The therapeutic potential of ribonucleolytic enzymes as antitumor and antiviral agents is well known. Currently, the cytotoxic and cytostatic properties of RNase A from bovine pancreas, ranpirnase (onconase), its analog amphinase from frog oocytes, and binase from Bacillus pumilus are being intensively studied. However, data on the side effects of RNases are currently insufficient. Detection of the genotoxic effect of RNases is important to justify the prospects for using RNases in antitumor therapy. Identification of possible changes in contractile activity of an isolated segment of the mouse colon, under the influence of RNases may also contribute to the justification of their subsequent application. Both of these indicators characterize frequent consequences of cancer radiotherapy and chemotherapy. In this regard, the aim of this work was to determine the clastogenic and/or aneugenic effect of RNase A and binase in in vivo micronucleus test and evaluate their effect on contractions of mouse proximal colon segments. It has been shown that RNases in therapeutic concentrations are not genotoxic and do not cause changes in spontaneous and carbochol-induced intestinal contractility, which indicates the safety of their use in the treatment of oncological diseases.

Irritable bowel syndrome (IBS) is a multifactorial disorder, with altered intestinal motility, visceral hypersensitivity, and dysfunction of the gut-brain axis. The aim of our study was to analyze the role of nitric oxide (NO) in the inhibitory effects of sodium butyrate on spontaneous contractility of proximal colon in a mouse model of IBS. IBS was induced by intracolonic infusion of acetic acid in the early postnatal period. Spontaneous contractions of proximal colon segments were studied in isometric conditions. The amplitude and frequency of colon contractions were higher in the IBS group. Sodium butyrate exerted inhibitory effects on colon contractions, which were less pronounced in IBS group. NO donors decreased spontaneous colon contractility and prevented the inhibitory effects of sodium butyrate in control and IBS groups. Nitric oxide synthase (NOS) inhibition by L-NAME increased contractile activity more effective in the control group and decreased the inhibitory action of sodium butyrate. In IBS group, preliminary application of L-NAME did not prevent sodium butyrate action. Our data indicate that butyrate exerts its inhibitory effects on colon motility at least partially through activation of NO synthesis. In the IBS model group, the NO-dependent mechanisms were less effective probably due to downregulation of NOS.

Increasing evidence suggests that the gut microbiota, through the “microbiota–gut–brain axis”, can regulate anxiety, mood, and cognitive abilities such as memory and learning processes. Consistently with this, treatments altering the gut microbiota, such as antibiotics and probiotics, may influence brain function and impact behavior. The mechanisms that underlie the interplay between the intestinal microbiota and the brain have been intensively studied. We aimed to investigate the effects of two probiotic lactobacilli strains, Lacticaseibacillus rhamnosus 12L and Lactiplantibacillus plantarum 8PA3, on behavioral disorders in mice induced by a two-week parenteral treatment with broad-spectrum antibiotics. On completion of the treatment, the mice were subjected to behavioral tests, including the open field test (OFT), novel object recognition test (ORT), and T-maze test. Antibiotic-treated mice demonstrated anxiety-related behavior, decreased cognition, and retarded exploratory activity that were ameliorated by the administration of probiotics. As was determined by high-performance liquid chromatography (HPLC), both tested strains produced serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), as well as dopamine, which was further metabolized into norepinephrine by L. plantarum 8PA3 and epinephrine by L. rhamnosus 12L. Moreover, these lactobacilli were found to harbor catecholamines and 3,4-dihydroxyphenylacetic acid (DOPAC) in their biomass when grown on MRS broth. Additionally, L. plantarum 8PA3 and L. rhamnosus 12L were able to impact oxidative stress via H2O2 production and antioxidant activity, as determined in this study by the ferrous oxidation–xylenol orange (FOX) assay and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, respectively. The results obtained in this study support the role of probiotics as a promising therapeutic for neurological disorders. However, more investigations are required to confirm the clinical significance of this finding.

