Muscarinic inhibition of adenylyl cyclase regulates intracellular calcium in single airway smooth muscle cells

1996 ◽  
Vol 270 (2) ◽  
pp. L208-L214 ◽  
Author(s):  
J. M. Madison ◽  
H. Yamaguchi
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Intracellular Calcium ◽  
Adenylyl Cyclase ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Inhibitory Effect ◽  
Airway Smooth Muscle Cells ◽  
Muscarinic Agonists ◽  
Cyclic Monophosphate

To determine whether muscarinic agonists attenuated isoproterenol-stimulated decreases in intracellular calcium concentration ([Ca2+]i), changes in [Ca2+]i were measured in single airway smooth muscle cells using ratiometric analysis of fura 2 fluorescence. Isoproterenol (10(-5) M) and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) decreased [Ca2+]i by 24 +/- 3% (P < 0.05, n= 28) and 17 +/- 1% (P < 0.05, n = 6), respectively. The decreased [Ca2+]i in response to isoproterenol was inhibited by propranolol (10(-6) M) and Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) (10-40 microM). In subsequent experiments assessing the effects of muscarinic agonists, isoproterenol did not decrease [Ca2+]i in the presence of carbachol (5 x 10(-8) M) (6 +/- 8% increase; NS, n = 8). To determine the mechanism underlying this inhibitory effect of carbachol, cells were loaded with 4,5-dimethoxy-2-nitrobenzyl adenosine-3',5'-cyclic monophosphate (caged cAMP). For cells loaded with 20 microM caged cAMP, photolysis of caged cAMP decreased basal [Ca2+]i by 28 +/- 3% (P < 0.05, n = 12). In the presence of carbachol (5 x 10(-8) M), photolysis of caged cAMP still induced a 27 +/- 4% decrease in [Ca2+]i (P < 0.05, n = 12). We concluded that a low concentration of carbachol did attenuate isoproterenol-stimulated decreases in [Ca2+]i. Because low concentrations of carbachol attenuated the decreases in [Ca2+]i stimulated by isoproterenol but not the comparable decreases stimulated by cAMP directly, we concluded that the inhibition of adenylyl cyclase activity by muscarinic agonists contributed to the regulation of [Ca2+]i in airway smooth muscle cells. The findings suggested that physiological levels of cholinergic stimulation inhibit adenylyl cyclase, thereby attenuating the effects that beta-adrenergic agonists have on [Ca2+]i.

scholarly journals Altered CD38/Cyclic ADP-Ribose Signaling Contributes to the Asthmatic Phenotype

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Joseph A. Jude ◽  
Mythili Dileepan ◽  
Reynold A. Panettieri ◽  
Timothy F. Walseth ◽  
Mathur S. Kannan
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Intracellular Calcium ◽  
Airway Smooth Muscle ◽  
Airway Hyperresponsiveness ◽  
Map Kinases ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Cd38 Expression ◽  
Cyclic Adp Ribose

CD38 is a transmembrane glycoprotein expressed in airway smooth muscle cells. The enzymatic activity of CD38 generates cyclic ADP-ribose from β-NAD. Cyclic ADP-ribose mobilizes intracellular calcium during activation of airway smooth muscle cells by G-protein-coupled receptors through activation of ryanodine receptor channels in the sarcoplasmic reticulum. Inflammatory cytokines that are implicated in asthma upregulate CD38 expression and increase the calcium responses to contractile agonists in airway smooth muscle cells. The augmented intracellular calcium responses following cytokine exposure of airway smooth muscle cells are inhibited by an antagonist of cyclic ADP-ribose. Airway smooth muscle cells from CD38 knockout mice exhibit attenuated intracellular calcium responses to agonists, and these mice have reduced airway response to inhaled methacholine. CD38 also contributes to airway hyperresponsiveness as shown in mouse models of allergen or cytokine-induced inflammatory airway disease. In airway smooth muscle cells obtained from asthmatics, the cytokine-induced CD38 expression is significantly enhanced compared to expression in cells from nonasthmatics. This differential induction of CD38 expression in asthmatic airway smooth muscle cells stems from increased activation of MAP kinases and transcription through NF-κB, and altered post-transcriptional regulation through microRNAs. We propose that increased capacity for CD38 signaling in airway smooth muscle in asthma contributes to airway hyperresponsiveness.

