A Randomized Clinical Trial Comparing Dubuisson Laparoscopic Lateral Suspension with Laparoscopic Sacropexy for Pelvic Organ Prolapse: Short-Term Results.

BACKGROUND: Laparoscopic sacrocolpopexy (LSC) is the gold standard for the treatment of apical prolapse, although dissection of the promontory may be challenging. Laparoscopic lateral suspension (LLS) with mesh is an alternative technique for apical repair with similar anatomical and functional outcomes, according to recent studies. The purpose of this study was to compare these operative techniques. METHODS: Women with uterine Pelvic Organ Prolapse Quantification (POP-Q) stage 2 were enrolled in this prospective study and were randomly allocated to the LLS or LSC group. At the 12-month follow-up, primary measures included both anatomical and functional outcomes. Perioperative parameters and complications were recorded. RESULTS: A total of 93 women were randomized, 48 in the LLS group and 45 in the LSC group, with 2 women lost to follow-up in both groups. LSC anatomic success rates were 81.82% for the apical compartment and 95.22% for the anterior compartment. LLS anatomic success rates for the apical and anterior compartments were 90% and 92.30%, respectively. The mean operative time for LLS was 160.3 min, while for LSC it was 168.3 min. The mean blood loss was 100 mL in both procedures. Conversion to laparotomy was necessary in three women. Mesh erosion was not observed in any of the cases. In terms of the complication, Clavien-Dindo grade 1 was observed in two patients in the LLS group and a complication rated grade 3b was observed in one patient in LSC group. CONCLUSIONS: LLS is a good alternative to LSC, with promising anatomical and quality-of-life results.

Pico145 inhibits TRPC4-mediated mICAT and postprandial small intestinal motility.

In intestinal smooth muscle cells, receptor-operated TRPC4 are responsible for the majority of muscarinic receptor cation current (mICAT), which initiates cholinergic excitation-contraction coupling. Our aim was to examine the effects of the TRPC4 inhibitor Pico145 on mICAT and Ca2+ signalling in mouse ileal myocytes, and on intestinal motility. Ileal myocytes freshly isolated from two month-old male BALB/c mice were used for patch-clamp recordings of whole-cell currents and for intracellular Ca2+ imaging using Fura-2. Functional assessment of Pico145's effects was carried out by standard in vitro tensiometry, ex vivo video recordings and in vivo postprandial intestinal transit measurements using carmine red. Carbachol (50 µM)-induced mICAT was strongly inhibited by Pico145 starting from 1 pM. The IC50 value for the inhibitory effect of Pico145 on this current evoked by intracellularly applied GTPγS (200 µM), and thus lacking desensitisation, was found to be 3.1 pM, while carbachol-induced intracellular Ca2+ rises were inhibited with IC50 of 2.7 pM. In contrast, the current activated by direct TRPC4 agonist (-)-englerin A was less sensitive to the action of Pico145 that caused only ∼43 % current inhibition at 100 pM. The inhibitory effect developed rather slowly and it was potentiated by membrane depolarisation. In functional assays, Pico145 produced concentration-dependent suppression of both spontaneous and carbachol-evoked intestinal smooth muscle contractions and delayed postprandial intestinal transit. Thus, Pico145 is a potent GI-active small-molecule which completely inhibits mICAT at picomolar concentrations and which is as effective as trpc4 gene deficiency in in vivo intestinal motility tests.

General anaesthesia-related complications of gut motility with a focus on cholinergic mechanisms, TRP channels and visceral pain.

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 mI CAT) 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.

Bidirectional TRP/L Type Ca2+ Channel/RyR/BKCa Molecular and Functional Signaloplex in Vascular Smooth Muscles.

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.

Calcium-dependent modulation of BKCa channel activity induced by plasmonic gold nanoparticles in pulmonary artery smooth muscle cells and hippocampal neurons.

