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The effects of external H+ on the acetylcholine-induced inward current (nonselective cationic current; InsACh) in guinea pig ileal smooth muscle were investigated using the conventional whole cell patch-clamp technique. When the external pH (pHo) was lowered, the amplitude of InsACh was increased, with no significant change in the reversal potential or no detectable induction of other ionic permeabilities. The dose-response curve for this effect was best described by a Hill-type equation with an apparent pKa value of 7.4 and a Hill coefficient of approximately 1. The effect of pHo was associated with a shift of the steady-state activation curve for InsACh; the half-maximum activation potential became more negative on lowering pHo. Similar results were obtained when InsACh was activated by intracellularly applied guanosine 5'-O-(3-thiotriphosphate). These results indicate that the external H+ activity is an efficient regulator of InsACh channel, and this may have a physiological importance for controlling the muscarinic receptor-mediated contractions in this muscle.

TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P(2)-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P(2) regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.

The cardiac arrhythmia is a common cause of morbidity and mortality in the present world, especially in developed countries. The etiology of cardiac arrhythmia is quite broad involving both hereditary and secondary backgrounds. An increasing number of rare but lethal arrhythmogenic mutations have been identified by genome-wide association assays in genes associated with cardiac excitation, conduction and morphogenesis. Such well-characterized examples include causative mutations in voltagedependent Na+, Ca2+ or K+ channel (Nav, Cav, Kv) genes for the prolongation of QT interval which sometimes result in premature sudden death and may act as predisposing factors to drug-induced arrhythmia (Chen et al., 1998; Roden, 2004; Saenen & Vrints, 2008; Towbin & Vatta, 2001; Zareba et al., 2004). Furthermore, some of heart malformations (e.g. atrial septal defect) and idiopathic cardiomyopathy (e.g. dilated cardiomyopathy) with symptoms of arrhythmias have also proved to have some genetic linkage to impaired impulse conduction (Benson et al., 1999; Bonne, et al.,2001; Schott et al., 1998). However, much more prevalent are rhythm disturbances and conduction failures occurring in ‘remodeling’ hearts which progressively undergo structural and electrical changes in prolonged metabolically and mechanically stressed states, e.g. chronic heart failure and myocardial infarction (Nattel, 2007; Olson, 2005). The well-known consequences of cardiac remodeling are; altered expression and gating properties of ion channels shaping the action potential and generating pace-making activities; reduced electrical coupling which leads to aberrant conduction of excitation. These changes are thought to work as the ‘substrates’ increasing the risk of lethal cardiac arrhythmia (Janse, 2004; Nattel, 2007). It has also been known that abnormal handling of intracellular Ca2+ occurring in the remodeled heart may cause spontaneous membrane depolarizations triggering ectopic excitations (Dobrev, 2010; Janse, 2004; Nattel, 2007). However, the available knowledge is still limited largely to ion channels/transporters/pumps whose roles in the cardiac excitation-contraction cycle have relatively been well established. The transient receptor potential (TRP) channels constitute a newly-emerging non-selective cation channel (NSCC) superfamily activated by a plethora of physico-chemical stimuli other than voltage change. Because of this unique activation profile as well as the ability to permeate Ca2+ (except for TRPM4/TRPM5), TRP channels have attracted great attention as promising candidate molecules elucidating a variety of biological functions and disorders

The transient receptor potential (TRP) proteins form a large Ca2+-permeable nonselective cation channel superfamily activated by physicochemical stimuli, and participate in a wide array of biological functions including sensory signal transduction. Recent investigations have disclosed that many of TRP channels expressed in the cardiovascular system (CVS) are activated by mechanical stresses operating therein such as membrane stretch, hypoosmolarity and shear stress. Although mechanisms proposed for mechanical signal transduction are diverse, accumulating evidence suggests that lipid mediators derived from phospholipase C (PLC)- and phospholipase A2 (PLA2)-dependent pathways may play central roles in the activation and regulation of these TRP channels. In this review, we focus on the lipid-mediated regulation of two TRP channels abundantly expressed in the CVS, i.e. TRPC6 and TRPV4, with particular interest in the synergistic interaction between receptor-mediated and mechanical stimulations, and discuss about their complex functional antagonism in vascular tone and blood pressure regulation.

1. The effects of isoprenaline, a forskolin derivative NKH-477, and dibutyryl cyclic AMP (db cyclic AMP) on the membrane potential, conductance and cell volume of the dog non-pigmented ciliary epithelium (NPE) were investigated by intracellular potential recording, nystatin-perforated patch clamp technique and videomicroscopic cytometry. 2. The resting membrane potential of NPE was about -70 mV in physiological saline and was depolarized by isoprenaline in a dose-dependent manner with an ED50 of about 3 nM. This depolarization was competitively antagonized by the beta-adrenoceptor antagonist, timolol (pA2 = ca. 9) and almost completely blocked by the Cl transport blocker, DIDS. 3. In single dissociated NPE cells, 10 microM isoprenaline induced an inward current and caused a concomitant decrease in cell volume. The reversal potential measurement indicated that this inward current was carried mainly by Cl ion. DIDS (10 microM) abolished both the current and cell volume decrease. 4. NKH-477 (10 microM) or db cyclic AMP (1 mM) also induced an inward current together with a cell volume decrease, the properties of which were similar to those caused by isoprenaline. 5. These results suggest that beta-adrenoceptor stimulation in NPE leads to an increased rate of aqueous humour production by increasing Cl- efflux via an elevation of cyclic AMP and this effect is efficiently blocked by timolol.

Activation of transient receptor potential (TRP) canonical TRPC3/C6/C7 channels by diacylglycerol (DAG) upon stimulation of phospholipase C (PLC)-coupled receptors results in the breakdown of phosphoinositides (PIPs). The critical importance of PIPs to various ion-transporting molecules is well documented, but their function in relation to TRPC3/C6/C7 channels remains controversial. By using an ectopic voltage-sensing PIP phosphatase (DrVSP), we found that dephosphorylation of PIPs robustly inhibits currents induced by carbachol (CCh), 1-oleolyl-2-acetyl-sn-glycerol (OAG) or RHC80267 in TRPC3, TRPC6 and TRPC7 channels, though the strength of the DrVSP-mediated inhibition (VMI) varied among the channels with a rank order of C7 > C6 > C3. Pharmacological and molecular interventions suggest that depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is most likely the critical event for VMI in all three channels. When the PLC catalytic signal was vigorously activated through overexpression of the muscarinic type-I receptor (M1R), the inactivation of macroscopic TRPC currents was greatly accelerated in the same rank order as the VMI, and VMI of these currents was attenuated or lost. VMI was also rarely detected in vasopressin-induced TRPC6-like currents in A7r5 vascular smooth muscle cells, indicating that the inactivation by PI(4,5)P2 depletion underlies the physiological condition. Simultaneous fluorescence resonance energy transfer (FRET)-based measurement of PI(4,5)P2 levels and TRPC6 currents confirmed that VMI magnitude reflects the degree of PI(4,5)P2 depletion. These results demonstrate that TRPC3/C6/C7 channels are differentially regulated by depletion of PI(4,5)P2, and that the bimodal signal produced by PLC activation controls these channels in a self-limiting manner.

