Glyphosate-induced changes in the expression of galanin and GALR1, GALR2 and GALR3 receptors in the porcine small intestine wall.

Glyphosate is the active ingredient of glyphosate-based herbicides and the most commonly used pesticide in the world. The goal of the present study was to verify whether low doses of glyphosate (equivalent to the environmental exposure) evoke changes in galanin expression in intramural neurons in the small intestine in pigs and to quantitatively determine changes in the level of galanin receptor encoding mRNA (GALR1, GALR2, GALR3) in the small intestine wall. The experiment was conducted on 15 sexually immature gilts divided into three study groups: control (C)-animals receiving empty gelatin capsules; experimental 1 (G1)-animals receiving a low dose of glyphosate (0.05 mg/kg b.w./day); experimental 2 (G2)-animals receiving a higher dose of glyphosate (0.5 mg/kg b.w./day) orally in gelatine capsules for 28 days. Glyphosate ingestion led to an increase in the number of GAL-like immunoreactive intramural neurons in the porcine small intestine. The results of RT-PCR showed a significant increase in the expression of mRNA, which encodes the GAL-receptors in the ileum, a decreased expression in the duodenum and no significant changes in the jejunum. Additionally, intoxication with glyphosate increased the expression of SOD2-encoding mRNA in the duodenum and decreased it in the jejunum and ileum, but it did not affect SOD1 expression. The results suggest that it may be a consequence of the cytotoxic and/or neurotoxic properties of glyphosate and/or its ability to induce oxidative stress.

Effect of different glyphosate doses on the chemical coding of neurons of the enteric nervous system of the porcine descending colon.

BACKGROUND: Neurons of the enteric nervous system are characterised by high neuronal plasticity, with their number likely to change in response to various endogenous and exogenous substances. MATERIALS AND METHODS: Fifteen sexually immature gilts divided into 3 groups were used: control - animals receiving empty gelatin capsules; G1 - animals receiving a low dose of glyphosate - 0.05 mg/kg bw/day; G2 - animals receiving a higher dose of glyphosate-0.5 mg/kg/day in gelatin capsules orally for 28 days. Frozen sections were then subjected to the procedure of double immunofluorescent staining. RESULTS: With low-dose supplementation, no effect on the SP- and CART-positive neuron population was observed. However, a reduction in the number of VAChT-positive neurons in the internal submucosal plexus was described, while the number of CGRP-positive neurons increased in all enteric plexuses. In response to a high glyphosate dose, the quantitative variability of the neurons was significantly more pronounced than that for a low dose. There was an increase in the number of SP- and CGRP-positive neurons and a decrease in the number of VAChT-positive neurons in both the myenteric plexus and the submucosal plexuses. The response of CART-positive neurons was the weakest, as a high dose of glyphosate led to an increase in the number of neurons only in the myenteric plexus. CONCLUSIONS: The above data show that glyphosate is an exogenous substance that affects neuronal populations of the enteric nervous system, in this case, the descending colon.

Role of Noradrenaline and Adrenoreceptors in Regulating Prostaglandin E2 Synthesis Cascade in Inflamed Endometrium of Pigs.

