scholarly journals Airway smooth muscle CXCR3 ligand production: regulation by JAK-STAT1 and intracellular Ca2+

2013 ◽  
Vol 304 (11) ◽  
pp. L790-L802 ◽  
Author(s):  
X. Tan ◽  
Y. A. Alrashdan ◽  
H. Alkhouri ◽  
B. G. G. Oliver ◽  
C. L. Armour ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Immunoblot Analysis ◽  
Interferon Γ ◽  
Protein Activation ◽  
Early Activation ◽  
Plasmic Reticulum ◽  
Cxcr3 Ligand ◽  
Intracellular Ca ◽  
Jnk Activation

In asthma, airway smooth muscle (ASM) chemokine (C-X-C motif) receptor 3 (CXCR3) ligand production may attract mast cells or T lymphocytes to the ASM, where they can modulate ASM functions. In ASM cells (ASMCs) from people with or without asthma, we aimed to investigate JAK-STAT1, JNK, and Ca2+ involvement in chemokine (C-X-C motif) ligand (CXCL)10 and CXCL11 production stimulated by interferon-γ, IL-1β, and TNF-α combined (cytomix). Confluent, growth-arrested ASMC were treated with inhibitors for pan-JAK (pyridone-6), JAK2 (AG-490), JNK (SP-600125), or the sarco(endo)plasmic reticulum Ca2+ATPase (SERCA) pump (thapsigargin), Ca2+ chelator (BAPTA-AM), or vehicle before and during cytomix stimulation for up to 24 h. Signaling protein activation as well as CXCL10/CXCL11 mRNA and protein production were examined using immunoblot analysis, real-time PCR, and ELISA, respectively. Cytomix-induced STAT1 activation was lower and CXCR3 ligand mRNA production was more sensitive to pyridone-6 and AG-490 in asthmatic than nonasthmatic ASMCs, but CXCL10/CXCL11 release was inhibited by the same proportion. Neither agent caused additional inhibition of release when used in combination with the JNK inhibitor SP-600125. Conversely, p65 NF-κB activation was higher in asthmatic than nonasthmatic ASMCs. BAPTA-AM abolished early CXCL10/CXCL11 mRNA production, whereas thapsigargin reduced it in asthmatic cells and inhibited CXCL10/CXCL11 release by both ASMC types. Despite these inhibitory effects, neither Ca2+ agent affected early activation of STAT1, JNK, or p65 NF-κB. In conclusion, intracellular Ca2+ regulated CXCL10/CXCL11 production but not early activation of the signaling molecules involved. In asthma, reduced ASM STAT1-JNK activation, increased NF-κB activation, and altered Ca2+ handling may contribute to rapid CXCR3 ligand production and enhanced inflammatory cell recruitment.

Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle

2008 ◽  
Vol 294 (4) ◽  
pp. L787-L796 ◽  
Author(s):  
Venkatachalem Sathish ◽  
Figen Leblebici ◽  
Sertac N. Kip ◽  
Michael A. Thompson ◽  
Christina M. Pabelick ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Airway Smooth Muscle ◽  
Small Interfering Rna ◽  
Regulatory Protein ◽  
Plasmic Reticulum ◽  
Intracellular Ca ◽  
Subsequent Exposure ◽  
Interfering Rna ◽  
Serca Inhibition

Regulation of intracellular Ca2+ concentration ([Ca2+]i) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca2+ release and reuptake. The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is key to replenishment of SR Ca2+ stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca2+ indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca2+]i responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca2+]i transients to 1 μM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca2+]i oscillations (which were previously shown to result from repetitive SR Ca2+ release/reuptake). However, when ACh-induced [Ca2+]i oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca2+]i transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca2+]i oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca2+ release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca2+ reuptake, potentially through altered PLN phosphorylation.

