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Activation of hepatic stellate cells (HSCs) causes hepatic fibrosis and results in chronic liver diseases. Although activated HSC functions are facilitated by an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt), the pathophysiological roles of ion channels are largely unknown. In the present study, functional analyses of the two-pore domain K+ (K2P) channels, which regulate the resting membrane potential and [Ca2+]cyt, were performed using the human HSC line, LX-2. Expression analyses revealed that TREK1 (also known as KCNK2 and K2P2.1) channels are expressed in LX-2 cells. Whole-cell K+ currents were activated by 10 μM arachidonic acid and the activation was abolished by 100 μM tetrapentylammonium, which are pharmacological characteristics of TREK1 channels. The siRNA knockdown of TREK1 channels caused membrane depolarization and reduced [Ca2+]cyt. In addition, TREK1 knockdown downregulated the gene expression of collage type I and platelet-derived growth factor. Furthermore, TREK1 knockdown inhibited the proliferation of LX-2 cells. In conclusion, the activity of TREK1 channels determines the resting membrane potential and [Ca2+]cyt, which play a role in extracellular matrix production and cell proliferation in HSCs. This study may help elucidate the molecular mechanism underlying hepatic fibrosis in HSCs and provide a potential therapeutic target for hepatic fibrosis.

Pulmonary arterial hypertension (PAH) is a rare, progressive, and fatal cardiovascular/lung disease. The incidence rate is affected by age. Monocrotaline (MCT, 60 mg/kg)-treated rats are widely used as an experimental PAH model. Here, we found that young rats died at a mean of 23.4 days after MCT injection, whereas adult rats survived for over 42 days. However, young (7-week-old) and adult (20-week-old) MCT-treated rats developed PAH, and had upregulated Ca2+-sensing receptor and transient receptor potential canonical subfamily 6 channel expression in pulmonary arteries. The present study provides novel information for elucidating the mechanism underlying the age difference in PAH patients.

Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling of the pulmonary artery, which is mainly attributed to the excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) comprising the medial layer of pulmonary arteries. The activity of ion channels associated with cytosolic Ca

Hepatic stellate cells (HSCs) play a significant role in the development of chronic liver diseases. Hepatic damage activates HSCs and results in hepatic fibrosis. The functions of activated HSCs require an increase in the cytosolic Ca

Pulmonary arterial hypertension (PAH) is a progressive and lethal disease of the pulmonary artery. The pathogenesis of PAH is mainly sustained vasoconstriction and vascular remodeling of the pulmonary artery. These pathogeneses cause progressive elevations in pulmonary vascular resistance and pulmonary arterial pressure in PAH patients. Elevated pulmonary arterial pressure leads to right heart failure and finally death. The vascular remodeling is caused by the enhanced proliferation and reduced apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Excitable abnormality in the pulmonary artery of PAH patients is mostly mediated by an elevated cytosolic Ca2+ concentration. PASMCs express several Ca2+-permeable channels including voltage-dependent Ca2+ channels, store-operated Ca2+ (SOC) channels, and receptor-operated Ca2+ (ROC) channels. The activation and upregulation of these Ca2+ channels have been reported in PASMCs from PAH patients. Here, we analyzed pathophysiological functions of enhanced Ca2+ signaling mediated by SOC and ROC channels using PASMCs from idiopathic PAH patients and animal PAH models. Notch signal enhanced transient receptor potential canonical 6 (TRPC6) "SOC" channels via direct (non-genomic and stimulatory) and indirect (genomic and upregulating) effects in PAH. On the other hand, the activation of Ca2+-sensing receptors evoked Ca2+ influx through TRPC6 "ROC" channels in PAH. In addition, TRPC6 channel blocker and TRPC6 gene deletion inhibited the development of PAH. Specifically, TRPC6 channels potentially form both ROC and SOC channels in PASMCs, which are involved in the pathophysiological events in PAH. Therefore, targeting TRPC6 channels in PASMCs may help develop novel therapeutic approach for PAH.

In pulmonary arterial smooth muscle cells (PASMCs), an increase in the cytosolic Ca

Two-pore domain K+ (K2P) channels are thought to be druggable targets. However, only a few agents specific for K2P channels have been identified, presumably due to the lack of an efficient screening system. To develop a new high-throughput screening (HTS) system targeting these channels, we have established a HEK293-based "test cell" expressing a mutated Na+ channel (Nav1.5) with markedly slowed inactivation, as well as a K+ channel (Kir2.1) that sets the membrane potential quite negative, close to K+ equilibrium potential. We found in this system that Kir2.1 block by 100 μM Ba2+ application consistently elicited a large depolarization like a long-lasting action potential. This maneuver resulted in cell death, presumably due to the sustained Na+ influx. When either the TWIK-related acid-sensitive K+ (TASK)-1 or TASK-3 channel was expressed in the test cells, Ba2+-induced cell death was markedly weakened. Stronger activation of TASK-1 by extracellular acidification further decreased the cell death. In contrast, the presence of K2P channel blockers enhanced cell death. IC50 values for TASK-1 and/or TASK-3 blockers acquired by measurements of relative cell viability were comparable to those obtained using patch-clamp recordings. Both blockers and openers of K2P channels can be accurately assessed with high efficiency and throughput by this novel HTS system.

