Store-operated Ca2+Influx in Airway Smooth Muscle

Background Volatile anesthetics produce bronchodilation in part by depleting sarcoplasmic reticulum Ca stores in airway smooth muscle (ASM). Other bronchodilatory drugs are known to act via cyclic nucleotides (cyclic adenosine 3',5'-cyclic monophosphate, cyclic guanosine 3',5'-cyclic monophosphate). Intracellular Ca regulation in ASM involves plasma membrane Ca influx, including that triggered by sarcoplasmic reticulum Ca depletion (store-operated Ca entry [SOCE]). The authors hypothesized that anesthetics and bronchodilatory agents interact in inhibiting SOCE, thus enhancing ASM relaxation. Methods In enzymatically dissociated porcine ASM cells imaged using fluorescence microscopy, sarcoplasmic reticulum Ca was depleted by 1 microm cyclopiazonic acid in 0 extracellular Ca, nifedipine, and potassium chloride (preventing Ca influx through L-type channels and SOCE). Extracellular Ca was rapidly reintroduced to selectively activate SOCE in the presence or absence of 1 minimum alveolar concentration (MAC) halothane, isoflurane, or sevoflurane. Anesthetic interference with SOCE regulation by cyclic nucleotides was examined by activating SOCE in the presence of (1) 1 microm acetylcholine, (2) 100 microm dibutryl cyclic adenosine 3',5'-cyclic monophosphate, or (3) 100 microm 8-bromo-cyclic guanosine 3',5'-cyclic monophosphate. Results SOCE was enhanced by acetylcholine, whereas volatile anesthetics and both cyclic nucleotides partially inhibited Ca influx. Preexposure to 1 or 2 MAC anesthetic (halothane > isoflurane > sevoflurane) inhibited SOCE. Only halothane and isoflurane inhibited acetylcholine-induced augmentation of Ca influx, and significantly potentiated cyclic nucleotide inhibition such that no influx was observed in the presence of anesthetics and cyclic nucleotides. Conclusions These data indicate that volatile anesthetics prevent sarcoplasmic reticulum refilling by inhibiting SOCE and enhancing cyclic nucleotide blunting of Ca influx in ASM. Such interactions likely result in substantial airway relaxation in the presence of both anesthetics and bronchodilatory agents such as beta agonists or nitric oxide.

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Effects of Volatile Anesthetics on Store-operated Ca2+Influx in Airway Smooth Muscle

Anesthesiology ◽

10.1097/00000542-200408000-00018 ◽

2004 ◽

Vol 101 (2) ◽

pp. 373-380 ◽

Cited By ~ 22

Author(s):

