Search

Pulmonary arterial hypertension (PAH) is a multifactorial disease characterized by pulmonary arterial vasoconstriction and remodeling. Src family tyrosine kinases, including Fyn, play critical roles in vascular remodeling via the inhibition of STAT3 signaling. EPA is known to inhibit Fyn kinase activity. This study investigated the therapeutic potential and underlying mechanisms of EPA and its metabolite, resolvin E1 (RvE1), to treat PAH using monocrotaline-induced PAH model rats (MCT-PAH), human pulmonary artery endothelial cells (HPAECs), and human pulmonary artery smooth muscle cells (HPASMCs). Administration of EPA 1 and 2 weeks after MCT injection both ameliorated right ventricular hypertrophy, remodeling and dysfunction, and medial wall thickening of the pulmonary arteries and prolonged survival in MCT-PAH rats. EPA attenuated the enhanced contractile response to 5-hydroxytryptamine in isolated pulmonary arteries of MCT-PAH rats. Mechanistically, the treatment with EPA and RvE1 or the introduction of dominant-negative Fyn prevented TGF-β2-induced endothelial-to-mesenchymal transition and IL-6-induced phosphorylation of STAT3 in cultured HPAECs. EPA and RvE1 suppressed Src family kinases' activity as evaluated by their phosphorylation status in cultured HPAECs and HPASMCs. EPA and RvE1 suppressed vasocontraction of rat and human PA. Furthermore, EPA and RvE1 inhibited the enhanced proliferation and activity of Src family kinases in HPASMCs derived from patients with idiopathic PAH. EPA ameliorated PAH's pathophysiology by mitigating vascular remodeling and vasoconstriction, probably inhibiting Src family kinases, especially Fyn. Thus, EPA is considered a potent therapeutic agent for the treatment of PAH.

In the heart, TRPM4 is most abundantly distributed in the conduction system. Previously, a single mutation, ‘E7K’, was identified in its distal N-terminus to cause conduction disorder because of enhanced cell-surface expression. It remains, however, unclear how this expression increase leads to conduction failure rather than abnormally enhanced cardiac excitability. To address this issue theoretically, we mathematically formulated the gating kinetics of the E7K-mutant TRPM4 channel by a combined use of voltage jump analysis and ionomycin-perforated cell-attached recording technique and incorporated the resultant rate constants of opening and closing into a human Purkinje fiber single-cell action potential (AP) model (Trovato model) to perform 1D-cable simulations. The results from TRPM4 expressing HEK293 cells showed that as compared with the wild-type, the open state is much preferred in the E7K mutant with increased voltage-and Ca2+-sensitivities. These theoretical predictions were confirmed by power spectrum and single channel analyses of expressed wild-type and E7K-mutant TRPM4 channels. In our modified Trovato model, the facilitated opening of the E7K mutant channel markedly prolonged AP duration with concomitant depolarizing shifts of the resting membrane potential in a manner dependent on the channel density (or maximal activity). This was, however, little evident in the wild-type TRPM4 channel. Moreover, 1D-cable simulations with the modified Trovato model revealed that increasing the density of E7K (but not of wild-type) TRPM4 channels progressively reduced AP conduction velocity eventually culminating in complete conduction block. These results clearly suggest the brady-arrhythmogenicity of the E7K mutant channel which likely results from its pathologically enhanced activity.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The fast inhibitory actions of GABA are mainly mediated by GABAA receptors (GABAARs), which are widely recognized as clinically relevant drug targets. However, it remains difficult to create screening systems for drug candidates that act on GABAARs because of the existence of multiple ligand-binding sites and the delicate pentameric structures of GABAARs. We here developed the first turn-on fluorescent imaging probe for GABAARs, which can be used to quantitatively evaluate ligand–receptor interactions under live cell conditions. Using noncovalent labeling of GABAARs with this turn-on probe, a new imaging-based ligand assay system, which allows discovery of positive allosteric modulators (PAMs) for the GABAAR, was successfully constructed. Our system is applicable to high-throughput ligand screening, and we discovered new small molecules that function as PAMs for GABAARs. These results highlight the power of the use of a turn-on fluorescent probe to screen drugs for complicated membrane proteins that cannot be addressed by conventional methods., Using a newly found turn-on fluorescent imaging probe for GABAARs, we developed a new imaging-based ligand assay system, which allows for the discovery of positive allosteric modulators for GABAARs.

In the past few decades, solid evidence has been accumulated for the pivotal significance of immunoinflammatory processes in the initiation, progression, and exacerbation of many diseases and disorders. This groundbreaking view came from original works by Ross who first described that excessive inflammatory-fibroproliferative response to various forms of insult to the endothelium and smooth muscle of the artery wall is essential for the pathogenesis of atherosclerosis (Ross, Nature 1993; 362(6423): 801–9). It is now widely recognized that both innate and adaptive immune reactions are avidly involved in the inflammation-related remodeling of many tissues and organs. When this state persists, irreversible fibrogenic changes would occur often culminating in fatal insufficiencies of many vital parenchymal organs such as liver, lung, heart, kidney and intestines. Thus, inflammatory diseases are becoming the common life-threatening risk for and urgent concern about the public health in developed countries (Wynn et al., Nature Medicine 2012; 18(7): 1028–40). Considering this timeliness, we organized a special symposium entitled “Implications of immune/inflammatory responses in smooth muscle dysfunction and disease” in the 58th annual meeting of the Japan Society of Smooth Muscle Research. This symposium report will provide detailed synopses of topics presented in this symposium; (1) the role of inflammasome in atherosclerosis and abdominal aortic aneurysms by Fumitake Usui-Kawanishi and Masafumi Takahashi; (2) Mechanisms underlying the pathogenesis of hyper-contractility of bronchial smooth muscle in allergic asthma by Hiroyasu Sakai, Wataru Suto, Yuki Kai and Yoshihiko Chiba; (3) Vascular remodeling in pulmonary arterial hypertension by Keizo Hiraishi, Lin Hai Kurahara and Ryuji Inoue.