AIM: Gold nanoparticles are widely used for biomedical applications, but the precise molecular mechanism of their interaction with cellular structures is still unclear. Assuming that intracellular calcium fluctuations associated with surface plasmon-induced calcium entry could modulate the activity of potassium channels, we studied the effect of 5 nm gold nanoparticles on calcium-dependent potassium channels and associated calcium signaling in freshly isolated rat pulmonary artery smooth muscle cells and cultured hippocampal neurons. METHODS: Outward potassium currents were recorded using patch-clamp techniques. Changes in intracellular calcium concentration were measured using the high affinity Ca2+ fluorescent indicator fluo-3 and laser confocal microscope. RESULTS: In pulmonary artery smooth muscle cells, plasmonic gold nanoparticles increased the amplitude of currents via large-conductance Ca2+ -activated potassium channels, which was potentiated by green laser irradiation near plasmon resonance wavelength (532 nm). Buffering of intracellular free calcium with ethylene glycol-bis-N,N,N',N'-tetraacetic acid (EGTA) abolished these effects. Furthermore, using confocal laser microscopy it was found that application of gold nanoparticles caused oscillations of intracellular calcium concentration that were decreasing in amplitude with time. In cultured hippocampal neurons gold nanoparticles inhibited the effect of EGTA slowing down the decline of the BKCa current while partially restoring the amplitude of the slow after hyperpolarizing currents. CONCLUSION: We conclude that fluctuations in intracellular calcium can modulate plasmonic gold nanoparticles-induced gating of BKCa channels in smooth muscle cells and neurons through an indirect mechanism, probably involving the interaction of plasmon resonance with calcium-permeable ion channels, which leads to a change in intracellular calcium level.

Single-Walled Carbon Nanotubes Inhibit TRPC4-Mediated Muscarinic Cation Current in Mouse Ileal Myocytes.

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.

Electrophysiological characterization of the activating action of a novel liposomal nitric oxide carrier on Maxi-K channels in pulmonary artery smooth muscle cells.

The aim of this study was to establish the mechanisms of action of a novel liposomal nitric oxide (NO) carrier on large-conductance Ca2+-activated channels (BKCa or Maxi-K) expressed in vascular smooth muscle cells (VSMCs) isolated from the rat main pulmonary artery (MPA). Experimental design comprised of both whole-cell and cell-attached single-channel recordings using the patch-clamp techniques. The liposomal form of NO, Lip(NO), increased whole-cell outward K+ currents in a dose dependent manner while shifting the activation curve negatively by about 50 mV with respect to unstimulated cells with the EC50 value of 0.55 ± 0.17 µM. At the single channel level, Lip(NO) increased the probability of the open state (Po) of Maxi-K channels from 0.0020 ± 0.0008 to 0.74 ± 0.02 with half-maximal activation occurring at 4.91 ± 0.01 µM, while sub-maximal activation was achieved at 10-5 M Lip(NO). Channel activation was mainly due to significant decrease in the mean closed dwell time (about 500-fold), rather than an increase in the mean open dwell time, which was comparatively modest (about twofold). There was also a slight decrease in the amplitude of the elementary Maxi-K currents (approximately 15%) accompanied by an increase in current noise, which might indicate some non-specific effects of Lip(NO) on the plasma membrane itself and/or on the phospholipids environment of the channels. In conclusion, the activating action of Lip(NO) on the Maxi-K channel is due to the destabilization of the closed conformation of the channel protein, which causes its more frequent openings and, accordingly, increases the probability of channel transition to its open state.

Suppression of mICAT in Mouse Small Intestinal Myocytes by General Anaesthetic Ketamine and its Recovery by TRPC4 Agonist (-)-englerin A.

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.

Hypoxic pulmonary vasoconstriction is lacking in rats with type 1 diabetes.

Hypoxic pulmonary vasoconstriction (HPV) is the most important feature of intact lung circulation that matches local blood perfusion to ventilation. The main goal of this work was to study the effects of diabetes on the development of HPV in rats. The experimental design comprised diabetes mellitus induction by streptozotocin, video-morphometric measurements of the lumen area of intrapulmonary arteries (iPAs) using perfused lung tissue slices and patch-clamp techniques. It was shown that iPA lumen size was significantly reduced under physical and chemical hypoxia (7-10 mm Hg) in normal iPA, but, on the contrary, it clearly increased in diabetic lung slices. The amplitude of the outward K+ current in diabetic iPAs smooth muscle cells (SMCs) was two-fold greater than that seen in healthy cells. Chemical hypoxia led to significant decrease in the amplitude of the K+ outward current in healthy iPA SMCs while it was without effect in diabetic cells. The data obtained clearly indicate a significant dysregulation of vascular tone in pulmonary circulation under diabetes, ie diabetes damages the adaptive mechanism for regulating blood flow from poorly ventilated to better ventilated regions of the lung under hypoxia. This effect could be clinically important for patients with diabetes who have acute or chronic lung diseases associated with the lack of blood oxygenation.