In the inflamed uterus, the production and secretion of prostaglandins (PGs) and noradrenergic innervation pattern are changed. Receptor-based control of prostaglandin E2 (PGE2) production and secretion by noradrenaline during uterine inflammation is unknown. The aim of this study was to determine the role of α1-, α2- and ß-adrenoreceptors (ARs) in noradrenaline-influenced PG-endoperoxidase synthase-2 (PTGS-2) and microsomal PTGE synthase-1 (mPTGES-1) protein levels in the inflamed pig endometrium, and in the secretion of PGE2 from this tissue. E. coli suspension (E. coli group) or saline (CON group) was injected into the uterine horns. Eight days later, severe acute endometritis developed in the E. coli group. Endometrial explants were incubated with noradrenaline and/or α1-, α2- and ß-AR antagonists. In the CON group, noradrenaline did not significantly change PTGS-2 and mPTGES-1 protein expression and increased PGE2 secretion compared to the control values (untreated tissue). In the E. coli group, both enzyme expression and PGE2 release were stimulated by noradrenaline, and these values were higher versus the CON group. The antagonists of α1- and α2-AR isoforms and ß-AR subtypes do not significantly alter the noradrenaline effect on PTGS-2 and mPTGES-1 protein levels in the CON group, compared to noradrenaline action alone. In this group, α1A-, α2B- and ß2-AR antagonists partly eliminated noradrenaline-stimulated PGE2 release. Compared to the noradrenaline effect alone, α1A-, α1B-, α2A-, α2B-, ß1-, ß2- and ß3-AR antagonists together with noradrenaline reduced PTGS-2 protein expression in the E. coli group. Such effects were also exerted in this group by α1A-, α1D-, α2A-, ß2- and ß3-AR antagonists with noradrenaline on mPTGES-1 protein levels. In the E. coli group, the antagonists of all isoforms of α1-ARs and subtypes of ß-ARs as well as α2A-ARs together with noradrenaline decreased PGE2 secretion versus noradrenaline action alone. Summarizing, in the inflamed pig endometrium, α1(A, B)-, α2(A, B)- and ß(1, 2, 3)-ARs mediate the noradrenaline stimulatory effect on PTGE-2 protein expression, while noradrenaline via α1(A, D)-, α2A- and ß(2, 3)-ARs increases mPTGES-1 protein expression and α1(A, B, D)-, α2A- and ß(1, 2, 3)-ARs are involved in PGE2 release. Data suggest that noradrenaline may indirectly affect the processes regulated by PGE2 by influencing its production. Pharmacological modulation of particular AR isoforms/subtypes can be used to change PGE2 synthesis/secretion to alleviate inflammation and improve uterine function.

Effect of blocking of alpha1-adrenoreceptor isoforms on the noradrenaline-induced changes in contractility of inflamed pig uterus.

BACKGROUND: Disturbances in uterine contractility often lead to the origin, development and maintenance of endometritis and metritis, which are a very common and serious pathologies in domestic animals. Here we aimed to investigate the role of α1A-, α1B- and α1D-adrenoreceptors (ARs) in noradrenaline (NA)-induced contractility of inflammatory-changed porcine uterus. METHODS: On Day 3 of the estrous cycle, either Escherichia coli (E. coli) suspension (E. coli group) or saline (SAL group) was injected into uterine horns, or only laparotomy was performed (CON group). Eight days later, infected gilts developed severe acute endometritis. RESULTS: Compared to the period before NA application, NA reduced the contractile amplitude and frequency in myometrium (MYO) and endometrium (ENDO)/MYO strips from the CON, SAL and E. coli groups. In the last group, the amplitude in MYO and the frequency in ENDO/MYO were lowered versus other groups. After using α1A-ARs antagonist with NA, a greater decrease or occurrence of a drop in the amplitude and frequency in all groups (ENDO/MYO) were found compared to this neurotransmitter action alone. Such results were noted for NA action on the frequency after α1B-ARs blocking in the CON (both kinds of strips) and SAL (ENDO/MYO) groups. In response to α1D-ARs antagonist with NA, a greater decrease or occurrence of a drop in the amplitude was noted in the CON (both kinds of strips) and SAL and E. coli (MYO) groups. Use of these factors caused the similar changes in the frequency in CON and E. coli (MYO) and SAL (ENDO/MYO) groups. In response to NA, α1A,B,D-ARs antagonist led to a greater reduction or appearance of a drop in the amplitude in the CON and SAL (ENDO/MYO) and E. coli (both kinds of strips) as well as in the frequency in the CON and SAL (ENDO/MYO) and E. coli (MYO) groups. CONCLUSIONS: In conclusion, activation of α1A- and α1D-ARs by NA promotes the contractile amplitude and frequency in the inflamed pig uterus; pharmacological modulation of these receptors can be utilized to enhance systolic activity of myometrium.

Effects of Substance P and Neurokinin A on the Contractile Activity of Inflamed Porcine Uterus.