Background & Aims The transient receptor potential ankyrin 1 (TRPA1) channel is highly expressed in the intestinal lamina propria, but its contribution to gut physiology/pathophysiology is unclear. Here, we evaluated the function of myofibroblast TRPA1 channels in intestinal remodeling. Methods An intestinal myofibroblast cell line (InMyoFibs) was stimulated by transforming growth factor-β1 to induce in vitro fibrosis. Trpa1 knockout mice were generated using the Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. A murine chronic colitis model was established by weekly intrarectal trinitrobenzene sulfonic acid (TNBS) administration. Samples from the intestines of Crohn’s disease (CD) patients were used for pathologic staining and quantitative analyses. Results In InMyoFibs, TRPA1 showed the highest expression among TRP family members. In TNBS chronic colitis model mice, the extents of inflammation and fibrotic changes were more prominent in TRPA1-/- knockout than in wild-type mice. One-week enema administration of prednisolone suppressed fibrotic lesions in wild-type mice, but not in TRPA1 knockout mice. Steroids and pirfenidone induced Ca2+ influx in InMyoFibs, which was antagonized by the selective TRPA1 channel blocker HC-030031. Steroids and pirfenidone counteracted transforming growth factor-β1–induced expression of heat shock protein 47, type 1 collagen, and α-smooth muscle actin, and reduced Smad-2 phosphorylation and myocardin expression in InMyoFibs. In stenotic intestinal regions of CD patients, TRPA1 expression was increased significantly. TRPA1/heat shock protein 47 double-positive cells accumulated in the stenotic intestinal regions of both CD patients and TNBS-treated mice. Conclusions TRPA1, in addition to its anti-inflammatory actions, may protect against intestinal fibrosis, thus being a novel therapeutic target for highly incurable inflammatory/fibrotic disorders., Graphical abstract

Cancer is currently one of the major causes of death in patients with type 2 diabetes mellitus. We previously reported the beneficial effects of the glucagon-like peptide-1 receptor agonist exendin-4 against prostate and breast cancer. In the present study, we examined the anti-cancer effect of the sodium-glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin using a breast cancer model. In human breast cancer MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. Ipragliflozin at 1-50 μM significantly and dose-dependently suppressed the growth of MCF-7 cells. BrdU assay also revealed that ipragliflozin attenuated the proliferation of MCF-7 cells in a dose-dependent manner. Because the effect of ipragliflozin against breast cancer cells was completely canceled by knocking down SGLT2, ipragliflozin could act via inhibiting SGLT2. We next measured membrane potential and whole-cell current using the patch clamp technique. When we treated MCF-7 cells with ipragliflozin or glucose-free medium, membrane hyperpolarization was observed. In addition, glucose-free medium and knockdown of SGLT2 by siRNA suppressed the glucose-induced whole-cell current of MCF-7 cells, suggesting that ipragliflozin inhibits sodium and glucose cotransport through SGLT2. Furthermore, JC-1 green fluorescence was significantly increased by ipragliflozin, suggesting the change of mitochondrial membrane potential. These findings suggest that the SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation via membrane hyperpolarization and mitochondrial membrane instability.

BACKGROUND: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved. METHODS: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca(2+)-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes. RESULTS: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca(2+)-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca(2+) elevations and a disorganized cytoskeleton. CONCLUSIONS: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC’s coiled-coil assembly which is essential for CaM binding. The resulting excess Ca(2+) may contribute to structural damage in the podocytes.

We describe the case a 92-year-old woman who was admitted to our hospital with choledocholithiasis and periampullary diverticulum (PAD). Due to PAD, clear visualization of the ampulla of Vater could not be obtained. Although selective bile duct cannulation was difficult, a 7-Fr plastic stent was placed during the first session. Fifteen days later, endoscopic retrograde cholangiopancreatography was retried using traction devices, and the papilla became visible. Endoscopic sphincterotomy and stone extraction were performed without any complications. The application of traction devices in endoscopic submucosal dissection may be a promising technique in cases in which endoscopic biliary intervention is difficult due to PAD.

A Ca2+-activated monovalent cation-selective TRPM4 channel is abundantly expressed in the heart. Recently, a single gain-of-function mutation identified in the distal N-terminus of the human TRPM4 channel (Glu5 to Lys5, E7K) was found to be arrhythmogenic because of enhanced cell membrane expression. In this study, we conducted detailed analyses of this mutant channel from more functional aspects, in comparison with its wild type (WT). In an expression system, intracellular application of a short soluble PIP2 (diC8PIP2) restored the single-channel activities of both WT and E7K, which had quickly faded after membrane excision. The potency (Kd) of diC8PIP2 for this recovery was stronger in E7K than its WT (1.44 vs. 2.40 μM). FRET-based PIP2 measurements combined with the Danio rerio voltage-sensing phosphatase (DrVSP) and patch clamping revealed that lowering the endogenous PIP2 level by DrVSP activation reduced the TRPM4 channel activity. This effect was less prominent in E7K than its WT (apparent Kd values estimated from DrVSP-mediated PIP2 depletion: 0.97 and 1.06 μM, respectively), being associated with the differential PIP2-mediated modulation of voltage dependence. Moreover, intracellular perfusion of short N-terminal polypeptides containing either the ‘WT’ or ‘E7K’ sequences respectively attenuated the TRPM4 channel activation at whole-cell and single-channel levels, but in both configurations, the E7K polypeptide exerted greater inhibitory effects. These results collectively suggest that N-terminal interaction with endogenous PIP2 is essential for the TRPM4 channel to function, the extent of which may be abnormally strengthened by the E7K mutation through modulating voltage-dependent activation. The altered PIP2 interaction may account for the arrhythmogenic potential of this mutation.

Objectives Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. Methods Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. Results VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. Conclusions We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.