There is growing body of evidence that nitric oxide (NO)-cGMP-PKG signaling plays a central role in negative regulation of cardiovascular (CV) responses and its disorders through suppressed Ca(2+) dynamics. Other lines of evidence also reveal the stimulatory effects of this signaling on some CV functions. Recently, transient receptor potential (TRP) channels have received much attention as non-voltage-gated Ca(2+) channels involved in CV physiology and pathophysiology. Available information suggests that these channels undergo both inhibition and activation by NO via PKG-mediated phosphorylation and S-nitrosylation, respectively, and also act as upstream regulators to promote endothelial NO production. This review summarizes the roles of NO-cGMP-PKG signaling pathway, particularly in regulating TRP channel functions with their associated physiology and pathophysiology.

TNF-α-NF-κB signaling plays a central role in inflammation, apoptosis, and neoplasia. One major consequence of this signaling in the gut is increased production of prostaglandin E2(PGE2) via cyclooxygenase-2 (COX-2) induction in myofibroblasts, which has been reported to be dependent on Ca2+. In this study, we explored a potential role of canonical transient receptor potential (TRPC) proteins in this Ca2+-mediated signaling using a human colonic myofibroblast cell line CCD-18Co. In CCD-18Co cell, treatment with TNF-α greatly enhanced Ca2+influx induced by store depletion along with increased cell-surface expression of TRPC1 protein (but not of the other TRPC isoforms) and induction of a Gd3+-sensitive nonselective cationic conductance. Selective inhibition of TRPC1 expression by small interfering RNA (siRNA) or functionally effective TRPC1 antibody targeting the near-pore region of TRPC1 (T1E3) antagonized the enhancement of store-dependent Ca2+influx by TNF-α, whereas potentiated TNF-α induced PGE2production. Overexpression of TRPC1 in CCD-18Co produced opposite consequences. Inhibitors of NF-κB (curcumin, SN-50) attenuated TNF-α-induced enhancement of TRPC1 expression, store-dependent Ca2+influx, and COX-2-dependent PGE2production. In contrast, inhibition of calcineurin-nuclear factor of activated T-cell proteins (NFAT) signaling by FK506 or NFAT Activation Inhibitor III enhanced the PGE2production without affecting TRPC1 expression and the Ca2+influx. Finally, the suppression of store-dependent Ca2+influx by T1E3 antibody or siRNA knockdown significantly facilitated TNF-α-induced NF-κB nuclear translocation. In aggregate, these results strongly suggest that, in colonic myofibroblasts, NF-κB and NFAT serve as important positive and negative transcriptional regulators of TNF-α-induced COX-2-dependent PGE2production, respectively, at the downstream of TRPC1-associated Ca2+influx.

Calcium dynamics and its linked molecular interactions contribute to a variety of biological responses, thus it is essential to exploit techniques for detecting both concurrently. Here we describe a new method to image Ca2+ and protein-protein interaction on semi-simultaneous base through combined application of Fura-2 fluorescence imaging and Fluorescence or F[[Unsupported Character - Orster Resonance Energy Transfer (FRET) with genetically encoded fluorescence proteins in single living cells. By applying Ca2+-dependent corrections for Fura-2 fluorescence superposition on FRET images, we could semi-simultaneously analyze in vivo Fura-2 and FRET signals with high quantitative fidelity.Furthermore, by producing gradual stepwise increases in [Ca2+]i in HEK cells, we could obtain quantitative and synchronous dose-response relationships for [Ca2+]i versus protein-protein interaction between Ca2+ binding protein ‘calmodulin’ (CaM) and its binding domain from an enzyme, which were fused with fluorophores CFP and YFP, respectively. The same approach applied to voltage-gated Na+, Ca2+ and TRP channels revealed their distinct sensitivities to basal Ca2+ through CaM binding.This semi-simultaneous imaging system could be applicable to the identification of Ca2+-dependent protein interactions with further potential application to elucidating the interrelations between Ca2+ signals and cellular functions such as enzyme activities, signal transductions, and membrane protein regulations in various living cells.

1. The excitatory actions of phenylephrine (Phe) and acetylcholine (ACh) on the smooth muscle of rabbit portal vein were investigated and compared by using whole-cell and single channel configurations of the patch clamp technique, in combination with a modified concentration jump method. 2. At negative holding potentials with KCl (0.1 mM EGTA) electrodes, rapid applications of Phe (> 1 microM) and ACh (> 10 microM) both resulted in biphasic responses consisting of fast outward or inward currents and a long-lasting inward current with an increased noise level. 3. The slow inward current was still recorded when the cell was dialysed with caesium aspartate solution complemented with 10 mM EGTA, in order to eliminate contributions of calcium-dependent conductances (K+ and Cl- currents). Phe was more potent at activating the current than ACh. Ion replacement experiments revealed that both the Phe- and ACh-induced slow inward currents are cation-selective conductances (CS currents). 4. The I-V relationships of the Phe- or ACh-induced CS currents were similar both for the instantaneous peak and the steady state. The tail current analysis over a wide range of membrane potentials (-150 to +100 mV) showed that depolarizations to very positive potentials (> +50 mV) from near the resting membrane potential (-40 mV) can produce a several-fold increase in the steady-state activation of the CS currents, but the maximal activations were in most cases not observed even at +100 mV. 5. Externally applied Cd2+ produced a quick and reversible inhibition of both the Phe- and ACh-induced CS currents. This inhibition seemed almost voltage independent and the concentrations of half-inhibition were 100 and 129 microM for Phe and ACh, respectively. 6. Single channel activities were recorded in the presence of Phe or ACh using the outside-out membrane patches. The unitary conductances and reversal potentials of the Phe- and ACh-activated channels were 23 pS, +5.2 mV and 25 pS, +4.1 mV, respectively, and the open lifetimes evaluated for 50 microM Phe and 500 microM ACh were of similar order (the longer open times at -60 mV for Phe and ACh were 4.3 and 4.4 ms, respectively). In addition, the relative open probability (Po,rel) was no more than 0.2 in the voltage range of -100 to -30 mV for both 50 microM Phe and 500 microM ACh, suggesting low open probabilities of the Phe- and ACh-activated CS channels near the resting membrane potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of pinacidil were investigated on the resting membrane potential and excitatory neuro-effector transmission in the smooth muscle tissue of dog trachea using tension recording, microelectrode and double sucrose gap methods. Pinacidil (>5×10 -7 M) increased the ionic conductance and hyperpolarized smooth muscle cell membranes in dog trachea, in a dose-dependent manner. This pinacidil-induced hyperpolarization was antagonized by glibenclamide (2×10 -6 M)