Articular chondrocytes are exposed to dynamic osmotic environments during normal joint loading, and thus, require effective volume regulatory mechanisms. A regulatory volume decrease (RVD) is one of the mechanisms for protecting chondrocytes from swelling and damage. Swelling-activated Cl

Large conductance Ca2+-activated K+ (BKCa) channels are ubiquitously expressed in plasma membrane of both excitable and non-excitable cells and possess significant physiological functions. A tetrameric complex of α subunit (BKα) forms a functional pore of BKCa channel. The properties of BKCa channel, such as voltage-dependence, Ca2+ sensitivity and pharmacological responses, are extensively modulated by co-expressing accessory β subunits (BKβ), which can associate with BKα in one to one manner. Although the functional significance of newly identified γ subunits (BKγ) has been revealed, the stoichiometry between BKα and BKγ1 remains unclear. In the present study, we utilized a single molecule fluorescence imaging with a total internal reflection fluorescence (TIRF) microscope to directly count the number of green fluorescent protein (GFP)-tagged BKγ1 (BKγ1-GFP) within a single BKCa channel complex in HEK293 cell expression system. BKγ1-GFP significantly enhanced the BK channel activity even when the intracellular Ca2+ concentration was kept lower, i.e., 10 nM, than the physiological resting level. BKγ1-GFP stably formed molecular complexes with BKα-mCherry in the plasma membrane. Counting of GFP bleaching steps revealed that a BKCa channel can contain up to four BKγ1 per channel at the maximum. These results suggest that BKγ1 forms a BKCa channel complex with BKα in a 1 : 1 stoichiometry in a human cell line.

An improved understanding of fundamental physiological principles and progressive pathophysiological processes in human articular joints (e.g., shoulders, knees, elbows) requires detailed investigations of two principal cell types: synovial fibroblasts and chondrocytes. Our studies, done in the past 8–10 years, have used electrophysiological, Ca2+ imaging, single molecule monitoring, immunocytochemical, and molecular methods to investigate regulation of the resting membrane potential (ER) and intracellular Ca2+ levels in human chondrocytes maintained in 2-D culture. Insights from these published papers are as follows: (1) Chondrocyte preparations express a number of different ion channels that can regulate their ER. (2) Understanding the basis for ER requires knowledge of (a) the presence or absence of ligand (ATP/histamine) stimulation and (b) the extraordinary ionic composition and ionic strength of synovial fluid. (3) In our chondrocyte preparations, at least two types of Ca2+-activated K+ channels are expressed and can significantly hyperpolarize ER. (4) Accounting for changes in ER can provide insights into the functional roles of the ligand-dependent Ca2+ influx through store-operated Ca2+ channels. Some of the findings are illustrated in this review. Our summary diagram suggests that, in chondrocytes, the K+ and Ca2+ channels are linked in a positive feedback loop that can augment Ca2+ influx and therefore regulate lubricant and cytokine secretion and gene transcription.

Pancreatic neuroendocrine tumors (pNETs) occur due to the abnormal growth of pancreatic islet cells and predominantly develop in the duodenal-pancreatic region. Somatostatinoma is one of the pNETs associated with tumors of pancreatic δ cells, which produce and secrete somatostatin. Limited information is currently available on the pathogenic mechanisms of somatostatinoma. The large-conductance Ca

In visceral smooth muscle cells (SMCs), the large-conductance Ca(2+)-activated K(+) (BK) channel is one of the key elements underlying a negative feedback mechanism that is essential for the regulation of intracellular Ca(2+) concentration. Although leucine-rich repeat-containing (LRRC) proteins have been identified as novel auxiliary γ-subunits of the BK channel (BKγ) in several cell types, its physiological roles in SMCs are unclear. The BKγ expression patterns in selected SM tissues were examined using real-time PCR analyses and Western blotting. The functional contribution of BKγ1 to BK channel activity was examined by whole cell patch-clamp in SMCs and heterologous expression systems. BKγ1 expression in mouse bronchial SMCs (mBSMCs) was higher than in other several SMC types. Coimmunoprecipitation and total internal reflection fluorescence imaging analyses revealed molecular interaction between BKα and BKγ1 in mBSMCs. Under voltage-clamp, steady-state activation of BK channel currents at pCa 8.0 in mBSMCs occurred in a voltage range comparable to that of reconstituted BKα/BKγ1 complex. However, this range was much more negative than in mouse aortic SMCs (mASMCs) or in HEK293 cells expressing BKα alone and β-subunit (BKβ1). Mallotoxin, a selective activator of BK channel that lacks BKγ1, dose-dependently activated BK currents in mASMCs but not in mBSMCs. The abundant expression of BKγ1 in mBSMCs extensively facilitates BK channel activity to keep the resting membrane potential at negative values and prevents contraction under physiological conditions.

Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) and mitochondria play a critical role in growth factor-induced switch from a quiescent to an angiogenic phenotype in endothelial cells (ECs). However, how highly diffusible ROS produced from different sources can coordinate to stimulate VEGF signaling and drive the angiogenic process remains unknown. Using the cytosol- and mitochondria-targeted redox-sensitive RoGFP biosensors with real-time imaging, here we show that VEGF stimulation in human ECs rapidly increases cytosolic RoGFP oxidation within 1 min, followed by mitochondrial RoGFP oxidation within 5 min, which continues at least for 60 min. Silencing of Nox4 or Nox2 or overexpression of mitochondria-targeted catalase significantly inhibits VEGF-induced tyrosine phosphorylation of VEGF receptor type 2 (VEGFR2-pY), EC migration and proliferation at the similar extent. Exogenous hydrogen peroxide (H2O2) or overexpression of Nox4, which produces H2O2, increases mitochondrial ROS (mtROS), which is prevented by Nox2 siRNA, suggesting that Nox2 senses Nox4-derived H2O2to promote mtROS production. Mechanistically, H2O2increases S36 phosphorylation of p66Shc, a key mtROS regulator, which is inhibited by siNox2, but not by siNox4. Moreover, Nox2 or Nox4 knockdown or overexpression of S36 phosphorylation-defective mutant p66Shc(S36A) inhibits VEGF-induced mtROS, VEGFR2-pY, EC migration, and proliferation. In summary, Nox4-derived H2O2in part activates Nox2 to increase mtROS via pSer36-p66Shc, thereby enhancing VEGFR2 signaling and angiogenesis in ECs. This may represent a novel feed-forward mechanism of ROS-induced ROS release orchestrated by the Nox4/Nox2/pSer36-p66Shc/mtROS axis, which drives sustained activation of angiogenesis signaling program.

