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Rhabdomyolysis, as an acute stage of myopathy is known to be associated with the accumulation of muscle breakdown products, acute renal failure and oxidative stress. The goal of the study was to evaluate the effect of C60 fullerene as an antioxidant on kidney damage in the model of glycerol-induced rhabdomyolysis in rats. The study was conducted on male Wistar rats, divided into the following experimental groups: control animals, animals intramuscularly injected with glycerol in a doses of 5, 10 and 15 mg/kg and those intraperitoneally injected daily with C60 fullerene aqueous solution (C60FAS) in a dose of 1 or 2 mg/kg at 48 h after glycerol administration­. Monitoring of the biochemical and morphological indicators was carried out on 3rd, 6th and 9th days of the experiment. A close correlation between the acute renal damage severity, increased creatinine and urea level, superoxide dismutase (SOD) and catalase (CAT) activity in the blood of rats was observed. It was shown that in rats which received 2 mg/kg of C60FAS the renal glomeruli size and necrosis manifestations were attenuated, whereas SOD and CAT activity in the blood was significantly decreased. The results obtained may be useful for developing approaches to the treatment of pathological conditions of the muscular system caused by rhabdomyolysis and associated oxidative stress.

General anesthesia produces multiple side effects. Notably, it temporarily impairs gastrointestinal motility following surgery and causes the so-called postoperative ileus (POI), a multifactorial and complex condition that develops secondary to neuromuscular failure and mainly affects the small intestine. There are currently limited medication options for POI, reflecting a lack of comprehensive understanding of the mechanisms involved in this complex condition. Notably, although acetylcholine is one of the major neurotransmitters initiating excitation-contraction coupling in the gut, cholinergic stimulation by prokinetic drugs is not very efficient in case of POI. Acetylcholine when released from excitatory motoneurones of the enteric nervous system binds to and activates M2 and M3 types of muscarinic receptors in smooth muscle myocytes. Downstream of these G protein-coupled receptors, muscarinic cation TRPC4 channels act as the major focal point of receptor-mediated signal integration, causing membrane depolarisation accompanied by action potential discharge and calcium influx via L-type Ca2+ channels for myocyte contraction. We have recently found that both inhalation (isoflurane) and intravenous (ketamine) anesthetics significantly inhibit this muscarinic cation current (termed mICAT) in ileal myocytes, even when G proteins are activated directly by intracellular GTPγS, i.e., bypassing muscarinic receptors. Here we aim to summarize Transient Receptor Potential channels and calcium signalling-related aspects of the cholinergic mechanisms in the gut and visceral pain, discuss exactly how these may be negatively impacted by general anaesthetics, while proposing the receptor-operated TRPC4 channel as a novel molecular target for the treatment of POI.

Parkinson’s disease (PD) is neurodegenerative disease, which is accompanied by degeneration of dopaminergic neurons in subtantia nigra. Non-motor symptoms, in particular, disorders of the gastrointestinal (GI) tract are observed in 20-80% of patients some 15-20 years before clinically diagnosed PD and are not a least important feature of PD pathogenesis. The transient receptor potential (TRP) channels are expressed throughout the GI tract, where they play an important role in taste, thermoregulation, pain, mucosal function and homeostasis, control of interstitial motility etc. The aim of this study was to investigate the contribution of TRPV4 and TRPM8 channels in the GI motor function in the colon of rats with PD, incduced by injection of the 12 μg 6-hydroxydopamine (6-OHDA). The studies were performed on the 4th week and the 7th month after PD induction The rats were randomly divided into: I group – the sham-lesioned rats, 4 μl 0.9% NaCl, autopsy 4 weeks after injection (n = 5); II group – the 6-OHDA-PD rats, 4 μl 12 μg of 6-OHDA, autopsy 4 weeks after injection (n = 5); III group – the sham-lesioned rats, 4 μl 0.9% NaCl, autopsy 7 months after injection (n = 4); IV group – the 6-OHDA-PD rats, 4 μl 12 μg of 6-OHDA, autopsy 7 months after injection (n = 5). We evaluated the body weight of rats, GI transit time, the cecum weight index and immunohistochemical identification of tyrosine hydroxylase (TH) -positive cells, and TRPV4, TRPM8 expression in rat’s colon. We showed that on the 7th month of the experiment, the GI transit time doubles over time; the cecum weight index of 6-OHDA rats increased by 57%; the number of TH-positive cells in colon rats decreased 2-fold, while TRPM8 ion channels were downregulated in PD rats and TRPV4 ion channels were upregulated in the colon of rats with 6-OHDA-PD. It was concluded that TRPV4 and TRPM8 ion channels may be considered pharmacological targets in the progression of PD pathology.

