Materials and methods
we used 38 adult male Wistar rats weighing 250–320 g in the experiments. The requirements set out in Directive 86/609/EEC for accommodation and care of experimental animals were fully met. The animals were housed under standard living conditions: temperature 23±1°C, change of light and dark – 12/12 hours, relative humidity about 45%. Approximately 12 hours before the start of experiments, the rats were separated from food.
We obtained permission for the study from the Bulgarian Food Safety Agency: Permission No. 116 for the use of animals in experiments. The experimental work was approved by the Commission for Scientific Ethics at the Medical University of Plovdiv with protocol No. 2/13.06.2019.
Smooth muscle preparations
SM preparations were excised in situ, separating the muscle tissue without mucosa as follows: a section with a length of about 3-4 cm was previously isolated from a proximal colon (2.5 cm after caecum). The longitudinal preparations of the colon used to record the contractile activity were 15-18 mm long and 1.0-1.1 mm wide. The circular intestinal strips had a length equal to the circumference of the intestinal wall in the respective region of the tract and a width of 1.0-1.2 mm. During dissection, SM tissues were washed with a preparation solution containing NaCl/ KCl/CaCl2 in the ratio of 27.2:1.1:1.
During the experiment, the SM preparations were fixed in a tissue bath with Krebs solution (pH=7.4, t°=37°C) with the following content (mmol/L): NaCl – 120; KCl – 5.9; CaCl2 – 2.5; MgCl2 – 1.2; NaH2PO4 – 1.2; NaHCO – 15.4, and glucose – 11.5. All chemicals used to make the solution are manufactured by Merck. The pH of the solution was measured using a pH meter (HANNA). Krebs solution in contact with the SM preparations was aerated continuously during the experiment with a gas mixture O2/CO2 in the ratio of 19/1 (v/v).
Treatment of muscle preparations
Isolated SM were exogenously treated with ACh, 5-HT, dopamine, and norepinephrine by adding a precisely defined volume of concentrated solution of the respective substance necessary to achieve the desired concentration in the tissue bath (1.10-6 mol/L). The volume did not exceed 1/100 of the volume of the solution in the tissue bath. The vitality of SM tissue was tested by exposure to 1.10-6 mol/L ACh at the beginning of each experiment twice, after adaptation period of 60 minutes.
Assay of mechanical activity of smooth muscle preparations
Mechanical activity was recorded isometrically quantifying the contractile reactions in mN. The SM preparations were fixed to a glass holder at one end and to Swema strain gauges (Stockholm, Sweden) at the other by surgical sutures, with the gauges converting the mechanical deformation produced by contractile activity into a proportional electrical signal.
The initial mechanical stress for the preparations achieved by tensioning had a value corresponding to a force of 10 mN. The adaptation period to establish a baseline level of tone and regular spontaneous contractile activity was 60 minutes during which time the Krebs solution was changed two or three times. Changes in spontaneous mechanical activity and tone caused by exposure to various substances were reported relative to the corresponding baseline. Strength and frequency of phasic activity were defined as the mean of a large number of consecutive contractions (about 10).
The electrical signal from the strain gauges was amplified by K. Tesar-D 486 (Germany). The recording of mechanical activity on paper tape was performed using a Linseis recorder (Selb, Germany).
Assay of smooth muscle tissue response to <italic>Clostridium difficile</italic> toxins
Pre-dissolved in distilled water, the toxins were added to the Krebs solution (at standard conditions) in the tissue bath at the appropriate volume ratio to achieve the required concentration of TcdA – 1.10-8 mol/L and TCdB – 1.10-8 mol/L. After that, the incubation lasted 3 hours, and the incubation medium was replaced with fresh Krebs solution.
Presentation of results and statistical processing
The results obtained from the experiments were statistically analyzed using STATISTICA 12.0 (StatSoft, Tulsa, Oklahoma). The analysis was performed on groups of independent or dependent variables determined by the type of experiment. The final values were presented as mean ± standard error. The value of Student’s coefficient p<0.05 is accepted as criterion for significant difference.
Discussion
Because the effects of Clostridium difficile toxins on smooth muscle contractility are a component of their overall impact on clinical phenomena, the possible mechanisms of affecting the gastrointestinal tract are of critical importance. The effects on intestinal muscle involved in the formation of segments along the small intestine (assuming that the contractility of the longitudinal muscles is not affected) should cause a change in the frequency and amplitude of segmentation. The above would lead to some disruption of the synchrony of movement of intestinal contents. This would inevitably cause atypical retention of intestinal contents in some areas and premature evacuation of others, combined with difficulty in homogenizing the masses in the intestinal lumen.[6]
The obtained results showed significant differences in the magnitude of the contractile responses to ACh (main parasympathetic regulator and major testing agent for muscle contractile vitality) in SM preparations incubated with TCdA compared to the control ones, while no differences were found in the reactions of the preparations treated with TCdB compared to the control ones. The contractile effects caused by ACh in the SM of gastrointestinal tract are mainly the result of activation of the M3-cholinergic receptors.[7] Thus, the increase in SM tone is due to the release of Ca2+ from intracellular depots.[8, 9] The observed increase in contractile response in colon preparations incubated with TCdA could be a consequence of the SM membrane depolarization observed by Gilbert upon action with TcdA. [10] In the causal aspect, this is related to the enhancement of Ca2+ influx, provoked indirectly by exogenously applied ACh. An argument in favor of our hypothesis is the reported increase in carbachol-induced contractions in SM treated with TCdA.[10] The above data suggest the presence of prerequisites for enhanced sensitivity of the muscles of the gastrointestinal tract to the regulatory function of ACh.
