Received for publication March 18, 2008.
Revised August 3, 2008.
Accepted for publication August 6, 2008.

The ₃-adrenoceptor agonist L755507 and antagonist L748337 activate different signalling pathways in CHO-K1 cells stably expressing the human ₃-adrenoceptor

Abstract

This study identifies signaling pathways activated by the ₂-/₃-AR agonist zinterol, the selective ₃-AR agonist L755507 and the selective ₃-AR antagonist L748337 in CHO-K1 cells expressing human ₃-adrenoceptors. Zinterol and L755507 caused a robust concentration-dependent increase in cAMP accumulation (pEC₅₀ 8.5 and 12.3 respectively), while L748337 had low efficacy. Maximal cAMP accumulation with zinterol and L755507 was increased after pre-treatment with pertussis toxin, indicating that the human ₃-AR couples to Gi as well as Gs. In contrast to cAMP, zinterol, L755507 and L748337 increased phosphorylation of Erk1/2 with very high potency (pEC₅₀ 10.9, 11.7 and 11.6). These compounds also stimulated phosphorylation of p38 MAPK, but with much lower potency than Erk1/2 (pEC₅₀ 5.9, 5.5 and 5.7 respectively). Pertussis toxin completely blocked Erk1/2 and p38 MAPK phosphorylation in response to L748337, demonstrating a requirement for Gi/o coupling, whereas L755507-stimulated p38 MAPK phosphorylation was not inhibited by pertussis toxin, and Erk1/2 phosphorylation was inhibited by only 30%. We found that high levels of cAMP interfered with agonist-activated p38 MAPK phosphorylation. L748337 increased extracellular acidification rate (ECAR) in the cytosensor microphysiometer with efficacy similar to zinterol and L755507, albeit with lower potency (pEC₅₀ 7.2 compared to zinterol, 8.1, and L755507, 8.6). The ECAR response to L748337 was largely via activation of p38 MAPK, demonstrated by 65% inhibition with RWJ67657. We conclude that the ₃-AR agonist L755507 couples to both Gs and Gi to activate adenylate cyclase and MAPK signaling, whereas the ₃-AR antagonist L748337 couples predominantly to Gi to activate MAPK signaling.

Key words: Adrenergic, Gi family, Gs family, cAMP, MAP Kinase