Intestinal fibrosis is an intractable complication of Crohn's disease (CD), and, when occurring excessively, causes severe intestinal obstruction that often necessitates surgical resection. The fibrosis is characterized by an imbalance in the turnover of extracellular matrix (ECM) components, where intestinal fibroblasts/myofibroblasts play active roles in ECM production, fibrogenesis and tissue remodeling, which eventually leads to the formation of stenotic lesions. There is however a great paucity of knowledge about how intestinal fibrosis initiates and progresses, which hampers the development of effective pharmacotherapies against CD. Recently, we explored the potential implications of transient receptor potential (TRP) channels in the pathogenesis of intestinal fibrosis, since they are known to act as cellular stress sensors/transducers affecting intracellular Ca2+ homeostasis/dynamics, and are involved in a broad spectrum of cell pathophysiology including inflammation and tissue remodeling. In this review, we will place a particular emphasis on the intestinal fibroblast/myofibroblast TRPC6 channel to discuss its modulatory effects on fibrotic responses and therapeutic potential for anti-fibrotic treatment against CD-related stenosis.

It is now widely accepted that advanced fibrosis underlies many chronic inflammatory disorders and is the main cause of morbidity and mortality of the modern world. The pathogenic mechanism of advanced fibrosis involves diverse and intricate interplays between numerous extracellular and intracellular signaling molecules, among which the non-trivial roles of a stress-responsive Ca2+/Na+-permeable cation channel superfamily, the transient receptor potential (TRP) protein, are receiving growing attention. Available evidence suggests that several TRP channels such as TRPC3, TRPC6, TRPV1, TRPV3, TRPV4, TRPA1, TRPM6 and TRPM7 may play central roles in the progression and/or prevention of fibroproliferative disorders in vital visceral organs such as lung, heart, liver, kidney, and bowel as well as brain, blood vessels and skin, and may contribute to both acute and chronic inflammatory processes involved therein. This short paper overviews the current knowledge accumulated in this rapidly growing field, with particular focus on cardiac and intestinal fibrosis, which are tightly associated with the pathogenesis of atrial fibrillation and inflammatory bowel diseases such as Crohn's disease.

Currently, cancer is one of major cause of death in patients with type 2 diabetes. We have previously reported the anti-prostate and anti-breast cancer effect of GLP-1R agonist Exendin-4 (Diabetes 2014, Endocrinology 2017). In the present study, we examined the anti-cancer effect of SGLT2 inhibitor ipragliflozin (Ipra) using a breast cancer model. In human breast cancer cell line, MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. 1-50nM Ipra significantly and dose-dependently suppressed the growth curve of MCF-7 cells. BrdU assay revealed that Ipra attenuates the proliferation rate of MCF-7 in a dose dependent manner. Further, apoptosis was also induced by Ipra in Tunel assay. Because the anti-breast cancer effect of Ipra was completely canceled by knocking down of SGLT2, this effect could be induced by SGLT2 inhibition by Ipra. We next measured membrane potential and whole cell current using the patch clamp technique. When we treated MCF-7 cell with Ipra or glucose free medium, membrane hyperpolarization was observed. In addition, the replacement of sodium with NMDG and knock-down of SGLT2 by siRNA suppressed glucose induced whole cell current of MCF-7 cell, suggesting that Ipra inhibits sodium and glucose incorporation through SGLT2. Further, mitochondrial membrane protein Bcl-2 was decreased and Bax was increased in western blotting, and JC-1 fluorescence was significantly increased, suggesting the change of mitochondrial membrane potential. These data suggest that SGLT2 inhibitor Ipra induces apoptosis in the breast cancer cell via membrane hyperpolarization and mitochondria dysfunction. Disclosure S. Komatsu: None. T. Nomiyama: Research Support; Self; MSD K.K., Sanofi-Aventis, Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan.T. Numata: None.T. Kawanami: None.Y. Hamaguchi: None.T. Tanaka: None.R. Inoue: None. T. Yanase: Research Support; Self; MSD K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan.

Background Intestinal fibrosis is a frequent complication of Crohn's disease (CD) and often leads to detrimental stricture formation. Myofibroblasts play active roles in mediating fibrotic changes in various tissues. We investigated whether transient receptor potential channels in intestinal myofibroblasts are involved in CD-associated intestinal fibrosis. Methods An intestinal myofibroblast cell line (InMyoFibs) was stimulated with transforming growth factor-β1 (TGF-β1) to model excessive fibrosis. Biopsy samples from nonstenotic or stenotic intestinal regions from patients with CD were used for quantitative comparisons of transient receptor potential channel and fibrosis-associated factor expression levels. Results TGF-β1 treatment transformed spindle-shaped InMyoFibs into filament-shaped cells with enhanced α-actin stress fiber formation, transient receptor potential canonical (TRPC) 4 and TRPC6 messenger RNA and protein expression, and basal- and agonist-induced Ca influxes. TGF-β1 also enhanced the formation of TRPC6/smooth muscle α-actin, TRPC6/N-cadherin, and TRPC4/N-cadherin coimmunoprecipitates. Inhibition of TRPC6 in InMyoFibs by RNA interference or dominant-negative mutations suppressed TGF-β1-induced Ca influxes, stress fiber formation, and smooth muscle α-actin expression, but increased COL1A1, interleukin (IL)-10, and IL-11 expression, as well as Smad-2, ERK, and p38-MAPK phosphorylation. Similar increases in phosphorylation levels were observed with TRPC and calcineurin inhibitors. In stenotic areas in patients with CD, TRPC6, ACTA2 (smooth muscle α-actin), CDH2 (N-cadherin), COL1A1, IL-10, and IL-11 were significantly increased. Conclusions These results suggest that augmented Ca influxes due to TRPC6 upregulation facilitate stress fiber formation and strengthen cell-cell interactions by negatively regulating the synthesis of antifibrotic factors in TGF-β1-treated myofibroblasts. Similar changes observed in stenotic areas of patients with CD suggest the therapeutic significance of targeting TRPC6.

