Rieske Iron-Sulfur Protein Mediates Pulmonary Hypertension Following Nicotine/Hypoxia Coexposure.

The high mortality rate in patients with chronic obstructive pulmonary disease (COPD) may be due to pulmonary hypertension (PH). These diseases are highly associated with cigarette smoke and its key component nicotine. Here, we created a novel animal model of PH using coexposure to nicotine (or cigarette smoke) and hypoxia. This heretofore unreported model showed significant early-onset pulmonary vasoremodeling and PH. Using newly generated mice with complementary smooth muscle-specific Rieske iron-sulfur protein (RISP) gene knockout and overexpression, we demonstrate that RISP is critically involved in promoting pulmonary vasoremodeling and PH, which are implemented by oxidative ataxia telangiectasia-mutated-mediated DNA damage and NF-κB-dependent inflammation in a reciprocal positive mechanism. Together, our findings establish for the first time an animal model of hypoxia-induced early-onset PH in which mitochondrial RISP-dependent DNA damage and NF-κB inflammation play critical roles in vasoremodeling. Specific therapeutic targets for RISP and related oxidative stress-associated signaling pathways may create unique and effective treatments for PH, chronic obstructive pulmonary disease, and their complications.

Inhibition of big-conductance Ca2+-activated K+ channels in cerebral artery (vascular) smooth muscle cells is a major novel mechanism for tacrolimus-induced hypertension.

Tacrolimus (TAC, also called FK506), a common immunosuppressive drug used to prevent allograft rejection in transplant patients, is well known to alter the functions of blood vessels. In this study, we sought to determine whether chronic treatment of TAC could inhibit the activity of big-conductance Ca2+-activated K+ (BK) channels in vascular smooth muscle cells (SMCs), leading to hypertension. Our data reveal that the activity of BK channels was inhibited in cerebral artery SMCs (CASMCs) from mice after intraperitoneal injection of TAC once a day for 4 weeks. The voltage sensitivity, Ca2+ sensitivity, and open time of single BK channels were all decreased. In support, BK channel ß1-, but not α-subunit protein expression was significantly decreased in cerebral arteries. In TAC-treated mice, application of norepinephrine induced stronger vasoconstriction in both cerebral and mesenteric arteries as well as a larger [Ca2+]i in CASMCs. Chronic treatment of TAC, similar to BK channel ß1-subunit knockout (KO), resulted in hypertension in mice, but did not cause a further increase in blood pressure in BK channel ß1-subunit KO mice. Moreover, BK channel activity in CASMCs was negatively correlated with blood pressure. Our findings provide novel evidence that TAC inhibits BK channels by reducing the channel ß1-subunit expression and functions in vascular SMCs, leading to enhanced vasoconstriction and hypertension.

Interactive Roles of CaMKII/Ryanodine Receptor Signaling and Inflammation in Lung Diseases.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase and has been recently recognized to play a vital role in pathological events in the pulmonary system. CaMKII has diverse downstream targets that promote vascular disease, asthma, and cancer, so improved understanding of CaMKII signaling has the potential to lead to new therapies for lung diseases. Multiple studies have demonstrated that CaMKII is involved in redox modulation of ryanodine receptors (RyRs). CaMKII can be directly activated by reactive oxygen species (ROS) which then regulates RyR activity, which is essential for Ca2+-dependent processes in lung diseases. Furthermore, both CaMKII and RyRs participate in the inflammation process. However, their role in the pulmonary physiology in response to ROS is still an ambiguous one. Because CaMKII and RyRs are important in pulmonary biology, cell survival, cell cycle control, and inflammation, it is possible that the relationship between ROS and CaMKII/RyRs signal complex will be necessary for understanding and treating lung diseases. Here, we review roles of CaMKII/RyRs in lung diseases to understand with how CaMKII/RyRs may act as a transduction signal to connect prooxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of pulmonary disease.

Reciprocal Correlations of Inflammatory and Calcium Signaling in Asthma Pathogenesis.

