Question: 

NANC induced relaxation of gastric fundus is mediated by cGMP which is known to disinhibit myosin phosphatase (MLCP) in vascular smooth muscle by dephosphorylating its regulatory subunit, MYPT1 at Thr696 and Thr853(Kitazawa et al., 2009). By simultaneously determining force and MYPT1 phosphorylation in gastric fundus smooth muscle strips we investigated the contribution of respectively activation and inhibition of MLCP to NANC induced relaxation and subsequent force recovery.

Methods: 

Endothelin-1 preconstricted endothelin-1 strips were relaxed with electrical field stimulation (EFS, 10 Hz, for 30 s). At distinct times during the relaxation/force recovery cycle phosphorylation of MLC-20, of MYPT1 at Ser695, Thr696, and Thr853, and of telokin was determined in Western blots with phosphospecific antibodies.

Results: 

Relaxation amounting to 80% was associated with dephosphorylation of MLC-20 by 75% and of the inhibitory phosphorylations of MYTP1 at Thr696/Thr853 by 50% and 60% respectively (n=5–10). Phosphorylation of the PKG sites of MYPT1 (Ser695) and of the putative MLCP activator, telokin increased 3 and 1.7 fold respectively. Termination of EFS initiated force recovery after a delay of ~1 min. Force recovered completely within 9 min while MLC-20 returned to ~60% of its initial level. Force recovery was preceded by rephosphorylation of MYPT1-Thr696/Thr853 and dephosphorylation of MYPT1-Ser695 and telokin. Dephosphorylation of MYPT1-Ser695 preceded rephosphorylation of MYPT1-Thr696 corroborating the hypothesis that phosphorylation of Thr696 and Ser695 is mutally exclusive.

Conclusions: 

In conclusion we propose that NANC relaxation is at least in part mediated by an increased activity of MLCP whereas force recovery can be ascribed to re-inhibition of MLCP mediated by respective changes in MYPT1 phosphorylation.