation for 16 h in a shaking water bath at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Incubation was with fresh medium (as controls) or with the addition of 10⁻⁵ M of ACh alone, 10⁻⁴ M of AChR antagonists alone for M2R, M3R and α-7 nAChR, or with ACh together with particular antagonists all in the same amounts as when used alone. The ACh was catalogue No. A6625 (Sigma-Aldrich); the M2R antagonist was AF-DX 116/11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one, catalogue No. 1105; the M3R antagonist was 4-DAMP/1,1-dimethyl-4-diphenylacetoxypiperidinium iodide, catalogue No. 0482; and the α-7 nAChR antagonist was 03B1-bungarotoxin, catalogue No. 2133 (all from Tocris Bioscience, Bristol, UK). Initial dilutions of the antagonists were made in accordance with the manufacturer’s instructions, in dimethyl sulfoxide (catalogue No. W387509; Sigma-Aldrich) for the antagonists for M2R and M3R, and in 0.2 mm filtered distilled water for the antagonist for α-7 nAChR, and the solutions were then stored at −20°C. The final antagonist solutions and ACh solution were prepared using the same medium as for preincubation and incubation of explants. Each separate study (one per gilt, n = 5 in each group) was performed in triplicate. Prostaglandin F2α (PGF2α) secretion after exposure to 10⁻⁴ M of a nitric oxide donor (NONOate, catalogue No. 82150; Cayman Chemical, Ann Arbor, MI, USA) was measured to evaluate the reactivity of the endometrial explants. The doses of ACh, antagonists and NONOate and the time of incubation were selected based on the findings from preliminary studies or according to methodology in the authors’ previous experiments. After the end of incubation, the endometrial explants were blotted with a paper filter, weighed and stored at −80°C until estimation of 5-LO, LTAH and LTCS protein abundances was carried out. The medium was placed into tubes with a solution of 5% ethylenediaminetetraacetic acid (EDTA, catalogue No. 118798103; Chempur, Piekary Śląskie, Poland), 1% acetylsalicylic acid (catalogue No. 107140422; POCH, Gliwice, Poland) at pH 7.4 and stored at −20°C until determination of LTB4 and LTC4 concentrations was made.

For endometrial explant homogenisation, a buffer with the following composition was used: 50 mM Tris–HCl at pH 7.4, 10 mM EDTA, 150 mM NaCl, 1% Triton X100 with 1 mM pepstatin A, 5 mg/mL of leupeptin, 5 mg/mL of aprotinin and 1 mM phenylmethylsulphonyl fluoride. All reagents were purchased from Sigma-Aldrich. The obtained homogenates were centrifuged for 10 min at 2,500 × g and 4°C. The supernatants were centrifuged for 1 h at 17,500 × g and 4°C, and pellets were frozen at −80°C for enzyme estimation. The total protein concentration was determined (4). Equal 20 μg amounts of protein isolate were dissolved in sodium dodecyl sulphate (SDS, catalogue No. L3771; Sigma-Aldrich) and a gel-loading buffer, heated for 4 min at 95°C and separated by 12% SDS-polyacrylamide gel electrophoresis. The separated proteins were then electroblotted onto 0.45 μm pore size Immobilon-P PVDF membranes (catalogue No. IOVH00010; Sigma-Aldrich) in a transfer buffer. In order to block nonspecific binding sites, incubation was carried out with 5% fat-free dry milk (Spółdzielnia Mleczarska, Gostyń, Poland) in Tris -buffered saline with Tween 20 (respective catalogue Nos T1503 and P1379; Sigma-Aldrich) for 1.5 h at 21°C. Next, the membranes were incubated for 18 h at 4°C with polyclonal rabbit antibodies for 5-LO at 1:200 dilution (catalogue No. 160402; Cayman Chemical Co.), polyclonal rabbit antibody for LTAH at 1:200 dilution (catalogue No. 1600250; Cayman Chemical Co.) and polyclonal rabbit antibody for LTCS at 1:1000 dilution (catalogue No. DF14129; Affinity Biosciences, Changzhou, China). Subsequently, the membranes were incubated for 1.5 h at 21°C with alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies for all enzymes at 1:10,000 dilution (catalogue No. 111-055-003; Jackson ImmunoResearch, West Grove, PA, USA). Protein immune complexes were visualised with a standard alkaline phosphatase method (nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP), catalogue No. 72091; Sigma-Aldrich). Analysis was made three times. To demonstrate the specificity of the primary antibodies, specific binding peptides to 5-LO (catalogue No. 360402; Cayman Chemical Co.) and to LTCS (catalogue no. DF14129-BP; Affinity Biosciences) were used as negative controls (Supplementary Fig. 1). The specificity of the LTAH antibody was previously reported for the porcine uterus (14). Polyclonal rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, at 1:5,000 dilution, catalogue No. G9545; Sigma-Aldrich) was used as an internal control for protein loading. Images were acquired and quantified using a ChemiDoc Imaging System (Image Lab Touch 5.2; Bio-Rad Laboratories, Hercules, CA, USA).