In this study we investigated the role of voltage dependent (K V ), Ca 2+ -activated (K Ca ), and inward rectifier (K ir ) potassium channels in the effects of hydrogen sulfide donor (H 2 S) sodium hydrosulfide (NaHS) on spontaneous contractile activity of rat jejunum. It was shown that NaHS dose-dependently (10–500 μM) reduced the tonus of the preparation, as well as the amplitude and frequency of spontaneous contractions of jejunum preparations under isometric conditions; the half-maximal effective concentration (EC 50 ) of the inhibitory effect of NaHS on the amplitude of contractions was 165 μM. The blocker of K V channels 4-AP (200 μM) caused an increase in the amplitude of spontaneous contractions. NaHS (200 μM) decreased the amplitude and frequency of spontaneous activity of the preparation in the presence of 4-AP as well as in the control, and the effect on basal tonus was less pronounced. Blockers of large conductance K Ca channels (BK), non-specific TEA (3 mM) and specific paxillin (1 μM), increased the amplitude of spontaneous contractions, while the depressing effect of NaHS was completely preserved. The selective blocker of small conductance K Ca channels (SK) NS8593 (4 μM) did not affect the tonus of the preparation and the parameters of spontaneous contractions; it did not prevent the effect of NaHS. The activator of K ATP channels diazoxide (100 μM) caused a decrease in the basal tonus of the preparation, as well as the amplitude and frequency of spontaneous contractions. Diazoxide and the K ATP channel blocker glibenclamide (50 μM) prevented the effect of NaHS on the tonus of the preparation. BaCl 2 , the K ir channel blocker (30 μM), caused an increase in the amplitude of spontaneous contractions and prevented the development of the NaHS inhibitory effects on the frequency and amplitude of spontaneous contractions; the decrease in tonus was less pronounced than in the control. Thus, a decrease in the basal tonus of the rat jejunum preparation under the action of the H 2 S donor was associated with activation of K ir channels, including K ATP channels, whereas the effect of H 2 S on amplitude and frequency was mediated by an increase in Ba 2+ -sensitive conductivity.

Nitric oxide is one of the endogenous molecules that play a key role in migraine. However, the interaction between NO and the main players in the nociceptive activity of the meningeal trigeminal afferents-TRPV1 and P2X3 receptors-remains unstudied. In the current project, the effects of acute and chronic NO administration on the activity of TRPV1 and P2X3 receptors in the peripheral afferents were studied using electrophysiological recording of action potentials of the trigeminal nerve in the rat hemiskull preparations. The data obtained indicate that exogenous and endogenous NO increased the activity of the trigeminal nerve independent on the inhibition of the TRPV1 and P2X3 receptors. The activity of the trigeminal nerve triggered by ATP changed neither in acute incubation in the NO donor-sodium nitroprusside (SNP) nor in the chronic nitroglycerine (NG)-induced migraine model. Moreover, the chronic NG administration did not increase in the number of degranulated mast cells in the rat meninges. At the same time, the capsaicin-induced activity of the trigeminal nerve was higher with chronic NO administration or after acute NO application, and these effects were prevented by N-ethylmaleimide. In conclusion, we suggested that NO positively modulates the activity of TRPV1 receptors by S-nitrosylation, which may contribute to the pro-nociceptive action of NO and underlie the sensitization of meningeal afferents in chronic migraine.

In this work, we analyzed the role of voltage-gated (KV), calcium-activated (KCa), and inward-rectifier potassium channels (Kir) in the effects of hydrogen sulphide (H2S) donor sodium hydrosulphide (NaHS) on the spontaneous contractile activity of the rat jejunum. Experiments were performed on jejunum segments under isometric contraction conditions. It was shown that NaHS reduced the basal tension of the segments, the amplitude, and the frequency of spontaneous contractions in a dose-dependent manner (10–500 μM); the half-effective concentration (EC50) of the inhibitory effect of NaHS on amplitude was 165 μM. The KV channel blocker 4-AP (200 µM) increased the amplitude of spontaneous contractions and subsequent application of NaHS (200 μM) suppressed the amplitude and frequency of spontaneous activity as well as in the control; the effect on tonic tension was less pronounced. TEA (3 mM), a non-specific blocker, and paxillin (1 µM), a specific blocker of large conductance KСа (ВK) channels, increased the amplitude of spontaneous contractions, while the inhibitory effect of NaHS was completely preserved. The selective blocker of small conductance KCa (SK) channels NS8593 (4 μM) did not affect the tension and the parameters of spontaneous contractions and did not prevent the effects of NaHS. Diazoxide (100 μM), the opener of КATP channels, caused a decrease in the basal tone, the amplitude and frequency of spontaneous contractions. Diazoxide and KATP channel blocker glibenclamide (50 μM) prevented the effects of NaHS on the basal tone. The Kir-channel blocker BaCl2 (30 µM) increased the amplitude of spontaneous contractions and eliminated the inhibitory effects of NaHS on the frequency and amplitude of spontaneous contractions, and the basal tension decrease was less pronounced compared to control. Thus, a decrease in the tonic tension of a rat jejunum preparation under the action of an H2S donor is associated with the activation of Kir, including КATP channels, while the effects of H2S on the amplitude and frequency of spontaneous contractions are mediated by an increase in Ba2+-sensitive conductance.