Asthma is a chronic disease characterized by airway hyperresponsiveness, which can be caused by exposure to an allergen, spasmogen, or be induced by exercise. Despite its prevalence, the exact mechanisms by which the airway becomes hyperresponsive in asthma are not fully understood. There is evidence that myosin light-chain kinase is overexpressed, with a concomitant downregulation of myosin light-chain phosphatase in the airway smooth muscle, leading to sustained contraction. Additionally, the sarco/endoplasmic reticulum ATPase may be affected by inflammatory cytokines, such as IL-4, IL-5, IL-13, and TNF-α, which are all associated with asthmatic airway inflammation. IL-13 and TNF-α seem to promote sodium/calcium exchanger 1 overexpression as well. Anyhow, the exact mechanisms beyond these dysregulations need to be clarified. Of note, multiple studies show an association between asthma and the ORMLD3 gene, opening new perspectives to future potential gene therapies. Currently, several treatments are available for asthma, although many of them have systemic side effects, or are not effective in patients with severe asthma. Furthering our knowledge on the molecular and pathophysiological mechanisms of asthma plays a pivotal role for the development of new and more targeted treatments for patients who cannot totally benefit from the current therapies.

Overview on Interactive Role of Inflammation, Reactive Oxygen Species, and Calcium Signaling in Asthma, COPD, and Pulmonary Hypertension.

Inflammatory signaling is a major component in the development and progression of many lung diseases, including asthma, chronic obstructive pulmonary disorder (COPD), and pulmonary hypertension (PH). This chapter will provide a brief overview of asthma, COPD, and PH and how inflammation plays a vital role in these diseases. Specifically, we will discuss the role of reactive oxygen species (ROS) and Ca2+ signaling in inflammatory cellular responses and how these interactive signaling pathways mediate the development of asthma, COPD, and PH. We will also deliberate the key cellular responses of pulmonary arterial (PA) smooth muscle cells (SMCs) and airway SMCs (ASMCs) in these devastating lung diseases. The analysis of the importance of inflammation will shed light on the key questions remaining in this field and highlight molecular targets that are worth exploring. The crucial findings will not only demonstrate the novel roles of essential signaling molecules such as Rieske iron-sulfur protein and ryanodine receptor in the development and progress of asthma, COPD, and PH but also offer advanced insight for creating more effective and new therapeutic targets for these devastating inflammatory lung diseases.

Cellular and Molecular Processes in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a progressive lung disease characterized by persistent pulmonary vasoconstriction. Another well-recognized characteristic of PH is the muscularization of peripheral pulmonary arteries. This pulmonary vasoremodeling manifests in medial hypertrophy/hyperplasia of smooth muscle cells (SMCs) with possible neointimal formation. The underlying molecular processes for these two major vascular responses remain not fully understood. On the other hand, a series of very recent studies have shown that the increased reactive oxygen species (ROS) seems to be an important player in mediating pulmonary vasoconstriction and vasoremodeling, thereby leading to PH. Mitochondria are a primary site for ROS production in pulmonary artery (PA) SMCs, which subsequently activate NADPH oxidase to induce further ROS generation, i.e., ROS-induced ROS generation. ROS control the activity of multiple ion channels to induce intracellular Ca2+ release and extracellular Ca2+ influx (ROS-induced Ca2+ release and influx) to cause PH. ROS and Ca2+ signaling may synergistically trigger an inflammatory cascade to implicate in PH. Accordingly, this paper explores the important roles of ROS, Ca2+, and inflammatory signaling in the development of PH, including their reciprocal interactions, key molecules, and possible therapeutic targets.

Mitochondrial Rieske iron-sulfur protein in pulmonary artery smooth muscle: A key primary signaling molecule in pulmonary hypertension.

Rieske iron-sulfur protein (RISP) is a catalytic subunit of the complex III in the mitochondrial electron transport chain. Studies for years have revealed that RISP is essential for the generation of intracellular reactive oxygen species (ROS) via delicate signaling pathways associated with many important molecules such as protein kinase C-ε, NADPH oxidase, and ryanodine receptors. More significantly, mitochondrial RISP-mediated ROS production has been implicated in the development of hypoxic pulmonary vasoconstriction, leading to pulmonary hypertension, right heart failure, and death. Investigations have also shown the involvement of RISP in ROS-dependent cardiac ischemic/reperfusion injuries. Further research may provide novel and valuable information that can not only enhance our understanding of the functional roles of RISP and the underlying molecular mechanisms in the pulmonary vasculature and other systems, but also elucidate whether RISP targeting can act as preventative and restorative therapies against pulmonary hypertension, cardiac diseases, and other disorders.

Canonical Transient Potential Receptor-3 Channels in Normal and Diseased Airway Smooth Muscle Cells.