Disturbances in uterine contractile activity contribute to the development of inflammation, and recent evidence indicates that tachykinins, including substance P (SP) and neurokinin A (NKA), are involved in controlling uterine function. Here, we determined the effect of Escherichia coli (E. coli)-induced inflammation on expression of protein receptor subtypes for substance P (NK1R) and neurokinin A (NK2R) in the pig myometrium as well as their role in contractility of inflamed uterus. The severe acute endometritis developed in the E. coli group and the expression of NK1R and NK2R proteins increased in the myometrium. Compared to the pre-administration period, SP (10-6 M) reduced the amplitude and frequency in the myometrium of the E. coli group and the amplitude was higher and the frequency was lower versus other groups. NKA reduced the amplitude and increased the frequency in endometrium/myometrium of the E. coli group. In this group, the amplitude was lower and the frequency was higher than in the CON and SAL groups. Our research showed that NK2R (10-6 M) antagonist application abolished the NKA inhibitory effect on uterine amplitude. The application of the NK1R (10-5 M) antagonist together with SP revealed that the inhibitory effect of SP on uterine contractility is achieved independently of the NKR1. Additionally, taking into account the fact that NKA shows an inhibitory effect with the use of NK2R on uterine amplitude suggests the possibility of therapeutic use of the antagonist as a drug increasing uterine contractility in inflammation.

Involvement of the calcitonin gene-related peptide system in the modulation of inflamed uterus contractile function in pigs.

This study analyzed severe acute endometritis action on myometrial density and distribution of protein gene product (PGP)9.5- and calcitonin gene-related peptide (CGRP)-like immunoreactive nerve fibers and calcitonin receptor-like receptor (CLR) expression, and on CGRP receptor (CGRPR) participation in uterine contractility in pigs. E. coli suspension (E. coli group) or saline (SAL group) were injected into the uteri, or only laparotomy was performed (CON group). In the E. coli group myometrium, a lack of significant changes in PGP9.5 and CGRP innervation patterns and increased CLR protein level were revealed. In all groups, compared to the pretreatment period, human αCGRP increased amplitude in the myometrium, while reducing it in endometrium/myometrium. In the E. coli group endometrium/myometrium, human αCGRP lowered amplitude vs other groups. Human αCGRP reduced frequency in CON and SAL groups and enhanced it in the E. coli group endometrium/myometrium. The frequency in E. coli group increased vs other groups. CGRPR antagonist, human αCGRP8-37, reversed (CON, SAL groups) and eliminated (E. coli group) the rise in human αCGRP-induced myometrial amplitude. In endometrium/myometrium, human αCGRP8-37 abolished (CON group) and reversed (SAL group) a decrease in frequency, and reduced the rise in frequency (E. coli group) caused by human αCGRP. Collectively, in the myometrium, endometritis did not change PGP9.5 and CGRP innervation patterns and enhanced CLR protein level. CGRPR also mediated in CGRP action on inflamed uterus contractility.

Uterine Inflammation Changes the Expression of Cholinergic Neurotransmitters and Decreases the Population of AChE-Positive, Uterus-Innervating Neurons in the Paracervical Ganglion of Sexually Mature Gilts.