The lack of significant changes in ACh-induced responses in TCdB-incubated preparations should be analyzed on the background of data existing[11], namely: significant likelihood of TcdB inactivating Rho proteins involved in modulation of the contractile apparatus; regulating the activity of phospholipase D, with a negligible effect on the activity of protein kinase C as well as pronounced inhibitory effect on muscarinic receptor activity.[12]
The tests to determine the effect of TCdA and TCdB on 5-HT mediation were performed according to the same scheme as in the experiments with ACh. It was established that circular SM colon preparations demonstrated resistance to 5-HT-induced reactions in the presence of TCdA and TCdB. Rise of contractile reaction in the presence of TCdA was observed in longitudinal preparations.
Enteroendocrine cells are known to act as pressure sensors secreting 5-HT that initiates peristaltic reflexes.[13] The contraction-relaxation processes provoked by 5-HT through interneuronal interactions involve acetylcholine, substance P, nitric oxide (NO), vasoactive intestinal peptide (VIP), and calcitonin gene-regulating peptide.[14] The activation of Rho-protein and Rho-kinase is most probably a leading factor in the generation of 5-HT-induced contractions.[15] Meanwhile, a major regulatory link in the contractility chain – protein kinase C, plays a minor role in the development of these processes.[15] Our observations in this aspect contradict Lucius’ hypothesis about the negative effect of 5-HT on the contractile activity of the longitudinal intestinal muscles in experimental animals.[16]
Gastrointestinal expression and function of 5-HT receptors are specific on different levels of the gastrointestinal tract in rats. 5-HT7-receptor activation causes relaxation of SM probably through activation of cyclic nucleotides, whereas 5-HT2B-receptors mediate the contractions. The other expressed type – 5-HT4-receptor is likely to participate in both inhibition and activation of SM. Parallel neural and myogenic processes are involved in above mentioned effects.[17]
It remains unclear why the contractile reactivity of the longitudinal muscles in the area of the proximal colon is affected by TCdA intoxication while the contractile reactivity of the circular ones is not. It is likely that there are differences in the interneuronal interactions of serotonergic mediation leading to the release of ACh, substance P, NO, and VIP in the submucosal, and myenteric intramural plexuses.[3]
In contrast to the contractile character of effects caused by ACh and 5-HT, exogenously administered dopamine relaxes the SM of the colon. The magnitude of the effect is different in circular and longitudinal preparations; it is significantly weaker reaction in the latter. Applied to isolated SM from rat colon, dopamine induces a relaxation, which at high concentrations (10-4 mol/L) is accompanied by a complete loss of spontaneous contractile activity.[10] The effect is not related to direct activation of dopamine receptors but is probably mediated by β-adrenoceptors.
According to other evidence of a noncholinergic neuronal origin of its relaxation effect, dopamine inhibits SM contractions in the rat colon by activating β1-adrenoceptors on intramural plexuses and by β2-receptors expressed on SM itself.[18] Analogous experiments have shown that the inhibitory action of dopamine involves inhibition of ACh release from enteric neurons mediated by D1 and D2-receptors, as well as influencing NO activity and purinergic mediation. [19] Probably the dominant role in these processes is played by activation of adenylate cyclase and consequent increase in cyclic adenosine monophosphate concentration.
The lack of influence of TCdA and TCdB on dopamine-induced relaxations in our experiments allows some speculations: on the one hand, pronounced “non-competitiveness” in the involvement of the adenylate cyclase mechanism of toxins and catecholamines, and on the other, inability to stop relaxing SM processes due to the wide range of receptor and interneuronal interactions that have been described, dopamine activates at the enteric plexus.
We do not rule out that preservation of the relaxation effect caused by dopamine is the result of membrane hyperpolarization[20] of SM cells of the colon (concomitant enhanced K+ current), which in itself turns the effect independent of specific intracellular contractile mechanisms. It was commented above that activation of β-adrenoceptors is one of the pathways leading to suppression of SM activity in the colon wall. The effect is due to increased synthesis of cyclic adenosine monophosphate, activation of protein kinase A, and blocking the ability of myosin light-chain kinase to initiate contractions.
As a neuronal regulator of enteric contractile activity, the sympathetic part of the autonomic nervous system interacts in a complex pattern, both with the parasympathetic part and with the structures of the nonadrenergic and noncholinergic innervating systems. An important element of that interaction at the rat colon level is the antagonism of noradrenaline and VIP activity.[21] In addition, noradrenaline and adrenaline have been shown to significantly reduce the effectiveness of ACh in the longitudinal intestinal layer excitation. The effect is receptor-mediated (α-adrenoceptors), and it does not involve dopamine, the other catecholamine that is important for the motility.[22]
As commented above, conditions such as colitis, dysbacteremia, gastrointestinal intoxications (including Clostridium difficile intoxication), and infections affect in a similar way certain units and agents of the SM contractile system of the tract, such as VIP and ACh. A study using a model of chemically-induced colitis[23] demonstrated a significant increase in acetylcholinesterase and VIP levels in the SM wall of the colon in rats.
Our experimental data related to the effect of exogenous norepinephrine on isolated SMs show a weak relaxation effect in both circular and longitudinal preparations. Preliminary treatment with TcdA and TcdB did not result in changes in responses, except in the case of a longitudinal colon preparation treated with TcdB in which no reaction at all was present.
An acceptable explanation for this is the above-mentioned complex intraplex interactions of sympathetic innervation, as well as the fact that the presence of norepinephrine is associated with a decrease in VIP efficacy, while when the intestinal function is impaired, the peptide levels are significantly elevated. Such antagonism of influences can serve to extrapolate the direction of development of contraction and relaxation processes. In purely quantitative terms, it should be noted that exogenously administered norepinephrine is about four times less active than adrenaline.[22]