Background and Purpose Ca2+ influx is important for cell cycle progression, but the mechanisms involved seem to vary. We investigated the potential roles of transient receptor potential (TRP) channels and store-operated Ca2+ entry (SOCE)-related molecules STIM (stromal interaction molecule)/Orai in the cell cycle progression of rat bone marrow stromal cells (BMSCs), a reliable therapeutic resource for regenerative medicine.

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca(2+) elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.

Myocardial infarction (MI) results in the generation of dead cells in the infarcted area. These cells are swiftly removed by phagocytes to minimize inflammation and limit expansion of the damaged area. However, the types of cells and molecules responsible for the engulfment of dead cells in the infarcted area remain largely unknown. In this study, we demonstrated that cardiac myofibroblasts, which execute tissue fibrosis by producing extracellular matrix proteins, efficiently engulf dead cells. Furthermore, we identified a population of cardiac myofibroblasts that appears in the heart after MI in humans and mice. We found that these cardiac myofibroblasts secrete milk fat globule-epidermal growth factor 8 (MFG-E8), which promotes apoptotic engulfment, and determined that serum response factor is important for MFG-E8 production in myofibroblasts. Following MFG-E8-mediated engulfment of apoptotic cells, myofibroblasts acquired antiinflammatory properties. MFG-E8 deficiency in mice led to the accumulation of unengulfed dead cells after MI, resulting in exacerbated inflammatory responses and a substantial decrease in survival. Moreover, MFG-E8 administration into infarcted hearts restored cardiac function and morphology. MFG-E8-producing myofibroblasts mainly originated from resident cardiac fibroblasts and cells that underwent endothelial-mesenchymal transition in the heart. Together, our results reveal previously unrecognized roles of myofibroblasts in regulating apoptotic engulfment and a fundamental importance of these cells in recovery from MI.

BACKGROUND: Our aim was to assess whether cardiac hypertrophy is associated with cognitive function independently of office, 24-h, or sleep blood pressure (BP) levels in older hypertensive patients treated with antihypertensive medications. METHODS: In this cross-sectional study, we recruited 443 hypertensive patients aged over 60 years (mean age: 73.0 years; 41% men) who were ambulatory, lived independently, and were without clinically overt dementia. They underwent measurements of 24-h BP monitoring, echocardiographic left ventricular mass index (LVMI), and cognitive function (mini-mental state examination, MMSE). RESULTS: MMSE score was inversely associated with office, 24-h, awake, and sleep systolic BP (SBP) (each, P < 0.05). There was a close association between MMSE score and LVMI (ρ = -0.32; P < 0.001). Using multiple logistic regression analysis including numerous covariates (i.e., age, sex, obesity, current smoking, educational level, duration of antihypertensive medications, renal dysfunction, statin use, and previous history of cardiovascular disease), the odds ratio (OR) for the presence of cognitive dysfunction, defined as the lowest quartile of MMSE score (median MMSE score: 23 points; n = 115), was estimated; the presence of cardiac hypertrophy (LVMI ≥125 kg/m(2) in men and ≥110 kg/m(2) in women) as well as uncontrolled 24-h BP (mean 24-h SBP/diastolic BP (DBP) ≥130/80 mm Hg) or sleep BP (mean sleep SBP/DBP ≥120/70 mm Hg), but not uncontrolled office BP (mean office SBP/DBP ≥140/90 mm Hg), were independently associated with cognitive dysfunction (all P < 0.05). CONCLUSIONS: Among older hypertensive patients with antihypertensive medications, those who had echocardiographically determined cardiac hypertrophy may be at high risk for cognitive dysfunction, irrespective of their office BP and 24-h BP levels.

Decidualization is an ovarian steroid-induced remodeling/differentiation process of uterus essential for embryo implantation and placentation. Here, we investigated the possible involvement of enhanced Ca2+ dynamics in the decidualization process in human endometrial stromal cells (hESC) in its connection with a recently emerging nonvoltage-gated Ca2+ entry channel superfamily, the transient receptor potential (TRP) protein. Combined application of 17β-estradiol (E2) (10 nm) and progesterone (P4) (1 μm) for 7–14 d resulted in morphological changes of hESC characteristic of decidualization (i.e. cell size increase), whereas sole application of E2 exerted little effects. A 7- to 14-d E2/P4 treatment greatly increased the expression level of decidualization markers IGF binding protein-1 (IGFBP-1) and prolactin and also up-regulated the expression of TRPC1, a canonical TRP subfamily member that has been implicated in store-operated Ca2+ influx (SOC) in other cell types. In parallel with this up-regulation, SOC activity in hESC, the nuclear translocation of phosphorylated cAMP responsive element binding protein (p-CREB) and the expression of Forkhead box protein 01 were enhanced significantly. Small interfering RNA knockdown of TRPC1 counteracted the E2/P4-induced up-regulation of IGFBP-1 and prolactin and enhancement of SOC activity together with the inhibition of hESC size increase, p-CREB nuclear translocation, and FOXO1 up-regulation. Coadministration of SOC inhibitors SK&F96365 or Gd3+ with E2/P4 also suppressed the up-regulation of IGFBP-1 and hESC size increase. Similar inhibitory effects were observed with extracellularly applied TRPC1 extracellular loop 3-directed antibody, which is known to bind a near-pore domain of TRPC1 channel and block its Ca2+ transporting activity. These results strongly suggest that up-regulation of TRPC1 protein and consequent enhancement of SOC-mediated Ca2+ influx may serve as a crucial step for the decidualization process of hESC probably via p-CREB-dependent transcriptional activity associated with FOXO1 activation.

