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Mitochondria buffer cytosolic Ca2+ increases following Ca2+ influx from extracellular spaces, and Ca2+ release from intracellular Ca2+ store sites under physiological circumstances. Therefore, close contact of mitochondria with the sarcoplasmic reticulum (SR) is required for maintaining Ca2+ homeostasis. Mitofusin 2 (Mfn2) localizes in both mitochondrial and SR membranes and is hypothesized to optimize the distance and Ca2+ transfer between these organelles. However, the physiological significance of Mfn2 in vascular smooth muscle cells (VSMCs) is poorly understood. In the present study, the role of Mfn2 in the physical and functional couplings between SR and mitochondria was examined in rat aortic smooth muscle cells (rASMCs) by confocal and electron microscope imaging. When Mfn2 was knocked down using siRNA in rASMCs, the mean distance between these organelles was extended from 16.2 to 21.6 nm. The increase in the cytosolic Ca2+ concentration ([Ca2+]cyt) induced by 100 nM arginine vasopressin (AVP) was not affected by Mfn2 siRNA knockdown, whereas cytosolic Ca2+ removal was slower after Mfn2 knockdown. Following the AVP-induced [Ca2+]cyt increase, mitochondrial Ca2+ uptake and Ca2+ refill into the SR were attenuated by Mfn2 knockdown. In addition, Mfn2-knockdown cells exhibited a loss of mitochondrial membrane potential (ΔΨmito) and lower ATP levels in mitochondria. Moreover, Mfn2 knockdown inhibited cell proliferation. In contrast, Mfn2 overexpression increased ΔΨmito and cell growth. This study strongly suggests that Mfn2 is responsible for SR-mitochondria Ca2+ signaling by tethering mitochondria to SR, thereby regulating ATP production and proliferation of VSMCs.

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.

Ca

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

Mitochondria buffer cytosolic Ca

Male penis is required to become erect during copulation. In the upper (dorsal) part of penis, the erectile tissue termed corpus cavernosum (CC) plays fundamental roles for erection by regulating the inner blood flow. When blood flows into the CC, the microvascular complex termed sinusoidal space is reported to expand during erection. A novel in vitro explant system to analyze the dynamic erectile responses during contraction/relaxation is established. The current data show regulatory contraction/relaxation processes induced by phenylephrine (PE) and nitric oxide (NO) donor mimicking dynamic erectile responses by in vitro CC explants. Two-photon excitation microscopy (TPEM) observation shows the synchronous movement of sinusoidal space and the entire CC. By taking advantages of the CC explant system, tadalafil (Cialis) was shown to increase sinusoidal relaxation. Histopathological changes have been generally reported associating with erection in several pathological conditions. Various stressed statuses have been suggested to occur in the erectile responses by previous studies. The current CC explant model enables to analyze such conditions through directly manipulating CC in the repeated contraction/relaxation processes. Expression of oxidative stress marker and contraction-related genes, Hypoxia-inducible factor 1-alpha (Hif1a), glutathione peroxidase 1 (Gpx1), Ras homolog family member A (RhoA), and Rho-associated protein kinase (Rock), was significantly increased in such repeated contraction/relaxation. Altogether, it is suggested that the system is valuable for analyzing structural changes and physiological responses to several regulators in the field of penile medicine.

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− currents (ICl,swell) are responsible for the RVD, but the molecular identity in chondrocytes is largely unknown. In this study, we reveal that in human OUMS-27 chondrocytes, ICl,swell can be elicited by hypoosmotic stimulation (180 mOsm) and be inhibited by classical Cl− channel blockers, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and niflumic acid, and be attenuated by siRNA knockdown of ClC-3. Our molecular analyses revealed that ClC-3A is expressed as a major splice variant in both human articular chondrocytes and OUMS-27 cells. The onset and early phase of RVD following hypoosmotic stress in OUMS-27 cells were affected by DIDS and ClC-3 knockdown. Hypoosmotic stimulation caused Ca2+ influx and subsequent release of prostaglandin E2 (PGE2) in OUMS-27 cells, and both of these responses were reduced by DIDS and ClC-3 knockdown. These results strongly suggest that ClC-3 is responsible for ICl,swell and RVD under the hypoosmotic environments. It is likely that ClC-3 is associated with the pathogenesis of cartilage degenerative diseases including osteoarthritis via PGE2 release.