C60 fullerenes selectively inhibit BKCa but not Kv channels in pulmonary artery smooth muscle cells.

Possessing unique physical and chemical properties, C60 fullerenes are arising as a potential nanotechnological tool that can strongly affect various biological processes. Recent molecular modeling studies have shown that C60 fullerenes can interact with ion channels, but there is lack of data about possible effects of C60 molecule on ion channels expressed in smooth muscle cells (SMC). Here we show both computationally and experimentally that water-soluble pristine C60 fullerene strongly inhibits the large conductance Ca2+-dependent K+ (BKCa), but not voltage-gated K+ (Kv) channels in pulmonary artery SMC. Both molecular docking simulations and analysis of single channel activity indicate that C60 fullerene blocks BKCa channel pore in its open state. In functional tests, C60 fullerene enhanced phenylephrine-induced contraction of pulmonary artery rings by about 25% and reduced endothelium-dependent acetylcholine-induced relaxation by up to 40%. These findings suggest a novel strategy for biomedical application of water-soluble pristine C60 fullerene in vascular dysfunction.

Liposomal quercetin potentiates maxi-K channel openings in smooth muscles and restores its activity after oxidative stress.

The effects of quercetin-loaded liposomes (PCL-Q) and their constituents, that is, free quercetin (Q) and 'empty' phosphatidylcholine vesicles (PCL), on maxi-K channel activity were studied in single mouse ileal myocytes before and after H2O2-induced oxidative stress. Macroscopic Maxi-K channel currents were recorded using whole-cell patch clamp techniques, while single BKCa channel currents were recorded in the cell-attached configuration. Bath application of PCL-Q (100 µg/ml of lipid and 3 µg/ml of quercetin) increased single Maxi-K channel activity more than threefold, from 0.010 ± 0.003 to 0.034 ± 0.004 (n = 5; p < 0.05), whereas single-channel conductance increased non-significantly from 138 to 146 pS. In the presence of PCL-Q multiple simultaneous channel openings were observed, with up to eight active channels in the membrane patch. Surprisingly, 'empty' PCL (100 µg/ml) also produced some channel activation, although it was less potent compared to PCL-Q, that is, these increased NPo from 0.010 ± 0.003 to 0.019 ± 0.003 (n = 5; p < 0.05) and did not affect single-channel conductance (139 pS). Application of PCL-Q restored macroscopic Maxi-K currents suppressed by H2O2-induced oxidative stress in ileal smooth muscle cells. We conclude that PCL-Q can activate Maxi-K channels in ileal myocytes mainly by increasing channel open probability, as well as maintain Maxi-K-mediated whole-cell current under the conditions of oxidative stress. While fusion of the 'pure' liposomes with the plasma membrane may indirectly activate Maxi-K channels by altering channel's phospholipids environment, the additional potentiating action of quercetin may be due to its better bioavailability.

Inhalation anaesthetic isoflurane inhibits the muscarinic cation current and carbachol-induced gastrointestinal smooth muscle contractions.

Gastrointestinal tract motility may be demoted significantly after surgery operations at least in part due to anaesthetic agents, but there is no comprehensive explanation of the molecular mechanism(s) of such adverse effects. Anesthetics are known to interact with various receptors and ion channels including several subtypes of transient receptor potential (TRP) channels. Two members of the canonical subfamily of TRP channels (TRPC), TRPC4 and TRPC6 are Ca2+-permeable cation channels involved in visceral smooth muscle contractility induced by acetylcholine, the primary excitatory neurotransmitter in the gut. In the present study, we aimed to study the effect of anesthetics on muscarinic receptor-mediated excitation and contraction of intestinal smooth muscle. Here we show that muscarinic cation current (mICAT) mediated by TRPC4 and TRPC6 channels in mouse ileal myocytes was strongly inhibited by isoflurane (0.5mM), one of the most commonly used inhalation anesthetics. Carbachol-activated mICAT was reduced by 63 ± 11% (n = 5), while GTPγS-induced (to bypass muscarinic receptors) current was inhibited by 44 ± 9% (n = 6). Furthermore, carbachol-induced ileum and colon contractions were inhibited by isoflurane by about 30%. We discuss the main sites of isoflurane action, which appear to be G-proteins and muscarinic receptors, rather than TRPC4/6 channels. These results contribute to our better understanding of the signalling pathways affected by inhalation anesthetics, which may cause ileus, and thus may be important for the development of novel treatment strategies during postoperative recovery.