Voltage-gated K+ (KV) channels, which control firing and shape of action potentials in excitable cells, are supposed to be potential therapeutic targets in many types of diseases. Pimaric acid (PiMA) is a unique opener of large conductance Ca2+-activated K+ channel. Here, we report that PiMA modulates recombinant rodent KV channel activity. The enhancement was significant at low potentials (

The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca

Downregulated expression of K(+) channels and decreased K(+) currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K(+) channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3′-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K(+) channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K(+) channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K(+) currents and downregulated voltage-gated K(+) channel 1.5 (K(V)1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K(+) channel function and K(V)1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that K(V)1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel currents and downregulated BK(Ca) channel β1 subunit (BK(Ca)β1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BK(Ca) channel activity and BK(Ca)β1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of K(V) and BK(Ca) channels in PASMC from patients with IPAH.

The mechanisms underlying neuronal cell death induced by the rise of intracellular Na

Conversion of intracellular Ca

K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.

Articular chondrocytes are embedded in the cartilage of diarthrodial joints and responsible for the synthesis and secretion of extracellular matrix. The extracellular matrix mainly contains collagens and proteoglycans, and covers the articular cartilage to protect from mechanical and biochemical stresses. In mammalian chondrocytes, various types of ion channels have been identified: e.g., voltage-dependent K

Brain capillary endothelial cells (BCECs) play a central role in maintenance of blood-brain barrier (BBB) function and, therefore, are essential for central nervous system homeostasis and integrity. Although brain ischemia damages BCECs and causes disruption of BBB, the related influence of hypoxia on BCECs is not well understood. Hypoxic stress can upregulate functional expression of specific K

Articular chondrocytes in osteoarthritis (OA) patients are exposed to hypoosmotic stress because the osmolality of this synovial fluid is significantly decreased. Hypoosmotic stress can cause an efflux of Cl(-) and an associated decrease of cell volume. We have previously reported that a Cl(-) conductance contributes to the regulation of resting membrane potential and thus can alter intracellular Ca(2+) concentration ([Ca(2+)]i) in human chondrocytes. The molecular identity and pathologic function of these Cl(-) channels, however, remained to be determined. Here, we show that the ClC-7 Cl(-) channel is strongly expressed in a human chondrocyte cell line (OUMS-27) and that it is responsible for Cl(-) currents that are activated by extracellular acidification (pH 5.0). These acid-sensitive currents are inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; IC50 = 13 μM) and are markedly reduced by small-interfering RNA-induced knockdown of ClC-7. DIDS hyperpolarized these chondrocytes, and this was followed by an increase in [Ca(2+)]i. ClC-7 knockdown caused a similar hyperpolarization of the membrane potential. Short-term culture (48 hours) in hypoosmotic medium (270 mOsm) reduced the expression of ClC-7 and decreased the acid-sensitive currents. Interestingly, these hypoosmotic culture conditions, or ClC-7 knockdown, resulted in enhanced cell death. Taken together, our results show that the significant hyperpolarization due to ClC-7 impairment in chondrocytes can significantly increase [Ca(2+)]i and cell death. Thus, downregulation of ClC-7 channels during the hypoosmotic stress that accompanies OA progression is one important concept of the complex etiology of OA. These findings suggest novel targets for therapeutic intervention(s) and drug development for OA.

Ca(2+) influx via store-operated Ca(2+) entry (SOCE) plays critical roles in many essential cellular functions. The Ca(2+) release-activated Ca(2+) (CRAC) channel complex, consisting of Orai and STIM, is one of the major components of store-operated Ca(2+) (SOC) channels. Our previous study demonstrated that histamine can cause sustained Ca(2+) entry through SOC channels in OUMS-27 cells derived from human chondrosarcoma. This SOCE was increased by low- and decreased by high-concentrations of 2-aminoethoxydiphenyl borate. Quantitative reverse transcription PCR and Western blot analyses revealed abundant expressions of Orai1, Orai2 and STIM1. Introduction of dominant negative mutant of Orai1, or siOrai1 knockdown significantly attenuated SOCE. Following histamine application, single molecule imaging using total internal reflection fluorescence (TIRF) microscopy demonstrated punctate Orai1-STIM1 complex formation in plasma membrane. In contrast, knockdown or over-expression of Orai2 resulted in an increase or a decrease in SOCE, respectively. Finally, TIRF imaging revealed direct coupling between Orai1 and Orai2, and suggested that Orai2 reduces Orai1 function by formation of a hetero-tetramer. These results provide substantial evidence that Orai1, Orai2 and STIM1 form functional CRAC channels in OUMS-27 cells and that these complexes are responsible for sustained Ca(2+) entry in response to agonist stimulation.

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease of the pulmonary artery resulting from a currently unidentified etiology. IPAH is pathologically characterized as sustained vasoconstriction and vascular remodeling of the pulmonary artery. Vascular remodeling is mediated by enhanced proliferation and reduced apoptosis in pulmonary arterial smooth muscle cells (PASMCs). Based on its pathological mechanism, specific phosphodiesterase type 5 (PDE5) inhibitors have been used in the treatment of IPAH. In addition to sildenafil, tadalafil has been approved for the treatment of IPAH. However, the effects of tadalafil on excessive proliferation of IPAH-PASMCs currently remain unknown. In the present study, the in vitro pharmacological profiles of tadalafil for cell proliferation and apoptosis were assessed in IPAH-PASMCs using MTT, BrdU incorporation, and caspase 3/7 assays. Expression analyses revealed that PDE5 mRNA and protein expression levels were markedly higher in IPAH-PASMCs than in normal-PASMCs. The treatment with tadalafil inhibited the excessive proliferation of IPAH-PASMCs in a concentration-dependent manner with an IC50 value of 4.5μM. On the other hand, tadalafil (0.03-100μM) did not affect cell growth of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). In addition, tadalafil induced apoptosis in IPAH-PASMCs. The antiproliferative and apoptotic effects of tadalafil were markedly stronger than those of sildenafil and vardenafil. The upregulated expression of PDE5 in IPAH-PASMCs was significantly attenuated by a long-term treatment with tadalafil. Taken together, these results indicate that tadalafil attenuates vascular remodeling by inhibiting cell proliferation, promoting apoptosis, and downregulating PDE5 in IPAH-PASMCs, thereby ameliorating IPAH.