A 37-year-old man was referred to our hospital with a cough. His blood test result and chest radiography findings were normal. High resolution chest computed tomography (HRCT) showed diffuse ground glass attenuation (GGA) with centrilobular distribution in the bilateral upper lung fields. As diagnosis could not be made using transbronchial lung biopsy and bronchoalveolar lavage, video-assisted lung biopsy was performed. The histological findings were a thickening of the alveolar walls with infiltration of lymphocyte-dominant inflammatory cells, and exudation of pigmented macrophages in the air spaces of the respiratory bronchioles and alveolis. We diagnosed his illness as respiratory bronchiolitis-associated interstitial lung disease. Six months after stopping smoking, his symptoms, and the GGA on chest HRCT improved.

Ca(2+) signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca(2+) entry activated by Ins(1,4,5)P(3)-induced Ca(2+) release from endoplasmic reticulum, the importance of DAG-activated Ca(2+) entry remains elusive. Here, we describe the physiological role of DAG-activated Ca(2+) entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cbeta (PKCbeta) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCbeta that maintained localization of PKCbeta at the PM. Thus, TRPC3 functions as both a Ca(2+)-permeable channel and a protein scaffold at the PM for downstream PKCbeta activation in B cells.

Activation of Ca(2+) signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca(2+) signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-, diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca(2+) influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca(2+) spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca(2+) influx itself or molecule(s) upstream of Ca(2+) influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr(69) and prevented TRPC6-mediated Ca(2+) influx. Substitution of Ala for Thr(69) in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce G alpha(q)-mediated signaling, did not affect the suppression of receptor-activated Ca(2+) influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition.

TRPC6 is a ubiquitous and predominant isoform expressed in vascular smooth muscle cells (VSMCs) and has been implicated in the regulation of vascular tone and remodelling via neurohormonal and mechanosensitive mechanisms. Although these two mechanisms are generally thought to work independently, our recent investigation has revealed that TRPC6 channel is synergistically activated by receptor and mechanical stimulations via the phospholipase C/Gq-protein/diacylglycerol and phospholipase A2/ω-hydroxylase/20-hydroxyeicosatetraenoic acid pathways. Experiments with pressurized mesenteric artery have suggested that this synergism likely contributes to enhanced myogenic responsiveness under near-threshold receptor stimulation. Different lines of evidence also suggest that TRPC6 channel is subject to both positive and negative regulation via activation of Ca2+/ calmodulin-dependent kinase (CAMKII), and protein kinases C (PKC) and G (PKG); while CAMKII- and PKC-mediated phosphorylation accelerates the time courses of TRPC6 activation and inactivation during receptor stimulation respectively, activation of PKG via stimulation of the nitric oxide or atrial natriuretic peptide/guanylate cyclase/cGMP pathway tonically suppresses TRPC6 channel activity through its phosphorylation on T69, regardless of receptor or mechanical stimulation[1]. Interestingly, prolonged activation of PKG (10–20 min) reactivated TRPC6 channel rendering it spontaneously active with loss of receptor- and mechano-sensitivities. Disruption of actin cytoskeleton by cytochalasin D treatment induced similar consequences. It can be speculated that phosphorylation of TRPC6 channel by PKG may not only cause an acute tonic inhibition of the channel activation by neurohormonal and mechanical stimuli, but also induce a slow transition between differential activation modes, presumably altering the VSMC phenotype from 'contractile' to 'proliferative' ones.

TRPC6 is a non–voltage-gated Ca 2+ entry/depolarization channel associated with vascular tone regulation and remodeling. Expressed TRPC6 channel responds to both neurohormonal and mechanical stimuli, the mechanism for which remains controversial. In this study, we examined the possible interactions of receptor and mechanical stimulations in activating this channel using the patch clamp technique. In HEK293 cells expressing TRPC6, application of mechanical stimuli (hypotonicity, shear, 2,4,6-trinitrophenol) caused, albeit not effective by themselves, a prominent potentiation of cationic currents ( I TRPC6 ) induced by a muscarinic receptor agonist carbachol. This effect was insensitive to a tarantula toxin GsMTx-4 (5 μmol/L). A similar extent of mechanical potentiation was observed after activation of I TRPC6 by GTPγS or a diacylglycerol analog 1-oleoyl-2-acetyl- sn -glycerol (OAG). Single TRPC6 channel activity evoked by carbachol was also enhanced by a negative pressure added in the patch pipette. Mechanical potentiation of carbachol- or OAG-induced I TRPC6 was abolished by small interfering RNA knockdown of cytosolic phospholipase A 2 or pharmacological inhibition of ω-hydroxylation of arachidonic acid into 20-HETE (20-hydroxyeicosatetraenoic acid). Conversely, direct application of 20-HETE enhanced both OAG-induced macroscopic and single channel TRPC6 currents. Essentially the same results were obtained for TRPC6-like cation channel in A7r5 myocytes, where its activation by noradrenaline or Arg8 vasopressin was greatly enhanced by mechanical stimuli via 20-HETE production. Furthermore, myogenic response of pressurized mesenteric artery was significantly enhanced by weak receptor stimulation dependently on 20-HETE production. These results collectively suggest that simultaneous operation of receptor and mechanical stimulations may synergistically amplify transmembrane Ca 2+ mobilization through TRPC6 activation, thereby enhancing the vascular tone via phospholipase C/diacylglycerol and phospholipase A 2 /ω-hydroxylase/20-HETE pathways.