All seven canonical transient potential receptor (TRPC1-7) channel members are expressed in mammalian airway smooth muscle cells (ASMCs). Among this family, TRPC3 channel plays an important role in the control of the resting [Ca2+]i and agonist-induced increase in [Ca2+]i. This channel is significantly upregulated in molecular expression and functional activity in airway diseases. The upregulated channel significantly augments the resting [Ca2+]i and agonist-induced increase in [Ca2+]i, thereby exerting a direct and essential effect in airway hyperresponsiveness. The increased TRPC3 channel-mediated Ca2+ signaling also results in the transcription factor nuclear factor-κB (NF-κB) activation via protein kinase C-α (PKCα)-dependent inhibitor of NFκB-α (IκBα) and calcineurin-dependent IκBß signaling pathways, which upregulates cyclin-D1 expression and causes cell proliferation, leading to airway remodeling. TRPC3 channel may further interact with intracellular release Ca2+ channels, Orai channels and Ca2+-sensing stromal interaction molecules, mediating important cellular responses in ASMCs and the development of airway diseases.

Mitochondrial Rieske iron-sulfur protein in pulmonary artery smooth muscle: A key primary signaling molecule in pulmonary hypertension.

Rieske iron-sulfur protein (RISP) is a catalytic subunit of the complex III in the mitochondrial electron transport chain. Studies for years have revealed that RISP is essential for the generation of intracellular reactive oxygen species (ROS) via delicate signaling pathways associated with many important molecules such as protein kinase C-ε, NADPH oxidase, and ryanodine receptors. More significantly, mitochondrial RISP-mediated ROS production has been implicated in the development of hypoxic pulmonary vasoconstriction, leading to pulmonary hypertension, right heart failure, and death. Investigations have also shown the involvement of RISP in ROS-dependent cardiac ischemic/reperfusion injuries. Further research may provide novel and valuable information that can not only enhance our understanding of the functional roles of RISP and the underlying molecular mechanisms in the pulmonary vasculature and other systems, but also elucidate whether RISP targeting can act as preventative and restorative therapies against pulmonary hypertension, cardiac diseases, and other disorders.

Azithromycin inhibits muscarinic 2 receptor-activated and voltage-activated Ca2+ permeant ion channels and Ca2+ sensitization, relaxing airway smooth muscle contraction.

Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch-clamp techniques. AZM completely inhibited acetylcholine (ACH)-induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L-type voltage-dependent Ca2+ channels (LVDCCs)), Pyr3 (selective inhibitor of TRPC3 and/or STIM/Orai channels, which are nonselective cation channels (NSCCs)), and Y-27632 (selective inhibitor of Rho-associated kinase (ROCK)). Moreover, LVDCC- and NSCC-mediated currents were inhibited by AZM, and the latter were suppressed by the muscarinic (M) 2 receptor inhibitor methoctramine. AZM inhibited LVDCC Ca2+ permeant ion channels, M2 receptors, and TRPC3 and/or STIM/Orai, which decreased cytosolic Ca2+ concentrations and led to muscle relaxation. This relaxation was also enhanced by the inhibition of Ca2+ sensitization. Therefore, AZM has potential as a novel and potent bronchodilator. The findings of this study improve the understanding of the effects of AZM on asthma and COPD.

PLCγ1-PKCε-IP3R1 signaling plays an important role in hypoxia-induced calcium response in pulmonary artery smooth muscle cells.

Hypoxia-induced pulmonary vasoconstriction (HPV) is attributed to an increase in intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs). We have reported that phospholipase C-γ1 (PLCγ1) plays a significant role in the hypoxia-induced increase in [Ca2+]i in PASMCs and attendant HPV. In this study, we intended to determine molecular mechanisms for hypoxic Ca2+ and contractile responses in PASMCs. Our data reveal that hypoxic vasoconstriction occurs in pulmonary arteries, but not in mesenteric arteries. Hypoxia caused a large increase in [Ca2+]i in PASMCs, which is diminished by the PLC inhibitor U73122 and not by its inactive analog U73433 . Hypoxia augments PLCγ1-dependent inositol 1,4,5-trisphosphate (IP3) generation. Exogenous ROS, hydrogen peroxide (H2O2), increases PLCγ1 phosphorylation at tyrosine-783 and IP3 production. IP3 receptor-1 (IP3R1) knock-down remarkably diminishes hypoxia- or H2O2-induced increase in [Ca2+]i. Hypoxia or H2O2 increases the activity of IP3Rs, which is significantly reduced in protein kinase C-ε (PKCε) knockout PASMCs. A higher PLCγ1 expression, activity, and basal [Ca2+]i are found in PASMCs, but not in mesenteric artery smooth muscle cells from mice exposed to chronic hypoxia (CH) for 21 days. CH enhances H2O2- and ATP-induced increase in [Ca2+]i in PASMCs and PLC-dependent, norepinephrine-evoked pulmonary vasoconstriction. In conclusion, acute hypoxia uniquely causes ROS-dependent PLCγ1 activation, IP3 production, PKCε activation, IP3R1 opening, Ca2+ release, and contraction in mouse PASMCs; CH enhances PASM PLCγ1 expression, activity, and function, playing an essential role in pulmonary hypertension in mice.