Objective— The goal of this study was to determine whether inhibition of transient receptor potential canonical (TRPC) channels underlies attenuation of angiotensin II (Ang II)–induced vasoconstriction by phosphodiesterase (PDE) 3 inhibition. Methods and Results— Pretreatment of rat thoracic aorta with cilostazol, a selective PDE3 inhibitor, suppressed vasoconstriction induced by Ang II but not that induced by KCl. The Ang II–induced contraction was largely dependent on Ca 2+ influx via receptor-operated cation channels. Cilostazol specifically suppressed diacylglycerol-activated TRPC channels (TRPC3/TRPC6/TRPC7) through protein kinase A (PKA)–dependent phosphorylation of TRPC channels in HEK293 cells. In contrast, we found that phosphorylation of TRPC6 at Thr69 was essential for the suppression of Ang II–induced Ca 2+ influx by PDE3 inhibition in rat aortic smooth muscle cells (RAoSMCs). Cilostazol specifically induced phosphorylation of endogenous TRPC6 at Thr69. The endogenous TRPC6, but not TRPC3, formed a ternary complex with PDE3 and PKA in RAoSMCs, suggesting the specificity of TRPC6 phosphorylation by PDE3 inhibition. Furthermore, inhibition of PDE3 suppressed the Ang II–induced contraction of reconstituted ring with RAoSMCs, which were abolished by the expression of a phosphorylation-deficient mutant of TRPC6. Conclusion— PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II.

We investigated actions of uridine 5′-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 μM) elicited an inward-rectifying current strongly reminiscent of activation of P2X1 receptor, and a similar current was also induced by α,β-methylene adenosine 5′-triphosphate (ATP) (≥100 nM). UTP desensitized α,β-methylene ATP–evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X1-receptor antibody. Both UTP (1 mM) and α,β-methylene ATP (10 μM) elicited similar conductance single channel activities. UTP (≥10 μM) provoked a dose-dependent contraction of deendothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca2+ or bath-applied 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 μM) or nifedipine (10 μM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 μM). Similar biphasic rises in [Ca2+]i were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X1 receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X1-like and P2Y receptors, but not TRPC3.[Supplementary materials: available only at http://dx.doi.org/10.1254/jphs.10281FP] Keywords:: uridine 5′-triphosphate (UTP), P2X receptor, TRPC3, non-selective cation channel

Rationale : Atrial and brain natriuretic peptides (ANP and BNP, respectively) exert antihypertrophic effects in the heart via their common receptor, guanylyl cyclase (GC)-A, which catalyzes the synthesis of cGMP, leading to activation of protein kinase (PK)G. Still, much of the network of molecular mediators via which ANP/BNP-GC-A signaling inhibit cardiac hypertrophy remains to be characterized. Objective : We investigated the effect of ANP-GC-A signaling on transient receptor potential subfamily C (TRPC)6, a receptor-operated Ca 2+ channel known to positively regulate prohypertrophic calcineurin–nuclear factor of activated T cells (NFAT) signaling. Methods and Results : In cardiac myocytes, ANP induced phosphorylation of TRPC6 at threonine 69, the PKG phosphorylation site, and significantly inhibited agonist-evoked NFAT activation and Ca 2+ influx, whereas in HEK293 cells, it dramatically inhibited agonist-evoked TRPC6 channel activity. These inhibitory effects of ANP were abolished in the presence of specific PKG inhibitors or by substituting an alanine for threonine 69 in TRPC6. In model mice lacking GC-A, the calcineurin-NFAT pathway is constitutively activated, and BTP2, a selective TRPC channel blocker, significantly attenuated the cardiac hypertrophy otherwise seen. Conversely, overexpression of TRPC6 in mice lacking GC-A exacerbated cardiac hypertrophy. BTP2 also significantly inhibited angiotensin II–induced cardiac hypertrophy in mice. Conclusions : Collectively, these findings suggest that TRPC6 is a critical target of antihypertrophic effects elicited via the cardiac ANP/BNP-GC-A pathway and suggest TRPC6 blockade could be an effective therapeutic strategy for preventing pathological cardiac remodeling.

TRPタンパク質は電位変化以外の種々の物理化学刺激で活性化されるCa2+透過型非特異的陽イオンチャネルであり，感覚伝導を始め種々の生体機能とその破綻による病態に関与していると推測されている．最近の研究成果から，TRPタンパク質（チャネル）のうちの多くが，低浸透圧，ずり応力などの機械刺激で活性化されることが明らかとなってきた．興味深いことに，そのうちの多くが，血圧・血流変化等の機械刺激に暴露されている心血管系に発現している．しかし，これらの機械刺激感受性TRPチャネルがどのような分子機序によって活性化されるのか不明であった．本稿では，心血管系全般に亘って高レベルで発現し，血管緊張度，心血管のリモデリング等に密接に関与していることが明らかとなってきたTRPC6タンパク質に焦点を絞り，このチャネルタンパク質が受容体刺激，機械刺激の協働作用によって極めて効率的に活性化され，その分子機序として，PLC/ジアシルグリセロール（DAG）系とPLA2/ω-hydroxylase/20-HETE系のクロストークが重要であるという我々の仮説を中心に，心血管TRPタンパク質を巡る機械情報伝達のトピックスを紹介する．

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 ∼70%) by a NO donor SNAP (100 μm) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 μm) or after G-protein activation with intracellular perfusion of GTPγS (100 μm). A similar extent of suppression was also observed with a membrane-permeable analogue of cGMP, 8Br-cGMP (100 μm). 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 32P 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 Arg8-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.