Transient receptor potential (TRP) proteins constitute a large non-voltage-gated cation channel superfamily, activated polymodally by various physicochemical stimuli, and are implicated in a variety of cellular functions. Known activators for TRP include not only chemical stimuli such as receptor stimulation, increased acidity and pungent/cooling agents, but temperature change and various forms of mechanical stimuli such as osmotic stress, membrane stretch, and shear force. Recent investigations have revealed that at least ten mammalian TRPs exhibit mechanosensitivity (TRPC1, 5, 6; TRPV1, 2, 4; TRPM3, 7; TRPA1; TRPP2), but the mechanisms underlying it appear considerably divergent and complex. The proposed mechanisms are associated with lipid bilayer mechanics, specialized force-transducing structures, biochemical reactions, membrane trafficking and transcriptional regulation. Many of mechanosensitive (MS)-TRP channel likely undergo multiple regulations via these mechanisms. In the cardiovascular system in which hemodynamic forces constantly operate, the impact of mechanical stress may be particularly significant. Extensive morphological and functional studies have indicated that several MS-TRP channels are expressed in cardiac muscle, vascular smooth muscle, endothelium and vasosensory neurons, each differentially contributing to cardiovascular (CV) functions. To further complexity, the recent evidence suggests that mechanical stress may synergize with neurohormonal mechanisms thereby amplifying otherwise marginal responses. Furthermore, the currently available data suggest that MS-TRP channels may be involved in CV pathophysiology such as cardiac arrhythmia, cardiac hypertrophy/myopathy, hypertension and aneurysms. This review will overview currently known mechanisms for mechanical activation/modulation of TRPs and possible connections of MS-TRP channels to CV disorders.

Recent investigations have revealed that mammalian homologues of transient receptor potential (TRP) protein (TRP1-7) are promising candidates for Ca2+ entry mechanisms (or channels) associated with various metabotropic G protein-coupled receptors (GPCRs) in smooth muscle, stimulation of which generates lipid second messengers and depletes internal stores. RT-PCR and immunocytochemical experiments have demonstrated that although the level of expression varies depending on tissues, the major TRP isoforms expressed in smooth muscle are TRP4, 6 and 7. In some vascular preparations, the significant expression of TRP1 mRNA and protein is also detected. Consistent with these findings, recent functional studies using TRP6- and TRP1-specific antisense oligonucleotides and antibodies have suggested that TRP6 is the essential component of alpha1-adrenoceptor activated, store depletion-independent Ca2+ entry channels, while TRP1 is partly involved in Ca2+ entry associated with store depletion or capacitative Ca2+ entry. In addition, coexpression of different TRP isoforms results in the appearance of cation channels showing novel properties reminiscent of some native GPCR-activated Ca2+-permeable non-selective cation channels. Thus, at present, TRP proteins may be the most important clues for elucidating the molecular entities of receptor- and store-operated Ca2+ entry mechanisms in smooth muscle and their roles in smooth muscle functions.

We investigated the inhibitory role of the nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway on receptor-activated TRPC6 channels in both a heterologous expression system (HEK293 cells) and A7r5 vascular myocytes. Cationic currents due to TRPC6 expression were strongly suppressed (by approximately 70%) by a NO donor SNAP (100 microm) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 microm) or after G-protein activation with intracellular perfusion of GTPgammaS (100 microm). A similar extent of suppression was also observed with a membrane-permeable analogue of cGMP, 8Br-cGMP (100 microm). The inhibitory effects of SNAP and 8Br-cGMP on TRPC6 channel currents were strongly attenuated by the presence of inhibitors for guanylyl cyclase and PKG such as ODQ, KT5823 and DT3. Alanine substitution for the PKG phosphorylation candidate site at T69 but not at other sites (T14A, S28A, T193A, S321A) of TRPC6 similarly attenuated the inhibitory effects of SNAP and 8Br-cGMP. SNAP also significantly reduced single TRPC6 channel activity recorded in the inside-out configuration in a PKG-dependent manner. SNAP-induced PKG activation stimulated the incorporation of (32)P into wild-type and S321A-mutant TRPC6 proteins immunoprecipitated by TRPC6-specific antibody, but this was greatly attenuated in the T69A mutant. SNAP or 8Br-cGMP strongly suppressed TRPC6-like cation currents and membrane depolarization evoked by Arg(8)-vasopressin in A7r5 myocytes. These results strongly suggest that TRPC6 channels can be negatively regulated by the NO-cGMP-PKG pathway, probably via T69 phosphorylation of the N-terminal. This mechanism may be physiologically important in vascular tissues where NO is constantly released from vascular endothelial cells or nitrergic nerves.

1. The actions of exogenously applied ATP were investigated with the whole-cell patch clamp method in single cells isolated from guinea-pig urinary bladder with a modified concentration jump technique. 2. Rapid application of ATP (threshold ca. 100 nM) depolarized the cell membrane with superimposition of action potentials which was followed by transient hyperpolarization. In the presence of D600, the amplitude of the ATP-induced depolarization was a function of the ATP concentration (EC50: 0.5-1 microM). 3. ATP activated a dose-dependent inward current with a short latency (18 ms with 10 microM ATP; measured as the time between the start of application and 10% of the peak). The relationship of the peak current versus ATP concentration was well fitted by a Michaelis-Menten equation with a Hill coefficient (n) of 1.7 and a dissociation constant (Kd) of 2.3 microM. The current desensitized rapidly and the time course of desensitization was a function of the ATP concentration and could be fitted by two exponentials. 4. The reversal potential of the ATP-activated current was near 0 mV. Replacement of extracellular Na by other monovalent or divalent cations indicated that the current flows through nonselective cation channels. 5. alpha,beta-Methylene ATP also produced a dose-dependent inward current but was less potent than ATP (n: 1.6, Kd: 10.4 microM). alpha,beta-Methylene ATP blocked the response to ATP by desensitization of the receptor. 6. alpha,beta-Methylene ATP was 50-100 times more potent than ATP at eliciting a contractile response of strips of detrusor smooth muscle. 7. The relevance of the above results to the possible role of ATP as the fast excitatory transmitter is discussed.

