β-adrenoceptor-mediated down-regulation of M2 muscarinic receptors: Role of cyclic adenosine 5′-monophosphate-dependent protein kinase and protein kinase C

Stimulation of β2-adrenoceptors with the selective β2 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 β2-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.link_to_subscribed_fulltex

Conventional protein kinase C and atypical protein kinase Cζ differentially regulate macrophage production of tumour necrosis factor-α and interleukin-10

In chronic inflammatory diseases such as rheumatoid arthritis, joint macrophages/monocytes are the major source of pro- and anti-inflammatory cytokines. Little is understood regarding the signalling pathways which determine the production of the pro-inflammatory cytokine, tumour necrosis factor-α (TNF-α) and the anti-inflammatory cytokine, interleukin-10 (IL-10). Two pathways integral to macrophage function are the protein kinase C (PKC)- and the cAMP-dependent pathways. In this report, we have investigated the involvement of PKC and cAMP in the production of TNF-α and IL-10 by peripheral blood monocyte-derived macrophages. The utilization of the PKC inhibitors Go6983, Go6976 and RO-32-0432 demonstrated a role for conventional PKCs (α and β) in the production of TNF-α in response to stimulation by lipopolysaccharide and phorbol 12-myristate 13-acetate (PMA)/ionomycin. PKC stimulation resulted in the downstream activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway which differentially regulates TNF-α and IL-10. The addition of cAMP however, suppressed activation of this MAPK and TNF-α production. Cyclic-AMP augmented IL-10 production and cAMP response element binding protein activation upon stimulation by PMA/ionomycin. In addition, cAMP activated PKCζ; inhibition of which, by a dominant negative adenovirus construct, selectively suppressed IL-10 production. These observations suggest that pro-inflammatory and anti-inflammatory cytokines are differentially regulated by PKC isoforms; TNF-α being dependent on conventional PKCs (α and β) whereas IL-10 is regulated by the cAMP-regulated atypical PKCζ