C60 fullerenes disrupt cellular signalling leading to TRPC4 and TRPC6 channels opening by the activation of muscarinic receptors and G-proteins in small intestinal smooth muscles.

The effect of water-soluble pristine C60 fullerene nanoparticles (C60NPs) on receptor-operated cation channels formed by TRPC4/C6 proteins in ileal smooth muscle cells was investigated for the first time. Activation of these channels subsequent to acetylcholine binding to the expressed in these cells M2 and M3 muscarinic receptors represents the key event in the parasympathetic control of gastrointestinal smooth muscle motility and cholinergic excitation-contraction coupling. Experiments were performed on single collagenase-dispersed mouse ileal myocytes using patch-clamp techniques with symmetrical 125mM Cs+ solutions and [Ca2+]i 'clamped' at 100nM in order to isolate the muscarinic cation current (mICAT). The current was induced by intracellular infusion of 200µM GTPγS, which activates G-proteins directly, i.e. bypassing the muscarinic receptors. C60NPs applied at 10-6M at peak response to activation of G-proteins caused mICAT inhibition by 47.0±3.5% (n=9). The inhibition developed rather slowly, with the time constant of 119±16s, was voltage-independent and irreversible. Thus, C60NPs are unlikely to cause any direct block of TRPC4/C6 channels; rather, they may accumulate in the membrane and disrupt G-protein signalling leading to mICAT generation. C60NPs may represent a novel class of biocompatible molecules for the treatment of disorders associated with enhanced gastrointestinal motility.

Comparison of biological effects of electromagnetic fields with pulse frequencies of 8 and 50 Hz on gastric smooth muscles.

The influence of electromagnetic fields (EMFs) with rectangular pulse frequencies of 8 and 50 Hz and flux density of 25 µT on contraction, nitric oxide/nitrite synthesis, and intracellular calcium concentration in the gastric smooth muscles of rats was investigated. An approximately 8-Hz field reduced the fast component of contraction induced by KCl depolarization and slowed down the time to reach the maximum of the slow component of contraction, whereas the 50-Hz field increased the fast and slow components and accelerated the time to reach the maximum of the slow component of contraction. After turning off the EMF, the force and character of contraction returned to the control values. In addition, the 8-Hz field increased nitric oxide/nitrite synthesis in the excited smooth muscle tissue with KCl depolarization, while the 50-Hz field had no significant effect. 8- and 50-Hz fields had no significant effects on nitric oxide/nitrite production in non-stimulated tissue. However, the 50-Hz field significantly increased the basic intracellular calcium concentration in smooth muscle cells (SMC) in a time-dependent manner, whereas the 8-Hz field only slightly increased calcium levels. Thus, we showed that responses of gastric smooth muscles to EMFs are pulse-frequency dependent.

THE ROLE OF TRPV4 CATION CHANNELS IN THE REGULATION OF PHENYLEPHRINE-INDUCED CONTRACTION OF RAT PULMONARY ARTER.

The aim of our study was to investigate the role of mechanosensitive TRPV4 channels in the regulation of rat pulmonary artery smooth muscle (PASM) contractile activity induced by the activation of α-adrenoceptors and the possibility of their use as novel pharmacological targets in pulmonary hypertension. TRPV4 selective agonist, GSK1016790A, in the presence of the agonist of α-adrenoceptors phenylephrine (PhE) evoked biphasic contractile reaction with initial relaxation (63,5% ± 7,1) followed by significant vasoconstriction (142% ± 17,9). GSK1016790A evoked similar effects in PASM rings with and without endothelium, indicating that its main site of action was TRPV4 expressed in smooth muscle cells. TRPV4 selective blocker, HC-067047, completely inhibited the effects of GSK1016790A confirming the specific role of TRPV4 in these vascular responses. Application of Ca2+-free external solution reduced the relaxation phase and completely abolished the sustained contractile response to GSK1016790A (from 43,9 % to 0,3 %). The biphasic reaction could be explained as an initial calcium store depletion by PhE and further calciuminduced calcium release activated by TRPV4 that causes BKCa activation, membrane hyperpolarisation and vasorelaxation, followed by Ca2+ entry via TRPV4 and contraction. We conclude that TRPV4 channels play an important role in the regulation of the adrenergic vascular tone of PASM cells, but TRPV4 activation mechanism(s) and signaling pathways remain unclear.