Aim: The aim of the present study was to explore the mechanism for the Ca 2+ -dependent inactivation of the canonical transient receptor potential (TRPC) 7 channel expressed in human embryonic kidney 293 cells. Method: The whole-cell patch-clamp technique was used in the study. Results: With Ca 2+ -free external solution, the perfusion of 100 μmol/L carbachol to, or dialysis of the cell with 100 μmol/L guanosine 5'-3- O -(thio)triphosphate (GTPgS), induced large inward currents, respectively. These currents were rapidly inhibited by the addition of 1 mmol/L Ca 2+ into the bath, and recovery from this inhibition was only partial after the Ca 2+ removal, unless vigorous intracellular Ca 2+ buffering with 10 mmol/L 1,2 bis (2-aminophenoxy)ethane- N,N,N’,N’ -tetraacetic acid (BAPTA) (plus 4 mmol/L Ca 2+ ) was employed. In contrast, the current induced by a membrane-permeable analog of diacylglycerol (DAG), 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 μmol/L) did not undergo the inhibition persisting after Ca 2+ removal. Interestingly, the inclusion of inositol 1,4,5 trisphosphate (IP 3 ; 100 μmol/L) in the patch pipette rendered the OAG-induced current susceptible to the persistent Ca 2+ -mediated inhibition independent of the IP3 receptor in the majority of the tested cells, as evidenced by the inability of heparin and thapsigargin in reversing the effect of IP 3 . Conclusion: The present results suggest that Ca 2+ entry via the activated TRPC7 channel plays a critical role in inactivating the channel where the cooperative actions of DAG and IP 3 are essentially involved.

Background/Aims: The roles of intercellular communication and T-type versus L-type voltage-dependent Ca2+ channels (VDCCs) in conducted vasoconstriction to local KCl-induced depolarization were investigated in mesenteric arterioles. Methods: Ratiometric Ca2+ imaging (R) using Fura-PE3 with micro-ejection of depolarizing KCl solution and VDCC blockers, and immunohistochemical and RT-PCR techniques were applied to isolated rat mesenteric terminal arterioles (n = 71 from 47 rats; intraluminal diameter: 24 ± 1 μm; length: 550–700 μm). Results: Local application of KCl (at 0 μm) led to local (ΔR = 0.54) and remote (ΔR = 0.17 at 500 μm) increases in intracellular Ca2+. Remote Ca2+ responses were inhibited by the gap junction uncouplers carbenoxolone and palmitoleic acid. CaV1.2, CaV3.1 and CaV3.2 channels were immunolocalized in vascular smooth muscle cells and CaV3.2 in adjacent endothelial cells. Local and remote Ca2+ responses were inhibited by bath application of L- and T-type blockers [nifedipine, NNC 55-0396 and R(–)-efonidipine]. Remote Ca2+ responses (500 μm) were not affected by abolishing Ca2+ entry at an intermediate position on the arterioles (at 200–300 μm) using micro-application of VDCC blockers. Conclusion: Both L- and T-type channels mediate Ca2+ entry during conducted vasoconstriction to local KCl in mesenteric arterioles. However, these channels do not participate in the conduction process per se.

A 39-year-old man was referred to our hospital with anterior chest discomfort, dry cough and shortness of breath. His blood test revealed mild inflammatory change and high serum KL-6 levels. Chest radiograph and computed tomography (CT) showed ground glass attenuation with volume loss in both lower lung fields, and in particular a reversed halo sign was shown on high-resolution CT (HRCT). As transbronchial lung biopsy and bronchoalveolar lavage did not enable a diagnosis, video-assisted thoracic surgery was performed. The histological findings of the resected specimen showed cellular nonspecific interstitial pneumonia. This suggested the possibility of collagen vascular disorder (CVD) associated with interstitial pneumonitis, but no criteria of CVD were fulfilled. Although the reversed halo sign is relatively specific for cryptogenic organizing pneumonia, we report a case of cellular nonspecific interstitial pneumonia showing this sign on chest HRCT.

Calcium ions (Ca(2+)) are particularly important in cellular homeostasis and activity. To elicit physiologically relevant timing and spatial patterns of Ca(2+) signaling, ion channels in the surface of each cell precisely control Ca(2+) influx across the plasma membrane. A group of surface membrane ion channels called receptor-activated cation/Ca(2+) channels (RACCs) are activated by diverse cellular stimuli from the surrounding extracellular environment via receptors and other pathways such as heat, osmotic pressure, and mechanical and oxidative stress. An important clue to understanding the molecular mechanisms underlying the functional diversity of RACCs was first attained by molecular identification of the transient receptor potential (trp) protein (TRP), which mediates light-induced depolarization in Drosophila photoreceptor cells, and its homologues from various biological species. Recent studies have revealed that respective TRP channels are indeed activated by characteristic cellular stimuli. Furthermore, the involvement of TRP channels has been demonstrated in the signaling pathways essential for tissue-specific functions as well as ubiquitous biological responses, such as cell proliferation, differentiation, and death. These findings encourage the usage of TRP channels and their signalplexes as powerful tools for developing novel pharmaceutical targets.

Sustained elevation in the intracellular Ca 2+ concentration via Ca 2+ influx, which is activated by a variety of mechanisms, plays a central regulatory role for cardiovascular functions. Recent molecular biological research has disclosed an unexpectedly diverse array of Ca 2+ -entry channel molecules involved in this Ca 2+ influx. These include more than ten transient receptor potential (TRP) superfamily members such as TRPC1, TRPC3–6, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, and polycystin (TRPP2). Most of them appear to be multimodally activated or modulated and show relevant features to both acute hemodynamic control and long-term remodeling of the cardiovascular system, and many of them have been found to respond not only to receptor stimulation but also to various forms of stimuli. There is good evidence to implicate TRPC1 in neointimal hyperplasia after vascular injury via store-depletion–operated Ca 2+ entry. TRPC6 likely contributes to receptor-operated and mechanosensitive Ca 2+ mobilizations, being involved in vasoconstrictor and myogenic responses and pulmonary arterial proliferation and its associated disease (idiopathic pulmonary arterial hypertension). Considerable evidence has also been accumulated for unique involvement of TRPV1 in blood flow/pressure regulation via sensory vasoactive neuropeptide release. New lines of evidence suggest that TRPV2 may act as a Ca 2+ -overloading pathway associated with dystrophic cardiomyopathy, TRPV4 as a mediator of endothelium-dependent hyperpolarization, TRPM7 as a proproliferative vascular Mg 2+ entry channel, and TRPP2 as a Ca 2+ -entry channel requisite for vascular integrity. This review attempts to provide an overview of the current knowledge on TRP proteins and discuss their possible roles in cardiovascular functions and diseases.

