Chronic carbachol pretreatment decreases adenylyl cyclase activity in airway smooth muscle

1997 ◽  
Vol 273 (3) ◽  
pp. L640-L647 ◽  
Author(s):  
G. Schears ◽  
J. Clancy ◽  
C. A. Hirshman ◽  
C. W. Emala
Keyword(s):  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
Airway Smooth Muscle ◽  
Muscarinic Receptors ◽  
Cyclase Activity ◽  
Adenylyl Cyclase Activity ◽  
Chronic Activation ◽  
A Site ◽  
M3 Muscarinic Receptor ◽  
G Protein Coupled

In airway smooth muscle, the regulation of adenylyl cyclase, the enzyme that synthesizes adenosine 3',5'-cyclic monophosphate, is under dual regulation by G protein-coupled receptors. It is unknown if chronic activation of muscarinic receptors in airway smooth muscle alters the stimulatory adenylyl cyclase cascade to decrease airway relaxation. Bovine airway smooth muscle pretreated with carbachol for 18 h, but not for 30 min or 2 h, showed decreased adenylyl cyclase activity under basal conditions and in response to isoproterenol, prostaglandin E1, GTP, and forskolin. The quantity of beta-adrenergic receptors or of Gi alpha proteins was unaffected by carbachol pretreatment. The effect of carbachol pretreatment was blocked by the inclusion of atropine or the protein kinase C (PKC) inhibitor staurosporine. These results suggest that chronic but not acute agonist pretreatment of muscarinic receptors decreases in adenylyl cyclase stimulation at a site distal to receptors and that this effect is mediated by the chronic activation of PKC via the M3 muscarinic receptor.

scholarly journals Decreased adenylyl cyclase protein and function in airway smooth muscle by chronic carbachol pretreatment

2000 ◽  
Vol 279 (4) ◽  
pp. C1008-C1015 ◽  
Author(s):  
Charles W. Emala ◽  
Judith Clancy-Keen ◽  
Carol A. Hirshman
Keyword(s):  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
Airway Smooth Muscle ◽  
Muscle Tone ◽  
Cyclase Activity ◽  
Binding Assays ◽  
Adenylyl Cyclase Activity ◽  
Protein Kinase C Inhibitor ◽  
Chronic Activation ◽  
And Function

Cellular levels of cAMP are an important determinant of airway smooth muscle tone. We have previously shown that chronic (18 h) but not acute (30 min or 2 h) pretreatment with the muscarinic receptor agonist carbachol resulted in decreased adenylyl cyclase activity in response to GTP, isoproterenol, or forskolin via a pathway blocked by the protein kinase C inhibitor staurosporine. The present study was designed to determine if carbachol-induced decreases in adenylyl cyclase activity were due to regulatory events at the level of either Gsα or adenylyl cyclase. Detergent-solubilized Gsα from control or carbachol-pretreated bovine airway smooth muscle had similar adenylyl cyclase activity in response to either NaF or guanosine 5′- O-(3-thiotriphosphate) (GTPγS) when reconstituted into S49 cyc− membranes that lack endogenous Gsα (carbachol pretreated: GTPγS, 93 ± 13% of control; NaF/AlCl3, 99 ± 8.6% of control; n = 4). Exogenous Gsα solubilized from red blood cells failed to restore normal adenylyl cyclase activity when reconstituted into carbachol-pretreated bovine airway smooth muscle (carbachol pretreated: GTP, 36 ± 10% of control; NaF/AlCl3, 54 ± 11% of control; n = 4). [3H]forskolin radioligand saturation binding assays revealed a decreased quantity of total adenylyl cyclase protein after carbachol pretreatment (maximal binding: 152 ± 40 and 107 ± 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, respectively). These results suggest that chronic activation of muscarinic receptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle.

scholarly journals The dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle

2012 ◽  
Vol 302 (3) ◽  
pp. L316-L324 ◽  
Author(s):  
Kentaro Mizuta ◽  
Yi Zhang ◽  
Dingbang Xu ◽  
Eiji Masaki ◽  
Reynold A. Panettieri ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Adenylyl Cyclase ◽  
Airway Smooth Muscle ◽  
Dopamine D2 Receptor ◽  
Inositol Phosphate ◽  
Cyclase Activity ◽  
Receptor Protein ◽  
Receptor Subtypes ◽  
Adenylyl Cyclase Activity

Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, “D1-like” receptors (D1 and D5) that couple to the Gs protein and “D2-like” receptors (D2, D3, and D4) that couple to Gi. Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of Gi-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of Gi-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D2-like receptor is expressed on ASM, whether it exhibits classical Gi-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D2 receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D2 receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D2 receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D2 receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D2 receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D2 receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.

TNF-alpha inhibits isoproterenol-stimulated adenylyl cyclase activity in cultured airway smooth muscle cells

1997 ◽  
Vol 272 (4) ◽  
pp. L644-L650 ◽  
Author(s):  
C. W. Emala ◽  
J. Kuhl ◽  
C. L. Hungerford ◽  
C. A. Hirshman
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
Airway Smooth Muscle ◽  
Adrenergic Receptor ◽  
Cyclase Activity ◽  
Tnf Alpha ◽  
Adenylyl Cyclase Activity ◽  
Beta Adrenergic Receptor ◽  
Beta Adrenergic

Inflammation, increased cytokine production, and decreased responsiveness of airway smooth muscle (ASM) to beta-adrenergic agonists are characteristics of asthma. We questioned whether the cytokine tumor necrosis factor-alpha (TNF-alpha) directly impaired beta-adrenergic signal transduction in cultured canine ASM cells. Confluent ASM cells exposed to TNF-alpha (0.1-10 ng/ml) for 72 h showed lower maximal levels of adenylyl cyclase activity in response to isoproterenol (10 ng/ml; 14 +/- 4.3 vs. 7.5 +/- 1.3 pmol adenosine 3',5'-cyclic monophosphate x well(-1) x 20 min(-1), control vs. treated, respectively), despite no changes in beta-adrenergic receptor numbers (maximum number of binding sites = 4.8 +/- 0.72 vs. 4.5 +/- 0.81 fmol/mg protein, control vs. treated, respectively). Adenylyl cyclase activities in response to prostaglandin E1, NaF, or forskolin were not different in treated and untreated cells. These results demonstrate that a cytokine known to be increased during exacerbation of asthmatic symptoms directly impairs beta-adrenergic function in ASM cells and suggests a mechanism by which inflammation impairs beta-adrenergic receptor signal transduction in asthma.

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation

2004 ◽  
Vol 286 (2) ◽  
pp. L312-L319 ◽  
Author(s):  
J. K. L. Walker ◽  
R. R. Gainetdinov ◽  
D. S. Feldman ◽  
P. K. McFawn ◽  
M. G. Caron ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
G Protein ◽  
Muscarinic Receptor ◽  
Airway Smooth Muscle ◽  
Muscarinic Receptors ◽  
Muscle Relaxation ◽  
Receptor Activation ◽  
Smooth Muscle Relaxation ◽  
Airway Responses ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M2 muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene ( GRK5- /-) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5- /- and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from β2-adrenergic receptor activation were diminished in GRK5- /- mice. These data suggest that M2 muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5- /- mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.

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