Christina M. Pabelick ◽

Binnaz Ay ◽

Yedatore S. Prakash ◽

Gary C. Sieck

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Sarcoplasmic Reticulum ◽

Airway Smooth Muscle ◽

Minimum Alveolar Concentration ◽

Cyclopiazonic Acid ◽

Volatile Anesthetics ◽

Membrane Channels

Background In airway smooth muscle (ASM), volatile anesthetics deplete sarcoplasmic reticulum (SR) Ca(2+) stores by increasing Ca(2+) "leak." Accordingly, SR replenishment becomes dependent on Ca(2+) influx. Depletion of SR Ca(2+) stores triggers Ca(2+) influx via specific plasma membrane channels, store-operated Ca(2+) channels (SOCC). We hypothesized that anesthetics inhibit SOCC triggered by increased SR Ca(2+) "leak," preventing SR replenishment and enhancing ASM relaxation. Methods In porcine ASM cells, SR Ca was depleted by cyclopiazonic acid or caffeine in 0 extracellular Ca(2+), nifedipine and KCl (preventing Ca(2+) influx through L-type and SOCC channels). Extracellular Ca(2+) was rapidly introduced to selectively activate SOCC. After SOCC activation, SR was replenished and the protocol repeated in the presence of 1 or 2 minimum alveolar concentration halothane, isoflurane, or sevoflurane. In other cells, characteristics of SOCC and interactions between acetylcholine (Ach) and volatile anesthetics were examined. Results Cyclopiazonic acid produced slow SR leak, whereas the caffeine response was transient in ASM cells. Reintroduction of extracellular Ca(2+) rapidly increased [Ca(2+)]i. This influx was insensitive to nifedipine, SKF-96365, and KBR-7943, inhibited by Ni and blockade of inositol 1,4,5-triphosphate-induced SR Ca(2+) release, and enhanced by ACh. Preexposure to 1 or 2 minimum alveolar concentration halothane completely inhibited Ca(2+) influx when extracellular Ca(2+) was reintroduced, whereas isoflurane and sevoflurane produced less inhibition. Only halothane and isoflurane inhibited ACh-induced augmentation of Ca(2+) influx. Conclusion Volatile anesthetics inhibit a Ni/La-sensitive store-operated Ca(2+) influx mechanism in porcine ASM cells, which likely helps maintain anesthetic-induced bronchodilation.

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Mechanisms of cyclic nucleotide-induced relaxation in canine tracheal smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.268.3.l407 ◽

1995 ◽

Vol 268 (3) ◽

pp. L407-L413 ◽

Cited By ~ 2

Author(s):

I. McGrogan ◽

S. Lu ◽

S. Hipworth ◽

L. Sormaz ◽

R. Eng ◽

...

Keyword(s):

Smooth Muscle ◽

Cyclic Nucleotide ◽

Cyclopiazonic Acid ◽

Inhibitory Effect ◽

Beta Agonist ◽

Cyclic Monophosphate ◽

Median Effective Concentration ◽

Camp Levels ◽

Ca2 Channels

The effects of exogeneous cyclopiazonic acid (CPA, 10 microM), a selective inhibitor of the sarcoplasmic reticulum (SR) Ca2+ adenosinetriphosphatase, on cyclic nucleotide-induced relaxations of canine airway smooth muscle were examined. Strips of tracheal muscle were precontracted with carbachol (50% median effective concentration, 0.1 microM) or with 60 mM KCl. The beta-agonist isoproterenol (ISO, 10 microM) relaxed the tissue by approximately 50%. The relaxation was reduced in the presence of CPA when L-type Ca2+ channels were available but not when these were blocked by 0.1 microM nifedipine. Forskolin (1.0 microM), an adenylate cyclase activator, was less effective at inhibiting the contraction than ISO, and addition of CPA did not block its inhibitory effect as effectively as when ISO was used. Radioimmunoassay indicated that both these agents raised adenosine 3',5'-cyclic monophosphate (cAMP) levels to the same degree. Very little relaxation of the precontracted smooth muscle was elicited by 3 mM 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BrcAMP), and addition of CPA had no effect. Sodium nitroprusside (100 microM) and 8-bromo-guanosine 3',5'-cyclic monophosphate (10 mM) inhibited contraction to a greater degree than any agent that raised cAMP. These inhibitions were greatly reduced in the presence of CPA when L-type Ca2+ channels were available. We conclude that pumping of Ca2+ into SR plays a major role guanosine 3',5'-cyclic monophosphate-produced but not cAMP-induced relaxation; L-type Ca2+ channels must be available for the relaxant role of Ca2+ pumping into the SR to be expressed; and ISO-induced relaxation may not involve primarily elevation of the cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

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Superficial buffer barrier and preferentially directed release of Ca2+ in canine airway smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.276.5.l744 ◽

1999 ◽

Vol 276 (5) ◽

pp. L744-L753 ◽

Cited By ~ 21

Author(s):

Luke J. Janssen ◽

Pierre A. Betti ◽

Stuart J. Netherton ◽

Denise K. Walters

Keyword(s):