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

Elevation of intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as excitation–transcription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts [Ca2+]i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.

Significance Excitation–transcription (E-T) coupling can initiate and modulate essential physiological or pathological responses in cells, such as neurons and cardiac myocytes. Although vascular myocytes also exhibit E-T coupling in response to membrane depolarization, the underlying molecular mechanisms are unknown. Our study reveals that E-T coupling in vascular myocytes converts intracellular Ca 2+ signals into selective gene transcription related to chemotaxis, leukocyte adhesion, and inflammation. Our discovery identifies a mechanism for vascular remodeling as an adaptation to increased circumferential stretch.

Melatonin secretion from the pineal glands regulates circadian rhythms in mammals. Melatonin production is decreased by an increase in cytosolic Ca

Portal hypertension is defined as an increased pressure in the portal venous system and occurs as a major complication in chronic liver diseases. The pathological mechanism underlying the pathogenesis and development of portal hypertension has been extensively investigated. Vascular tone of portal vein smooth muscles (PVSMs) is regulated by the activities of several ion channels, including Ca2+-activated Cl− (ClCa) channels. TMEM16A is mainly responsible for ClCa channel conductance in vascular smooth muscle cells, including portal vein smooth muscle cells (PVSMCs). In the present study, the functional roles of TMEM16A channels were examined using two experimental portal hypertensive models, bile duct ligation (BDL) mice with cirrhotic portal hypertension and partial portal vein ligation (PPVL) mice with non-cirrhotic portal hypertension. Expression analyses revealed that the expression of TMEM16A was downregulated in BDL-PVSMs, but not in PPVL-PVSMs. Whole-cell ClCa currents were smaller in BDL-PVSMCs than in sham- and PPVL-PVSMCs. The amplitude of spontaneous contractions was smaller and the frequency was higher in BDL-PVSMs than in sham- and PPVL-PVSMs. Spontaneous contractions sensitive to a specific inhibitor of TMEM16A channels, T16Ainh-A01, were reduced in BDL-PVSMs. Furthermore, in normal PVSMs, the downregulation of TMEM16A expression was mimicked by the exposure to angiotensin II, but not to bilirubin. This study suggests that the activity of ClCa channels is attenuated by the downregulation of TMEM16A expression in PVSMCs associated with cirrhotic portal hypertension, which is partly mediated by increased angiotensin II in cirrhosis.

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 Ca2+-activated K+ (BKCa) channel is expressed in several types of cancer cells and regulates cell proliferation, migration, invasion, and metastasis. In the present study, the functional expression of the BKCa channel was examined in a human somatostatinoma QGP-1 cell line. In QGP-1 cells, outward currents were elicited by membrane depolarization at pCa 6.5 (300 nM) in the pipette solution and inhibited by the specific BKCa channel blocker, paxilline. Paxilline-sensitive currents were detected, even at pCa 8.0 (10 nM) in the pipette solution, in QGP-1 cells. In addition to the α and β2-4 subunits of the BKCa channel, the novel regulatory γ1 subunit (BKCaγ1) was co-localized with the α subunit in QGP-1 cells. Paxilline-sensitive currents at pCa 8.0 in the pipette solution were reduced by the siRNA knockdown of BKCaγ1. Store-operated Ca2+ entry was smaller in BKCaγ1 siRNA-treated QGP-1 cells. The proliferation of QGP-1 cells was attenuated by paxilline or the siRNA knockdown of BKCaγ1. These results strongly suggest that BKCaγ1 facilitates the proliferation of human somatostatinoma cells. Therefore, BKCaγ1 may be a novel therapeutic target for somatostatinoma.

