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.

Multifunctional TRPV1 Ion Channels in Physiology and Pathology with Focus on the Brain, Vasculature, and Some Visceral Systems.

TRPV1 has been originally cloned as the heat and capsaicin receptor implicated in acute pain signalling, while further research has shifted the focus to its importance in chronic pain caused by inflammation and associated with this TRPV1 sensitization. However, accumulating evidence suggests that, apart from pain signalling, TRPV1 subserves many other unrelated to nociception functions in the nervous system. In the brain, TRPV1 can modulate synaptic transmission via both pre- and postsynaptic mechanisms and there is a functional crosstalk between GABA receptors and TRPV1. Other fundamental processes include TRPV1 role in plasticity, microglia-to-neuron communication, and brain development. Moreover, TRPV1 is widely expressed in the peripheral tissues, including the vasculature, gastrointestinal tract, urinary bladder, epithelial cells, and the cells of the immune system. TRPV1 can be activated by a large array of physical (heat, mechanical stimuli) and chemical factors (e.g., protons, capsaicin, resiniferatoxin, and endogenous ligands, such as endovanilloids). This causes two general cell effects, membrane depolarization and calcium influx, thus triggering depending on the cell-type diverse functional responses ranging from neuronal excitation to secretion and smooth muscle contraction. Here, we review recent research on the diverse TRPV1 functions with focus on the brain, vasculature, and some visceral systems as the basis of our better understanding of TRPV1 role in different human disorders.

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.

Curcuminoids and Novel Opportunities for the Treatment of Alzheimer's Disease: Which Molecules are Actually Effective?

BACKGROUND: Millions of people worldwide are suffering from Alzheimer's disease (AD), and there are only symptomatic treatments available for this disease. Thus, there is a great need to identify drugs capable of arresting or reversing AD. Constituents of the spice turmeric, in particular, curcuminoids, seem to be very promising, as evident from in vitro experiments and tests using animal models of AD. However, most of the clinical trials did not reveal any beneficial effects of curcuminoids in the treatment of AD. These controversies, including conflicting results of clinical trials, are thought to be related to bioavailability of curcuminoids, which is low unless it is enhanced by developing a special formulation. However, there is growing evidence suggesting that other reasons may be of even greater importance, but these avenues are less explored. OBJECTIVE: Review relevant literature, and analyze potential reasons for the controversial results. METHODOLOGY: Recent in vitro and preclinical studies; clinical trials (without a limiting period) were searched in PubMed and Google Scholar. RESULTS: While recent in vitro and preclinical studies confirm the therapeutic potential of curcuminoids in the treatment of AD and cognitive dysfunctions, results of corresponding clinical trials remain rather controversial. CONCLUSION: The controversial results obtained in the clinical trials may be in part due to particularities of the curcuminoid formulations other than bioavailability. Namely, it seems likely that the various formulations differ in terms of their minor turmeric constituent(s). We hypothesize that these distinctions may be of key importance for efficacy of the particular formulation in clinical trials. A testable approach addressing this hypothesis is suggested.

TRPV4 Channel Signaling in Macrophages Promotes Gastrointestinal Motility via Direct Effects on Smooth Muscle Cells.

Intestinal macrophages are critical for gastrointestinal (GI) homeostasis, but our understanding of their role in regulating intestinal motility is incomplete. Here, we report that CX3C chemokine receptor 1-expressing muscularis macrophages (MMs) were required to maintain normal GI motility. MMs expressed the transient receptor potential vanilloid 4 (TRPV4) channel, which senses thermal, mechanical, and chemical cues. Selective pharmacologic inhibition of TRPV4 or conditional deletion of TRPV4 from macrophages decreased intestinal motility and was sufficient to reverse the GI hypermotility that is associated with chemotherapy treatment. Mechanistically, stimulation of MMs via TRPV4 promoted the release of prostaglandin E2 and elicited colon contraction in a paracrine manner via prostaglandin E receptor signaling in intestinal smooth muscle cells without input from the enteric nervous system. Collectively, our data identify TRPV4-expressing MMs as an essential component required for maintaining normal GI motility and provide potential drug targets for GI motility disorders.