Эксперименты проводили на мышах возрастом 45 дней, где в опытной группе СРК индуцировали путем неонатальной сенсибилизации. Оценивали силу сокращения сегментов проксимального отдела толстой кишки мыши в изометрических условиях и оказалось, что у мышей с моделью СРК повышена амплитуда сокращений. Применение бутирата натрия приводило к значительному снижению амплитуды сокращений препарата, однако данные эффекты были менее выражены в опытной группе. L-NAME в контрольной группе приводило к повышению амплитуды, а в модели СРК напротив добавление L-NAME не приводило к достоверным изменениям параметров сокращения. При этом в обеих исследуемых группах угнетающее действие бутирата натрия на параметры сократимости на фоне аппликации L-NAME были выражены в меньшей степени чем в контроле. Ключевые слова: синдром раздраженного кишечника, короткоцепочечные жирные кислоты, оксид азота, сократимость, толстая кишка.

Short-chain fatty acids (which include acetic, propionic, and butyric acids) are metabolites of microbiota that can have diverse physiological effects both in the intestine and outside it, including in the central nervous system. The aim of our work was to study the effect of sodium acetate, propionate, and butyrate on the activity of large conductance Ca2+-activated K+ channels and calcium oscillations in the culture of rat pituitary GH3 cells. It was demonstrated that the studied fatty acids cause a dose-dependent increase in the amplitude of the integral outward potassium currents; these effects are prevented by tetraethylammonium, which indicates the involvement of large conductance Ca2+-activated K+ channels in the effects of fatty acids. Indeed, fatty acids increased open probability of single channels without a change in the amplitude and mean channel open time (dwell time). In addition, they led to a significant decrease in the amplitude and frequency of Ca2+ oscillations in GH3 cells. An increase in potassium conductance and a decrease in the level of intracellular Ca2+ under the effect of short-chain fatty acids can mediate their effects in different excitable structures, such as a decrease in the contractility of intestinal and vascular smooth muscle cells, hyperpolarization of neurons, and regulation of the release of hormones and neurotransmitters.

The roles of nitric oxide, intra- and extracellular calcium in the effects of hydrogen sulfide on spontaneous and carbachol-induced contractions of rat jejunal preparation under conditions of isometric contraction were studied. A donor of H2S (sodium hydrosulfide) led to a decrease in the preparation tone, amplitude and frequency of spontaneous contractions, as well as parameters of the contractions caused by carbachol, a nonspecific agonist of acetylcholine receptors. The effect of the H2S donor persisted under conditions of the inhibition of endogenous NO synthesis with L-NAME, while in the presence of the action of SNAP (an NO donor), the effects of NaHS on the amplitudes of spontaneous and carbachol-induced contractions were less pronounced. A decrease in preparation tone under the effect of NaHS was prevented by dantrolene, a blocker of ryanodine receptors. The calcium-free solution decreased the inhibitory effect of NaHS on the contractions induced by the application of carbachol. It is suggested that the inhibitory effect of H2S is associated with the dynamics of intracellular concentration of calcium ions, and the interaction of NO and H2S occurs at the level of common targets of the effect of the two gases.

Background Irritable bowel syndrome (IBS) is defined as a multifactorial disorder associated with visceral hypersensitivity, altered gut motility and dysfunction of the brain-gut axis. Gut microbiota and its metabolites are proposed as possible etiological factors of IBS. Short chain fatty acids (SCFAs) induce both inhibitory and stimulatory action on colon motility, however, their effects on the IBS model were not investigated. The aim of our study was to investigate the level of SFCAs in feces and their effects on colon motility in a mouse model of IBS. Methods IBS model was induced in mice by intracolonic infusion of 1% acetic acid during the early postnatal period. Mice colon hypersensitivity was assessed by the threshold of the abdominal withdrawal reflex in response to colorectal distention. Colon contractility was studied using proximal colon specimens in isometric conditions. Transit rates were assessed by the pellet propulsion in the isolated colon. Concentrations of SCFAs in feces were measured using gas–liquid chromatography. Results The concentration of SCFAs in feces of IBS model mice was higher compared to the control group. Visceral sensitivity to colorectal distension and colonic transit rate were increased indicating IBS with predominant diarrhea. The frequency and amplitude of spontaneous contractions of proximal colon segments from IBS mice were higher, but carbachol induced contractions were lower compared to control. During acute application of SCFAs (sodium propionate, sodium acetate or butyric acid) dose-dependently (0.5–30 mM) decreased tonic tension, frequency and amplitude of spontaneous and carbachol-evoked contractions. In the mouse IBS group the inhibitory effects SCFAs on spontaneous and carbachol-evoked contractions were less pronounced. At the same time intraluminal administration of butyrate (5 mM) increased the transit rate in the colon of both groups, but its stimulatory effect was more pronounced in mouse IBS model group. Conclusion Our data indicate that the increased transit rate in the mouse IBS model group is associated with a disbalance of activating and inhibiting action of SCFAs due to chronically elevated SCFA levels, which may impact the pathogenesis of IBS with predominant diarrhea syndrome.