The focus of this study was based on examining the impact of endometritis on the chemical coding of the paracervical ganglion (PCG) perikaryal populations supplying pig uterus. Four weeks after the injection of Fast Blue retrograde tracer into uterine horns, either the Escherichia coli (E. coli) suspension or saline solution was applied to both horns. Laparotomy treatment was performed for the control group. Uterine cervices containing PCG were extracted on the eighth day after previous treatments. Subsequent macroscopic and histopathologic examinations acknowledged the severe form of acute endometritis in the E. coli-treated gilts, whereas double-labeling immunofluorescence procedures allowed changes to be analyzed in the PCG perikaryal populations coded with vesicular acetylcholine transporter (VAChT) and/or somatostatin (SOM), vasoactive intestinal polypeptide (VIP), a neuronal isoform of nitric oxide synthase (nNOS), galanin (GAL). The acetylcholinesterase (AChE) detection method was used to check for the presence and changes in the expression of this enzyme and further confirm the presence of cholinergic perikarya in PCG. Treatment with E. coli resulted in an increase in VAChT+/VIP+, VAChT+/VIP-, VAChT+/SOM+, VAChT+/SOM-, VAChT+/GAL- and VAChT+/nNOS- PCG uterine perikarya. An additional increase was noted in the non-cholinergic VIP-, SOM- and nNOS-immunopositive populations, as well as a decrease in the number of cholinergic nNOS-positive perikarya. Moreover, the population of cholinergic GAL-expressing perikarya that appeared in the E. coli-injected gilts and E. coli injections lowered the number of AChE-positive perikarya. The neurochemical characteristics of the cholinergic uterine perikarya of the PCG were altered and influenced by the pathological state (inflammation of the uterus). These results may indicate the additional influence of such a state on the functioning of this organ.

Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs.

Uterine inflammation is a common pathology in animals, leading to disturbances in reproductive processes and reduced production profitability. Pituitary adenylate cyclase-activating peptide (PACAP) effects at the uterine level during inflammation are not known. In the current study, we analyzed the relative PACAP type 1 receptor (PAC1R) mRNA transcript and protein abundances in the myometrium (MYO), as well s PACAP and PAC1R involvement in the contractile function of inflamed pig uterus. To that end, E. coli suspension (E. coli group) or saline (SAL group) was injected into the uterine horns or laparotomy was performed (CON group). Eight days after the bacteria injections, severe acute endometritis and a reduced relative abundance of PAC1R protein in the MYO were observed. Compared to the period before PACAP in vitro administration, PACAP (10-7 M) in the CON and SAL groups decreased in amplitude in the MYO and endometrium (ENDO)/MYO, whereas in the E. coli group, increased amplitude in the MYO and reduced amplitude in the ENDO/MYO were observed. In the E. coli group, PACAP enhanced the amplitude in the MYO (10-7 M) and decreased the amplitude in the ENDO/MYO (10-8 M) compared with other groups. PACAP (10-7 M) increased the frequency of both kinds of strips in the CON and SAL groups compared with the pretreatment period. PACAP (both doses) did not significantly change the frequency in the E. coli group, whereas in response to PACAP (10-7 M), the frequency was reduced compared to other groups. In the MYO, PAC1R antagonist decreased the amplitude reduction (CON and SAL groups) and reversed a rise in PACAP (10-7 M)-evoked amplitude (E. coli group). PAC1R blocking reversed (MYO) and abolished (ENDO/MYO) the stimulatory effect of PACAP (10-7 M) on the frequency (CON and SAL groups). PAC1R antagonist and PACAP (10-7 M) evoked the appearance of frequency depression in both kinds of strips (E. coli group). In summary, in pigs, severe acute endometritis reduces the relative abundance of PAC1R protein in the MYO, and PAC1R mediates the influence of PACAP on inflamed uterus contractility.

Roles of alpha-2-adrenergic receptor isoforms in inflamed pig uterus contractility in vitro.

Noradrenergic control is very significant for the contractility of healthy and inflamed uteri. The receptor mechanisms of noradrenaline (NA) influencing the contractile activity of an inflamed uterus are poorly recognized. This study was undertaken to determine the participation of α2-adrenergic receptors (ARs) isoforms (A, B, C) in NA-evoked contractility of the pig uterus with severe acute endometritis. Saline (SAL group) or E.coli suspension (E.coli group) were injected into uterine horns, while only laparotomy was performed in the CON group. In relation to the period before NA application, NA decreased the tension, amplitude and frequency in the uterine strips of the CON and SAL groups, and the amplitude and frequency in the E.coli group. In the E.coli group, the amplitude and frequency were lower than in other groups. Compared to NA effect alone, a greater reduction or appearance of a decrease in the amplitude and frequency were noted in all groups following the use of selective α2A- and α2C-ARs antagonists together with NA as well as in the tension in the CON and SAL groups in response to an α2C-ARs antagonist and NA. Such effects were also exerted by an α2B-ARs antagonist with NA on the frequency in all groups and on the amplitude in the CON and SAL groups. To sum up, in an inflammatory-changed porcine uterus, three α2-AR isoforms mediate the effect of NA on contractile frequency, while α2A- and α2C-AR are involved in the control of contractile amplitude. These results could offer new targets for drugs against decreased uterine contractility in inflammation.

