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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.

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.

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.

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

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 (

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.

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.

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+]cytin 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+]cytwas 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+]cytincreases 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+]cytwas 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+]cytincreases 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+]cytincrease due to upregulated TRPV1 may be a critical pathogenic mechanism that contributes to augmented Ca2+influx and excessive PASMC proliferation in patients with IPAH.

Maintaining Ca2+ homeostasis in lymphatic fluids is necessary for proper hearing. Despite its significance, the mechanisms that maintain the cochlear lymphatic Ca2+ concentrations within a certain range are not fully clarified. We investigated the functional expression of calcium-sensing receptor (CaSR), which plays a pivotal role in sensing extracellular Ca2+ concentrations for feedback regulations. Western blotting for CaSR revealed an approximately 130-kDa protein expression in cochlear tissue extracts and immunohistochemical analysis revealed its expression specifically in type I fibrocytes in the spiral ligament, fibrocytes in the supralimbal and limbal regions, the epithelium of the osseous spiral lamina, and the smooth muscle cells of the spiral modiolar arteries. Ca2+ imaging demonstrated that extracellular Ca2+ increased the levels of intracellular Ca2+ in CaSR-expressing fibrocytes in the spiral ligament, and that this was suppressed by the CaSR inhibitor, NPS2143. Furthermore, hearing thresholds were moderately elevated by intracochlear application of the CaSR inhibitors NPS2143 and Calhex231, across a range of frequencies (8–32 kHz). These results demonstrate the functional expression of CaSR in the cochlear perilymphatic compartment. In addition, the elevated hearing thresholds that are achieved by inhibiting CaSR suggest this is a required mechanism for normal hearing, presumably by sensing perilymphatic Ca2+ to stabilize Ca2+ concentrations within a certain range. These results provide novel insight into the mechanisms regulating Ca2+ homeostasis in the cochlea and provide a new perspective on cochlear physiology.

The pineal glands regulate circadian rhythm through the synthesis and secretion of melatonin. The stimulation of nicotinic acetylcholine receptor due to parasympathetic nerve activity causes an increase in intracellular Ca2+concentration and eventually downregulates melatonin production. Our previous report shows that rat pinealocytes have spontaneous and nicotine-induced Ca2+oscillations that are evoked by membrane depolarization followed by Ca2+influx through voltage-dependent Ca2+channels (VDCCs). These Ca2+oscillations are supposed to contribute to the inhibitory mechanism of melatonin secretion. Here we examined the involvement of large-conductance Ca2+-activated K+(BKCa) channel conductance on the regulation of Ca2+oscillation and melatonin production in rat pinealocytes. Spontaneous Ca2+oscillations were markedly enhanced by BKCachannel blockers (1 μM paxilline or 100 nM iberiotoxin). Nicotine (100 μM)-induced Ca2+oscillations were also augmented by paxilline. In contrast, spontaneous Ca2+oscillations were abolished by BKCachannel opener [3 μM 12,14-dichlorodehydroabietic acid (diCl-DHAA)]. Under whole cell voltage-clamp configurations, depolarization-elicited outward currents were significantly activated by diCl-DHAA and blocked by paxilline. Expression analyses revealed that the α and β3 subunits of BKCachannel were highly expressed in rat pinealocytes. Importantly, the activity of BKCachannels modulated melatonin secretion from whole pineal gland of the rat. Taken together, BKCachannel activation attenuates these Ca2+oscillations due to depolarization-synchronized Ca2+influx through VDCCs and results in a recovery of reduced melatonin secretion during parasympathetic nerve activity. BKCachannels may play a physiological role for melatonin production via a negative-feedback mechanism.

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.

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.

Store-operated Ca(2+) entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca(2+) influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells.