Canonical transient receptor potential 3 channels activate NF-κB to mediate allergic airway disease via PKC-α/IκB-α and calcineurin/IκB-ß pathways.

The purpose of this study was to determine the role of canonical transient receptor potential 3 (TRPC3) channel in allergen-induced airway disease (AIAD) and its underlying signaling mechanisms. The procedures included (1) intravenous injection of lentiviral TRPC3 channel or nonsilencing short hairpin ribonucleic acid (shRNA) to make the channel knockdown (KD) or control mice, (2) allergen sensitization/challenge to induce AIAD, (3) patch-clamp recording and Ca(2+) imaging to examine the channel activity, and (4) gene manipulations and other methods to determine the underlying signaling mechanisms. The findings are that (1) intravenous or intranasal delivery of TRPC3 channel lentiviral shRNAs or blocker 1-[4-[(2,3,3-trichloro-1-oxo-2-propen-1-yl)amino]phenyl]-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid prevents AIAD in mice, (2) TRPC3 channel KD and overexpression, respectively, blocks and augments protein kinase C-α/nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor-α (PKC-α/IκB-α)-mediated or calcineurin/IκB-ß-dependent, NF-κB-dependent allergen-induced airway smooth muscle cell (ASMC) hyperproliferation and cyclin D1 (an important cell proliferation molecule) induction, and (3) the changes of the major molecules of the PKC-α/IκBα- and calcineurin/IκB-ß-dependent NF-κB signaling pathways are also observed in asthmatic human ASMCs. The conclusions are that TRPC3 channels plays an essential role in AIAD via the PKC-α/IκB-α- and calcineurin/IκB-ß-dependent NF-κB signaling pathways, and lentiviral shRNA or inhibitor of TRPC3 channels may become novel and effective treatments for AIAD.

Role of Transcription Factors in Pulmonary Artery Smooth Muscle Cells: An Important Link to Hypoxic Pulmonary Hypertension.

Hypoxia, namely a lack of oxygen in the blood, induces pulmonary vasoconstriction and vasoremodeling, which serve as essential pathologic factors leading to pulmonary hypertension (PH). The underlying molecular mechanisms are uncertain; however, pulmonary artery smooth muscle cells (PASMCs) play an essential role in hypoxia-induced pulmonary vasoconstriction, vasoremodeling, and PH. Hypoxia causes oxidative damage to DNAs, proteins, and lipids. This damage (oxidative stress) modulates the activity of ion channels and elevates the intracellular calcium concentration ([Ca2+]i, Ca2+ signaling) of PASMCs. The oxidative stress and increased Ca2+ signaling mutually interact with each other, and synergistically results in a variety of cellular responses. These responses include functional and structural abnormalities of mitochondria, sarcoplasmic reticulum, and nucleus; cell contraction, proliferation, migration, and apoptosis, as well as generation of vasoactive substances, inflammatory molecules, and growth factors that mediate the development of PH. A number of studies reveal that various transcription factors (TFs) play important roles in hypoxia-induced oxidative stress, disrupted PAMSC Ca2+ signaling and the development and progress of PH. It is believed that in the pathogenesis of PH, hypoxia facilitates these roles by mediating the expression of multiple genes. Therefore, the identification of specific genes and their transcription factors implicated in PH is necessary for the complete understanding of the underlying molecular mechanisms. Moreover, this identification may aid in the development of novel and effective therapeutic strategies for PH.

Cross Talk Between Mitochondrial Reactive Oxygen Species and Sarcoplasmic Reticulum Calcium in Pulmonary Arterial Smooth Muscle Cells.