An increase in cytosolic Ca2+concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for pulmonary arterial medial hypertrophy in patients with idiopathic pulmonary arterial hypertension (IPAH). Vascular smooth muscle cells (SMC) sense the blood flow shear stress through interstitial fluid driven by pressure or direct exposure to blood flow in case of endothelial injury. Mechanical stimulus can increase [Ca2+]cyt. Here we report that flow shear stress raised [Ca2+]cytin PASMC, while the shear stress-mediated rise in [Ca2+]cytand the protein expression level of TRPM7 and TRPV4 channels were significantly greater in IPAH-PASMC than in normal PASMC. Blockade of TRPM7 by 2-APB or TRPV4 by Ruthenium red inhibited shear stress-induced rise in [Ca2+]cytin normal and IPAH-PASMC, while activation of TRPM7 by bradykinin or TRPV4 by 4αPDD induced greater increase in [Ca2+]cytin IPAH-PASMC than in normal PASMC. The bradykinin-mediated activation of TRPM7 also led to a greater increase in [Mg2+]cytin IPAH-PASMC than in normal PASMC. Knockdown of TRPM7 and TRPV4 by siRNA significantly attenuated the shear stress-mediated [Ca2+]cytincreases in normal and IPAH-PASMC. In conclusion, upregulated mechanosensitive channels (e.g., TRPM7, TRPV4, TRPC6) contribute to the enhanced [Ca2+]cytincrease induced by shear stress in PASMC from IPAH patients. Blockade of the mechanosensitive cation channels may represent a novel therapeutic approach for relieving elevated [Ca2+]cytin PASMC and thereby inhibiting sustained pulmonary vasoconstriction and pulmonary vascular remodeling in patients with IPAH.

Notch signaling plays a critical role in controlling proliferation and differentiation of pulmonary arterial smooth muscle cells (PASMC). Upregulated Notch ligands and Notch3 receptors in PASMC have been reported to promote the development of pulmonary vascular remodeling in patients with pulmonary arterial hypertension (PAH) and in animals with experimental pulmonary hypertension. Activation of Notch receptors by their ligands leads to the cleavage of the Notch intracellular domain (NICD) to the cytosol by γ-secretase; NICD then translocates into the nucleus to regulate gene transcription. In this study, we examined whether short-term activation of Notch functionally regulates store-operated Ca2+entry (SOCE) in human PASMC. Treatment of PASMC with the active fragment of human Jagged-1 protein (Jag-1) for 15–60 min significantly increased the amplitude of SOCE induced by passive deletion of Ca2+from the intracellular stores, the sarcoplasmic reticulum (SR). The Jag-1-induced enhancement of SOCE was time dependent: the amplitude was maximized at 30 min of treatment with Jag-1, which was closely correlated with the time course of Jag-1-mediated increase in NICD protein level. The scrambled peptide of Jag-1 active fragment had no effect on SOCE. Inhibition of γ-secretase by N-[ N-(3,5-difluorophenacetyl-l-alanyl)]- S-phenylglycine t-butyl ester (DAPT) significantly attenuated the Jag-1-induced augmentation of SOCE. In addition to the short-term effect, prolonged treatment of PASMC with Jag-1 for 48 h also markedly enhanced the amplitude of SOCE. These data demonstrate that short-term activation of Notch signaling enhances SOCE in PASMC; the NICD-mediated functional interaction with store-operated Ca2+channels (SOC) may be involved in the Jag-1-mediated enhancement of SOCE in human PASMC.

Caveolin family is supposed to be essential molecules for the formation of not only caveola structure on cell membrane but also functional molecular complexes in them with direct and/or indirect interaction with other membrane and/or submembrane associated proteins. The direct coupling of caveolin-1 (cav1) with large conductance Ca(2+)-activated K(+) channel, KCa1.1 has been established in several types of cells and in expression system as well. The possible interaction of caveolin-3 (cav3), which shows expression in some differential tissues from cav1, with KCa1.1 remains to be determined. In the present study, the density of KCa1.1 current expressed in HEK293 cells was significantly reduced by the co-expression of cav3, as well as cav1. The co-localization and direct interaction of GFP- or CFP-labeled cav3 (GFP/CFP-cav3) with YFP- or mCherry-labeled KCa1.1 (KCa1.1-YFP/mCherry) were clearly demonstrated by single molecular image analyses using total internal reflection fluorescence (TIRF) microscopy and fluorescence resonance energy transfer (FRET) analyses with acceptor photobleaching method. The deletion of suggested cav1-binding motif in C terminus region of KCa1.1 (KCa1.1ΔCB-YFP) resulted in the marked decrease in cell surface expression, co-localization and FRET efficiency with CFP-cav3 and CFP-cav1. The FLAG-KCa1.1 co-immunoprecipitation with GFP-cav3 or GFP-cav1 also supported their direct molecular interaction. These results strongly suggest that cav3 possesses direct interaction with KCa1.1, presumably at the same domain for cav1 binding. This interaction regulates KCa1.1 expression to cell surface and the formation of functional molecular complex in caveolae in living cells.