Accumulating clinical and preclinical data indicate a prominent role of gut microbiota in regulation of physiological functions. The gut-brain axis imbalance due to gut dysbiosis is associated with a range of neurodegenerative diseases. Probiotics were suggested not only to restore intestinal dysbiosis but also modulate stress response and improve mood and anxiety symptoms. In this study, we assessed the effects of probiotic lactobacilli on behavioral reactions, the level of oxidative stress and microbiota content in mice administered to broad-spectrum antibiotics. Our study demonstrates that antibiotic treatment of adolescent mice for two weeks resulted in higher mortality and lower weight gain and induced significant changes in behavior including lower locomotor and exploratory activity, reduced muscle strength, visceral hypersensitivity, higher level of anxiety and impaired cognitive functions compared to the control group. These changes were accompanied by decreased diversity and total amount of bacteria, abundance of Proteobacteria and Verrucomicrobia phyla, and reduced Firmicutes/Bacteroides ratio in the gut microbiota. Moreover, a higher level of oxidative stress was found in brain and skeletal muscle tissues of mice treated with antibiotics. Oral administration of two Lactobacillus strains prevented the observed changes and improved not only microbiota content but also the behavioral alterations, suggesting a neuroprotective and antioxidant role of probiotics.

Chronic constipation (CC) is one of the most common gastrointestinal disorders worldwide. Its pathogenesis, however, remains largely unclear. The purpose of the present work was to gain an insight into the role of contractility and microbiota in the etiology of CC. To this end, we studied spontaneous and evoked contractile activity of descending colon segments from patients that have undergone surgery for refractory forms of CC. The juxta-mucosal microbiota of these colon samples were characterized with culture-based and 16S rRNA sequencing techniques. In patients with CC the spontaneous colonic motility remained unchanged compared to the control group without dysfunction of intestinal motility. Moreover, contractions induced by potassium chloride and carbachol were increased in both circular and longitudinal colonic muscle strips, thus indicating preservation of contractile apparatus and increased sensitivity to cholinergic nerve stimulation in the constipated intestine. In the test group, the gut microbiota composition was assessed as being typically human, with four dominant bacterial phyla, namely Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, as well as usual representation of the most prevalent gut bacterial genera. Yet, significant inter-individual differences were revealed. The phylogenetic diversity of gut microbiota was not affected by age, sex, or colonic anatomy (dolichocolon, megacolon). The abundance of butyrate-producing genera Roseburia, Coprococcus, and Faecalibacterium was low, whereas conventional probiotic genera Lactobacillus and Bifidobacteria were not decreased in the gut microbiomes of the constipated patients. As evidenced by our study, specific microbial biomarkers for constipation state are absent. The results point to a probable role played by the overall gut microbiota at the functional level. To our knowledge, this is the first comprehensive characterization of CC pathogenesis, finding lack of disruption of motor activity of colonic smooth muscle cells and insufficiency of particular members of gut microbiota usually implicated in CC.

Acetaldehyde and acetic acid/acetate, the active metabolites of alcohol (ethanol, EtOH), generate actions of their own ranging from behavioral, physiological, to pathological/cancerogenic effects. EtOH and acetaldehyde have been studied to some depth, whereas the effects of acetic acid have been less well explored. In this study, we investigated the effect of acetic acid on big conductance calcium-activated potassium (BK) channels present in GH3 rat pituitary tumor cells in more detail. In whole cell voltage clamp recordings, extracellular application of acetic acid increased total outward currents in a dose-dependent manner. This effect was prevented after the application of the specific BK channel blocker paxilline. Acetic acid action was pH-dependent-in whole cell current and single BK channel recordings, open probability (Po) was significantly increased by extracellular pH reduction and decreased by neutral or base pH. Acetic acid hyperpolarized the membrane potential, whereas acidic physiological solution had a depolarizing effect. Moreover, acetic acid reduced calcium (Ca2+) oscillations and exocytosis of growth hormone contained secretory granules from GH3 cells. These effects were partially prevented by BK inhibitors-tetraethylammonium or paxillin. In conclusion, our experiments indicate that acetic acid activates BK channels in GH3 cells which eventually contribute to acetic acid-induced membrane hyperpolarization, cessation of Ca2+ oscillations, and decrease of growth hormone release.

1. On page 400. The name of the third author L. M. Gabitova should read D. M. Gabitova. 2. On page 400, in running head. The name of the third author L.M. Gabitova should read D.M. Gabitova.