Qualitative immunoblot analysis of PKC isoforms expressed in airway smooth muscle

1997 ◽  
Vol 272 (4) ◽  
pp. L603-L607 ◽  
Author(s):  
H. Togashi ◽  
C. A. Hirshman ◽  
C. W. Emala
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Airway Smooth Muscle ◽  
Intracellular Signaling ◽  
Immunoblot Analysis ◽  
Calcium Dependent ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Dependent Activity ◽  
Trachealis Muscle

Protein kinase C (PKC) was originally identified as a single serine/ threonine protein kinase with calcium- and phospholipid-dependent activity, but more recently PKC has been found to consist of a family of multiple isoenzymes with different biochemical characteristics, substrates, and cofactor requirements. PKC is particularly important in regulating airway smooth muscle (ASM) tone. Although a previous investigation has demonstrated PKC-beta, -delta, -epsilon, -theta and -zeta in canine trachealis muscle, additional PKC isoforms have not been characterized in ASM. Therefore, immunoblot analysis using nine isotype-specific antibodies was used to further characterize the expression of PKC isoforms in porcine ASM. In addition to the previously described beta-, delta-, epsilon-, and zeta-isoforms in ASM, the calcium-dependent alpha-isoform, and the calcium- and diacylglycerol-independent isoforms iota/lambda and mu were identified. This study demonstrates multiple PKC isoforms in porcine ASM that can participate in intracellular signaling pathways in this tissue.

scholarly journals Soluble guanylate cyclase modulators blunt hyperoxia effects on calcium responses of developing human airway smooth muscle

2015 ◽  
Vol 309 (6) ◽  
pp. L537-L542 ◽  
Author(s):  
Rodney D. Britt ◽  
Michael A. Thompson ◽  
Ine Kuipers ◽  
Alecia Stewart ◽  
Elizabeth R. Vogel ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Guanylate Cyclase ◽  
Therapeutic Strategy ◽  
Soluble Guanylate Cyclase ◽  
Novel Drugs ◽  
Intracellular Ca ◽  
Recurrent Wheeze ◽  
Airway Tone ◽  
Sgc Stimulators

Exposure to moderate hyperoxia in prematurity contributes to subsequent airway dysfunction and increases the risk of developing recurrent wheeze and asthma. The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic GMP (cGMP) axis modulates airway tone by regulating airway smooth muscle (ASM) intracellular Ca2+ ([Ca2+]i) and contractility. However, the effects of hyperoxia on this axis in the context of Ca2+/contractility are not known. In developing human ASM, we explored the effects of novel drugs that activate sGC independent of NO on alleviating hyperoxia (50% oxygen)-induced enhancement of Ca2+ responses to bronchoconstrictor agonists. Treatment with BAY 41–2272 (sGC stimulator) and BAY 60-2770 (sGC activator) increased cGMP levels during exposure to 50% O2. Although 50% O2 did not alter sGCα1 or sGCβ1 expression, BAY 60-2770 did increase sGCβ1 expression. BAY 41-2272 and BAY 60-2770 blunted Ca2+ responses to histamine in cells exposed to 50% O2. The effects of BAY 41-2272 and BAY 60-2770 were reversed by protein kinase G inhibition. These novel data demonstrate that BAY 41-2272 and BAY 60-2770 stimulate production of cGMP and blunt hyperoxia-induced increases in Ca2+ responses in developing ASM. Accordingly, sGC stimulators/activators may be a useful therapeutic strategy in improving bronchodilation in preterm infants.