Pinealocytes regulate circadian rhythm by synthesizing and secreting melatonin. These cells generate action potentials; however, the contribution of specific ion channels to melatonin secretion from pinealocytes remains unclear. In this study, the involvement and molecular identity of Ca2+-activated Cl− (ClCa) channels in the regulation of melatonin secretion were examined in rat pineal glands. Treatment with the ClCa channel blockers, niflumic acid or T16Ainh-A01, significantly reduced melatonin secretion in pineal glands. After pineal K+ currents were totally blocked under whole-cell patch clamp conditions, depolarization and subsequent repolarization induced a slowly activating outward current and a substantial inward tail current, respectively. Both of these current changes were dependent on intracellular Ca2+ concentration and inhibited by niflumic acid and T16Ainh-A01. Quantitative real-time PCR, Western blotting, and immunocytochemical analyses revealed that TMEM16A and TMEM16B were highly expressed in pineal glands. siRNA knockdown of TMEM16A and/or TMEM16B showed that both channels contribute to ClCa currents in pinealocytes. Conversely, co-expression of TMEM16A and TMEM16B channels or the expression of this tandem channel in HEK293 cells mimicked the electrophysiological characteristics of ClCa currents in pinealocytes. Moreover, bimolecular fluorescence complementation, FRET, and co-immunoprecipitation experiments suggested that TMEM16A and TMEM16B can form heteromeric channels, as well as homomeric channels. In conclusion, pineal ClCa channels are composed of TMEM16A and TMEM16B subunits, and these fluxes regulate melatonin secretion in pineal glands.

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.

The pineal gland regulates circadian rhythm through the synthesis and secretion of melatonin. The rise of intracellular Ca2+ concentration ([Ca2+]i) following nicotinic acetylcholine receptor (nAChR) stimulation due to parasympathetic nerve activity downregulates melatonin production. Important characteristics and roles of Ca2+ mobilization due to nAChR stimulation remain to be clarified. We report here that spontaneous Ca2+ oscillations can be observed in ∼15% of the pinealocytes in slice preparations from rat pineal glands when this dissociation procedure is done within 6 h from a dark-to-light change. The frequency and half-life of [Ca2+]i rise were 0.86 min−1 and 19 s, respectively. Similar spontaneous Ca2+ oscillations were recorded in 17% of rat pinealocytes that were primary cultured for several days. Simultaneous measurement of [Ca2+]i and membrane potential revealed that spontaneous Ca2+ oscillations were triggered by periodic membrane depolarizations. Spontaneous Ca2+ oscillations in cultured pinealocytes were abolished by extracellular Ca2+ removal or application of nifedipine, a blocker of voltage-dependent Ca2+ channel (VDCC). In contrast, blockers of intracellular Ca2+-release channels, 2-aminoethoxydiphenylborate and ryanodine, have no effect. Our results also reveal that, in 23% quiescent pinealocytes, Ca2+ oscillations were observed following the withdrawal of nicotine. Norepinephrine-induced melatonin secretion from whole pineal glands was significantly decreased by the coapplication of acetylcholine (ACh). This inhibitory effect of ACh was attenuated by nifedipine. In conclusion, both spontaneous and evoked Ca2+ oscillations are due to membrane depolarization following activation of VDCCs. This consists of VDCC α1F subunit, and the associated Ca2+ influx can strongly regulate melatonin secretion in pineal glands.

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.

Ca 2+ -activated Cl − channel (CaCC) often plays substantial roles in the regulation of membrane excitability in smooth muscle cells (SMCs). TMEM16A, a member of the TMEM16 family, has been suggested as the molecular entity responsible for CaCC in several types of SMCs. In this study, the expression of TMEM16A splicing variants and their contribution to CaCC activity were examined in murine portal vein SMCs (mPVSMCs). Four transcripts of TMEM16A splicing variants, which include four alternatively spliced segments (“ a ” and “ b ” in N-terminus and “ c ” and “ d ” in the first intracellular loop), were identified; the expression ratio of four transcripts of “ abc ”, “ acd ”, “ abcd ” and “ ac ” was 64.5, 25.8, 4.8 and 4.8%, respectively. The immunostaining of isolated mPVSMCs with anti-TMEM16A antibody indicates the abundant expression of TMEM16A on the cell membrane. CaCC currents recorded in mPVSMCs were markedly reduced by T16A inh -A01, a specific TMEM16A inhibitor. When the two major TMEM16A splicing variants, abc and acd isoforms, were expressed separately in HEK293 cells, the CaCC currents, which possess similar electrophysiological characteristics to those in mPVSMCs were observed. The single-molecule photobleaching analyses using total internal reflection fluorescence (TIRF) microscope indicated that the distribution of stepwise photobleaching events was fit well with a binomial distribution for homodimer. Additionally, the heterodimer formation was suggested by fluorescence resonance energy transfer (FRET) analyses in HEK293 cells co-expressing CFP- or YFP-tagged variants. In conclusion, alternatively spliced variants of TMEM16A abc and acd in mPVSMCs are two major molecular entities of CaCC and may form hetero-/homo-dimers to be functional as CaCC in the regulation of membrane excitability and contractility in mPVSMCs.