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)

Concentrations of LTB4 and LTC4 in the incubation medium were estimated with ELISA kits (catalogue Nos 502390 and 501070, respectively; Cayman Chemical Co.) according to the manufacturer’s instructions. The standard curve for LTB4 ranged from 1.96 to 1,000 pg/mL, and the assay’s effective concentration for 50% inhibition (IC₅₀) was 2.5 pg/mL. The intra- and interassay coefficients of variation were 4.5% and 7.2%, respectively. The standard curve for LTC4 ranged from 0.98 to 500 pg/mL, and the assay’s IC50 was 1.85 pg/mL. The intra- and interassay coefficients of variation were 4.7% and 6.2%, respectively.

The only results obtained from the incubation of endometrium which were taken into account were those for which the PGF2α release under the influence of NONOate was statistically significant. A two-way analysis of variance (group and treatment) followed by the Bonferroni test (InStat; Graph Pad, San Diego, CA, USA) was applied to compare the mean ± SEM values. Differences with P-values < 0.05 were considered significant.

In the control group endometria, the 5-LO enzyme abundance under the influence of ACh was higher than it was under the influence of M2R, M3R or α-7 nAChR antagonists (P-value < 0.01 for M2R and P-value < 0.05 for M3R and α-7 nAChR) (Table 1). This enzyme’s abundance in the E. coli-challenged endometria in response to ACh was increased versus the antagonists’ influences (P-value < 0.001). A rise in the enzyme abundance was also caused by the exposure of the E. coli group tissue to M2R, M3R and α-7 nAChR compared to the control group (P-value < 0.001 for M2R and P-value < 0.01 for M3R and α-7 nAChR).

In the control and E. coli groups, ACh used alone increased the 5-LO abundances versus the control values obtained from endometria of the control or E. coli groups that had not undergone any in vitro treatment (P-value < 0.01 for the control group and P-value 0.001 for the E. coli group) (Fig. 1A). In the E. coli group, the control value of the protein abundance and the value in response to ACh were higher than the value in the control group (P-value < 0.001). In both groups, when the M2R antagonist was added together with ACh, it did not significantly change the 5-LO abundances in relation to these abundances produced by the ACh action alone (Fig. 1A). In the E. coli group, the enzyme abundance was increased by the M2R antagonist with ACh versus the control group abundance (P-value < 0.001). In the control and the E. coli groups, when the M3R antagonist was added together with ACh, it diminished the 5-LO enzyme abundances compared to the 5-LO abundance produced by ACh alone (P-value < 0.05 for the control group and P-value < 0.001 for the E. coli group) (Fig. 1A). In both groups, the pairing of the α-7 nAChR antagonist with ACh did not significantly alter 5-LO enzyme abundances from their levels when ACh influenced them alone (Fig. 1A). The enzyme abundance was increased by the α-7 nAChR antagonist in concert with ACh in the E. coli group compared to the control group (P-value < 0.001).

The LTAH abundances in the control and E. coli groups were reduced in response to the M2R, M3R and α-7 nAChR antagonists versus the ACh action (P-value < 0.001) (Table 1). In the E. coli group, the enzyme abundance after using the M2R antagonist was higher than in the control group (P-value < 0.01). Acetylcholine without the antagonists increased the LTAH abundances in the control and E. coli groups compared to the control values (P-value < 0.001) (Fig. 1B). In both groups, the enzyme abundances in response to the M2R antagonist with ACh did not differ significantly from the abundance produced by the ACh action singly (Fig. 1B). In both groups, the M3R antagonist with ACh decreased the LTAH protein abundances compared to the effect of ACh alone (P-value < 0.001) (Fig. 1B). In the control group, the LTAH protein abundance in response to the α-7 nAChR antagonist with ACh did not differ significantly from that resulting from the action of ACh in isolation (Fig. 1B). In the E. coli group, the α-7 nAChR antagonist with ACh increased the LTAH enzyme abundance from the level it reached through ACh action (P-value < 0.05). The enzyme abundance in this group was also greater after the α-7 nAChR antagonist and ACh had acted than it was in the control group (P-value < 0.001).