Lysophosphatidylcholine (LPC) is metabolized from a membrane phospholipid and modulates a variety of channels in the plasma membrane (PM). We examined LPC modulation of cardiac ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR) using the planar lipid bilayer method to measure the single-channel currents. Micromolar concentrations of LPC increased the open probability of the reconstituted RyR channels irrespective of whether LPC was added to the cis or trans chamber. LPC also increased the membrane capacitance of the bilayer. The effects of LPC contrasted well with those of sphingosylphosphorylcholine (SPC). Taken together, these results suggest that amphipathic lipid LPC does not bind directly to the RyR channel protein, but rather, is incorporated into the bilayer membrane and activates the channel. Thus, we consider cell membrane-derived LPC to be a putative endogenous mediator that activates not only plasma membrane channels but also RyR channels and induces arrhythmogenic Ca(2+) mobilization in cardiomyocytes.

The mechanisms of intracellular calcium store depletion and store-related Ca(2+) dysregulation in relation to apoptotic cell death in PC12 cells were investigated at physiological temperatures with a leak-resistant fluorescent indicator dye Fura-PE3/AM by a cooled CCD imaging analysis system. Electron microscopic observations have shown thapsigargin (TG; 100 nM)-induced apoptosis in PC12 cells. Thorough starvation of stored Ca(2+) by BAPTA/AM (50 microM), or La(3+) (100 microM) enhanced while dantrolene (100 microM) attenuated the TG-induced apoptosis by preventing a calcium release from internal stores. An immunoblotting analysis revealed an enhanced expression of GRP78, the hallmark of endoplasmic reticulum (ER) stress when cells were treated by TG along with BAPTA/AM. These results indicate that the depletion of the intracellular Ca(2+) stores itself induces the ER stress and apoptosis in PC12 cells without any involvement of the capacitative calcium entry (CCE) or a sustained elevation of intracellular Ca(2+) concentrations ([Ca(2+)](i)).

Angiotensin II (Ang II) activates multiple signaling pathways leading to hyperplasia of cardiac fibroblasts. Reactive oxygen species (ROS) produced by Ang II stimulation are assumed to play pivotal roles in this process. Here, we show that ROS mediate Ang II-induced activation of nuclear factor of activated T cells (NFAT) in rat cardiac fibroblasts. Ang II-induced NFAT activation was suppressed by diphenyleneiodonium (an NADPH oxidase inhibitor), dominant negative (DN)-Rac, DN-p47(phox), and an inhibitor of Galpha(12/13) (Galpha(12/13)-specific regulator of G protein signaling domain of p115RhoGEF, p115-regulator of G protein signaling (RGS)). Stimulation of Ang II receptor increased the intracellular ROS level in a Rac- and p47(phox)-dependent manner. Because p115-RGS suppressed Ang II-induced Rac activation, Ang II receptor-coupled Galpha(12/13) mediated NFAT activation through ROS production by Rac activation. Ang II-induced nuclear translocation of the green fluorescent protein (GFP)-tagged amino-terminal region of NFAT4 (GFP-NFAT4) was suppressed by p115-RGS or BAPTA but not by diphenyleneiodonium. The expression of constitutively active (CA)-Galpha(12/13), CA-G translocation alpha(13), or CA-Rac increased the nuclear of GFP-NFAT4. These results suggest that NFAT activity is regulated by both Ca(2+)-dependent and ROS-dependent pathways. Furthermore, activation of c-Jun NH(2)-terminal kinase (JNK) induced by Ang II stimulation is required for NFAT activation because Ang II-induced NFAT activation was inhibited by SP600125, a selective JNK inhibitor. These results indicate that Ang II stimulates the nuclear translocation and activation of NFAT by integrated pathways including the activation of Galpha(12/13), Rac, NADPH oxidase, and JNK and that Galpha(12/13)-mediated ROS production is essential for NFAT transcriptional activation.

For effective operation of PEFC (Proton Exchange Fuel Cell), it is desired to measure hydrogen concentration in anode side gas stream. We have developed a linear Hydrogen Sensor, which comprises a pair of Pt supported carbon electrodes and a proton exchange membrane (PEM). The sensor has limited current sensing characteristics. The concentration of hydrogen supplied from the gas diffusing area at the anode side is measured by detecting the current of the selective hydrogen pumping. The carbon electrode deterioration occurred when little hydrogen existed in the sensing area. By controlling an applying voltage to the PEM electrodes when the pumping current is lowered to a certain limit, this issue has been solved; no deterioration occurred even after 2500 hours continuous operation under ambient atmospheric conditions. This sensor is suitable for anode side gas purge control of automotive hydrogen FC and for power management of reformed gas FC for home co-generation.

Although plasma B-type natriuretic peptide (BNP) levels increase with age, the mechanisms responsible for this increase are unknown. We investigated the predictors of elevated BNP in older subjects without cardiac systolic dysfunction and overt renal dysfunction. Furthermore, we analyzed the relations between BNP and its second messenger, cyclic guanosine monophosphate (cGMP), to aging. In 252 subjects (mean age 69 +/- 12 years) with left ventricular ejection fraction/=50% and creatinine levels==1.5 mg/dl, plasma levels of BNP, cGMP, blood urea nitrogen, creatinine, and beta2-microglobulin (an endogenous marker of renal function), estimated glomerular filtration rate, and echocardiographic data were prospectively evaluated. Plasma BNP levels increased with age (r = 0.4, p0.0001). With use of multivariate analysis, predictors of elevated BNP levels were age, use of beta blockers, and serum beta2-microglobulin levels. The molar ratio of cGMP to BNP significantly decreased with aging (r = 0.55, p0.0001). Elevated BNP in older subjects with normal cardiac systolic function may be due in part to renal impairment. With aging, biologic compensation of the cardiac natriuretic peptide system may be attenuated.

We investigated the possible sources of intracellular ATP which was previously shown essential for maintaining the muscarinic cationic channel activities (or currents; I(cat)) in guinea-pig ileal myocytes, using two variants of patch clamp techniques. Deprivation of external glucose or its replacement with 2-deoxyglucose significantly reduced the magnitude of I(cat), recorded with nystatin-perforated method, with greater efficacy than for voltage-dependent Ca2+ current Intracellular dialysis of ileal myocytes with key substrates for glycolysis, oxidative metabolism and creatine-phosphocreatine system all resulted in a comparably effective maintenance of I(cat), which was abolished by inhibitors for these ATP-producing systems, 3-bromopyruvate, cyanide and 2,4-dinitrofluorobenzene (DNFB), respectively. However, amongst these inhibitors, only 3-bromopyruvate effectively reduced I(cat) recorded with the nystatin-perforated method. These results strongly suggest the exclusive physiological importance of glycolytic ATP production in maintaining I(cat), activity, and thus this mechanism may play a role in the regulation of gut motility.