Raf-1 kinase mediates adenylyl cyclase sensitization by TNF-α in human airway smooth muscle cells

2007 ◽  
Vol 292 (6) ◽  
pp. L1414-L1421 ◽  
Author(s):  
Yoko Osawa ◽  
Peter D. Yim ◽  
Dingbang Xu ◽  
Reynold A. Panettieri ◽  
Charles W. Emala
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Adenylyl Cyclase ◽  
Airway Smooth Muscle ◽  
Egf Receptor ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Human Airway ◽  
Tnf Α

Tumor necrosis factor (TNF)-α is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF-α has been reported to induce G protein-coupled receptor desensitization, but adenylyl cyclase sensitization, in airway smooth muscle cells by an unknown mechanism. Cyclic AMP, which is synthesized by adenylyl cyclases in response to G protein-coupled receptor signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration, and tone. In other cell types, TNF-α receptors transactivate the EGF receptor, which activates raf-1 kinase. Further studies in transfected cells show that raf-1 kinase can phosphorylate and activate some isoforms of adenylyl cyclase. Cultured human airway smooth muscle cells were treated with TNF-α in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or Gi proteins. TNF-α caused a significant dose- (1–10 ng/ml) and time-dependent (24 and 48 h) increase in forskolin-stimulated adenylyl cyclase activity, which was abrogated by pretreatment with GW5074 (a raf-1 kinase inhibitor), was partially inhibited by an EGF receptor inhibitor, but was unaffected by pertussis toxin. TNF-α also increased phosphorylation of Ser338 on raf-1 kinase, indicative of activation. IL-1β and EGF sensitization of adenylyl cyclase activity was also sensitive to raf-1 kinase inhibition by GW5074. Taken together, these studies link two signaling pathways not previously characterized in human airway smooth muscle cells: TNF-α transactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase.

Pharmacological activation changes stiffness of cultured human airway smooth muscle cells

1996 ◽  
Vol 271 (5) ◽  
pp. C1660-C1668 ◽  
Author(s):  
R. D. Hubmayr ◽  
S. A. Shore ◽  
J. J. Fredberg ◽  
E. Planus ◽  
R. A. Panettieri ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
High Density ◽  
Cell Stiffness ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Cyclic Monophosphate ◽  
Human Airway

Using magnetic twisting cytometry (MTC), we measured the cytoskeletal stiffness of adherent human airway smooth muscle (HASM) cells. We hypothesized that modulation of actin-myosin interactions by application of contractile agonists would induce changes in cytoskeletal stiffness. In cells plated on high-density collagen, bradykinin (10(-6) M) and histamine (10(-4) M) increased stiffness by 85 +/- 15 and 68 +/- 16%, respectively. Increases in cell stiffness were also consistently observed after acetylcholine, substance P, and KCl. The bronchodilator agonists isoproterenol, prostaglandin E2, forskolin, dibutryl adenosine 3', 5'-cyclic monophosphate, and 8-bromoguanosine 3', 5'-cyclic monophosphate each caused a dose-dependent decrease in cell stiffness in unstimulated as well as bradykinin-treated cells. HASM cells plated on high-density collagen were stiffer than cells plated on low-density collagen (126 +/- 16 vs. 43 +/- 3 dyn/cm2) and developed more pronounced increases in stiffness in response to bradykinin as well as more pronounced decreases in stiffness in response to isoproterenol. These results are consistent with the hypothesis that modulation of actin-myosin interactions by application of contractile agonists causes changes in cytoskeletal stiffness of HASM cells. MTC may be a valuable tool for evaluating the mechanisms of pharmacomechanical coupling in airway smooth muscle cells in culture.

Human airway smooth muscle expresses 7 isoforms of adenylyl cyclase: a dominant role for isoform V

2001 ◽  
Vol 281 (4) ◽  
pp. L832-L843 ◽  
Author(s):  
Dingbang Xu ◽  
Cary Isaacs ◽  
Ian P. Hall ◽  
Charles W. Emala
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Adenylyl Cyclase ◽  
Airway Smooth Muscle ◽  
Primary Cultures ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Functional Assay ◽  
Human Airway Smooth Muscle ◽  
Human Airway

Adenylyl cyclases are a nine-member family of differentially regulated enzymes responsible for the synthesis of cAMP. cAMP is an important second messenger that contributes to the regulation of airway smooth muscle tone. However, little is known regarding the expression and regulation of adenylyl cyclase isoforms in airway smooth muscle cells. Nondegenerate specific primers were designed for all nine known isoforms of human adenylyl cyclase. RT-PCR experiments were performed using total RNA extracted from whole human brain (positive control), whole rat brain (negative control), whole human trachea, human airway smooth muscle, and primary cultures of human airway smooth muscle cells. Seven of the nine known isoforms of adenylyl cyclase (isoforms I, III–VII, and IX) were expressed at the mRNA level in both human airway smooth muscle and primary cultures of human airway smooth muscle cells. Immunoblot and adenylyl cyclase functional assay indicated that isoform V is likely among the functionally predominant isoforms of adenylyl cyclase in human airway smooth muscle. These results suggest that multiple isoforms of adenylyl cyclase enzymes are coexpressed in human airway smooth muscle cells and that isoform V is among the functionally important isoforms.

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