TRP channels are expressed both in vascular myocytes and endothelial cells, but knowledge of their operational mechanisms in vascular tissue is particularly limited. Here, we show for the first time the biphasic contractile reaction with relaxation followed by a contraction in response to TRPV4 agonist, GSK1016790A, in a rat pulmonary artery preconstricted with phenylephrine. Similar responses were observed both with and without endothelium, and these were abolished by the TRPV4 selective blocker, HC067047, confirming the specific role of TRPV4 in vascular myocytes. Using selective blockers of BKCa and L-type voltage-gated Ca2+ channels (CaL), we found that the relaxation phase was inducted by BKCa activation generating STOCs, while subsequent slowly developing TRPV4-mediated depolarisation activated CaL, producing the second contraction phase. These results are compared to TRPM8 activation using menthol in rat tail artery. Activation of both types of TRP channels produces highly similar changes in membrane potential, namely slow depolarisation with concurrent brief hyperpolarisations due to STOCs. We thus propose a general concept of bidirectional TRP-CaL-RyR-BKCa molecular and functional signaloplex in vascular smooth muscles. Accordingly, both TRPV4 and TRPM8 channels enhance local Ca2+ signals producing STOCs via TRP–RyR–BKCa coupling while simultaneously globally engaging BKCa and CaL channels by altering membrane potential.

Abstract. There is a general lack of research on the long-term effects of acute kidney injury (AKI) on oxalate-degrading bacteria (ODB) and their total oxalate-degrading activity (ODA) in fecal microbiota. In the present pilot study, we separately evaluated the changes in the ODB number and their total ODA in fecal microbiota at 3-time points after glycerol-induced AKI. In addition, we assessed the interactions between AKI-induced renal histopathological changes and ODB, total fecal ODA, and plasma and urine oxalate concentrations in rats. Methods. The male Wistar rats (200-300 g, n = 20) on oxalate-free diet were randomly divided into 2 groups. After 24-h of water deprivation, experimental group 1 (n = 10) received an intramuscular injection of 50% glycerol (10 ml/kg of body weight), and group 2 (n = 10) served as a control. The numbers of ODB (incubated in a highly selective Oxalate Medium and determined using the culture method), total fecal ODA and urinary oxalate (UOx) excretion were measured after injection on days 8, 22 and 70. The method of redoximetric titration with a KMnO4 solution was adopted to evaluate total ODA in fecal microbiota. Renal injury was assessed by histopathology examination, serum creatinine plasma oxalic acid (POx) concentration and daily proteinuria levels after removing the animals from the experiment on day 70. Results. After glycerol injection on days 8 and 22, no differences were found in the numbers of ODB, their total fecal ODA, and UOx excretion level between the experimental and control groups. However, after AKI initiation on day 70, the numbers of ODB, total fecal ODA, and daily UOx excretion were significantly lower in the experimental group as compared with the control group. In addition, in 10 weeks following AKI, the number of ODB had a direct correlation with UOx excretion and an inverse correlation with POx and serum creatinine concentrations and daily proteinuria. Total ODA in fecal microbiota was directly associated with the percentage of renal interstitial fibrosis and the average glomerular volumes in the experimental rats. Conclusions: AKI had long-term negative effects on the quantitative and qualitative characteristics of ODB in fecal microbiota in rats. Moreover, the results of our study confirmed an increasing trend in total fecal ODA according to the aggravation of renal interstitial fibrosis and glomerular volume in rats’ kidneys. Further studies are warranted to gain more insight into the mechanism of oxalate homeostasis impairment in AKI.