Cellular stimulation from the surrounding extracellular environment via receptors and other pathways evoke activation of Ca(2+)-permeable cation channels. An important clue to understand the molecular mechanisms underlying these receptor-activated cation channels (RACC) was first provided through molecular studies of the transient receptor potential (trp) protein (TRP), which controls light-induced deporlarization in Drosophila photoreceptor cells. Recent studies have revealed that these TRP channels are also activated by diverse stimuli such as heat, osmotic stress, and oxidative stress. Furthermore, involvement of TRP channels has been demonstrated in signaling pathways essential for biological responses, such as proliferation, differentiation, and cell death. These findings encourage usage of TRP channels and their signalplexes as powerful tools for the development of novel pharmaceutical targets.

TRPC3/C6/C7 channels are activated by diacylglycerol (DAG) upon the phospholipase C (PLC) coupled receptor stimulation which results in the breakdown of phosphoinositides (PIPs). Although the critical function of PIPs on various ion transporting molecules has been demonstrated, their roles in TRPC3/C6/C7 channels remain controversial. Here, by ectopical usage of voltage-sensing PIPs phosphatase (DrVSP), we found that dephosphorylation of PIPs produces robust inhibition of currents induced by carbachol (CCh), OAG and RHC80267, for all TRPC3/C6/C7 channels. The strength of inhibition was, however, variable with the rank order of DrVSP-mediated inhibition (VMI): C3 < C6 < C7. Pharmacological and molecular interventions most likely suggest that depletion of PI(4,5)P2 is a critical event for VMI of all three channels. As predicted, when PLC catalytic signal was vigorously activated through overexpressed muscarinic type-I receptor (M1R), macroscopic kinetics of TRPC currents were greatly accelerated as similar the rank order of VMI, and it was simultaneously dismissed or attenuated. Likewise, VMI was rarely detected in vasopressin-induced TRPC6-like currents from smooth muscle cells (A7r5), supporting a severe or various degree of PI(4,5)P2 depletion in the physiological conditions. We also employed simultaneous detection of FRET-based measurement of PI(4,5)P2 and TRPC6 current, confirming that VMI reflects the degree of PI(4,5)P2 depletion. These results thus demonstrate that TRPC3/C6/C7 channels are differentially regulate by depletion of PI(4,5)P2, and bimodal signal produced by PLC activation (i.e. depletion of PIP2 and production of DAG) simultaniously controls these channels in a self-limiting manner.

We investigated a possible role of nifedipine-insensitive high voltage-activated (NI-HVA) Ca 2+ channels in arterial diameter regulation in the semi-terminal branches of rabbit mesenteric artery (RMA). From these branches, NI-HVA Ca 2+ currents showing almost identical properties to those previously identified in a similar region of guinea-pig [Circulation Research 1999;85:596–605] were recorded with whole-cell patch clamp recording. With video-microscopic measurement, the diameter of RMA segments perfused intraluminally at a constant rate (2–6 mL/h; 269 ± 9 μm, n = 27) decreased by 50–60% by raising the external K + concentration ([K + ] o ) to 75 mM, a substantial part of which remained after addition of 1–10 μM nifedipine (44 ± 5% of initial diameter, n = 27). This nifedipine-insensitive diameter decrease (NI-DD) appeared to consist of initial transient and subsequent tonic phases (this separation was, however, not always clear), was resistant to tetrodotoxin, and was completely abolished in Ca 2+ -free or 100 μM Cd 2+ -containing bath solutions. The magnitude of NI-DD increased depending on [K + ] o with a threshold concentration of 20–40 mM. Raising the external Ca 2+ concentration dose-dependently increased the magnitude of NI-DD, the extent being more prominent in the late tonic phase. Combined application of caffeine (10 mM) with ryanodine (3 μM) produced a large transient NI-DD, which strongly attenuated the NI-DD evoked by a subsequent elevation in [K + ] o . Using the fura-2 spectrofluorimetric Ca 2+ imaging technique, a nifedipine-insensitive [Ca 2+ ] i increase showing similar [K + ] o -dependence and Cd 2+ sensitivity to NI-DD was observed. These properties of NI-DD accord with those of NI-HVA Ca 2+ channels, thus suggesting their contribution to small arterial diameter regulation in RMA.

Nowadays an idea of tele-existence or tele-presence that is based on the virtual reality technology has been proposed and is expected to be very effective for performing tasks in hazardous environments, such as a nuclear power plant. In our research we study the fundamental technique of tele-operation using virtual reality technology to manipulate the robot-arm in the nuclear power plant. This paper discusses quantitative evaluation of the properties of human in virtual space, i.e., the aggravation of the operability of the robot-arm manipulation system caused by the time lag in manipulating it, and the accuracy of stereoscopic viewing in virtual space.

1. Pharmacological properties of nifedipine-insensitive, high voltage-activated Ca(2+) channels in rat mesenteric terminal arteries (NICCs) were investigated and compared with those of alpha1E and alpha1G heterologously expressed in BHK and HEK293 cells respectively, using the patch clamp technique. 2. With 10 mM Ba(2+) as the charge carrier, rat NICCs (unitary conductance: 11.5 pS with 110 mM Ba(2+)) are almost identical to those previously identified in a similar region of guinea-pig, such as in current-voltage relationship, voltage dependence of activation and inactivation, and divalent cation permeability. However, these properties are considerably different when compared with alpha1E and alpha1G. 3. SNX-482(200 nM and sFTX3.3 (1 micro M), in addition to omega-conotoxin GVIA (1 micro M) and omega-agatoxin IVA (100 nM), were totally ineffective for rat NICC currents, but significantly suppressed alpha1E (by 82% at 200 nM; IC(50)=11.1 nM) and alpha1G (by 20% at 1 micro M) channel currents, respectively. A non-specific T-type Ca(2+) channel blocker nimodipine (10 micro M) differentially suppressed these three currents (by 40, 3 and 85% for rat NICC, alpha1E and alpha1G currents, respectively). 4. Mibefradil, the widely used T-type channel blocker, almost equally inhibited rat NICC and alpha1G currents in a voltage-dependent fashion with similar IC(50) values (3.5 and 0.3 micro M and 2.4 and 0.14 micro M at -100 and -60 mV, respectively). Furthermore, other organic T-type channel blockers such as phenytoin, ethosuximide, an arylpiperidine derivative SUN N5030 (IC(50)=0.32 micro M at -60 mV for alpha1G) also exhibited comparable inhibitory efficacies for NICC currents (inhibited by 22% at 100 micro M; IC(50)=27.8 mM; IC(50)=0.53 micro M, respectively). 5. These results suggest that despite distinctive biophysical properties, the rat NICCs have indistinguishable pharmacological sensitivities to many organic blockers compared with T-type Ca(2+) channels.

