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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.

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.

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.

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.

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 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.

The intermediate-conductance Ca2+-activated K+ channel KCa3.1/KCNN4 plays an important role in the modulation of Ca2+ signaling through the control of the membrane potential in T lymphocytes. Here, we study the involvement of KCa3.1 in the enlargement of the mesenteric lymph nodes (MLNs) in a mouse model of inflammatory bowel disease (IBD). The mouse model of IBD was prepared by exposing male C57BL/6J mice to 5% dextran sulfate sodium for 7 days. Inflammation-induced changes in KCa3.1 activity and the expressions of KCa3.1 and its regulators in MLN CD4+ T lymphocytes were monitored by real-time PCR, Western blot, voltage-sensitive dye imaging, patch-clamp, and flow cytometric analyses. Concomitant with an upregulation of KCa3.1a and nucleoside diphosphate kinase B (NDPK-B), a positive KCa3.1 regulator, an increase in KCa3.1 activity was observed in MLN CD4+ T lymphocytes in the IBD model. Pharmacological blockade of KCa3.1 elicited the following results: 1) a significant decrease in IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation, and crypt damage of the colon and MLN enlargement compared with control mice, and 2) the restoration of the expression levels of KCa3.1a, NDPK-B, and Th1 cytokines in IBD model MLN CD4+ T lymphocytes. These findings suggest that the increase in KCa3.1 activity induced by the upregulation of KCa3.1a and NDPK-B may be involved in the pathogenesis of IBD by mediating the enhancement of the proliferative response in MLN CD4+ T lymphocyte and, therefore, that the pharmacological blockade of KCa3.1 may decrease the risk of IBD.

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.

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.

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

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

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

The transient receptor potential melastatin subfamily (TRPM), which is a mammalian homologue of cell death-regulated genes in Caenorhabditis elegans and Drosophila, has potential roles in the process of the cell cycle and regulation of Ca2+signaling. Among this subfamily, TRPM8 (also known as Trp-p8) is a Ca2+-permeable channel that was originally identified as a prostate-specific gene upregulated in tumors. Here we showed that the TRPM8 channel was expressed in human melanoma G-361 cells, and activation of the channel produced sustainable Ca2+influx. The application of menthol, an agonist for TRPM8 channel, elevated cytosolic Ca2+concentration in a concentration-dependent manner with an EC50value of 286 μM in melanoma cells. Menthol-induced responses were significantly abolished by the removal of external Ca2+. Moreover, inward currents at a holding potential of −60 mV in melanoma cells were markedly potentiated by the addition of 300 μM menthol. The most striking finding was that the viability of melanoma cells was dose-dependently depressed in the presence of menthol. These results reveal that a functional TRPM8 protein is expressed in human melanoma cells to involve the mechanism underlying tumor progression via the Ca2+handling pathway, providing us with a novel target of drug development for malignant melanoma.