Effect of NSAIDs Supplementation on the PACAP-, SP- and GAL-Immunoreactive Neurons in the Porcine Jejunum.

Side effects associated with nonsteroidal anti-inflammatory drugs (NSAIDs) treatment are a serious limitation of their use in anti-inflammatory therapy. The negative effects of taking NSAIDs include abdominal pain, indigestion nausea as well as serious complications such as bleeding and perforation. The enteric nervous system is involved in regulation of gastrointestinal functions through the release of neurotransmitters. The present study was designed to determine, for the first time, the changes in pituitary adenylate cyclase-activating polypeptide (PACAP), substance P (SP) and galanin (GAL) expression in porcine jejunum after long-term treatment with aspirin, indomethacin and naproxen. The study was performed on 16 immature pigs. The animals were randomly divided into four experimental groups: control, aspirin, indomethacin and naproxen. Control animals were given empty gelatin capsules, while animals in the test groups received selected NSAIDs for 28 days. Next, animals from each group were euthanized. Frozen sections were prepared from collected jejunum and subjected to double immunofluorescence staining. NSAIDs supplementation caused a significant increase in the population of PACAP-, SP- and GAL-containing enteric neurons in the porcine jejunum. Our results suggest the participation of the selected neurotransmitters in regulatory processes of the gastrointestinal function and may indicate the direct toxic effect of NSAIDs on the ENS neurons.

Profile for mRNA transcript abundances in the pig endometrium where inflammation was induced by Escherichia coli.

Uterine inflammation is a common reproductive disorder in domestic animals, leading to disturbances in many reproductive processes and economic losses. More information on inflammatory pathways, however, is needed to understand mechanisms of uterine inflammation. The aim of the study was to investigate transcriptomic profiles of the pig endometrium affected by inflammation. On day 3 of the estrous cycle (day 0 = initial day of study), saline or Escherichia coli suspension were injected into uterine horns. In endometrial tissues collected 8 days later, microarray analysis results indicated there were 189 differentially abundant mRNA transcripts (DEGs, 95 in relatively greater and 94 in lesser abundance) after saline injections compared with samples where there was severe acute inflammation. Relative abundance of mRNA transcripts for proteins assigned to inflammatory response, movement of phagocytes, quantity of phagocytes, leukocyte migration and adhesion of immune cells and many other functions related to inflammation were different in the Escherichia coli-treated endometrium than in samples from gilts treated with saline. Among others, S100A9, SLC11A1, CCL15, CCL3L3, CCR1, CD48, CD163, THBS1, KIT, ITGB3, JAK3 and NFKB2 mRNA transcripts were in relatively greater abundance and there were those in relatively lesser abundance including IL24, FGG, SST, CXCL16 and CREB. In this study, for the first time, there was detection of alterations in the transcriptome of the inflamed pig endometrium which may be an important finding for maintaining uterine homeostasis and functions. Results form the basis for future studies focusing on regulation of uterine inflammation in animals and women.

Endometritis decreases the population of uterine neurons in the paracervical ganglion and changes the expression of sympathetic neurotransmitters in sexually mature gilts.