TRP Channels as Novel Targets for Endogenous Ligands: Focus on Endocannabinoids and Nociceptive Signalling.

BACKGROUND: Chronic pain is a significant clinical problem and a very complex pathophysiological phenomenon. There is growing evidence that targeting the endocannabinoid system may be a useful approach to pain alleviation. Classically, the system includes G protein-coupled receptors of the CB1 and CB2 subtypes and their endogenous ligands. More recently, several subtypes of the large superfamily of cation TRP channels have been coined as "ionotropic cannabinoid receptors", thus highlighting their role in cannabinoid signalling. Thus, the aim of this review was to explore the intimate connection between several "painful" TRP channels, endocannabinoids and nociceptive signalling. METHODS: Research literature on this topic was critically reviewed allowing us not only summarize the existing evidence in this area of research, but also propose several possible cellular mechanisms linking nociceptive and cannabinoid signaling with TRP channels. RESULTS: We begin with an overview of physiology of the endocannabinoid system and its major components, namely CB1 and CB2 G protein-coupled receptors, their two most studied endogenous ligands, anandamide and 2-AG, and several enzymes involved in endocannabinoid biosynthesis and degradation. The role of different endocannabinoids in the regulation of synaptic transmission is then discussed in detail. The connection between the endocannabinoid system and several TRP channels, especially TRPV1-4, TRPA1 and TRPM8, is then explored, while highlighting the role of these same channels in pain signalling. CONCLUSION: There is increasing evidence implicating several TRP subtypes not only as an integral part of the endocannabinoid system, but also as promising molecular targets for pain alleviation with the use of endo- and phytocannabinoids, especially when the function of these channels is upregulated under inflammatory conditions.

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.

Sensory TRP channels contribute differentially to skin inflammation and persistent itch.

Although both persistent itch and inflammation are commonly associated with allergic contact dermatitis (ACD), it is not known if they are mediated by shared or distinct signaling pathways. Here we show that both TRPA1 and TRPV1 channels are required for generating spontaneous scratching in a mouse model of ACD induced by squaric acid dibutylester (SADBE), a small molecule hapten, through directly promoting the excitability of pruriceptors. TRPV1 but not TRPA1 channels protect the skin inflammation, as genetic ablation of TRPV1 function or pharmacological ablation of TRPV1-positive sensory nerves promotes cutaneous inflammation in the SADBE-induced ACD. Our results demonstrate that persistent itch and inflammation are mediated by distinct cellular and molecular mechanisms in a mouse model of ACD. Identification of distinct roles of TRPA1 and TRPV1 in regulating itch and inflammation may provide new insights into the pathophysiology and treatment of chronic itch and inflammation in ACD patients.

Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition.

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 µM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 µM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon).

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.

Functional and Modeling Studies of the Transmembrane Region of the TRPM8 Channel.

Members of the transient receptor potential (TRP) ion channel family act as polymodal cellular sensors, which aid in regulating Ca(2+) homeostasis. Within the TRP family, TRPM8 is the cold receptor that forms a nonselective homotetrameric cation channel. In the absence of TRPM8 crystal structure, little is known about the relationship between structure and function. Inferences of TRPM8 structure have come from mutagenesis experiments coupled to electrophysiology, mainly regarding the fourth transmembrane helix (S4), which constitutes a moderate voltage-sensing domain, and about cold sensor and phosphatidylinositol 4,5-bisphosphate binding sites, which are both located in the C-terminus of TRPM8. In this study, we use a combination of molecular modeling and experimental techniques to examine the structure of the TRPM8 transmembrane and pore helix region including the conducting conformation of the selectivity filter. The model is consistent with a large amount of functional data and was further tested by mutagenesis. We present structural insight into the role of residues involved in intra- and intersubunit interactions and their link with the channel activity, sensitivity to icilin, menthol and cold, and impact on channel oligomerization.