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 Ca2+-activated Cl- (ClCa) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl- currents were sensitive to the ClCa channel blockers, 100 μM niflumic acid and 10 μM T16Ainh-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of ClCa currents with ClCa channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca2+ concentration ([Ca2+]cyt). T16Ainh-A01 reduced cell viability in a concentration-dependent manner. Either ClCa channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, ClCa channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to ClCa channel conductance and can regulate both the resting membrane potential and [Ca2+]cyt in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE STATEMENT: In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca2+-activated Cl- channels and can regulate both the resting membrane potential and cytosolic Ca2+ concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) and play an essential role in the regulation of its functions. Oxidative stress accumulates excessive reactive oxygen species (ROS) and facilitates the death of BCECs, leading to a dysfunctional BBB. However, the mechanisms underlying the death of BCECs under oxidative stress remain unclear. In the present study, the effects of oxidative stress on cell viability, ROS production, intracellular Ca2+ concentration, and protein expression were examined using a cell line derived from bovine BCECs, t-BBEC117. When t-BBEC117 cells were exposed to oxidative stress induced by hydrogen peroxide (H2O2, 10–100 μM), cell growth was inhibited in a dose-dependent manner. Oxidative stress by 30 μM H2O2 increased the production of ROS and its effects were blocked by the ROS scavenger, 10 mM N-acetyl- l -cysteine (NAC). In addition, oxidative stress reduced store-operated Ca2+ entry (SOCE) and this decrease was recovered by NAC or the Orai channel activator, 5 μM 2-aminoethyl diphenylborinate (2-APB). The siRNA knockdown of Orai1 revealed that Orai1 was mainly responsible for SOCE channels and its activity was decreased by oxidative stress. However, the protein expression of Orai1 and STIM1 was not affected by oxidative stress. Oxidative stress-induced cell death was rescued by 2-APB, NAC, or the STIM-Orai activating region. In conclusion, oxidative stress reduces Orai1-mediated SOCE and, thus, facilitates the death of BCECs.

In visceral smooth muscle cells (SMCs), the large-conductance Ca2+-activated K+ (BK) channel is one of the key elements underlying a negative feedback mechanism that is essential for the regulation of intracellular Ca2+ 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.

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.

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 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel β1 subunit (BKCaβ1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaβ1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.

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

Portal hypertension is defined as an increased pressure in the portal venous system and occurs as a major complication in chronic liver diseases. The pathological mechanism underlying the pathogenesis and development of portal hypertension has been extensively investigated. Vascular tone of portal vein smooth muscles (PVSMs) is regulated by the activities of several ion channels, including Ca

Functional coupling between large-conductance Ca2+-activated K+ (BKCa) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues. Spontaneous Ca2+ release through RyRs (Ca2+ sparks) and subsequent BKCa channel activation occur within the PM-SR junctional sites. We report here that a molecular interaction of caveolin-1 (Cav1), a caveola-forming protein, with junctophilin-2 (JP2), a bridging protein between PM and SR, positions BKCa channels near RyRs in SM cells (SMCs) and thereby contributes to the formation of a molecular complex essential for Ca2+ microdomain function. Approximately half of all Ca2+ sparks occurred within a close distance (

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.

The mechanisms underlying neuronal cell death induced by the rise of intracellular Na+ concentration ([Na+]i) following abnormal hyperexcitation are not fully understood. Previously, we have established a recombinant cell line derived from HEK293 cells, in which the occurrence of a sustained action potential (AP) induces cell death. Mutated voltage-gated Nav1.5 channel (IFM/QQQ) lacking inactivation, and inward rectifying K+ channel (Kir2.1) were co-expressed in HEK293 cells (IFM/QQQ + Kir2.1 cells). In this cell line, the rise of [Na+]i due to a sustained AP reached maximum within 15 min without concomitant [Ca2+]i rise, and then elicited significant externalization of phosphatidylserine and enhancement of caspase activity. Marked decreases in mitochondrial transmembrane potential and ATP concentration were also detected. The significant cell death occurred at 3 h from the AP onset and reached a steady state at around 12 h. The significant release of lactate dehydrogenase was not detected even after 12 h. These results provide novel findings that Na+ accumulation or/and possibly concomitant K+ loss elicits apoptosis presumably due to the mitochondrial dysfunction, which is attributable to neither the membrane depolarization nor [Ca2+]i change. This apoptotic mechanism may be involved, at least in part, in neuronal cell death under pathophysiological settings with abnormal hyperexcitability.