Коротке повідомлення, присвячене Восьмому з’їзду Українського біофізичного товариства.

A better understanding of the negative impact of general anesthetics on gastrointestinal motility requires thorough knowledge of their molecular targets. In this respect the muscarinic cationic current (mICAT carried mainly via TRPC4 channels) that initiates cholinergic excitation-contraction coupling in the gut is of special interest. Here we aimed to characterize the effects of one of the most commonly used “dissociative anesthetics”, ketamine, on mICAT. Patch-clamp and tensiometry techniques were used to investigate the mechanisms of the inhibitory effects of ketamine on mICAT in single mouse ileal myocytes, as well as on intestinal motility. Ketamine (100 µM) strongly inhibited both carbachol- and GTPγS-induced mICAT. The inhibition was slow (time constant of about 1 min) and practically irreversible. It was associated with altered voltage dependence and kinetics of mICAT. In functional tests, ketamine suppressed both spontaneous and carbachol-induced contractions of small intestine. Importantly, inhibited by ketamine mICAT could be restored by direct TRPC4 agonist (-)-englerin A. We identified mICAT as a novel target for ketamine. Signal transduction leading to TRPC4 channel opening is disrupted by ketamine mainly downstream of muscarinic receptor activation, but does not involve TRPC4 per se. Direct TRPC4 agonists may be used for the correction of gastrointestinal disorders provoked by general anesthesia.

Single-walled carbon nanotubes (SWCNTs) are characterized by a combination of rather unique physical and chemical properties, which makes them interesting biocompatible nanostructured materials for various applications, including in the biomedical field. SWCNTs are not inert carriers of drug molecules, as they may interact with various biological macromolecules, including ion channels. To investigate the mechanisms of the inhibitory effects of SWCNTs on the muscarinic receptor cation current (mICAT), induced by intracellular GTPγs (200 μM), in isolated mouse ileal myocytes, we have used the patch-clamp method in the whole-cell configuration. Here, we use molecular docking/molecular dynamics simulations and direct patch-clamp recordings of whole-cell currents to show that SWCNTs, purified and functionalized by carboxylation in water suspension containing single SWCNTs with a diameter of 0.5–1.5 nm, can inhibit mICAT, which is mainly carried by TRPC4 cation channels in ileal smooth muscle cells, and is the main regulator of cholinergic excitation–contraction coupling in the small intestinal tract. This inhibition was voltage-independent and associated with a shortening of the mean open time of the channel. These results suggest that SWCNTs cause a direct blockage of the TRPC4 channel and may represent a novel class of TRPC4 modulators.

Allergic contact dermatitis is associated both with persistent itch and inflammation, but it is not known if these are mediated by shared signaling pathways. The authors show that persistent itch requires both TRPA1 and TRPV1, while TRPV1 has a protective role against skin inflammation in mice.

TRP channels are expressed both in vascular myocytes and endothelial cells, but knowledge of their operational mechanisms in vascular tissue is particularly limited. Here, we show for the first time the biphasic contractile reaction with relaxation followed by a contraction in response to TRPV4 agonist, GSK1016790A, in a rat pulmonary artery preconstricted with phenylephrine. Similar responses were observed both with and without endothelium, and these were abolished by the TRPV4 selective blocker, HC067047, confirming the specific role of TRPV4 in vascular myocytes. Using selective blockers of BKCa and L-type voltage-gated Ca2+ channels (CaL), we found that the relaxation phase was inducted by BKCa activation generating STOCs, while subsequent slowly developing TRPV4-mediated depolarisation activated CaL, producing the second contraction phase. These results are compared to TRPM8 activation using menthol in rat tail artery. Activation of both types of TRP channels produces highly similar changes in membrane potential, namely slow depolarisation with concurrent brief hyperpolarisations due to STOCs. We thus propose a general concept of bidirectional TRP-CaL-RyR-BKCa molecular and functional signaloplex in vascular smooth muscles. Accordingly, both TRPV4 and TRPM8 channels enhance local Ca2+ signals producing STOCs via TRP–RyR–BKCa coupling while simultaneously globally engaging BKCa and CaL channels by altering membrane potential. [ABSTRACT FROM AUTHOR]