Stretch-activated cation channels (SACs) have been observed in many types of smooth muscle cells. However, the molecular identity and activation mechanisms of SACs remain poorly understood. We report that TRPM4-like cation channels are activated by membrane stretch in rat cerebral artery myocytes (CAMs). Negative pressure (≥20 mmHg, cell-attached mode) activated single channels (approximately 20 pS) in isolated CAMs. These channels were permeable to Na+ and Cs+ and inhibited by Gd3+ (30 µM) and DIDS (100 µM). The effect of negative pressure was abolished by membrane excision, but subsequent application of Ca2+ (>100 nM) to the intracellular side of the membrane restored single channel activity that was indistinguishable from SACs. Caffeine (5 mM), which depletes SR Ca2+-stores, first activated and then abolished SACs. Tetracaine (100 µM), a ryanodine receptor antagonist, inhibited SACs. Overexpression of hTRPM4B in HEK293 cells resulted in the appearance of cation channels that were activated by both negative pressure and Ca2+ and which had very similar biophysical and pharmacological properties as compared with SACs in CAMs. These studies indicate that TRPM4-like channels in CAMs can be activated by membrane stretch, possibly through ryanodine receptor activation, and this may contribute to the depolarization and concomitant vasoconstriction of intact cerebral arteries following mechanical stimulation. Keywords:: TRPM4, stretch, cation channel, cerebral artery, smooth muscle

Despite considerable advances in the research and treatment, the precise relationship between inflammation and cardiovascular (CV) disease remains incompletely understood. Therefore, understanding the immunoinflammatory processes underlying the initiation, progression, and exacerbation of many cardiovascular diseases is of prime importance. The innate immune system has an ancient origin and is well conserved across species. Its activation occurs in response to pathogens or tissue injury. Recent studies suggest that altered ionic balance, and production of noxious gaseous mediators link to immune and inflammatory responses with altered ion channel expression and function. Among plausible candidates for this are transient receptor potential (TRP) channels that function as polymodal sensors and scaffolding proteins involved in many physiological and pathological processes. In this review, we will first focus on the relevance of TRP channel to both exogenous and endogenous factors related to innate immune response and transcription factors related to sustained inflammatory status. The emerging role of inflammasome to regulate innate immunity and its possible connection to TRP channels will also be discussed. Secondly, we will discuss about the linkage of TRP channels to inflammatory CV diseases, from a viewpoint of inflammation in a general sense which is not restricted to the innate immunity. These knowledge may serve to provide new insights into the pathogenesis of various inflammatory CV diseases and their novel therapeutic strategies.

Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca(2+)-responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP(3)) is responsible for Ca(2+) increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP(3) is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca(2+) oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca(2+) channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca(2+) signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy.

Local blood flow is critically determined by the arterial tone in which sustained Ca(2+) influx, activated by a variety of mechanisms, plays a central regulatory role. Recent progress in molecular biological research has disclosed unexpectedly diverse and complex facets of Ca(2+) entry channel molecules involved in this Ca(2+) influx. Candidates include several transient receptor potential (TRP) superfamily members such as TRPC1, TRPC4, TRPC6, TRPV2, TRPV4 and TRPM4, none of which exhibit simple properties attributable to a single particular role. Rather, they appear to be multimodally activated or modulated by receptor stimulation, temperature, mechanical stress or lipid second messengers generated from various sources, and may be involved in both acute vasomotor control and long-term vascular remodelling. This paper provides an overview of existing knowledge of TRP proteins, and their possible relationships with principal factors regulating the arterial tone (i.e., autonomic nerves, various autocrine and paracrine factors, and intravascular pressure).

We investigated the possible involvement of the melastatin family protein TRPM7 in Ca2+-mediated proliferative control of human retinoblastoma (RB) cells. The growth of RB cell was facilitated by elevating the extracellular Ca2+ concentration with a parallel increase in the magnitude of spontaneous Ca2+ influx. Under nystatin-perforated voltage-clamp, RB cells exhibited an outward-rectifying, spontaneous cation current (Ispont) having Ca2+/Mg2+-inhibited but -permeating properties. Various cation channel blockers inhibiting Ispont (Gd3+, La3+, LOE908, 2-APB) suppressed the spontaneous Ca2+ influx and decelerated the growth of RB cells with similar efficacies. Excision of the RB cell membrane (inside-out) into MgATP-free solution induced a 70pS single channel activity, which was effectively inhibited by millimolar concentrations of Mg2+ or MgATP. RT-PCR and immunocytochemical experiments revealed the expression of TRPM7 mRNA and protein in RB cells, and heterologous expression of TRPM7 in HEK293 cells reproduced the key features of Ispont. In contrast, elimination of this protein from RB cells by siRNA silencing markedly reduced Ispont density and the magnitude of spontaneous Ca2+ influx, which was paralleled by decreased TRPM7 immunoreactivity, decelerated cell proliferation, and retarded G1/S cell cycle progression. These results suggest a significant regulatory role of TRPM7 for RB cell proliferation as a spontaneously activated Ca2+ influx pathway. Keywords:: cell proliferation, basal Ca2+ influx, transient receptor potential protein, melastatin family

The molecular mechanisms underlying Ca2+ entry evoked by cell surface receptors in smooth muscle have long been enigmatic, but an important breakthrough has been made by recent investigations on mammalian homologues of Drosophila transient receptor potential (TRP) protein. There is now growing evidence that TRPC6 plays an integrative role in vascular tone regulation, Ca2+ entry channels activated by the sympathetic nerve stimulation, vasoactive peptides, and mechanosensitive mechanisms. Other TRPC isoforms, such as TRPC1 and TRPC4 (and perhaps TRPC5), are also expressed abundantly in smooth muscle and may contribute to muscle contraction, cell proliferation, and cholinergic control of the gut motility. This paper briefly overviews the current knowledge about these TRP proteins in smooth muscle physiology.

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.