Calcium sensitization produced by G protein activation in airway smooth muscle

2001 ◽  
Vol 281 (3) ◽  
pp. L631-L638 ◽  
Author(s):  
Hayashi Yoshimura ◽  
Keith A. Jones ◽  
William J. Perkins ◽  
Tetsuya Kai ◽  
David O. Warner
Keyword(s):  
Smooth Muscle ◽  
G Proteins ◽  
Myosin Light Chain ◽  
Calcium Sensitization ◽  
Protein Activation ◽  
Maximal Force ◽  
Acute Response ◽  
Regulatory Myosin Light Chain ◽  
G Protein Activation ◽  
Intracellular Ca

We determined whether activation of G proteins can affect the force developed for a given intracellular Ca2+ concentration ([Ca2+]; i.e., the Ca2+ sensitivity) by mechanisms in addition to changes in regulatory myosin light chain (rMLC) phosphorylation. Responses in α-toxin-permeabilized canine tracheal smooth muscle were determined with Ca2+ alone or in the presence of ACh, endothelin-1 (ET-1), or aluminum fluoride (AlF[Formula: see text]; acute or 1-h exposure). Acute exposure to each compound increased Ca2+sensitivity without changing the response to high [Ca2+] (maximal force). However, chronic exposure to AlF[Formula: see text], but not to chronic ACh or ET-1, increased maximal force by increasing the force produced for a given rMLC phosphorylation. Studies employing thiophosphorylation of rMLC showed that the increase in force produced by chronic AlF[Formula: see text] exposure required Ca2+during activation to be manifest. Unlike the acute response to receptor agonists, which is mediated solely by increases in rMLC phosphorylation, chronic direct activation of G proteins further increases Ca2+ sensitivity in airways by additional mechanisms that are independent of rMLC phosphorylation.

scholarly journals Effect of proinflammatory cytokines on regulation of sarcoplasmic reticulum Ca2+ reuptake in human airway smooth muscle

2009 ◽  
Vol 297 (1) ◽  
pp. L26-L34 ◽  
Author(s):  
Venkatachalem Sathish ◽  
Michael A. Thompson ◽  
Jeffrey P. Bailey ◽  
Christina M. Pabelick ◽  
Y. S. Prakash ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Airway Inflammation ◽  
Airway Smooth Muscle ◽  
Proinflammatory Cytokines ◽  
Cell Types ◽  
Human Airway Smooth Muscle ◽  
Inhibitory Protein ◽  
Intracellular Ca ◽  
Additional Exposure

Airway inflammation leads to increased intracellular Ca2+ ([Ca2+]i) levels in airway smooth muscle (ASM) cells. Sarcoplasmic reticulum Ca2+ release and reuptake are key components of ASM [Ca2+]i regulation. Ca2+ reuptake occurs via sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) and is regulated by the inhibitory protein phospholamban (PLB) in many cell types. In human ASM, we tested the hypothesis that inflammation increases PLB, thus inhibiting SERCA function, and leading to maintained [Ca2+]i levels. Surprisingly, we found that human ASM does not express PLB protein (although mRNA is detectable). Overnight exposure to the proinflammatory cytokines TNFα and IL-13 did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (via CaMKII) occurs in human ASM. In fura-2-loaded human ASM cells, we found that the CaMKII antagonist KN-93 significantly slowed the rate of fall of [Ca2+]i transients induced by ACh or bradykinin (in zero extracellular Ca2+), suggesting a role for CaMKII-mediated SERCA regulation. SERCA expression was decreased by cytokine exposure, and the rate of fall of [Ca2+]i transients was slowed in cells exposed to TNFα and IL-13. Cytokine effects on Ca2+ reuptake were unaffected by additional exposure to KN-93. These data indicate that in human ASM, SERCA is regulated by mechanisms such as CaMKII and that airway inflammation maintains [Ca2+]i levels by decreasing SERCA expression and slowing Ca2+ reuptake.