Hypoxic pulmonary vasoconstriction (HPV) occurs during both fetal and postnatal development and plays a critical role in matching regional alveolar perfusion with ventilation in humans and animals. HPV also contributes significantly to the development of pulmonary hypertension. Although the molecular mechanisms of HPV and pulmonary hypertension remain incompletely understood, increasing evidence demonstrates that hypoxia induces an elevated intracellular reactive oxygen species concentration ([ROS]i) in pulmonary artery smooth muscle cells (PASMCs). The increased [ROS]i is attributed to the mitochondrial electron transport chain (ETC) and plasmalemmal NADPH oxidase (NOX); however, the mitochondrial ETC is a primary source for the elevated [ROS]i. Our studies reveal that mitochondrial ROS can specifically increase the activity of protein kinase C-ε, activate NOX, and then induce more ROS production (i.e., ROS-induced ROS production, RIRP). Mitochondrial ROS production is principally mediated by Rieske iron-sulfur protein (RISP) at the complex III. The increased [ROS]i causes an elevation of intracellular Ca2+ concentration ([Ca2+]i), thereby leading to HPV and associated pulmonary hypertension. Ryanodine receptor-2 (RyR2)/Ca2+ release channel on the sarcoplasmic reticulum (SR) serves as a most valuable player in the elevated [Ca2+]i. Our recent data indicate that RyR2-induced Ca2+ release can enhance RISP-mediated increase in mitochondrial ROS concentration ([ROS]mito), and that the mitochondrial Ca2+ uniporter is involved in elevating [ROS]mito. Based on the existing reports and our unpublished data, we conclude that the cross talk between [ROS]mito and [Ca2+]i, that is RISP-dependent mitochondrial ROS-induced RyR2-mediated SR Ca2+ release (ROS-induced Ca2+ release, RICR) and RyR2-mediated SR Ca2+ release-induced RISP-dependent mitochondrial ROS production (Ca2+-induced ROS production, CIRP), may form a positive reciprocal loop in mediating HPV and also possibly pulmonary hypertension.

Inositol 1,4,5-trisphosphate activates TRPC3 channels to cause extracellular Ca2+ influx in airway smooth muscle cells.

Transient receptor potential-3 (TRPC3) channels play a predominant role in forming nonselective cation channels (NSCCs) in airway smooth muscle cells (ASMCs) and are significantly increased in their activity and expression in asthmatic ASMCs. To extend these novel findings, we have explored the regulatory mechanisms that control the activity of TRPC3 channels. Our data for the first time reveal that inositol 1,4,5-trisphosphate (IP3), an important endogenous signaling molecule, can significantly enhance the activity of single NSCCs in ASMCs. The analog of diacylglycerol (DAG; another endogenous signaling molecule), 1-oleyl-2-acetyl-sn-glycerol (OAG), 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), and 1-stearoyl-2-linoleoyl-sn-glycerol (SLG) all augment NSCC activity. The effects of IP3 and OAG are fully abolished by lentiviral short-hairpin (sh)RNA-mediated TRPC3 channel knockdown (KD). The stimulatory effect of IP3 is eliminated by heparin, an IP3 receptor (IP3R) antagonist that blocks the IP3-binding site, but not by xestospongin C, the IP3R antagonist that has no effect on the IP3-binding site. Lentiviral shRNA-mediated KD of IP3R1, IP3R2, or IP3R3 does not alter the excitatory effect of IP3. TRPC3 channel KD greatly inhibits IP3-induced increase in intracellular Ca(2+) concentration. IP3R1 KD produces a similar inhibitory effect. TRPC3 channel and IP3R1 KD both diminish the muscarinic receptor agonist methacholine-evoked Ca(2+) responses. Taking these findings together, we conclude that IP3, the important intracellular second messenger, may activate TRPC3 channels to cause extracellular Ca(2+) influx, in addition to opening IP3Rs to induce intracellular Ca(2+) release. This novel extracellular Ca(2+) entry route may play a significant role in mediating IP3-mediated numerous cellular responses in ASMCs and other cells.

Calcineurin upregulates local Ca(2+) signaling through ryanodine receptor-1 in airway smooth muscle cells.