There is little information about the mechanisms by which G-protein-coupled receptors gate ion channels although many ionotropic receptors are well studied. We have investigated gating of the muscarinic cationic channel, which mediates the excitatory effect of acetylcholine in smooth muscles, and proposed a scheme consisting of four pairs of closed and open states. Channel kinetics appeared to be the same in cell-attached or outside-out patches whether the channel was activated by carbachol application or by intracellular dialysis with GTP[gamma]S. Since in the latter case G-proteins are permanently active, it is concluded that the cationic channel is the major determinant of its own gating, similarly to the [K.sub.Ach] channel (Ivanova-Nikolova, T.T., and G.E. Breitwieser. 1997. J. Gen. Physiol. 109:245-253). Analysis of adjacent-state dwell times revealed connections between the states that showed features conserved among many other ligand-gated ion channels (e.g., nAChR, B[K.sub.Ca] channel). Open probability ([P.sub.O]) of the cationic channel was increased by membrane depolarization consistent with the prominent U-shaped I-V relationship of the muscarinic whole-cell current at negative potentials. Membrane potential affected transitions within each closed-open state pair but had little effect on transitions between pairs; thus, the latter are likely to be caused by interactions of the channel with its ligands, e.g., [Ca.sup.2+] and G[alpha]o-GTP. Channel activity was highly heterogeneous, as was evident from the prominent cycling behavior when [P.sub.o] was measured over 5-s intervals. This was related to the variable frequency of openings (as in the [K.sub.Ach] channel) and, especially, to the number of long openings between consecutive long shuttings. Analysis of the underlying Markov chain in terms of probabilities allowed us to evaluate the contribution of each open state to the integral current (from shortest to longest open state: 0.1, 3, 24, and 73%) as [P.sub.O] increased 525-fold in three stages. KEY WORDS: Markov chain * channel kinetics * G-protein * carbachol * smooth muscle

In recent years, the transient receptor potential melastatin member 8 (TRPM8) channel has emerged as a promising prognostic marker and putative therapeutic target in prostate cancer (PCa). However, the mechanisms [...]

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [[Ca.sup.+]] in a [Ca.sup.2+]-free medium followed by a secondary [[Ca.sup.2+]] rise upon [Ca.sup.2+] readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as [Ca.sup.2+]-activated ([I.sub.Cl(Ca)]) and volume-regulated ([[I.sub.Cl, swell]) chloride currents. NPPB and DIDS more efficiently inhibited [I.sub.Cl(Ca)] and [[I.sub.Cl, swell] respectively. Cell swelling caused by hypotonic solution invariably activated [I.sub.Cl, swell] while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling-induced [I.sub.Cl, swell], while its inactive analogue U-73343 had no effect. [I.sub.Cl(Ca)] could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [[[Ca.sup.2+]].sub.i] in the pipette solution at 500 nM. In contrast, [I.sub.Cl, swell] could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by Ins[P.sub.3] infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced [I.sub.Cl, swell] in a nonadditive manner, suggesting their convergence on a common pathway. [I.sub.Cl, swell] and [I.sub.Cl(ca)] showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated [I.sub.Cl(Ca), but abolished [I.sub.Cl, swell], thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that [I.sub.Cl, swell] can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a 'selection' switch between closely localized channels. KEY WORDS: human keratinocytes * histamine receptors * cell swelling * chloride channels * store-operated [Ca.sup2+] entry