The LTCS abundances in both groups were reduced by M2R, M3R and α-7 nAChR antagonists compared to the ACh action (P-value < 0.001 for M2R and M3R and P-value < 0.01 for α-7 nAChR) (Table 1). In the E. coli group, this enzyme’s abundances after exposure to M2R, M3R and α-7 nAChR antagonists were higher than in the control group (P-value < 0.001 for M2R and M3R and P-value < 0.01 for α-7 nAChR). In the control and E. coli groups, ACh elevated the LTCS abundances over the control values (P-value < 0.01 for the control group and P-value < 0.001 for the E. coli group) (Fig. 1C). In the E. coli group, the control value of LTCS enzyme abundance and the value after using ACh were increased versus the control group value (P-value < 0.001). In the control group, the LTCS abundance after using the M2R antagonist with ACh did not differ significantly from the abundance when only ACh acted (Fig. 1C). The enzyme abundance in the E. coli group was lower under the influence of the M2R antagonist in combination with ACh than under the influence of ACh alone (P-value < 0.001). In the control and E. coli groups, the M3R antagonist with ACh reduced the LTCS enzyme abundances compared to those caused by ACh without the antagonist (P-value < 0.05 for the control group and P-value < 0.001 for the E. coli group) (Fig. 1C). In the E. coli group, higher LTCS abundance resulted from treatment with this combination than the abundance in the control group (P-value < 0.001). In the control group, the LTCS abundance produced by the action of the α-7 nAChR antagonist used with ACh did not differ significantly from what was observed by the action of ACh without the antagonist (Fig. 1C). In the E. coli group, the enzyme abundance was increased by the α-7 nAChR antagonist with ACh from what was caused by the ACh action (P-value < 0.01). After exposure to the α-7 nAChR antagonist with ACh, the LTCS abundance was higher in the E. coli group compared to the control group (P-value < 0.001).

In the control group, the endometrial LTB4 secretion under the influence of the M2R, M3R and α-7 nAChR antagonists was reduced versus the ACh action (P-value < 0.05 for M2R and M3R and P-value < 0.01 for α-7 nAChR) (Table 2).

The LTB4 release in the E. coli group was not as high after exposure to the antagonists for M2R, M3R and α-7 nAChR as it was after exposure to ACh alone (P-value < 0.05 for M2R and P-value < 0.01 for M3R and α-7 nAChR). The M2R antagonist induced more LTB4 release in the E. coli group than in the control group (P-value < 0.05).

The LTB4 release mediated by ACh in both groups was higher than the control values (P-value < 0.05) (Fig. 2A). In the E. coli group, the control value and the value in response to ACh exceeded that of the control group (P-value < 0.01 for the control value and P-value < 0.05 for ACh). The M2R antagonist in concert with ACh did not significantly change the LTB4 release in the control group (Fig. 2A), while in the E. coli group, it did so to diminish LTB4 release from that caused by ACh alone (P-value < 0.01). In both groups, the LTB4 secretion was reduced by the M3R antagonist with ACh versus the ACh action uncombined with the antagonist (P-value < 0.05) (Fig. 2A). In the control group, the LTB4 production mediated by the α-7 nAChR antagonist with ACh did not differ significantly from the production mediated by ACh alone (Fig. 2A). Leukotriene B4 was more heavily secreted from the E. coli group endometrium in response to the α-7 nAChR antagonist with ACh than in response only to ACh. In this group, the LTB4 release triggered by the α-7 nAChR antagonist with ACh was higher than the release in the control group (P-value < 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

In the control group, the LTC4 secretion in response to M2R, M3R and α-7 nAChR antagonists was lower versus the ACh action (P-value < 0.05 for M2R and P-value < 0.01 for M3R and α-7 nAChR) (Table 2). The LT release in the E. coli group by M2R, M3R and α-7 nAChR antagonists was reduced compared to the ACh effect (P-value < 0.05 for M2R and M3R and P-value < 0.01 for α-7 nAChR). After the M3R antagonist was used, the E. coli group endometrium secreted more LTC4 than that of the control group (P-value < 0.05). The LTC4 release by ACh in the control and E. coli groups was higher than the control values (P-value < 0.01 for the control group and P-value < 0.05 for the E. coli group) (Fig. 2B). In the E. coli group, the control value was increased versus the CON group (P-value < 0.05). In neither group did the LTC4 release by the M2R antagonist with ACh change significantly in relation to the influence of ACh alone (Fig. 2B). The LTC4 secretion in the control and E. coli groups was downregulated by the M3R antagonist with ACh compared to the ACh effect in isolation (P-value < 0.01 for the control group and P-value < 0.05 for the E. coli groups) (Fig. 2B). In the control group, the α-7 nAChR antagonist with ACh did not significantly change the LTC4 release from the release triggered by ACh (Fig. 2B). In the E. coli group, the LTC4 activity mediated by the α-7 nAChR antagonist with ACh was higher than that mediated by ACh (P-value < 0.001). The α-7 nAChR–ACh pair elevated the leukotriene’s activity in the E. coli group over this activity in the control group (P-value < 0.001).