Local Ca(2+) signals (Ca(2+) sparks) play an important role in multiple cellular functions in airway smooth muscle cells (ASMCs). Protein kinase Cϵ is known to downregulate ASMC Ca(2+) sparks and contraction; however, no complementary phosphatase has been shown to produce opposite effects. Here, we for the first time report that treatment with a specific calcineurin (CaN) autoinhibitory peptide (CAIP) to block CaN activity decreases, whereas application of nickel to activate CaN increases, Ca(2+) sparks in both the presence and absence of extracellular Ca(2+). Treatment with xestospogin-C to eliminate functional inositol 1,4,5-trisphosphate receptors does not prevent CAIP from inhibiting local Ca(2+) signaling. However, high ryanodine treatment almost completely blocks spark formation and prevents the nickel-mediated increase in sparks. Unlike CAIP, the protein phosphatase 2A inhibitor endothall has no effect. Local Ca(2+) signaling is lower in CaN catalytic subunit Aα gene knockout (CaN-Aα(-/-)) mouse ASMCs. The effects of CAIP and nickel are completely lost in CaN-Aα(-/-) ASMCs. Neither CAIP nor nickel produces an effect on Ca(2+) sparks in type 1 ryanodine receptor heterozygous knockout (RyR1(-/+)) mouse ASMCs. However, their effects are not altered in RyR2(-/+) or RyR3(-/-) mouse ASMCs. CaN inhibition decreases methacholine-induced contraction in isolated RyR1(+/+) but not RyR1(-/+) mouse tracheal rings. Supportively, muscarinic contractile responses are also reduced in CaN-Aα(-/+) mouse tracheal rings. Taken together, these results provide novel evidence that CaN regulates ASMC Ca(2+) sparks specifically through RyR1, which plays an important role in the control of Ca(2+) signaling and contraction in ASMCs.

Bitter tastants induce relaxation of rat thoracic aorta precontracted with high K(+).

It has been reported that bitter tastants decrease blood pressure and relax precontracted vascular smooth muscle. However, the underlying mechanisms remain unclear. The aim of the present study was to determine the mechanism underlying the vasorelaxant effect of the bitter tastants. Thoracic aortic rings were isolated from Wistar rats and contractions were measured using an isometric myograph. Intracellular Ca(2+) ([Ca(2+)]i) in single rat thoracic aortic smooth muscle cells was recorded by calcium imaging. Calcium currents in single cells were recorded using patch-clamp techniques. High K(+) (140 mmol/L) induced contractions in rat thoracic aortic rings that were inhibited by 3 mmol/L chloroquine, 3 mmol/L denatonium and 10 µmol/L nifedipine. In single rat thoracic aortic smooth muscle cells, high K(+) increased [Ca(2+)]i and this effect was also blocked by 3 mmol/L chloroquine and 10 µmol/L nifedipine. Under Ca(2+) -free conditions, high K(+) failed to induce contractions in rat thoracic aortic rings. On its own, chloroquine had no effect on the muscle tension of rat aortic rings and [Ca(2+) ]i. The vasorelaxant effects of chloroquine on precontracted rat thoracic aortic rings were not altered by either 1 µg/mL pertussis toxin (PTX), an inhibitor of Gαo/i-protein, or 1 mmol/L gallein, an inhibitor of Gßγ-protein. The results of patch-clamp analysis in single cells indicate that 1 mmol/L chloroquine blocks voltage-dependent L-type Ca(2+) channel (VDLCC) currents from both extracellular and intracellular sides. Together, the results indicate that chloroquine can block VDLCC, independent of PTX- and gallein-sensitive G-proteins, resulting in relaxation of high K(+)-precontracted thoracic aortic smooth muscle.

Distinct activity of BK channel ß1-subunit in cerebral and pulmonary artery smooth muscle cells.

This study was designed to test a hypothesis that the functional activity of big-conductance, Ca(2+)-activated K(+) (BK) channels is different in cerebral and pulmonary artery smooth muscle cells (CASMCs and PASMCs). Using patch-clamp recordings, we found that the activity of whole cell and single BK channels were significantly higher in CASMCs than in PASMCs. The voltage and Ca(2+) sensitivity of BK channels were greater in CASMCs than in PASMCs. Targeted gene knockout of ß(1)-subunits significantly reduced BK currents in CASMCs but had no effect in PASMCs. Western blotting experiments revealed that BK channel α-subunit protein expression level was comparable in CASMCs and PASMCs; however, ß(1)-subunit protein expression level was higher in CASMCs than in PASMCs. Inhibition of BK channels by the specific blocker iberiotoxin enhanced norepinephrine-induced increase in intracellular calcium concentration in CASMCs but not in PASMCs. Systemic artery blood pressure was elevated in ß(1)(-/-) mice. In contrast, pulmonary artery blood pressure was normal in ß(1)(-/-) mice. These findings provide the first evidence that the activity of BK channels is higher in cerebral than in PASMCs. This heterogeneity is primarily determined by the differential ß(1)-subunit function and contributes to diverse cellular responses in these two distinct types of cells.