BACKGROUND: The focus of the study was to examine the impact of the inflamed uterus on the population of the paracervical ganglion (PCG) uterus-innervating perikarya and their chemical coding. Fast Blue retrograde tracer was injected into the wall of uterine horns on the 17th day of the first studied estrous cycle. After 28 days, either Escherichia coli suspension or saline was applied to the horns of the uterus, whereas the control group received laparotomy only. Eight days after the above-mentioned procedures, uterine cervices with PCG were collected. Both macroscopic and histopathologic examinations confirmed severe acute endometritis in the Escherichia coli-injected uteri. The double immunofluorescence method was used to analyze changes in the PCG populations coded with dopamine-ß-hydroxylase (DßH) and/or neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP) and neuronal isoform of nitric oxide synthase (nNOS). RESULTS: The use of Escherichia coli lowered the total number of Fast Blue-positive neurons. Moreover, an increase in DßH+/VIP+, DßH+/NPY+, DßH+/SOM + and DßH+/nNOS + expressing perikarya was noted. A rise in non-noradrenergic VIP-, SOM- and nNOS-immunopositive populations was also recorded, as well as a drop in DßH-positive neurotransmitter-negative neurons. CONCLUSIONS: To sum up, inflammation of the uterus has an impact on the neurochemical properties of the uterine perikarya in PCG, possibly affecting the functions of the organ.

Regulatory Influence of Galanin and GALR1/GALR2 Receptors on Inflamed Uterus Contractility in Pigs.

Uterine inflammation is a very common and serious pathology in domestic animals, the development and progression of which often result from disturbed myometrial contractility. We investigated the effect of inflammation on the protein expression of galanin (GAL) receptor subtypes (GALR)1 and GALR2 in myometrium and their role in the contractile amplitude and frequency of an inflamed gilt uterus. The gilts of the E. coli and SAL groups received E. coli suspension or saline in their uteri, respectively, and only laparotomy was performed (CON group). Eight days later, the E. coli group developed severe acute endometritis and lowered GALR1 protein expression in the myometrium. Compared to the pretreatment period, GAL (10-7 M) reduced the amplitude and frequency in myometrium and endometrium/myometrium of the CON and SAL groups, the amplitude in both stripes and frequency in endometrium/myometrium of the E. coli group. In this group, myometrial frequency after using GAL increased, and it was higher than in other groups. GALR2 antagonist diminished the decrease in amplitude in myometrium and the frequency in endometrium/myometrium (SAL, E. coli groups) induced by GAL (10-7 M). GALR1/GALR2 antagonist and GAL (10-7 M) reversed the decrease in amplitude and diminished the decrease in frequency in both examined stripes (CON, SAL groups), and diminished the drop in amplitude and abolished the rise in the frequency in the myometrium (E. coli group). In summary, the inflammation reduced GALR1 protein expression in pig myometrium, and GALR1 and GALR2 participated in the contractile regulation of an inflamed uterus.

Role of beta-adrenergic receptor subtypes in pig uterus contractility with inflammation.

Uterine inflammation is a very common and serious condition in domestic animals. To development and progression of this pathology often lead disturbances in myometrial contractility. Participation of ß1-, ß2- and ß3-adrenergic receptors (ARs) in noradrenaline (NA)-influenced contractility of the pig inflamed uterus was studied. The gilts of SAL- and E.coli-treated groups were administered saline or E.coli suspension into the uterine horns, respectively. Laparotomy was only done in the CON group. Compared to the period before NA administration, this neurotransmitter reduced the tension, amplitude and frequency in uterine strips of the CON and SAL groups. In the E.coli group, NA decreased the amplitude and frequency, and these parameters were lower than in other groups. In the CON, SAL and E.coli groups, ß1- and ß3-ARs antagonists in more cases did not significantly change and partly eliminated NA inhibitory effect on amplitude and frequency, as compared to NA action alone. In turn, ß2-ARs antagonist completely abolished NA relaxatory effect on these parameters in three groups. Summarizing, NA decreases the contractile amplitude and frequency of pig inflamed uterus via all ß-ARs subtypes, however, ß2-ARs have the greatest importance. Given this, pharmacological modulation of particular ß-ARs subtypes can be used to increase inflamed uterus contractility.

Alterations in the relative abundance of the vasoactive intestinal peptide receptors (VPAC1 and VPAC2) and functions in uterine contractility during inflammation.