Smooth Muscle ◽

Sarcoplasmic Reticulum ◽

Airway Smooth Muscle ◽

Cyclopiazonic Acid ◽

Membrane Currents ◽

Global Changes ◽

Fura 2 ◽

Mechanical Responses ◽

Filling State

We examined cytosolic concentration of Ca2+([Ca2+]i) in canine airway smooth muscle using fura 2 fluorimetry (global changes in [Ca2+]i), membrane currents (subsarcolemmal [Ca2+]i), and contractions (deep cytosolic [Ca2+]i). Acetylcholine (10−4 M) elicited fluorimetric, electrophysiological, and mechanical responses. Caffeine (5 mM), ryanodine (0.1–30 μM), and 4-chloro-3-ethylphenol (0.1–0.3 mM), all of which trigger Ca2+-induced Ca2+ release, evoked Ca2+ transients and membrane currents but not contractions. The sarcoplasmic reticulum (SR) Ca2+-pump inhibitor cyclopiazonic acid (CPA; 10 μM) evoked Ca2+transients and contractions but not membrane currents. Caffeine occluded the response to CPA, whereas CPA occluded the response to acetylcholine. Finally, KCl contractions were augmented by CPA, ryanodine, or saturation of the SR and reduced when SR filling state was decreased before exposure to KCl. We conclude that 1) the SR forms a superficial buffer barrier dividing the cytosol into functionally distinct compartments in which [Ca2+]iis regulated independently; 2) Ca2+-induced Ca2+ release is preferentially directed toward the sarcolemma; and 3) there is no evidence for multiple, pharmacologically distinct Ca2+ pools.

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Cyclic nucleotide regulation of store-operated Ca2+ influx in airway smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00188.2005 ◽

2006 ◽

Vol 290 (2) ◽

pp. L278-L283 ◽

Cited By ~ 30

Author(s):

Binnaz Ay ◽

Adeyemi Iyanoye ◽

Gary C. Sieck ◽

Y. S. Prakash ◽

Christina M. Pabelick

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Protein Kinase ◽

Protein Kinases ◽

Airway Smooth Muscle ◽

Cyclic Nucleotides ◽

Cyclopiazonic Acid ◽

Nitric Oxide Donor ◽

Cgmp Analog ◽

Intracellular Ca

Sarcoplasmic reticulum (SR) Ca2+ release and plasma membrane Ca2+ influx are key to intracellular Ca2+ ([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+ depletion triggers influx via store-operated Ca2+ channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+ influx in enzymatically dissociated porcine ASM cells. SR Ca2+ was depleted by 1 μM cyclopiazonic acid in 0 extracellular Ca2+ ([Ca2+]o), nifedipine, and KCl (preventing Ca2+ influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 μM isoproterenol or 100 μM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 μM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 μM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+ influx was dependent on the extent of SR Ca2+ depletion, sensitive to Ni2+ and La3+, but not inhibitors of voltage-gated influx channels. cAMP as well as cGMP potently inhibited Ca2+ influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx in ASM triggered by SR Ca2+ depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., β-agonists vs. nitric oxide).

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Store-operated Ca2+ entry in porcine airway smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00317.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. L909-L917 ◽

Cited By ~ 73

Author(s):

Binnaz Ay ◽

Y. S. Prakash ◽

Christina M. Pabelick ◽

Gary C. Sieck

Keyword(s):