Important role of PLC-γ1 in hypoxic increase in intracellular calcium in pulmonary arterial smooth muscle cells.

An increase in intracellular calcium concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs) induces hypoxic cellular responses in the lungs; however, the underlying molecular mechanisms remain incompletely understood. We report, for the first time, that acute hypoxia significantly enhances phospholipase C (PLC) activity in mouse resistance pulmonary arteries (PAs), but not in mesenteric arteries. Western blot analysis and immunofluorescence staining reveal the expression of PLC-γ1 protein in PAs and PASMCs, respectively. The activity of PLC-γ1 is also augmented in PASMCs following hypoxia. Lentiviral shRNA-mediated gene knockdown of mitochondrial complex III Rieske iron-sulfur protein (RISP) to inhibit reactive oxygen species (ROS) production prevents hypoxia from increasing PLC-γ1 activity in PASMCs. Myxothiazol, a mitochondrial complex III inhibitor, reduces the hypoxic response as well. The PLC inhibitor U73122, but not its inactive analog U73433, attenuates the hypoxic vasoconstriction in PAs and hypoxic increase in [Ca(2+)](i) in PASMCs. PLC-γ1 knockdown suppresses its protein expression and the hypoxic increase in [Ca(2+)](i). Hypoxia remarkably increases inositol 1,4,5-trisphosphate (IP(3)) production, which is blocked by U73122. The IP(3) receptor (IP(3)R) antagonist 2-aminoethoxydiphenyl borate (2-APB) or xestospongin-C inhibits the hypoxic increase in [Ca(2+)](i). PLC-γ1 knockdown or U73122 reduces H(2)O(2)-induced increase in [Ca(2+)](i) in PASMCs and contraction in PAs. 2-APB and xestospongin-C produce similar inhibitory effects. In conclusion, our findings provide novel evidence that hypoxia activates PLC-γ1 by increasing RISP-dependent mitochondrial ROS production in the complex III, which causes IP(3) production, IP(3)R opening, and Ca(2+) release, playing an important role in hypoxic Ca(2+) and contractile responses in PASMCs.

Reactive oxygen species induce a Ca(2+)-spark increase in sensitized murine airway smooth muscle cells.

The level of reactive oxygen species (ROS) and the activity of spontaneous, transient, localized Ca(2+) increases (known as Ca(2+) sparks) in tracheal smooth muscle cells (TSMCs) in an experimental allergic asthma mouse model has not yet been investigated. We used laser confocal microscopy and fluorescent dyes to measure ROS levels and Ca(2+) sparks, and we found that both events were significantly increased in TSMCs obtained from ovalbumin (OVA)-sensitized/-challenged mice compared with control mice. ROS levels began to increase in TSMCs after the first OVA challenge, and this increase was sustained. However, this elevation and Ca(2+)-spark increase was abolished after the administration of the ROS scavenger N-acetylcysteine amide (NACA) for 5days. Furthermore, a similar inhibition was also observed following the direct perfusion of NACA into cells isolated from the (OVA)-sensitized mice that were not treated with NACA. Moreover, we used 0.1-mM caffeine treatment to increase the Ca(2+) sparks in single TSMCs and observed cell shortening. In addition, we did not find increases in the mRNA levels of ryanodine (RyRs) and inositol 1,4,5-trisphosphate (IP3Rs) receptors in the tracheal smooth muscle cells of (OVA)-sensitized mice compared with controls. We concluded that ROS and Ca(2+) sparks increased in (OVA)-sensitized TSMCs. We found that ROS induces Ca(2+) sparks, and increased Ca(2+) sparks resulted in the contraction of (OVA)-sensitized TSMCs, resulting in the generation of airway hyperresponsiveness (AHR). This effect may represent a novel mechanism for AHR pathogenesis and might provide insight into new methods for the clinical prevention and treatment of asthma and asthmatic AHR.