Capsaicin is an active component of chili pepper and a pain relief drug. Capsaicin can activate transient receptor potential vanilloid 1 (TRPV1) channels to increase cytosolic Ca2+ concentration ([Ca2+]cyt). A rise in [Ca2+]cyt in pulmonary artery smooth muscle cells (PASMCs) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. In this study, we observed that a capsaicin-induced increase in [Ca2+]cyt was significantly enhanced in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with normal PASMCs from healthy donors. In addition, the protein expression level of TRPV1 in IPAH PASMCs was greater than in normal PASMCs. Increasing the temperature from 23 to 43°C, or decreasing the extracellular pH value from 7.4 to 5.9 enhanced capsaicin-induced increases in [Ca2+]cyt; the acidity (pH 5.9)- and heat (43°C)-mediated enhancement of capsaicin-induced [Ca2+]cyt increases were greater in IPAH PASMCs than in normal PASMCs. Decreasing the extracellular osmotic pressure from 310 to 200 mOsmol/l also increased [Ca2+]cyt, and the hypo-osmolarity-induced rise in [Ca2+]cyt was greater in IPAH PASMCs than in healthy PASMCs. Inhibition of TRPV1 (with 5′-IRTX or capsazepine) or knockdown of TRPV1 (with short hairpin RNA) attenuated capsaicin-, acidity-, and osmotic stretch-mediated [Ca2+]cyt increases in IPAH PASMCs. Capsaicin induced phosphorylation of CREB by raising [Ca2+]cyt, and capsaicin-induced CREB phosphorylation were significantly enhanced in IPAH PASMCs compared with normal PASMCs. Pharmacological inhibition and knockdown of TRPV1 attenuated IPAH PASMC proliferation. Taken together, the capsaicin-mediated [Ca2+]cyt increase due to upregulated TRPV1 may be a critical pathogenic mechanism that contributes to augmented Ca2+ influx and excessive PASMC proliferation in patients with IPAH.

Large conductance Ca2+-activated K+ (BK) channels play essential roles in both excitable and non-excitable cells. For example, in chondrocytes, agonist-induced Ca2+ release from intracellular store activates BK channels, and this hyperpolarizes these cells, augments Ca2+ entry, and forms a positive feed-back mechanism for Ca2+ signaling and stimulation-secretion coupling. In the present study, functional roles of a newly identified splice variant in the BK channel α subunit (BKαΔe2) were examined in a human chondrocyte cell line, OUMS-27, and in a HEK293 expression system. Although BKαΔe2 lacks exon2, which codes the intracellular S0-S1 linker (Glu-127–Leu-180), significant expression was detected in several tissues from humans and mice. Molecular image analyses revealed that BKαΔe2 channels are not expressed on plasma membrane but can traffic to the plasma membrane after forming hetero-tetramer units with wild-type BKα (BKαWT). Single-channel current analyses demonstrated that BKα hetero-tetramers containing one, two, or three BKαΔe2 subunits are functional. These hetero-tetramers have a smaller single channel conductance and exhibit lower trafficking efficiency than BKαWT homo-tetramers in a stoichiometry-dependent manner. Site-directed mutagenesis of residues in exon2 identified Helix2 and the linker to S1 (Trp-158–Leu-180, particularly Arg-178) as an essential segment for channel function including voltage dependence and trafficking. BKαΔe2 knockdown in OUMS-27 chondrocytes increased BK current density and augmented the responsiveness to histamine assayed as cyclooxygenase-2 gene expression. These findings provide significant new evidence that BKαΔe2 can modulate cellular responses to physiological stimuli in human chondrocyte and contribute under pathophysiological conditions, such as osteoarthritis.

An increase in cytosolic free Ca2+concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and a critical stimulation for PASMC proliferation and migration. Previously, we demonstrated that expression and function of calcium sensing receptors (CaSR) in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) and animals with experimental pulmonary hypertension (PH) were greater than in PASMC from normal subjects and control animals. However, the mechanisms by which CaSR triggers Ca2+influx in PASMC and the implication of CaSR in the development of PH remain elusive. Here, we report that CaSR functionally interacts with TRPC6 to regulate [Ca2+]cytin PASMC. Downregulation of CaSR or TRPC6 with siRNA inhibited Ca2+-induced [Ca2+]cytincrease in IPAH-PASMC (in which CaSR is upregulated), whereas overexpression of CaSR or TRPC6 enhanced Ca2+-induced [Ca2+]cytincrease in normal PASMC (in which CaSR expression level is low). The upregulated CaSR in IPAH-PASMC was also associated with enhanced Akt phosphorylation, whereas blockade of CaSR in IPAH-PASMC attenuated cell proliferation. In in vivo experiments, deletion of the CaSR gene in mice ( casr−/−) significantly inhibited the development and progression of experimental PH and markedly attenuated acute hypoxia-induced pulmonary vasoconstriction. These data indicate that functional interaction of upregulated CaSR and upregulated TRPC6 in PASMC from IPAH patients and animals with experimental PH may play an important role in the development and progression of sustained pulmonary vasoconstriction and pulmonary vascular remodeling. Blockade or downregulation of CaSR and/or TRPC6 with siRNA or miRNA may be a novel therapeutic strategy to develop new drugs for patients with pulmonary arterial hypertension.

Rationale: A rise in cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca 2+ ] cyt and enhanced Ca 2+ influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). Objective: We examined whether the extracellular Ca 2+ -sensing receptor (CaSR) is involved in the enhanced Ca 2+ influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. Methods and Results: In normal PASMC superfused with Ca 2+ -free solution, addition of 2.2 mmol/L Ca 2+ to the perfusate had little effect on [Ca 2+ ] cyt . In IPAH-PASMC, however, restoration of extracellular Ca 2+ induced a significant increase in [Ca 2+ ] cyt . Extracellular application of spermine also markedly raised [Ca 2+ ] cyt in IPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca 2+ -induced [Ca 2+ ] cyt rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca 2+ -mediated [Ca 2+ ] cyt increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia. Conclusions: The extracellular Ca 2+ -induced increase in [Ca 2+ ] cyt due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca 2+ influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension.

To provide a high-throughput screening method for human ether-a-go-go-gene-related gene (hERG) K(+) channel inhibition, a new recombinant cell line, in which single action potential (AP)-induced cell death was produced by gene transfection. Mutated human cardiac Na(+) channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K(+) channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K(+) channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC(50) values to induce the cell death were 3 µM, 19 nM, 17 nM, 74 nM, and 3 µM, respectively, whereas 10 µM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K(+) channel safety assay.

The intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca) channel) encoded by K(Ca)3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K(Ca)3.1 (human (h) K(Ca)3.1b) from the human thymus, which were lacking the N-terminal domains of the original hK(Ca)3.1a as a result of alternative splicing events. hK(Ca)3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK(Ca)3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hK(Ca)3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking K(Ca)3.1 variants from mice and rat lymphoid tissues (mK(Ca)3.1b and rK(Ca)3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hK(Ca)3.1a and/or YFP-tagged hK(Ca)3.1b isoforms showed that hK(Ca)3.1b suppressed the localization of hK(Ca)3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hK(Ca)3.1b with hK(Ca)3.1a suppressed IK(Ca) channel activity of hK(Ca)3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mK(Ca)3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(-)CD8(-) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mK(Ca)3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of K(Ca)3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IK(Ca) channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

In articular cartilage inflammation, histamine release from mast cells is a key event. It can enhance cytokine production and matrix synthesis and also promote cell proliferation by stimulating chondrocytes. In this study, the functional impact of Ca2+-activated K+(KCa) channels in the regulation of intracellular Ca2+concentration ([Ca2+]i) in chondrocytes in response to histamine was examined using OUMS-27 cells, as a model of chondrocytes derived from human chondrosarcoma. Application of histamine induced a significant [Ca2+]irise and also membrane hyperpolarization, and both effects were mediated by the stimulation of H1receptors. The histamine-induced membrane hyperpolarization was attenuated to ∼50% by large-conductance KCa(BK) channel blockers, and further reduced by intermediate (IK) and small conductance KCa(SK) channel blockers. The tonic component of histamine-induced [Ca2+]irise strongly depended on the presence of extracellular Ca2+([Ca2+]o) and was markedly reduced by La3+or Gd3+but not by nifedipine. It was significantly attenuated by BK channel blockers, and further blocked by the cocktail of BK, IK, and SK channel blockers. The KCablocker cocktail also significantly reduced the store-operated Ca2+entry (SOCE), which was induced by Ca2+addition after store-depletion by thapsigargin in [Ca2+]ofree solution. Our results demonstrate that the histamine-induced membrane hyperpolarization in chondrocytes due to KCachannel activation contributes to sustained Ca2+entry mainly through SOCE channels in OUMS-27 cells. Thus, KCachannels appear to play an important role in the positive feedback mechanism of [Ca2+]iregulation in chondrocytes in the presence of articular cartilage inflammation.

The contribution of Cl− conductance relative to that of K+ in the regulation of membrane potential was examined using OUMS-27 cells, a model cell-line of human chondrocytes. Application of 100 μM niflumic acid (NFA) and other anion-channel blockers induced significant membrane hyperpolarization. The NFA-sensitive membrane current under voltage-clamp was predominantly Cl− current. Application of NFA induced small but significant increase in intracellular Ca2+ concentration ([Ca2+]i) and markedly enhanced the late component of [Ca2+]i rise induced by 1 μM histamine. In conclusion, Cl− conductance substantially contributes to the regulation of resting membrane potential and [Ca2+]i in OUMS-27 cells. Keywords:: chondrocyte, chloride channel, niflumic acid

The activation of Kv1.3 potassium channel has obligatory roles in immune responses of T lymphocytes. Stromal cell-derived factor-1α (SDF-1α) binds to C-X-C chemokine receptor type 4, activates phosphoinositide 3-kinase, and plays essential roles in cell migration of T lymphocytes. In this study, the effects of phosphoinositides and SDF-1α on Kv1.3 current activity were examined in the Jurkat T cell line using whole cell patch-clamp techniques. The internal application of 10 μM phosphatidylinositol 4,5-bisphosphate (PIP2) or 10 μM phosphatidylinositol-3,4,5-trisphosphate (PIP3) significantly reduced Kv1.3 current, but that of 10 μM phosphatidylinositol-4-monophosphate (PIP) did not. The coapplication of 10 μg/ml anti-PIP3antibody with PIP2from the pipette did not change the reduction of Kv1.3 current by PIP2, but the coapplication of the antibody with PIP3eliminated the reduction. The heat-inactivated anti-PIP3antibody had no effect on PIP3-induced inhibition. These results suggest that PIP2per se can reduce Kv1.3 current as well as PIP3. External application of 1 μM Akt-kinase inhibitor VIII did not reverse the effect of intracellular PIP3. External application of 10 and 30 ng/ml SDF-1α significantly reduced Kv1.3 current. Internal application of anti-PIP3antibody reversed the SDF-1α-induced reduction. These results suggest that, in Jurkat T cells, PIP2, PIP3, and SDF-1α reduce Kv1.3 channel activity and that the reduction by SDF-1α may be mediated by the enhancement of PIP3production. These novel inhibitory effects of phosphoinositides and SDF-1α on Kv1.3 current may have a significant function as a downregulation mechanism of Kv1.3 activity for the maintenance of T lymphocyte activation in immune responses.

Malignant melanoma is the most deadly form of skin cancer and its incidence is steadily increasing worldwide. The plasma membrane in melanoma cells possesses a variety of ion channels, so its profile is thought to lead to a novel target for medical treatment for malignant melanoma. Here we showed that human melanoma G-361 cells expressed the epithelial Na(+) channel delta subunit (ENaC delta), which is largely unknown in physiological and pathological functions in non-neuronal tissues. Expression analyses at the level of mRNA clearly revealed that ENaC delta transcript was strongly expressed in human melanoma cells using reverse transcription-polymerase chain reaction and cell-based in situ hybridization techniques. Other ENaC subunits (alpha, beta, and gamma) were also distributed in human melanoma cells. In addition, human melanoma cells possessed an abundant expression of ENaC delta protein by immunocytochemistry. These results provide an attractive target for drug development of malignant melanoma.