Vasoactive intestinal peptide (VIP) receptor (VPAC1, VPAC2) abundances in the myometrium and functions in the regulation of inflamed uterine contractility in pigs were studied. In the CON group with gilts, only laparotomy was performed. The gilts of SAL- and E. coli-treated groups were administered saline or E. coli into the uterine horns, respectively. The E. coli-induced endometritis resulted in a lesser myometrial relative abundance of VPAC1 and VPAC2 receptor mRNA transcripts and larger abundance of protein for these receptors. In the myometrium, treatment with VIP resulted in a lesser contractility amplitude than in the tissues of the CON- and SAL- and E. coli-treated groups and in frequency in the CON- and E.coli-treated group compared to the period before VIP treatment. Compared to when there was VIP treatment alone, treatment with VPAC1 and VPAC2 receptor antagonists resulted in a lesser inhibitory effect of VIP on contractility amplitude in the myometrium of the CON and SAL-treated groups and there was complete abolishment of the inhibitory VIP effect on frequency of myometrial contractility of the CON group. In the myometrium of E. coli-treated group, treatment with VPAC1 and VPAC2 receptor antagonists resulted in a reversal of the inhibitory effect of VIP on contractility amplitude, while treatment with VPAC2 receptor antagonist resulted in elimination of contractility and a lesser endometrium/myometrium inhibitory effect of VIP on frequency of these contractions. Results indicate VIP functions to decrease myometrial contractility of the inflamed pig uterus by having functions at VPAC1 and VPAC2 receptors.

Escherichia Coli -induced Inflammation Changes the Expression of Acetylcholine Receptors (M2R, M3R, and α-7 nAChR) in the Pig Uterus.

INTRODUCTION: The influence of inflammation on the patterns of muscarinic 2 and 3 receptor subtypes (M2R and M3R), and α-7 nicotinic acetylcholine receptor (α-7 nAChR) expression in the porcine uterus was investigated. MATERIAL AND METHODS: On day three of the oestrous cycle of gilts aged 7-8 months with body weight 90-120 kg, either an E. coli suspension (E. coli group, n = 5) or saline (Sal group, n = 5) was administered into the uterine horns via laparotomy or only laparotomy was performed on control swine (Ctrl group, n = 5). After eight days, and the onset of severe acute endometritis in the E. coli group, the uterine mRNA and protein receptor expression levels were determined using real-time RT-PCR and Western blotting, respectively, with receptor localisation by immunofluorescence. RESULTS: The studied receptors were in the luminal epithelium, glands, blood vessels, and myometrial muscle cells of all gilts. The M2R mRNA level was lower in the inflamed endometrium compared to the Ctrl and Sal groups. Also in this tissue, the expression of M3R mRNA and protein was lower than in the Ctrl and Sal groups. The M3R protein level in the bacterially challenged myometrium was found to be increased compared to unadministered groups. In the endometrium of the E. coli group, the α-7 nAChR protein level was lower than in the Sal group, and in the myometrium it was reduced in relation to both the other groups. P values were ≤ 0.05 in all cases. CONCLUSION: Inflammation causes alterations in the M2R, M3R, and α-7 nAChR expression in the pig uterus, suggesting their significance in the course and repercussions of uterine inflammation.

The role of somatostatin and its receptors (sstr2, sstr5) in the contractility of gilt inflamed uterus.

We studied the inflammation effect on somatostatin receptors subtypes 2 (sstr2) and 5 (sstr5) expression in myometrium and somatostatin influence alone or with sstr2 and sstr5 antagonists on the contractility of gilt inflamed uterus. On day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) were injected into uterine horns. In the control pigs (CON group), only laparotomy was performed. Eight days later, in the E.coli group developed severe acute endometritis. In this group, myometrial sstr2 mRNA expression lowered and protein expression increased compared to other groups. Compared to period before somatostatin administration, somatostatin did not change tension in myometrium and endometrium/myometrium of three groups, reduced amplitude and frequency in the CON and SAL groups, and increased amplitude and decreased frequency in the E.coli group. In this group, amplitude was increased by somatostatin compared to other groups. In the CON and SAL groups, sstr2 eliminated inhibitory somatostatin effect on amplitude, while sstr5 antagonist reversed inhibitory somatostatin effect on amplitude. In the E.coli group, sstr2 antagonist reversed stimulatory somatostatin effect on amplitude, while in sstr5 antagonist presence stimulatory somatostatin effect was more deepened compared to somatostatin action alone. After using sstr2 antagonist more deepened inhibitory somatostatin effect on frequency in the CON and E.coli groups was found. Sstr5 antagonist partly eliminated inhibitory somatostatin effect on frequency in the SAL group. Summarizing, the uterine inflammation increases the myometrial sstr2 protein expression; somatostatin raises amplitude of the inflamed uterus acting by sstr2, while drops this parameter by sstr5.