Conversion of intracellular Ca2+ signals to electrical activity results in multiple and differing physiological impacts depending on cell types. In some organs such as gastrointestinal and urinary systems, spontaneous Ca2+ oscillation in pacermaker cells can function essentially as a Ca2+ clock mechanism, which has been originally found in pacemaking in sinoatrial node cell of the heart. The conversion of discrete Ca2+ clock events to spontaneous electrical activity is an essential step for the initiation and propagation of pacemaker activity through the multicellular organs resulting in synchronized physiological functions. Here, a model of intracellular signal transduction from a Ca2+ oscillation to initiation of electrical slow waves and their propagation were reconstituted in HEK293 cells. This was accomplished based on ryanodine receptor (RyR) type 3, Ca2+-activated ion channels, i.e. small conductance Ca2+-activated K+ channel (SK2) or Ca2+-activated Cl− channel (TMEM16A), and connexin43 being heterologously co-expressed. The propagation of electrical waves was abolished or substantially reduced by treatment with selective blockers of the expressed channels and 18β-glycyrrhetinic acid, a gap junction inhibitor, respectively. Thus, we demonstrated that the conversion of Ca2+ oscillation to electrical signals with cell to cell propagation can be reconstituted as a model of Ca2+ clock pacemaker activity by combinational expression of critical elements in heterologous expression system.

The blood-brain barrier (BBB) is mainly formed by brain capillary endothelial cells (BCECs) and is exposed to hypoxic environments under pathological conditions. The effects of hypoxia on the expression and activity of Ca

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.

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+ currents in endothelial cells, e.g., Kir2.1 channels without any alterations in the mRNA level, in t-BBEC117, a cell line derived from bovine BCECs. The hyperpolarization of membrane potential due to Kir2.1 channel upregulation significantly facilitates cell proliferation. In the present study, the mechanisms underlying the hypoxia-induced Kir2.1 upregulation was examined. We emphasize the involvement of dynamin2, a protein known to be involved in a number of surface expression pathways. Hypoxic culture upregulated dynamin2 expression in t-BBEC117 cells. The inhibition of dynamin2 by Dynasore canceled hypoxia-induced upregulation of Kir2.1 currents by reducing surface expression. On the contrary, Kir2.1 currents and proteins in t-BBEC117 cultured under normoxia were increased by overexpression of dynamin2, but not by dominant-negative dynamin2. Molecular imaging based on bimolecular fluorescence complementation, double-immunostaining, and coimmunoprecipitation assays revealed that dynamin2 can directly bind to the Kir2.1 channel. Moreover, hypoxic culture downregulated hypoxic-inducible factor-1α (HIF-1α) expression. Knockdown of HIF-1α increased dynamin2 expression in t-BBEC117 cells, in both normoxic and hypoxic culture conditions. In summary, our results demonstrated that hypoxia downregulates HIF-1α, increases dynamin2 expression, and facilitates Kir2.1 surface expression, resulting in hyperpolarization of membrane potential and subsequent increase in Ca2+ influx in BCECs.

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+ channels, Ca2+-activated K+ channels, ATP-sensitive K+ channels, two-pore domain K+ channels, voltage-dependent Ca2+ channels, store-operated Ca2+ channels, epithelial Na+ channels, acid-sensing ion channels, transient receptor potential channels, and mechanosensitive channels. These channels play important roles for the regulation of resting membrane potential, Ca2+ signaling, pH sensing, mechanotransduction, and cell proliferation in articular chondrocytes. In addition to these cation channels, Cl- channels are known to be expressed in mammalian chondrocytes: e.g., voltage-dependent Cl- channels, cystic fibrosis transmembrane conductance regulator channels, swelling-activated Cl- channels, and Ca2+-activated Cl- channels. Although these chondrocyte Cl- channels are thought to contribute to the regulation of resting membrane potential, Ca2+ signaling, cell volume, cell survival, and endochondral bone formation, the physiological functions have not been fully clarified. Osteoarthritis (OA) is caused by the degradation of articular cartilage, resulting in inflammation and pain in the joints. Therefore the pathophysiological roles of Cl- channels in OA chondrocytes are of considerable interest. Elucidating the physiological and pathological functions of chondrocyte Cl- channels will provide us a more comprehensive understanding of chondrocyte functions and may suggest novel molecular targets of drug development for OA.

