No abstract

The inhibition of myosin light chain phosphatase (MLCP) enhances smooth muscle contraction at a constant [Ca2+]. There are two components, myosin-binding subunit of MLCP (MBS) and CPI17, thought to be responsible for the inhibition of MLCP by external stimuli. The phosphorylation of MBS at Thr-641 and of CPI17 at Thr-38 inhibits the MLCP activity in vitro. Here we determined the changes in the phosphorylation of MBS and CPI17 after agonist stimulation in intact as well as permeabilized smooth muscle strips using phosphorylation-site-specific antibodies as probes. The CPI17 phosphorylation transiently increased after agonist stimulation in both alpha-toxin skinned and intact fibres. The time course of the increase in CPI17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC) phosphorylation. The increase in CPI17 phosphorylation was significantly diminished by Y27632, a Rho kinase inhibitor, and GF109203x, a protein kinase C inhibitor, suggesting that both the protein kinase C and Rho kinase pathways influence the change in CPI17 phosphorylation. On the other hand, a significant level of MBS phosphorylation at Thr-641, an inhibitory site, was observed in the resting state for both skinned and intact fibres and the agonist stimulation did not significantly alter the MBS phosphorylation level at Thr-641. While the removal of the agonist markedly decreased MLC phosphorylation and induced relaxation, the phosphorylation of MBS was unchanged, while CPI17 phosphorylation markedly diminished. These results strongly suggest that the phosphorylation of CPI17 plays a more significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle than MBS phosphorylation in the Ca2+-independent activation mechanism of smooth muscle contraction.

No abstract

Dephosphorylation of the two key regulatory factors of myosin light chain phosphatase (MLCP), CPI17 and MBS (myosin binding subunit) of MLCP was studied. While Thr38 phosphorylated CPI17 is quite susceptible to protein phosphatases, phosphorylated MBS was highly resistant to dephosphorylation. Type 2A, 2B and 2C protein phosphatases (PP2A, PP2B and PP2C), but not type 1 (PP1), dephosphorylated CPI17. The majority of the CPI17 phosphatase activity in smooth muscle was attributed to PP2A and PP2C. Phospholipids inhibited dephosphorylation of MBS and arachidonic acid (AA) inhibited PP2A activity against both MBS and CPI17, raising the possibility that AA favors the preservation of active MLCP. Consistently, while the phosphorylation of CPI17 was promptly decreased when the agonist was removed, the phosphorylation of MBS was unchanged in intact smooth muscle fiber. The results suggest that MBS phosphorylation mediated regulation of MLCP is not suitable for regulating rapid change in myosin phosphorylation. On the other hand, phosphorylated CPI17 is readily dephosphorylated thus likely to play a role in regulating fast phosphorylation^dephosphorylation cycle in cells. ß

We concluded that beta3-ARs exist in rat detrusor smooth muscle based on both pharmacological and molecular biological studies. Based on these findings, beta-ARs of rat detrusor smooth muscle are considered to be mixed populations consisting of three subtypes which play an important role in relaxing smooth muscle in response to catecholamines.