TMEM16A is a major component of Ca2+-activated Cl− channel (CaCC) conductance in murine portal vein smooth muscle cells (mPVSMCs). Here, the regulation of CaCC activity by the actin cytoskeleton was examined in mPVSMCs. Actin disruption by cytochalasin D did not affect the current density, but increased the deactivation time constant in mPVSMCs. The elongated deactivation was recovered by jasplakinolide. When murine TMEM16A was transfected into HEK293 cells that have a poorly developed actin cytoskeleton, electrophysiological properties of CaCC currents were not changed by cytochalasin D. In conclusion, the CaCC activity in mPVSMCs is modified by the interaction of TMEM16A with abundant actin cytoskeleton. Keywords:: TMEM16A, actin cytoskeleton, portal vein

The Na+/Ca2+exchanger (NCX) is thought to be a key molecule in the regulation of cytosolic Ca2+dynamics. The relative importance of the two Ca2+transport modes of NCX activity leading to Ca2+efflux (forward) and influx (reverse) in smooth muscle, however, remains unclear. Unexpectedly, spontaneous contractions of urinary bladder smooth muscle (UBSM) were enhanced in transgenic mice overexpressing NCX1.3 (NCX1.3tg/tg). The enhanced activity was attenuated by KB-R7943 or SN-6. Whole cell outward NCX current sensitive to KB-R7943 or Ni2+was readily detected in UBSM cells from NCX1.3tg/tgbut not wild-type mice. Spontaneous Ca2+transients in myocytes of NCX1.3tg/tgwere larger and frequently resulted in propagating events and global elevations in cytosolic Ca2+concentration. Significantly, NCX1.3tg/tgmice exhibited a pattern of more frequent urination of smaller volumes and this phenotype was reversed by oral administration of KB-R7943. On the other hand, KB-R7943 did not improve it in KB-R7943-insensitive (G833C-)NCX1.3tg/tgmice. We conclude that NCX1.3 overexpression is associated with abnormal urination owing to enhanced Ca2+influx via reverse mode NCX leading to prolonged, propagating spontaneous Ca2+release events and a potentiation of spontaneous UBSM contraction. These findings suggest the possibility that NCX is a candidate molecular target for overactive bladder therapy.

Electromechanical coupling via membrane depolarization-mediated activation of voltage-dependent Ca2+ channels (VDCC) is an important mechanism in regulating pulmonary vascular tone, while mouse is an animal model often used to study pathogenic mechanisms of pulmonary vascular disease. The function of VDCC in mouse pulmonary artery (PA) smooth muscle cells (PASMC), however, has not been characterized, and their functional role in reactive oxygen species (ROS)-mediated regulation of vascular function remains unclear. In this study, we characterized the electrophysiological and pharmacological properties of VDCC in PASMC and the divergent effects of ROS produced by xanthine oxidase (XO) and hypoxanthine (HX) on VDCC in PA and mesenteric artery (MA). Our data show that removal of extracellular Ca2+ or application of nifedipine, a dihydropyridine VDCC blocker, both significantly inhibited 80 mM K+-mediated PA contraction. In freshly dissociated PASMC, the maximum inward Ca2+ currents were −2.6 ± 0.2 pA/pF at +10 mV (with a holding potential of −70 mV). Window currents were between −40 and +10 mV with a peak at −15.4 mV. Nifedipine inhibited currents with an IC50 of 0.023 μM, and 1 μM Bay K8644, a dihydropyridine VDCC agonist, increased the inward currents by 61%. XO/HX attenuated 60 mM K+-mediated increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) due to Ca2+ influx through VDCC in PASMC. Exposure to XO/HX caused relaxation in PA preconstricted by 80 mM K+ but not in aorta and MA. In contrast, H2O2 inhibited high K+-mediated increase in [Ca2+]cyt and caused relaxation in both PA and MA. Indeed, RT-PCR and Western blot analysis revealed significantly lower expression of CaV1.3 in MA compared with PA. Thus our study characterized the properties of VDCC and demonstrates that ROS differentially regulate vascular contraction by regulating VDCC in PA and systemic arteries.

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.

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.

The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4-5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba(2+)-sensitive inward rectifier K(+) current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca(2+) imaging study revealed that the hypoxic stress enhanced store-operated Ca(2+) (SOC) entry, which was significantly reduced in the presence of 100 μM Ba(2+). On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba(2+). We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca(2+) entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia.