Mechanisms of acute desensitization of the beta2AR-adenylyl cyclase pathway in human airway smooth muscle.
beta2-Adrenergic receptors (beta2ARs) are important regulators of airway smooth muscle tone, and beta-sympathomimetic drugs are the most widely used agents in asthma therapy and are universally recognized as the treatment of choice for acute asthma attacks. Despite the clinical importance of beta-agonists and a good understanding of their mechanism of action in airway smooth muscle relaxation, surprisingly little is known about the manner in which the beta2AR signaling pathway is regulated in human airway smooth muscle (HASM). In this communication, we characterize mechanisms underlying rapid desensitization of the HASM beta2AR-adenylyl cyclase (AC) pathway. Acute homologous desensitization of beta2AR-mediated cyclic adenosine monophosphate (cAMP) production was characterized by an approximately 60% loss of maximal responsiveness to isoproterenol (ISO) when cells were pretreated for 30 min with 1 microM ISO. Acute heterologous beta2AR desensitization was characterized by an approximately 20% and 30% loss of maximal responsiveness to ISO challenge when cells were pretreated with forskolin and prostaglandin E2 (PGE2), respectively. Each form of desensitization was also characterized by an increase in the EC50 for ISO. beta2AR sequestration was associated with but not required for homologous desensitization. However, sequestration was required for rapid resensitization. Minimal alterations in inherent AC activity were observed with both modes of desensitization, suggesting that the beta2AR is the principal locus of regulation. Protein kinase inhibition by staurosporine largely reversed heterologous beta2AR desensitization and had a small but significant effect on homologous desensitization. In contrast, bisindolylmaleimide IX, a specific PKC-inhibitor, had no effect on heterologous or homologous beta2AR desensitization, suggesting that staurosporine effects were mediated by PKA inhibition. Overexpression of the G protein-coupled receptor kinase GRK2 in HASM cultures enhanced homologous desensitization. These data suggest that HASM beta2ARs are highly susceptible to rapid desensitization by multiple agents, and identify both GRKs and PKA as important mediators of acute beta2AR desensitization.
Penn
R B
RB
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University; and Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Panettieri
R A
RA
Benovic
J L
JL
D000262:Adenylyl Cyclases / Q000378:metabolism*; D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D015398:Signal Transduction; D014132:Trachea / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*
D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000262:Adenylyl Cyclases