Beta-Adrenoceptor-medicated down-regulation of M2 muscarinic receptors: role of cyclic adenosine 5'-monophosphate-dependent protein kinase and protein kinase C.
Stimulation of beta2-adrenoceptors with the selective beta2 agonist procaterol caused a biphasic decrease in cell surface M2 muscarinic receptor number in human embryonic lung 299 cells when measured with the hydrophilic antagonist [3H]N-methylscopolamine. In contrast, total muscarinic receptor number, measured with the lipophilic antagonist [3H]quinuclidinylbenzilate, decreased after only 24-hr treatments with procaterol. The loss in receptor number at 24 hr was mimicked with the use of forskolin and the cAMP analogue 8-bromo-cAMP, indicating a cAMP-mediated mechanism. Northern blot analysis showed a small and transient increase in m2-receptor mRNA levels up to 2 hr but no long term (24 hr) effect. Chronic (24 hr) treatment with 8-bromo-cAMP also had no effect on m2 muscarinic receptor mRNA, whereas forskolin caused a 50% reduction in the steady state levels of m2 mRNA that could be only partially blocked by the cAMP-dependent protein kinase inhibitor H-8 and the protein kinase C inhibitor GF 109203X. Procaterol-induced down-regulation of M2 receptors was fully blocked by N-[2-(methylamino)ethyl]-5'-isoquinoline-sulfonamide and 2-[1-(3-dimethylaminopropyl)-inol-3-yl]-3-(indol-3-yl)maleimide, implicating both of these kinases in the M2 muscarinic receptor down-regulation. Conversely, the forskolin- and 8-bromo-cAMP-induced down-regulation was only partially inhibited and unaffected by these inhibitors, respectively. In control cells and those treated with procaterol for < / = 2 hr, cAMP generation was significantly inhibited by carbachol. The inhibitory effect of carbachol was, however, lost after 24-hr exposure to procaterol. This desensitization was partially reversed by preincubations with H-8 and GF 109203X. Collectively, these results suggest that transregulation of M2 muscarinic receptors by beta2-adrenoceptor stimulation can be demonstrated at the protein level in human embryonic lung 299 cells. Furthermore, a role is suggested for cAMP-dependent kinase and PKC in M2 muscarinic receptor down-regulation and their functional desensitization.
Rousell
J
J
Department of Thoracic Medicine, National Heart and Lung Institute, London, UK.
Haddad
E B
EB
Mak
J C
JC
Webb
B L
BL
Giembycz
M A
MA
Barnes
P J
PJ
D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D015536:Down-Regulation; D006801:Humans; D011493:Protein Kinase C / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D011869:Radioligand Assay; D018343:Receptors, Adrenergic, beta-2 / Q000502:physiology*; D011976:Receptors, Muscarinic / Q000032:analysis* / Q000235:genetics
D012333:RNA, Messenger; D018343:Receptors, Adrenergic, beta-2; D011976:Receptors, Muscarinic; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C