In this study, we provide evidence of critical changes in the expression of non-selective cation currents (NSCC) during culture in rat aortic myocytes. A selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4αPDD), had little effect on membrane currents and intracellular Ca2+ (Ca2+i) in freshly isolated cells from the aorta. In contrast, in cultured aortic myocytes with and without serum, 4αPDD at a concentration range between 0.3 and 3 μM effectively elevated Ca2+i, which was abolished in the absence of external Ca2+. Application of 4αPDD to cultured aortic myocytes also activated NSCC, which had a reversal potential of +3 mV. Both of these signals were blocked by ruthenium red (RuR), an effective blocker of TRPVs. Although the expression of TRPV4 mRNA transcript was found in cultured as well as non-cultured aortic myocytes, significant immunoreactivity to TRPV4 protein was only detected in cultured rat aortic myocytes. Moreover, cultured human pulmonary arterial smooth muscle cells (hPASM) had a substantial response to 4αPDD, which was susceptible to the removal of external Ca2+ and application of RuR. These results provide a strong basis for our proposal that endogenous TRPV4 functions as an important regulator of Ca2+i in vascular myocytes under some physiological and pathophysiological conditions. Keywords:: TRPV4, 4α-phorbol 12,13-didecanoate, cell-culture

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. Chronic hypoxia causes vascular remodeling, which is central to the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). We have previously shown that Notch3 is up-regulated in HPH and that activation of Notch signaling enhances store-operated Ca(2+) entry (SOCE), an important mechanism that contributes to pulmonary arterial smooth muscle cell (PASMC) proliferation and contraction. Here, we investigate the role of Notch signaling in HPV and hypoxia-induced enhancement of SOCE. We examined SOCE in human PASMCs exposed to hypoxia and pulmonary arterial pressure in mice using the isolated perfused/ventilated lung method. Wild-type and canonical transient receptor potential (TRPC) 6(-/-) mice were exposed to chronic hypoxia to induce HPH. Inhibition of Notch signaling with a γ-secretase inhibitor attenuates hypoxia-enhanced SOCE in PASMCs and hypoxia-induced increase in pulmonary arterial pressure. Our results demonstrate that hypoxia activates Notch signaling and up-regulates TRPC6 channels. Additionally, treatment with a Notch ligand can mimic hypoxic responses. Finally, inhibition of TRPC6, either pharmacologically or genetically, attenuates HPV, hypoxia-enhanced SOCE, and the development of HPH. These results demonstrate that hypoxia-induced activation of Notch signaling mediates HPV and the development of HPH via functional activation and up-regulation of TRPC6 channels. Understanding the molecular mechanisms that regulate cytosolic free Ca(2+) concentration and PASMC proliferation is critical to elucidation of the pathogenesis of HPH. Targeting Notch regulation of TRPC6 will be beneficial in the development of novel therapies for pulmonary hypertension associated with hypoxia.

The epithelial Na(+) channel (ENaC) regulates Na(+) homeostasis in cells and across epithelia. Four homologous ENaC subunits (alpha, beta, gamma, and delta) have been isolated in mammals. Combination of alpha-, beta-, and gamma-subunits or delta-, beta-, and gamma-subunits forms fully functional channels. Amiloride is a well known blocker of the ENaC family that inhibits both channel complexes. However, no specific antagonists are currently known that distinguish them. Here, we show that Evans blue, a diagnostic aid for the measurement of blood volume and vascular permeability, inhibits the activity of the delta-subunit expressed in Xenopus oocytes. The inward currents at a holding potential of -60 mV in human ENaCdeltabetagamma-expressing oocytes were inhibited by the application of Evans blue in a concentration-dependent manner with an IC(50) value of 143 muM. Evans blue markedly inhibited the delta-subunit current but did not block the alpha-subunit current. In conclusion, Evans blue is the first known delta-subunit-specific antagonist of ENaC. This finding provides us with a key compound for elucidating the physiological and pathological functions of ENaCdelta in humans and for drug development in the ENaC family.

The ubiquitin E3 protein ligase Nedd4-2 is a physiological regulator of the epithelial sodium channel ENaC, which is essential for transepithelial Na+ transport and is linked to Liddle's syndrome, an autosomal dominant disorder of human salt-sensitive hypertension. Nedd4-2 function is negatively regulated by phosphorylation via a serum- and glucocorticoid-inducible protein kinase (Sgk1), which serves as a mechanism to inhibit the ubiquitination-dependent degradation of ENaC. We report here that 14-3-3 proteins participate in this regulatory process through a direct interaction with a phosphorylated form of human Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2). The interaction is dependent on Sgk1-catalyzed phosphorylation of hNedd4-2 at Ser-468. We found that this interaction preserved the activity of the Sgk1-stimulated ENaC-dependent Na+ current while disrupting the interaction decreased ENaC density on the Xenopus laevis oocytes surface possibly by enhancing Nedd4-2-mediated ubiquitination that leads to ENaC degradation. Our findings suggest that 14-3-3 proteins modulate the cell surface density of ENaC cooperatively with Sgk1 kinase by maintaining hNedd4-2 in an inactive phosphorylated state.

The amiloride-sensitive epithelial Na+ channel (ENaC) regulates Na+ homeostasis into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. The chemical agents acting on ENaC are, however, largely unknown, except for amiloride and benzamil as ENaC inhibitors. In particular, there are no agonists currently known that are selective for ENaCdelta, which is mainly expressed in the brain. Here we demonstrate that capsazepine, a competitive antagonist for transient receptor potential vanilloid subfamily 1, potentiates the activity of human ENaCdeltabetagamma (hENaCdeltabetagamma) heteromultimer expressed in Xenopus oocytes. The inward currents at a holding potential of -60 mV in hENaCdeltabetagamma-expressing oocytes were markedly enhanced by the application of capsazepine (or =1 microM), and the capsazepine-induced current was mostly abolished by the addition of 100 microM amiloride. The stimulatory effects of capsazepine on the inward current were concentration-dependent with an EC50 value of 8 microM. Neither the application of other vanilloid compounds (capsaicin, resiniferatoxin, and olvanil) nor a structurally related compound (dopamine) modulated the inward current. Although hENaCdelta homomer was also significantly activated by capsazepine, unexpectedly, capsazepine had no effect on hENaCalpha and caused a slight decrease on the hENaCalphabetagamma current. In conclusion, capsazepine acts on ENaCdelta and acts together with protons. Other vanilloids tested do not have any effect. These findings identify capsazepine as the first known chemical activator of ENaCdelta.