Smooth Muscle ◽

Membrane Potential ◽

Sarcoplasmic Reticulum ◽

Airway Smooth Muscle ◽

Ryanodine Receptors ◽

Cyclopiazonic Acid ◽

Inositol 1,4,5 Trisphosphate ◽

Presence And Absence ◽

Induced Changes ◽

Subsequent Introduction

Ca2+ influx triggered by depletion of sarcoplasmic reticulum (SR) Ca2+ stores [mediated via store-operated Ca2+ channels (SOCC)] was characterized in enzymatically dissociated porcine airway smooth muscle (ASM) cells. When SR Ca2+ was depleted by either 5 μM cyclopiazonic acid or 5 mM caffeine in the absence of extracellular Ca2+, subsequent introduction of extracellular Ca2+ further elevated [Ca2+]i. SOCC was insensitive to 1 μM nifedipine- or KCl-induced changes in membrane potential. However, preexposure of cells to 100 nM–1 mM La3+ or Ni2+ inhibited SOCC. Exposure to ACh increased Ca2+ influx both in the presence and absence of a depleted SR. Inhibition of inositol 1,4,5-trisphosphate (IP)-induced SR Ca2+ release by 20 μM xestospongin D inhibited SOCC, whereas ACh-induced IP3 production by 5 μM U-73122 had no effect. Inhibition of Ca2+ release through ryanodine receptors (RyR) by 100 μM ryanodine also prevented Ca2+ influx via SOCC. Qualitatively similar characteristics of SOCC-mediated Ca2+ influx were observed with cyclopiazonic acid- vs. caffeine-induced SR Ca2+ depletion. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx via SOCC in porcine ASM cells involves SR Ca2+ release through both IP3 and RyR channels. Additional regulation of Ca2+ influx by agonist may be related to a receptor-operated, noncapacitative mechanism.

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Cyclic nucleotide-dependent protein kinases in airway smooth muscle.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)33806-7 ◽

1982 ◽

Vol 257 (19) ◽

pp. 11609-11616 ◽

Cited By ~ 4

Author(s):

T J Torphy ◽

W B Freese ◽

G A Rinard ◽

L L Brunton ◽

S E Mayer

Keyword(s):

Smooth Muscle ◽

Protein Kinases ◽

Airway Smooth Muscle ◽

Cyclic Nucleotide ◽

Dependent Protein

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VIP inhibits basal and histamine-stimulated proliferation of human airway smooth muscle cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.268.6.l1047 ◽

1995 ◽

Vol 268 (6) ◽

pp. L1047-L1051 ◽

Cited By ~ 9

Author(s):

K. Maruno ◽

A. Absood ◽

S. I. Said

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Thymidine Incorporation ◽

Inhibitory Effect ◽

Airway Smooth Muscle Cells ◽

Dependent Manner ◽

Cyclic Monophosphate ◽

Cell Counts ◽

Dependent Protein ◽

Camp Levels

Airway smooth muscle (ASM) cell proliferation contributes to increased airway resistance in bronchial asthma. We have examined the modulation of ASM proliferation by vasoactive intestinal peptide (VIP), a cotransmitter of airway relaxation. Human ASM cells were grown in culture as a monolayer. VIP (1.0 nM-1.0 microM) inhibited proliferation in a dose-dependent manner by up to 82% on day 2, but the related peptide glucagon had no effect. Histamine (100 nM-100 microM) increased cell counts by 66%, but in the presence of VIP, cell counts and [3H]thymidine incorporation were reduced by up to 55%. Adenosine 3',5'-cyclic monophosphate (cAMP)-promoting agents, including 3-isobutyl-1-methylxanthine, forskolin, and 8-bromo-adenosine 3',5'-cyclic monophosphate, alone and especially combined with VIP, reduced cell counts and [3H]thymidine incorporation, in correlation with cAMP levels. KT-5720 (1.0 nM-1.0 microM), a selective inhibitor of cAMP-dependent protein kinase A (PKA), abolished the inhibitory effect of VIP. The results show that VIP selectively and potently inhibits human ASM cell growth and multiplication, and nullifies the mitogenic effect of histamine, by a PKA-mediated mechanism. A deficiency of VIP may lead to ASM hyperplasia due to unopposed stimulation by endogenous mitogens.