Physiological functions of type 3 ryanodine receptors (RyR3) in smooth muscle (SM) tissues are not well understood, in spite of their wide expression. However, the short isoform of RyR3 is known to be a dominant-negative variant (DN-RyR3), which may negatively regulate functions of both RyR2 and full-length (FL) RyR3 by forming hetero-tetramers. Here, functional roles of RyR3 in the regulation of Ca2+signaling in mesenteric artery SM cells (MASMCs) were examined using RyR3 homozygous knockout mice (RyR3−/−). Quantitative PCR analyses suggested that the predominant RyR3 subtype in MASMs from wild-type mice (RyR3+/+) was DN-RyR3. In single MASMCs freshly isolated from RyR3−/−, the EC50of caffeine to induce Ca2+release was lower than that in RyR3+/+myocytes. The amplitude and frequency of Ca2+sparks and spontaneous transient outward currents in MASMCs from RyR3−/−were all larger than those from RyR3+/+. Importantly, mRNA and functional expressions of voltage-dependent Ca2+channel and large-conductance Ca2+-activated K+(BK) channel in MASMCs from RyR3−/−were identical to those from RyR3+/+. However, in the presence of BK channel inhibitor, paxilline, the pressure rises induced by BayK8644 in MA vascular beds of RyR3−/−were significantly larger than in those of RyR3+/+. This indicates that the negative feedback effects of BK channel activity on intracellular Ca2+signaling was enhanced in RyR3−/−. Thus, RyR3, and, in fact, mainly DN-RyR3, via a complex with RyR2 suppresses Ca2+release and indirectly regulated membrane potential by reducing BK channel activity in MASMCs and presumably can affect the regulation of intrinsic vascular tone.

Spatiotemporal changes in cytosolic Ca2+ concentration ([Ca2+]c) trigger a number of physiological functions in smooth muscle cells (SMCs). We previously imaged Ca2+-induced Ca2+ release following membrane depolarization as local Ca2+ transients, Ca2+ hotspots, in subplasmalemmal regions. In this study, the physiological significance of mitochondria on local Ca2+ signaling was examined. Cytosolic and mitochondrial Ca2+ images following depolarization or action potentials were recorded in single SMCs from the guinea pig urinary bladder using a fast-scanning confocal fluorescent microscope. Depolarization- and action potential-induced [Ca2+]c transients occurred at several discrete sites in subplasmalemmal regions, peaked within 30 ms, and then spread throughout the whole-cell. In contrast, Ca2+ concentration in the mitochondria matrix ([Ca2+]m) increased after a delay of ~50 ms from the start of depolarization, and then peaked within 500 ms. Following repolarization, [Ca2+]c returned to the resting level with a half-decay time of ~500 ms, while [Ca2+]m recovered more slowly (∼1.5 s). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, a mitochondrial uncoupler, abolished depolarization-induced [Ca2+]m elevations and slowed [Ca2+]c changes. Importantly, short depolarization-induced changes in [Ca2+]m and transmembrane potential in mitochondria coupled to Ca2+ hotspots were significantly larger than those in other mitochondria. Total internal reflection fluorescence imaging revealed that a subset of mitochondria closely localized with ryanodine receptors and voltage-dependent Ca2+ channels. These results indicate that particular mitochondria are functionally coupled to ion channels and sarcoplasmic reticulum fragments within the local Ca2+ microdomain, and thus, strongly contribute to [Ca2+]c regulation in SMCs.

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 (