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. 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. Whereas treatment with Y‐27632 (1 μM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) 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, Emax of methacholine became Rho‐kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. 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. 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. British Journal of Pharmacology (2006) 147, 737–743. doi:10.1038/sj.bjp.0706665

Preincubation (30 min) of bovine tracheal smooth muscle with various concentrations (0.1, 1 and 10 μM) of fenoterol decreased isoprenaline-induced maximal relaxation (Emax) of methacholine-contracted preparations in a concentration dependent fashion, indicating desensitization of the β2-adrenoceptor. Preincubation with 1 μM of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) caused a small but significant decrease in isoprenaline-induced Emax, indicating activated PKC-mediated heterologous β2-adrenoceptor desensitization. To investigate the capacity of activated PKC to regulate homologous desensitization, we incubated the smooth muscle strips with the combination of both 1 μM PMA and 1 μM fenoterol. This combined treatment synergistically decreased the isoprenaline-induced maximal relaxation, as compared to the individual effects of PMA and fenoterol alone, indicating a common pathway for heterologous and homologous desensitization. Moreover, the specific PKC-inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl) maleimide (GF 109203X) markedly increased the potency and Emax of isoprenaline for all conditions used, including control conditions, and the synergistic effects of PMA and fenoterol were completely prevented. In conclusion, the present study demonstrates that homologous desensitization of the β2-adrenergic receptor can be enhanced by PKC activation. For the first time we have provided evidence that this concept is functionally operative in airway smooth muscle, and it may explain the reduced bronchodilator response to β2-adrenoceptor agonists in patients with asthma during a severe exacerbation.

markedly amplified by muscarinic agonist-induced PKC activation.

To examine the role of contractile agonist-induced activation of protein kinase C (PKC) in functional antagonism of airway smooth muscle contraction by β-adrenoceptor agonists, we examined the effects of the specific PKC-inhibitor GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl) maleimide) on isoprenaline-induced relaxation of bovine tracheal smooth muscle contracted by various concentrations of methacholine and histamine. In the absence of GF 109203X, the potency of isoprenaline (pD2) was gradually reduced at increasing methacholine- and histamine-induced smooth muscle tones, but the maximal relaxation (Emax) was decreased only at higher concentrations of methacholine. In the presence of GF 109203X, pD2 values were significantly increased for both methacholine- and histamine-induced contractions. Moreover, isoprenaline Emax values in the presence of high concentrations of methacholine were also increased. Although both methacholine- and histamine-induced contractions were slightly reduced by GF 109203X, the changes in isoprenaline pD2 could only partially be explained by reduced contractile tone. In contrast to isoprenaline, forskolin-induced relaxations were not affected by GF 109203X. The results indicate that PKC activation contributes to the reduced β-adrenergic responsiveness induced by methacholine and histamine, which may involve uncoupling of the β-adrenoceptor from the effector system. Since many mediators and neurotransmitters in allergic airway inflammation can activate PKC, this cross talk may be important in the reduced bronchodilator response of patients with severe asthma.