We investigated the receptor-mediated regulation of nifedipine-insensitive, high voltage-activated Ca(2+) currents in guinea-pig terminal mesenteric arterioles (I(mVDCC)) using the whole-cell clamp technique. Screening of various vasoactive substances revealed that ATP, histamine and substance P exert modulatory effects on I(mVDCC). The effects of ATP on I(mVDCC) after complete P2X receptor desensitization exhibited a complex concentration dependence. With 5 mM Ba(2+), ATP potentiated I(mVDCC) at low concentrations (approximately 1-100 microM), but inhibited it at higher concentrations (100 microM). The potentiating effects of ATP were abolished by suramin (100 microM) and PPADS (10 microM) and by intracellular application of GDPbetaS (500 microM), whereas a substantial part of I(mVDCC) inhibition by milimolar concentrations of ATP remained unaffected; due probably to its divalent cation chelating actions. In divalent cation-free solution, I(mVDCC) was enlarged and underwent biphasic effects by ATPgammaS and ADP, while 2-methylthio ATP (2MeSATP) exerted only inhibition, and pyrimidines such as UTP and UDP were ineffective. ATP-induced I(mVDCC) potentiation was selectively inhibited by anti-Galpha(s) antibodies or protein kinase A (PKA) inhibitory peptides and mimicked by dibutyryl cAMP. In contrast, ATP-induced inhibition was selectively inhibited by Galpha(q/11) antibodies or protein kinase C (PKC) inhibitory peptides and mimicked by PDBu. Pretreatment with pertussis toxin was ineffective. The apparent efficacy for I(mVDCC) potentiation with PKC inhibitors was: ATPgammaSATP/=ADP and for inhibition with PKA inhibitors was: 2MeSATPATPgammaSATPADP. Neither I(mVDCC) potentiation nor inhibition showed voltage dependence. These results suggest that I(mVDCC) is multi-phasically regulated by external ATP via P2Y(11)-resembling receptor/G(s)/PKA pathway, P2Y(1)-like receptor/G(q/11)/PKC pathway, and metal chelation.

We investigated the effects of the novel gastroprokinetic agent Z-338 (N-(N-N'-diisopropylaminoethyl)-[2-(2-hydroxy-4,5-dimethoxybenzoylamino)-1,3-thiazole-4-yl] carboxyamide monohydrochloride trihydrate) on L-type voltage-dependent Ca2+ currents (ICa) in guinea pig gastric myocytes by using the whole-cell patch clamp technique. Bath-applied acetylcholine (ACh) produced biphasic effects on ICa, i.e., enhancement (1-100 nM) and inhibition (1-100 microM), both of which were abolished by pretreatment with atropine (10 microM) or intracellular perfusion of GDPbetaS (500 microM). Z-338 (> or = 1 nM, ED50: 120 nM) mimicked the enhancing effects of ACh, but did not inhibit ICa. The effects of Z-338 and ACh were non-additive and blocked by atropine and GDPbetaS, but not by pertussis toxin (PTX) pretreatment (500 ng/ml). ACh (> or = 1 microM) induced slow inward currents via activation of the muscarinic receptor/PTX-sensitive G-protein pathway, but Z-338 was devoid of these effects. Neither pirenzepine (1 microM), AF-DX116 (1 microM), nor oxybutynin (100 nM) could prevent Z-338 (1 microM) and ACh (10 nM) from enhancing ICa, whilst 4-DAMP (100 nM) blocked the effects of Z-338 and ACh. Bath-application of protein kinase C (PKC) activator PDBu (phorbol-12,13-dibutyrate) (250 nM) enhanced ICa, and conversely, pipette inclusion of PKC inhibitor peptide (150 microM) abolished the effects of ACh and Z-338 on ICa. These results collectively suggest that although contribution of the M3 receptor is not excluded, the major actions of Z-338 on gastric myocytes are potentiation of ICa through activation of M5-like receptor.

The effects of intracellular nucleotide triphosphates on time-dependent changes in muscarinic receptor cation currents ( Icat) were investigated using the whole cell patch-clamp technique in guinea pig ileal muscle. In the absence of nucleotide phosphates in the patch pipette, Icatevoked every 10 min decayed progressively. This decay was slowed dose dependently by inclusion of millimolar concentrations of ATP in the pipette. This required a comparable concentration of Mg2+, was mimicked by UTP and CTP, and was attenuated by simultaneous application of alkaline phosphatase or inhibitors of tyrosine kinase. In contrast, a sudden photolytic release of millimolar ATP (probably in the free form) caused a marked suppression of Icat. Submillimolar concentrations of GTP dose dependently increased the amplitude of Icatas long as ATP and Mg2+were in the pipette, but, in their absence, GTP was ineffective at preventing Icatdecay. The decay of Icatwas paralleled by altered voltage-dependent gating, i.e., a positive shift in the activation curve and reduction in the maximal conductance. It is thus likely that ATP exerts two reciprocal actions on Icat, through Mg2+-dependent and -independent mechanisms, and that the enhancing effect of GTP on Icatis essentially different from that of ATP.

Abstract —We have found nifedipine-insensitive (NI), rapidly inactivating, voltage-dependent Ca 2+ channels (current, NI- I Ca ) with unique biophysical and pharmacological properties in the terminal branches of guinea pig mesenteric artery, by using a whole-cell mode of the patch-clamp technique. The fraction of NI- I Ca appeared to increase dramatically along the lower branches of mesenteric artery, amounting to almost 100% of global I Ca in its periphery. With 5 mmol/L Ba 2+ as the charge carrier, NI- I Ca was activated with a threshold of −50 mV, peaked at −10 mV, and was half-activated and inactivated at −11 and −52 mV, respectively, generating a potential range of constant activation near the resting membrane potential. The NI- I Ca was rundown resistant, was not subject to Ca 2+ -dependent inactivation, and exhibited the pore properties typical for high voltage–activated Ca 2+ channels; Ba 2+ is ≈2-fold more permeable than Ca 2+ , and Cd 2+ is a better blocker than Ni 2+ (IC 50 , 6 and 68 μmol/L, respectively). Relatively specific blockers for N- and P/Q-type Ca 2+ channels such as ω-conotoxins GVIA and MVIIC (each 1 μmol/L) and ω-agatoxin IVA (1 μmol/L) were ineffective at inhibiting NI- I Ca , whereas nimodipine partially (10 μmol/L; ≈40%) and amiloride potently (≈75% with 1 mmol/L; IC 50 ; 107 μmol/L) blocked the current. Although these properties are reminiscent of R-type Ca 2+ channels, expression of the α 1E mRNA was not detected using reverse transcriptase–polymerase chain reaction. These results strongly suggest the predominant presence of NI, high voltage–activated Ca 2+ channels with novel properties, which may be abundantly expressed in peripheral small arterioles and contribute to their tone regulation.

