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NANC neuron mediated relaxation of murine gastric fundus smooth muscle: Phosphorylation of MYPT1 on T696 (ROK site) and S695 (PKG site) are mutually exclusive
Author(s) -
Pfitzer Gabriele,
Hunger Mona S.,
Puetz Sandra,
Metzler Doris,
Lubomirov Lubomir T,
Somlyo Avril V.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1164.1
Subject(s) - phosphorylation , dephosphorylation , chemistry , microbiology and biotechnology , phosphatase , biology
Myosin phosphatase is the target of activating and relaxing pathways which modulate its activity by phosphorylation of its regulatory subunit, MYPT1, at several sites, and by telokin. Here, we investigated whether phosphorylation of MYPT1‐S695 is involved in relaxation of murine gastric fundus by NANC neurons known to act through cAMP/cGMP. Endothelin‐1 (ET‐1) preconstricted SM strips were relaxed by electrical field stimulation (EFS, 10 Hz, 37°C, blocked adrenergic and cholinergic responses) and shock frozen for western blot analysis with phosphospecific antibodies at desired time points. EFS induced relaxation (half time ~8s) was preceded by dephosphorylation of MLC‐20 (S19) and of MYPT1 (pMYPT1) at T696/T853 indicating de‐inhibition of MLCP. Dephosphorylation of T696 preceded the EFS‐induced increase in pMYPT1‐S695. During force recovery (half time ~150s) pMYPT1‐S695 was dephosphorylated within 30s whereas re‐phosphorylation of T696/T853 paralleled force recovery. Relaxation was associated with phosphorylation of telokin and ablation of telokin reduced relaxation by ~25% (at 2 Hz).To conclude, our results indicate that phosphorylation of S695 is not involved in the initiation of PKA/PKG mediated relaxation but may prevent re‐phosphorylation of T696. Our results are consistent with the notion that phosphorylation of T696/S695 is mutually exclusive. Funding: German Research Foundation.