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Changes in cytosolic cGMP and calcium in airway smooth muscle relaxed by 3-morpholinosydnonimine

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.266.1.l9 ◽

1994 ◽

Vol 266 (1) ◽

pp. L9-L16 ◽

Cited By ~ 10

Author(s):

K. A. Jones ◽

R. R. Lorenz ◽

D. O. Warner ◽

Z. S. Katusic ◽

G. C. Sieck

Keyword(s):

Methylene Blue ◽

Smooth Muscle ◽

Airway Smooth Muscle ◽

Calcium Concentration ◽

Cytosolic Calcium ◽

Tracheal Smooth Muscle ◽

Relative Importance ◽

Cytosolic Calcium Concentration ◽

Fura 2 ◽

Cyclic Monophosphate

Nitrovasodilators relax airway smooth muscle by both guanosine 3',5'-cyclic monophosphate (cGMP)-dependent and cGMP-independent mechanisms and by mechanisms that reduce cytosolic calcium concentration ([Ca2+]i). This study was conducted to determine the relative importance of these mechanisms in relaxation of canine tracheal smooth muscle (CTSM) induced by 3-morpholinosydnonimine (SIN-1). We measured 1) the effect of SIN-1 on force, [cGMP]i, and [Ca2+]i, and 2) the ability of methylene blue (MB) to antagonize SIN-1-induced relaxation and cGMP accumulation. The ratio of fura 2 emission fluorescence intensities due to excitation at 340- and 380-nm wavelengths (F340/F380) was used as an index of [Ca2+]i. In strips contracted with 0.3 microM acetylcholine (ACh, n = 8) or 24 mM KCl (n = 8), SIN-1 (1-100 microM) caused a concentration-dependent decrease in force which was correlated with a concentration-dependent increase in [cGMP]i. MB (10 microM) proportionally attenuated both relaxation and cGMP accumulation. In fura 2-loaded strips contracted with 0.3 microM ACh (n = 7) or 30 mM KCl (n = 7), reductions in force induced by SIN-1 (1-100 microM) were accompanied by decreases in F340/F380. These findings suggest that in CTSM contracted with ACh or KCl, SIN-1 causes relaxation which appears to be mediated by cGMP-dependent mechanisms that reduce [Ca2+]i.

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Effects of cAMP on serotonin evoked calcium transients in cultured rat airway smooth muscle cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.5.l865 ◽

1997 ◽

Vol 272 (5) ◽

pp. L865-L871 ◽

Cited By ~ 1

Author(s):

B. Tolloczko ◽

Y. L. Jia ◽

J. G. Martin

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Airway Smooth Muscle ◽

Airway Smooth Muscle Cells ◽

Intracellular Camp ◽

Dependent Protein Kinase ◽

Cyclic Monophosphate ◽

Dependent Protein ◽

High Concentrations

Agents increasing intracellular adenosine 3',5'-cyclic monophosphate (cAMP) cause relaxation of airway smooth muscle. However, the mechanisms of their action are not fully understood. We investigated the role of cAMP in the modulation of intracellular Ca2+ concentration ([Ca2+]i) transients evoked by serotonin (5-HT) in cultured rat tracheal smooth muscle (TSM) cells. Forskolin (10(-7) M) caused a significant elevation of intracellular cAMP and a 60% relaxation of tracheal rings contracted with 5-HT but did not affect [Ca2+]i in TSM cells. Forskolin (10(-5) M) completely relaxed tracheal rings and significantly decreased [Ca2+]i during the sustained phase of the 5-HT response. Forskolin-induced relaxation was attenuated by the cAMP-dependent protein kinase A (PKA) inhibitor Rp diastereomer of cAMP (Rp-cAMPS; 10(-4) M) and by the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor [Rp isomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, 10(-4) M]. The effects of forskolin on [Ca2+]i were not altered by the PKA inhibitor but were abolished by the PKG inhibitor and thapsigargin. These results indicate that, in rat TSM, the relaxant effects of high concentrations of cAMP may be mediated, at least in part, by facilitating the sequestration of Ca2+ into intracellular stores by a mechanism involving PKG.

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