1. The effects of NO donors on Ca2+-dependent Cl- currents (ICl(Ca)) were investigated in freshly isolated cat tracheal myocytes using the whole-cell patch clamp technique. 2. With nystatin-perforated whole-cell recording, carbachol (CCh, >/= 1 microM) induced a transient inward current (ICCh) with a reversal potential of about -20 mV. Activation of ICCh probably occurred through the M3 muscarinic receptor, since nanomolar concentrations of 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) greatly inhibited this current, while 11-(2-(diethylamino)methyl)-1-piperidinylacetyl)-5, 11-dihydro-6H-pyrido (2,3beta) (1,4)benzodiazepine-6-one (AF-DX 116) or pirenzepine at concentrations of up to 1 microM were almost ineffective. 3. Chloride channel/transporter blockers such as DIDS (100 microM), anthracene-9-carboxylic acid (9-AC, 100 microM) and niflumic acid (100 microM) greatly inhibited ICCh, but cation channel blockers, such as nifedipine (10 microM), Zn2+ (500 microM) or Gd3+ (500 microM), were without effect. 4. Activation of ICCh was strongly attenuated by pretreatment with ryanodine (4 microM) plus caffeine (10 mM). Addition of neomycin (1 mM) into the bath or inclusion of heparin (3 mg ml-1) in the pipette abolished a substantial part of ICCh. These results suggest that ICCh is ICl(Ca), which is activated by inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release. 5. The nitric oxide donor S-nitroso-N-acetyl penicillamine (SNAP) reduced the amplitude of ICCh dose dependently (IC50, approximately 10 microM). Similar inhibition was also exerted by other types of NO donor such as glyceryl trinitrate (GTN) and (+/-)-E-methyl-2-(E-hydroxyimitol)-5-nitro-6-methoxy-3- hexeneamide (NO-R). 6. SNAP-induced ICCh inhibition was effectively antagonized by Methylene Blue (1-100 nM), and mimicked by dibutyryl cGMP (db-cGMP) (0.5-1 mM), whereas two structurally distinct types of cGMP-dependent (G)-kinase inhibitor, N-(2-aminoethyl)-5-isoquinilinesulphonamide (H-8, 2.5 microM) and KT5823 (1 microM), failed to counteract the inhibitory effects of SNAP or db-cGMP. Another G-kinase-specific inhibitor Rp-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS; 1 microM) itself caused a marked reduction in ICCh. 7. SNAP (100 microM) or db-cGMP (100 microM) exhibited no inhibitory actions, when caffeine (10 mM) or photolytically released IP3 were used instead of CCh to activate the inward current. 8. These results suggest that inhibition of ICCh by NO donors involves a cGMP-dependent but G-kinase-independent mechanism, which may operate at a site(s) between the muscarinic (M3) and IP3 receptors.

We have investigated a potential role of motilin in amplifying the postsynaptic muscarinic responses in the rabbit duodenal smooth muscle cells, using the whole cell variant of patch-clamp technique. Stimulation of motilin receptors by exogenously applied motilin (1 nM) resulted in a large increase in carbachol (CCh)-induced atropine-sensitive cation current ( ICCh) at threshold concentrations of CCh (0.3–1 μM) at 30°C. This potentiation was abolished in the presence of a specific blocker of motilin receptor (GM109) and was attenuated with increased concentrations of either motilin or CCh, being virtually absent with maximally effective concentrations of these agonists. Motilin failed to potentiate ICChwhen the ambient temperature was reduced to 20°C or if the cation current had been directly activated by internal perfusion with guanosine 5′- O-(3-thiotriphosphate) (50 μM) bypassing the muscarinic receptor. These results suggest that some biochemical processes, such as enzymatic reactions, might be involved in the motilin-induced potentiation and that its site of action might be the muscarinic receptor and/or associated G proteins.

The effects of hydrogen peroxide H2O2 (10−6 and 10−3 M) on membrane potential, membrane currents, intracellular calcium concentration, resting muscle tone and contractions elicited by electrical field stimulation (EFS) and carbachol were examined in cat tracheal strips and isolated smooth muscle cells. H2O2 (10−4 and 10−5 M) enhanced the amplitude of contractions and excitatory junction potentials (e.j.p.) evoked by EFS without changing muscle tone and resting membrane potential of the tracheal smooth muscle, and enhanced the contraction induced by carbachol (10−8 M). At an increased concentration (10−3 M), H2O2 elevated resting muscle tone and marginally hyperpolarized the membrane in the majority of the cells. In 51 out of 56 cells examined, H2O2 (10−6–10−3 M) elicited an outward current at a holding potential of −40 mV and enhanced the frequency of the spontaneous transient outward current (STOC). In 20 cells the outward current was preceded by a small inward current. In the other cells, H2O2 elicited only an inward current or did not affect the background current. In Ca2+ free solution the action of H2O2 on the resting muscle tone, STOCs, background current and on the current induced by ramp depolarization was significantly reduced. H2O2 (10−4 M) increased the intracellular ionized calcium concentration both in the absence and presence of external Ca2+. However, the effect developed faster and was of a higher amplitude in the presence of external Ca2+. These results suggest that H2O2 increases intracellular Ca2+, with a subsequent augmentation of stimulation-evoked contractions, and enhances Ca2+ and voltage-sensitive potassium conductance.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as I h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p.S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of I h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of I h during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.

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.

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

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.