Inflammation changes the expression of neuropeptide Y receptors in the pig myometrium and their role in the uterine contractility.

In the experiment the influence of inflammation on neuropeptide Y (NPY) receptor subtype 1 (Y1Rs) and 2 (Y2Rs) expression pattern in the gilt myometrium and NPY effect alone or with Y1Rs and Y2Rs antagonists on contractility of an inflamed uterus were recognized. On Day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) were administered into uterine horns. In the control gilts (CON group), only laparotomy was carried out. Eight days later, E.coli treatment evoked severe acute endometritis, significantly reduced Y1Rs mRNA and protein expression and increased Y2Rs protein expression in myometrium in relation to the CON and SAL groups. Compared to period before NPY application, NPY (10-7 M) significantly reduced amplitude in myometrium and endometrium/myometrium and frequency in myometrium of the CON and SAL groups and amplitude in endometrium/myometrium and frequency in strips of the E.coli group. In this group, after using NPY (10-7 M), the amplitude rose significantly in both kinds of strips, while frequency fell in endometrium/myometrium in relation to the SAL group. In the CON and SAL groups, NPY (10-7 M) with Y1Rs antagonist significantly increased the amplitude in endometrium/myometrium, while with Y2Rs antagonist caused a rise in myometrium. In the E.coli group after using Y1Rs or Y2Rs antagonist and NPY (10-7 M) amplitude did not significantly change in endometrium/myometrium, and this parameter was significantly lower after using the NPY and Y1Rs antagonist than in other groups. Y1Rs antagonist and NPY (10-8, 10-7 M) significantly increased frequency in endometrium/myometrium of the CON and SAL groups. In the E.coli group, Y1Rs or Y2Rs antagonists and NPY (10-7 M) significantly increased frequency in myometrium and it was significantly higher than in the CON group. Inflammation changes Y1Rs and Y2Rs expression in myometrium of the inflamed pig uterus and NPY reduces this organ contractility by Y1Rs and Y2Rs.

Endometritis Changes the Neurochemical Characteristics of the Caudal Mesenteric Ganglion Neurons Supplying the Gilt Uterus.

This study analyzed the influence of uterine inflammation on the neurochemical characteristics of the gilt caudal mesenteric ganglion (CaMG) uterus-supplying neurons. The horns of uteri were injected with retrograde tracer Fast Blue on day 17 of the first studied estrous cycle. Twenty-eight days later (the expected day 3 of the third studied estrous cycle), either saline or Escherichia coli suspension were administered into each uterine horn. Only the laparotomy was done in the control gilts. After 8 days, the CaMGs and uteri were harvested. The infected gilts presented a severe acute endometritis. In the CaMGs, the populations of uterine perikarya possessing dopamine-ß-hydroxylase (DßH) and/or neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL) and vasoactive intestinal polypeptide (VIP) were analyzed using the double immunofluorescence method. In the CaMG, bacterial injection decreased the total number of the perikarya (Fast Blue-positive), the small and large perikarya populations in the dorsal and central regions, and the small and large perikarya populations coded DßH+/GAL- and DßH-/NPY+. After bacterial treatment, there was an increase in the numbers of small and large perikarya coded DßH+/NPY+, small perikarya coded DßH+/GAL+ and DßH+/SOM- and large perikarya coded DßH+/VIP+. To summarize, uterine inflammation influences the neurochemical characteristics of the CaMG uterus-supplying neurons, which may be important for pathologically changed organ functions.