Differential Rho-kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction

1 In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. 2 To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase ( an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and - influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. 3 Whereas treatment with Y-27632 ( 1 mu M) significantly decreased potency (pEC(50)) for all agonists, maximal contraction (E-max) was reduced by 23.4 +/- 2.8 and 50.4 +/- 7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, E-max of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. 4 Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and - influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. 5 In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.