Recent investigations have revealed that mammalian homologues of transient receptor potential (TRP) protein (TRP1-7) are promising candidates for Ca2+ entry mechanisms (or channels) associated with various metabotropic G protein-coupled receptors (GPCRs) in smooth muscle, stimulation of which generates lipid second messengers and depletes internal stores. RT-PCR and immunocytochemical experiments have demonstrated that although the level of expression varies depending on tissues, the major TRP isoforms expressed in smooth muscle are TRP4, 6 and 7. In some vascular preparations, the significant expression of TRP1 mRNA and protein is also detected. Consistent with these findings, recent functional studies using TRP6- and TRP1-specific antisense oligonucleotides and antibodies have suggested that TRP6 is the essential component of alpha1-adrenoceptor activated, store depletion-independent Ca2+ entry channels, while TRP1 is partly involved in Ca2+ entry associated with store depletion or capacitative Ca2+ entry. In addition, coexpression of different TRP isoforms results in the appearance of cation channels showing novel properties reminiscent of some native GPCR-activated Ca2+-permeable non-selective cation channels. Thus, at present, TRP proteins may be the most important clues for elucidating the molecular entities of receptor- and store-operated Ca2+ entry mechanisms in smooth muscle and their roles in smooth muscle functions.

There is a rapidly expanding protein family which encompasses a broad repertoire of cation-selective channels serving as a continuous Ca2+ entry pathway into the cell. The transient receptor potential protein (TRP) and its relatives, which were originally thought to be Ca (2+)-permeable cation channels activated upon the stimulation of G-protein coupled and tyrosine kinase receptors, are now becoming promising candidates mediating a variety of cellular responses and functions such as mechano/chemo-transduction, oxidative stress, and cell survival/proliferation. This short paper briefly overviews the current knowledge about these proteins as a new target for drug discovery and development.

TRPC3, C6 and C7, which comprise a subfamily of the mammalian diacylglycerol (DAG)-activated TRPC cation channels, play key roles in cardiovascular, gastrointestinal, renal and nervous system physiology. We recently reported that the depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) dominantly inhibits these TRPC channels activity, irrespective of the presence of a DAG analogue. However, the functional significance of PI(4,5)P2 reduction/depletion during G(q) protein-coupled receptor-operated TRPC activation remains largely unknown. To address this question, we simultaneously measured TRPC current kinetics and PI(4,5)P2/DAG dynamics using Forster resonance energy transfer (FRET) at varying degrees of receptor stimulation. Our measurements demonstrated a clear correlation between the time-course of activation/inactivation in TRPC6/7 currents and the kinetics of PI(4,5)P2 reduction. In contrast, the time-course of DAG production is incompatible with TRPC6/7 current inactivation, indicating that reduction of PI(4,5)P2 is primarily responsible for this inhibition. After estimating the functional dissociation constant of PI(4,5)P2 binding to these DAG-sensitive TRPC channels, we used a model simulation to demonstrate the similarity of receptor-operated TRPC6/7 currents in both a HEK293 cell recombinant expression system and aortic smooth muscle cells (A7r5). Our findings demonstrate that G(q) protein-coupled receptor stimulation causes a reduction in PI(4,5)P2 levels that inhibits TRPC6/7 channels by accelerating their inactivation and perhaps limiting their maximum open probability.

Reconstitution of G-protein-coupled receptor activated cation channels into the lipid bilayer was attempted with plasma membrane vesicles prepared from guinea-pig ileal smooth muscle using the purification technique previously applied to the large conductance Ca2+-dependent and ATP-sensitive K+ channels (Toro et al., 1990). Under Na+-rich conditions, incorporation of plasma membrane vesicles into the bilayer produced GTPgammaS (100 microM)-activatable channel activities that are inhibited by GDPbetaS (1 mM), sensitive to Ca2+ and enhanced by depolarization. The reversal potential and unitary conductance (tens of picosiemens) of these channels varied in a manner dependent on Na+ concentration, but not affected by Cl-. These results strongly indicate that the reconstituted channels activated by GTPgammaS belong to a class of voltage-dependent, Ca2+-sensitive cation-selective channels that are activated through a G-protein, and correspond most likely to the muscarinic receptor-activated cation channels previously identified in the same preparation. These results also suggest potential usefulness of bilayer incorporation technique to investigate the receptor-operated cation channels in smooth muscle.

Abstract —The Drosophila transient receptor potential protein (TRP) and its mammalian homologues are thought to be Ca 2+ -permeable cation channels activated by G protein (G q/11 )–coupled receptors and are regarded as an interesting molecular model for the Ca 2+ entry mechanisms associated with stimulated phosphoinositide turnover and store depletion. However, there is little unequivocal evidence linking mammalian TRPs with particular native functions. In this study, we have found that heterologous expression of murine TRP6 in HEK293 cells reproduces almost exactly the essential biophysical and pharmacological properties of α 1 -adrenoceptor–activated nonselective cation channels (α 1 -AR–NSCC) previously identified in rabbit portal vein smooth muscle. Such properties include activation by diacylglycerol; S-shaped current-voltage relationship; high divalent cation permeability; unitary conductance of 25 to 30 pS and augmentation by flufenamate and Ca 2+ ; and blockade by Cd 2+ , La 3+ , Gd 3+ , SK&F96365, and amiloride. Reverse transcriptase–polymerase chain reaction and confocal laser scanning microscopy using TRP6-specific primers and antisera revealed that the level of TRP6 mRNA expression was remarkably high in both murine and rabbit portal vein smooth muscles as compared with other TRP subtypes, and the immunoreactivity to TRP6 protein was localized near the sarcolemmal region of single rabbit portal vein myocytes. Furthermore, treatment of primary cultured portal vein myocytes with TRP6 antisense oligonucleotides resulted in marked inhibition of TRP6 protein immunoreactivity as well as selective suppression of α 1 -adrenoceptor–activated, store depletion–independent cation current and Ba 2+ influx. These results strongly indicate that TRP6 is the essential component of the α 1 -AR–NSCC, which may serve as a store depletion–independent Ca 2+ entry pathway during increased sympathetic activity.