Selected Contribution: Tryptase-induced PAR-2-mediated Ca2+signaling in human airway smooth muscle cells

2001 ◽  
Vol 91 (2) ◽  
pp. 995-1003 ◽  
Author(s):  
Patrick Berger ◽  
J. Manuel Tunon-De-Lara ◽  
Jean-Pierre Savineau ◽  
Roger Marthan
Keyword(s):  
Smooth Muscle ◽  
Airway Inflammation ◽  
Airway Smooth Muscle ◽  
Airway Smooth Muscle Cells ◽  
Atpase Inhibitor ◽  
Human Airway Smooth Muscle ◽  
Human Airway ◽  
Transient Rise ◽  
Intracellular Ca ◽  
Protein Kinase C Inhibitor

Tryptase, the major mast cell product, is considered to play an important role in airway inflammation and hyperresponsiveness. Tryptase produces different, sometimes opposite, effects on airway responsiveness (bronchoprotection and/or airway contraction). This study was designed to examine the effect of human lung tryptase and activation of protease-activated receptor (PAR)-2 by synthetic activated peptide (AP) SLIGKV-NH2 on Ca2+ signaling in human airway smooth muscle (HASM) cells. Immunocytochemistry revealed that PAR-2 was expressed by HASM cells. Tryptase (7.5–30 mU/ml) induced a concentration-dependent transient relative rise in cytoplasmic Ca2+ concentration ([Ca2+]i) that reached 207 ± 32 nM ( n = 10) measured by indo 1 spectrofluorometry. The protease inhibitors leupeptin or benzamidine (100 μM) abolished tryptase-induced [Ca2+]iincrease. Activation of PAR-2 by AP (1–100 μM) also induced a concentration-dependent transient rise in [Ca2+]i, whereas the reverse peptide produced no effect. There was a homologous desensitization of the [Ca2+]i response on repeated stimulation with tryptase or AP. U-73122, a specific phospholipase C (PLC) antagonist, xestospongin, an inositol trisphosphate (IP3)-receptor antagonist, or thapsigargin, a sarcoplamic Ca2+-ATPase inhibitor, abolished tryptase-induced [Ca2+]iresponse, whereas Ca2+ removal, in the additional presence of EGTA, had no effect. Calphostin C, a protein kinase C inhibitor, increased PAR-2 [Ca2+]i response. Our results indicate that tryptase activates a [Ca2+]iresponse, which appears as PAR-2 mediated in HASM cells. Signal transduction implicates the intracellular Ca2+ store via PLC activation and thus via the IP3 pathway. This study provides evidence that tryptase, which is increasingly recognized as an important mediator in airway inflammation and hyperresponsiveness, is also a potent direct agonist at the site of airway smooth muscle.

Ionic mechanisms and Ca2+ regulation in airway smooth muscle contraction: do the data contradict dogma?

2002 ◽  
Vol 282 (6) ◽  
pp. L1161-L1178 ◽  
Author(s):  
Luke J. Janssen
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Smooth Muscle Contraction ◽  
Channel Blockers ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Spatial And Temporal Heterogeneity ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Ionic Mechanisms

In general, excitation-contraction coupling in muscle is dependent on membrane depolarization and hyperpolarization to regulate the opening of voltage-dependent Ca2+ channels and, thereby, influence intracellular Ca2+ concentration ([Ca2+]i). Thus Ca2+ channel blockers and K+ channel openers are important tools in the arsenals against hypertension, stroke, and myocardial infarction, etc. Airway smooth muscle (ASM) also exhibits robust Ca2+, K+, and Cl− currents, and there are elaborate signaling pathways that regulate them. It is easy, then, to presume that these also play a central role in contraction/relaxation of ASM. However, several lines of evidence speak to the contrary. Also, too many researchers in the ASM field view the sarcoplasmic reticulum as being centrally located and displacing its contents uniformly throughout the cell, and they have focused almost exclusively on the initial single [Ca2+] spike evoked by excitatory agonists. Several recent studies have revealed complex spatial and temporal heterogeneity in [Ca2+]i, the significance of which is only just beginning to be appreciated. In this review, we will compare what is known about ion channels in ASM with what is believed to be their roles in ASM physiology. Also, we will examine some novel ionic mechanisms in the context of Ca2+ handling and excitation-contraction coupling in ASM.

