Beta-Adrenoceptor-medicated down-regulation of M2 muscarinic receptors: role of cyclic adenosine 5'-monophosphate-dependent protein kinase and protein kinase C. 1996

J Rousell, and E B Haddad, and J C Mak, and B L Webb, and M A Giembycz, and P J Barnes
Department of Thoracic Medicine, National Heart and Lung Institute, London, UK.

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.

UI MeSH Term Description Entries
D011493 Protein Kinase C An serine-threonine protein kinase that requires the presence of physiological concentrations of CALCIUM and membrane PHOSPHOLIPIDS. The additional presence of DIACYLGLYCEROLS markedly increases its sensitivity to both calcium and phospholipids. The sensitivity of the enzyme can also be increased by PHORBOL ESTERS and it is believed that protein kinase C is the receptor protein of tumor-promoting phorbol esters. Calcium Phospholipid-Dependent Protein Kinase,Calcium-Activated Phospholipid-Dependent Kinase,PKC Serine-Threonine Kinase,Phospholipid-Sensitive Calcium-Dependent Protein Kinase,Protein Kinase M,Calcium Activated Phospholipid Dependent Kinase,Calcium Phospholipid Dependent Protein Kinase,PKC Serine Threonine Kinase,Phospholipid Sensitive Calcium Dependent Protein Kinase,Phospholipid-Dependent Kinase, Calcium-Activated,Serine-Threonine Kinase, PKC
D011869 Radioligand Assay Quantitative determination of receptor (binding) proteins in body fluids or tissue using radioactively labeled binding reagents (e.g., antibodies, intracellular receptors, plasma binders). Protein-Binding Radioassay,Radioreceptor Assay,Assay, Radioligand,Assay, Radioreceptor,Assays, Radioligand,Assays, Radioreceptor,Protein Binding Radioassay,Protein-Binding Radioassays,Radioassay, Protein-Binding,Radioassays, Protein-Binding,Radioligand Assays,Radioreceptor Assays
D011976 Receptors, Muscarinic One of the two major classes of cholinergic receptors. Muscarinic receptors were originally defined by their preference for MUSCARINE over NICOTINE. There are several subtypes (usually M1, M2, M3....) that are characterized by their cellular actions, pharmacology, and molecular biology. Muscarinic Acetylcholine Receptors,Muscarinic Receptors,Muscarinic Acetylcholine Receptor,Muscarinic Receptor,Acetylcholine Receptor, Muscarinic,Acetylcholine Receptors, Muscarinic,Receptor, Muscarinic,Receptor, Muscarinic Acetylcholine,Receptors, Muscarinic Acetylcholine
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012333 RNA, Messenger RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm. Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated
D015536 Down-Regulation A negative regulatory effect on physiological processes at the molecular, cellular, or systemic level. At the molecular level, the major regulatory sites include membrane receptors, genes (GENE EXPRESSION REGULATION), mRNAs (RNA, MESSENGER), and proteins. Receptor Down-Regulation,Down-Regulation (Physiology),Downregulation,Down Regulation,Down-Regulation, Receptor
D017868 Cyclic AMP-Dependent Protein Kinases A group of enzymes that are dependent on CYCLIC AMP and catalyze the phosphorylation of SERINE or THREONINE residues on proteins. Included under this category are two cyclic-AMP-dependent protein kinase subtypes, each of which is defined by its subunit composition. Adenosine Cyclic Monophosphate-Dependent Protein Kinases,Protein Kinase A,cAMP Protein Kinase,cAMP-Dependent Protein Kinases,Cyclic AMP-Dependent Protein Kinase,cAMP-Dependent Protein Kinase,Adenosine Cyclic Monophosphate Dependent Protein Kinases,Cyclic AMP Dependent Protein Kinase,Cyclic AMP Dependent Protein Kinases,Protein Kinase, cAMP,Protein Kinase, cAMP-Dependent,Protein Kinases, cAMP-Dependent,cAMP Dependent Protein Kinase,cAMP Dependent Protein Kinases
D018343 Receptors, Adrenergic, beta-2 A subclass of beta-adrenergic receptors (RECEPTORS, ADRENERGIC, BETA). The adrenergic beta-2 receptors are more sensitive to EPINEPHRINE than to NOREPINEPHRINE and have a high affinity for the agonist TERBUTALINE. They are widespread, with clinically important roles in SKELETAL MUSCLE; LIVER; and vascular, bronchial, gastrointestinal, and genitourinary SMOOTH MUSCLE. Adrenergic beta-2 Receptors,Receptors, beta-2 Adrenergic,beta-2 Adrenergic Receptors,Adrenergic Receptor, beta-2,Receptor, Adrenergic, beta-2,beta 2 Adrenergic Receptors,Adrenergic Receptor, beta 2,Adrenergic Receptors, beta-2,Adrenergic beta 2 Receptors,Receptor, beta-2 Adrenergic,Receptors, Adrenergic beta-2,Receptors, beta 2 Adrenergic,beta-2 Adrenergic Receptor,beta-2 Receptors, Adrenergic

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