The amiloride-sensitive epithelial Na(+) channel (ENaC) controls Na(+) transport into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. ENaCdelta can associate with beta and gamma subunits and generate a constitutive current that is 2 orders of magnitude larger than that of homomeric ENaCdelta. However, the distribution pattern of ENaCdelta is not consistent with that of the beta and gamma subunits. ENaCdelta is expressed mainly in the brain in contrast to beta and gamma subunits, which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaCdelta and investigated the detailed tissue distribution in humans. When human ENaCdelta was expressed in Xenopus oocytes and Chinese hamster ovary cells, a reduction of extracellular pH activated this channel (half-maximal pH for an activation of 5.0), and the acid-induced current was abolished by amiloride. The most striking finding was that the desensitization of the acid-evoked current was much slower (by approximately 10% 120 s later), dissociating from the kinetics of acid-sensing ion channels in the degenerin/epithelial Na(+) channel family, which were rapidly desensitized during acidification. RNA dot-blot analyses showed that ENaCdelta mRNA was widely distributed throughout the brain and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaCdelta was expressed in the cerebellum and the hippocampus. In conclusion, human ENaCdelta activity is regulated by protons, indicating that it may contribute to the pH sensation and/or pH regulation in the human brain.

Pulmonary arterial hypertension (PAH) is a rare, progressive, and fetal disease. The five-year survival rate after diagnosis is ～50%. In Japan, PAH is listed in the Specified Rare and Intractable Diseases. Pulmonary vascular remodeling and sustained pulmonary vasoconstriction are the major causes for the elevated pulmonary vascular resistance (PVR) in PAH. The pathogenic mechanisms involved in the pulmonary vascular abnormalities in PAH remain unclear. Sustained vasoconstriction and vascular remodeling owing to proliferation of pulmonary arterial smooth muscle cells (PASMCs) are key pathogenic events that lead to early morbidity and mortality. These events have been closely linked to Ca(2+) mobilization and signaling in PASMCs. An increase in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in PASMCs is an important stimulus for pulmonary vasoconstriction and cell proliferation which subsequently cause pulmonary vascular wall thickening followed by the increase in PVR. Increased resting [Ca(2+)]cyt and enhanced Ca(2+) influx have been implicated in PASMCs from PAH patients, but precise therapeutic targets to interrupt these signal pathways have not been identified. We recently found that the extracellular Ca(2+)-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), is upregulated in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH). In addition, blockage of the CaSR with an antagonist (NPS2143) prevents the development of pulmonary hypertension and right ventricular hypertrophy in animal models of pulmonary hypertension. The functionally upregulated CaSR in PASMCs is a novel pathogenic mechanism contributing to the augmented Ca(2+) signaling and excessive cell proliferation in IPAH. Targeting CaSR in PASMCs may help develop novel therapeutic approach for PAH.

L-type voltage-dependent Ca(2+) channels (LVDCC) and large conductance Ca(2+)-activated K(+) channels (BKCa) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca(2+) release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BKCa activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BKCa and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BKCa by Ca(2+) influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BKCa co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BKCa can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BKCa and Cav1.2 but also the functional coupling between BKCa and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K(+) revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BKCa-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca(2+) dynamics including the negative feedback, to control the arterial excitability and contractility.

Rationale: An increase in cytosolic free Ca 2+ concentration ([Ca 2+ ] cyt ) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca 2+ channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH). Objective: Our previous study demonstrated that the Ca 2+ -sensing receptor (CaSR) was upregulated and the extracellular Ca 2+ -induced increase in [Ca 2+ ] cyt was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca 2+ ] cyt by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC. Methods and Results: The nifedipine-mediated increase in [Ca 2+ ] cyt in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in [Ca 2+ ] cyt , whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca 2+ ] cyt . Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca 2+ ] cyt in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca 2+ ] cyt . Conclusions: The dihydropyridine derivatives increase [Ca 2+ ] cyt by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca 2+ channels; therefore, it is possible that the use of dihydropyridine Ca 2+ channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel encoded by K(Ca)1.1 plays an important role in the control of smooth muscle tone by modulating membrane potential and intracellular Ca(2+) mobilization. BK(Ca) channel is functionally expressed in prostatic smooth muscle cells, and is activated by α(1)-adrenoceptor agonists. The main objective of this study was to elucidate the pathophysiological significance of changes in prostatic K(Ca)1.1 expressions in benign prostatic hyperplasia (BPH). Our previous study has shown that K(Ca)3.1 encoding intermediate-conductance K(Ca) (IK(Ca)) channel is up-regulated in stromal cells of implanted urogenital sinuses (UGSs) of stromal hyperplasia BPH model rats and in those of prostatic tissues from BPH patients. In the present study, the results from real-time polymerase chain reaction (PCR), Western blot, and immunohistochemical analyses showed significant down-regulation of K(Ca)1.1 transcripts and proteins and negative correlation between K(Ca)1.1 and K(Ca)3.1 transcript expressions in prostatic stromal cells of both BPH model rats and BPH patients. Corresponding to down-regulation of K(Ca)1.1 expression in stromal cells of implanted UGSs, membrane depolarization by application of the BK(Ca) channel blocker was disappeared. Down-regulation of K(Ca)1.1 may be involved in the phenotype switch from contractile profile to proliferative one in prostatic stromal cells of BPH patients.