scholarly journals Characterization of adenylyl cyclase isoforms in rat peripheral pulmonary arteries

2001 ◽  
Vol 280 (6) ◽  
pp. L1359-L1369 ◽  
Author(s):  
Karen B. Jourdan ◽  
Nicola A. Mason ◽  
Lu Long ◽  
Peter G. Philips ◽  
Martin R. Wilkins ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
Pulmonary Arteries ◽  
Bronchial Epithelium ◽  
Rt Pcr ◽  
Bronchial Smooth Muscle ◽  
Functional Studies ◽  
Kinase C ◽  
Rat Lungs

Activation of adenylyl cyclase (AC), of which there are 10 diversely regulated isoforms, is important in regulating pulmonary vascular tone and remodeling. Immunohistochemistry in rat lungs demonstrated that AC2, AC3, and AC5/6 predominated in vascular and bronchial smooth muscle. Isoforms 1, 4, 7, and 8 localized to the bronchial epithelium. Exposure of animals to hypoxia did not change the pattern of isoform expression. RT-PCR confirmed mRNA expression of AC2, AC3, AC5, and AC6 and demonstrated AC7 and AC8 transcripts in smooth muscle. Western blotting confirmed the presence of AC2, AC3, and AC5/6 proteins. Functional studies provided evidence of cAMP regulation by Ca2+ and protein kinase C-activated but not Gi-inhibited pathways, supporting a role for AC2 and a Ca2+-stimulated isoform, AC8. However, NKH-477, an AC5-selective activator, was more potent than forskolin in elevating cAMP and inhibiting serum-stimulated [3H]thymidine incorporation, supporting the presence of AC5. These studies demonstrate differential expression of AC isoforms in rat lungs and provide evidence that AC2, AC5, and AC8 are functionally important in cAMP regulation and growth pathways in pulmonary artery myocytes.

scholarly journals Attenuation of relaxing response induced by pituitary adenylate cyclase-activating polypeptide in bronchial smooth muscle of experimental asthma

2020 ◽  
Vol 319 (5) ◽  
pp. L786-L793
Author(s):  
Yoshihiko Chiba ◽  
Chihiro Ueda ◽  
Naoko Kohno ◽  
Michio Yamashita ◽  
Yui Miyakawa ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Hyperresponsiveness ◽  
Receptor Expression ◽  
Rt Pcr ◽  
Bronchial Smooth Muscle ◽  
Binding Partner ◽  
Pituitary Adenylate Cyclase ◽  
Asthmatic Reaction ◽  
Contraction And Relaxation ◽  
Relaxing Response

Bronchomotor tone is regulated by contraction and relaxation of airway smooth muscle (ASM). A weakened ASM relaxation might be a cause of airway hyperresponsiveness (AHR), a characteristic feature of bronchial asthma. Pituitary adenylyl cyclase-activating polypeptide (PACAP) is known as a mediator that causes ASM relaxation. To date, whether or not the PACAP responsiveness is changed in asthmatic ASM is unknown. The current study examined the hypothesis that relaxation induced by PACAP is reduced in bronchial smooth muscle (BSM) of allergic asthma. The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. Tension study showed a BSM hyperresponsiveness to acetylcholine in the OA-challenged mice. Both quantitative RT-PCR and immunoblot analyses revealed a significant decrease in PAC1 receptor expression in BSMs of the diseased mice. Accordingly, in the antigen-challenged group, the PACAP-induced PAC1 receptor-mediated BSM relaxation was significantly attenuated, whereas the relaxation induced by vasoactive intestinal polypeptide was not changed. These findings suggest that the relaxation induced by PACAP is impaired in BSMs of experimental asthma due to a downregulation of its binding partner PAC1 receptor. Impaired BSM responsiveness to PACAP might contribute to the AHR in asthma.

Airway β-adrenoceptor number in cystic fibrosis and asthma

1990 ◽  
Vol 78 (4) ◽  
pp. 409-417 ◽  
Author(s):  
Rajiev K. Sharma ◽  
Peter K. Jeffery
Keyword(s):  
Cystic Fibrosis ◽  
Smooth Muscle ◽  
Binding Capacity ◽  
Statistical Significance ◽  
Bronchial Carcinoma ◽  
Bronchial Epithelium ◽  
Alveolar Wall ◽  
Primary Defect ◽  
Bronchial Smooth Muscle ◽  
Level 1

1. Maximal binding capacity (Bmax.) and the dissociation constant (KD) for the β-adrenoceptor antagonist 125I-cyanopindol were estimated in membrane preparations of hilar, lobar/main bronchi (level 1) and peripheral lung (level 11) of grossly normal lungs resected for bronchial carcinoma. The tissue distribution of 125I-cyanopindol-binding sites was assessed by autoradiography of complementary cryostat sections. The data obtained from the resections for carcinoma and bronchiectasis were used as disease controls for comparison with those obtained from patients with cystic fibrosis and asthma. 2. In carcinoma controls, mean Bmax. values (± sem) for airway levels 1 and 11 were 89 ± 4 and 133 ± 6 fmol/mg of protein, respectively (P <0.01). The corresponding KD values at each airway level were similar, i.e. 29 ± 2 and 33 ± 1 pmol/l, respectively. Autoradiography revealed that there was dense labelling of bronchial and bronchiolar epithelium and most strikingly of the alveolar wall. 3. Compared with carcinoma controls, mean Bmax. values in cystic fibrosis were significantly reduced in membrane preparations of both airway levels 1 and 11 (P <0.01). Autoradiography showed the reduction was most apparent in alveolar wall and bronchial epithelium. 4. There was a tendency to reduction of Bmax. in membrane preparations from patients with bronchiectasis at airway level I, but this failed to reach statistical significance. Autoradiography demonstrated that the density of labelling was significantly reduced in bronchial epithelium and bronchial smooth muscle as compared with carcinoma controls (P <0.01). 5. The Bmax. values were not significantly altered in membranes prepared from both airway levels in asthma, albeit there was a tendency towards reduction in Bmax. at level 1. At this level autoradiography demonstrated significantly reduced labelling of bronchial epithelium (P>0.01) and submucosal glands (P>0.05) but not of bronchial smooth muscle. 6. It is most likely that the reduction in β-adrenoceptor number that we have found in cystic fibrosis is secondary to pulmonary infection and airway inflammation. Its relationship to the primary defect remains unclear.

scholarly journals Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

2021 ◽  
Vol 118 (49) ◽  
pp. e2026668118
Author(s):  
Donghwa Kim ◽  
Alina Tokmakova ◽  
Lauren K. Lujan ◽  
Hannah R. Strzelinski ◽  
Nicholas Kim ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Chemical Space ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Airway Smooth Muscle Cell ◽  
Functional Studies ◽  
G Protein Coupled ◽  
Identification And Characterization

G protein–coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β2-adrenergic receptors (β2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling—a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S. These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S. C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β2AR actions favorable for treating obstructive lung disease.

CYP4A mRNA, protein, and product in rat lungs: novel localization in vascular endothelium

2002 ◽  
Vol 93 (1) ◽  
pp. 330-337 ◽  
Author(s):  
Daling Zhu ◽  
Chenyang Zhang ◽  
Meetha Medhora ◽  
Elizabeth R. Jacobs
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Epithelial Cells ◽  
Cellular Localization ◽  
Muscle Tone ◽  
Pulmonary Arteries ◽  
Cell Types ◽  
Male Rat ◽  
Type I ◽  
Rat Lungs

The vasodilatory effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on lung arteries is opposite to the constrictor effect seen in cerebral and renal vessels. These observations raise questions about the cellular localization of 20-HETE-forming isoforms in pulmonary arteries and other tissues. Using in situ hybridization, we demonstrate for the first time CYP4A (a family of cytochrome P-450 enzymes catalyzing formation of 20-HETE from the substrate arachidonic acid) mRNA in pulmonary arterial endothelial and smooth muscle cells, bronchial smooth muscle and bronchial epithelial cells, type I epithelial cells, and macrophages in adult male rat lungs. Moreover, we detect CYP4A protein in rat pulmonary arteries and bronchi as well as cultured endothelial cells. Finally, we identify endogenously formed 20-HETE by using fluorescent HPLC techniques, as well as the capacity to convert arachidonic acid into 20-HETE in pulmonary arteries, bronchi, and endothelium. These data show that 20-HETE is an endogenous product of several pulmonary cell types and is localized to tissues that optimally position it to modulate physiological functions such as smooth muscle tone or electrolyte flux.

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