Regulation of store-operated Ca2+ entry by CD38 in human airway smooth muscle

2008 ◽  
Vol 294 (2) ◽  
pp. L378-L385 ◽  
Author(s):  
Gary C. Sieck ◽  
Thomas A. White ◽  
Michael A. Thompson ◽  
Christina M. Pabelick ◽  
Mark E. Wylam ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Critical Role ◽  
Human Airway Smooth Muscle ◽  
Cd38 Expression ◽  
Adp Ribosyl Cyclase ◽  
Cyclic Adp Ribose ◽  
Intracellular Ca ◽  
Interfering Rna ◽  
Synthesis And Degradation

The ectoenzyme CD38 catalyzes synthesis and degradation of cyclic ADP ribose in airway smooth muscle (ASM). The proinflammatory cytokine TNFα, which enhances agonist-induced intracellular Ca2+ ([Ca2+]i) responses, has been previously shown to increases CD38 expression. In the present study, we tested the hypothesis that the effects of TNFα on CD38 expression vs. changes in [Ca2+]i regulation in ASM cells are linked. Using isolated human ASM cells, CD38 expression was either increased (transfection) or knocked down [small interfering RNA (siRNA)], and [Ca2+]i responses to sarcoplasmic reticulum depletion [i.e., store-operated Ca2+ entry (SOCE)] were evaluated in the presence vs. absence of TNFα. Results confirmed that TNFα significantly increased CD38 expression and ADP-ribosyl cyclase activity, an effect inhibited by CD38 siRNA, but unaltered by CD38 overexpression. CD38 suppression blunted, whereas overexpression enhanced, ACh-induced [Ca2+]i responses. TNFα-induced enhancement of [Ca2+]i response to agonist was blunted by CD38 suppression, but enhanced by CD38 overexpression. Finally, TNFα-induced increase in SOCE was blunted by CD38 siRNA and potentiated by CD38 overexpression. Overall, these results indicate a critical role for CD38 in TNFα-induced enhancement of [Ca2+]i in human ASM cells, and potentially to TNFα augmentation of airway responsiveness.

Caveolins and intracellular calcium regulation in human airway smooth muscle

2007 ◽  
Vol 293 (5) ◽  
pp. L1118-L1126 ◽  
Author(s):  
Y. S. Prakash ◽  
Michael A. Thompson ◽  
Brianna Vaa ◽  
Ihaab Matabdin ◽  
Timothy E. Peterson ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Scaffolding Protein ◽  
Membrane Microdomains ◽  
Caveolin 1 ◽  
Key Factor ◽  
Cellular Signal ◽  
Intracellular Ca ◽  
Interfering Rna ◽  
Intracellular Calcium Regulation

Regulation of intracellular Ca2+ concentration ([Ca2+]i) is a key factor in airway smooth muscle (ASM) tone. In vascular smooth muscle, specialized membrane microdomains (caveolae) expressing the scaffolding protein caveolin-1 are thought to facilitate cellular signal transduction. In human ASM cells, we tested the hypothesis that caveolae mediate Ca2+ responses to agonist stimulation. Fluorescence immunocytochemistry with confocal microscopy, as well as Western blot analysis, was used to determine that agonist receptors (M3 muscarinic, bradykinin, and histamine) and store-operated Ca2+ entry (SOCE)-regulatory mechanisms colocalize with caveolin-1. Although caveolin-2 coexpressed with caveolin-1, caveolin-3 was absent. In fura 2-loaded ASM cells, [Ca2+]i responses to 1 μM ACh, 10 μM histamine, and 10 nM bradykinin, as well as SOCE, were attenuated (each to a different extent) after disruption of caveolae by the cholesterol-chelating drug methyl-β-cyclodextrin. Transfection of ASM cells with 50 nM caveolin-1 small interfering RNA significantly weakened caveolin-1 expression and blunted [Ca2+]i responses to bradykinin and histamine, as well as SOCE, but the response to ACh was less intense. These results indicate that caveolae are present in ASM and that caveolin-1 contributes to regulation of [Ca2+]i responses to agonist.

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