Ca2+-independent, inhibitory effects of cyclic AMP on Ca2+ regulation of phosphoinositide 3-kinase C2, Rho and myosin phosphatase in vascular smooth muscle
Mohammed Ali Azam, Kazuaki Yoshioka, Shinsuke Ohkura, Noriko Takuwa, Naotoshi Sugimoto, Koichi Sato, and Yoh Takuwa
Dept. Physiol., Grad. Med., Kanazawa
Univ.
ABSTRACT
We
have recently demonstrated in vascular smooth muscle (VSM)
that membrane depolarization by high KCl induces
Ca2+-dependent
Rho activation and myosin phosphatase (MLCP) inhibition
(Ca2+-induced
Ca2+-sensitization)
through the mechanisms involving phosphorylation of MYPT1
and CPI-17. In the present study, we investigated whether
and how cyclic AMP affected Ca2+-dependent
MLCP inhibiton by examining the effects of forskolin, cell
permeable dibutyryl cyclic AMP (dbcAMP) and isoproterenol.
Forskolin, but not its inactive analogue
1,9-dideoxyforskolin, inhibited KCl-induced contraction and
the 20 kDa myosin light chain (MLC) phosphorylation without
inhibiting Ca2+
mobilization in rabbit aortic VSM. DbcAMP mimicked these
forskolin effects. We recently suggested that
Ca2+-mediated
Rho activation is dependent on class II -isoform of
phosphoinositide 3-kinase (PI3K-C2). Forskolin inhibited
KCl-induced stimulation of PI3K-C2 activity. KCl-induced
membrane depolarization stimulated Rho in a manner
dependent on a PI3K but not PKC (protein kinase C), and
stimulated phosphorylation of MYPT1 at
Thr850
and CPI-17 at Thr38
in manners dependent on both PI3K and Rho-kinase, but not
PKC. Forskolin, dbcAMP and isoproterenol inhibited
KCl-induced Rho activation and phosphorylation of MYPT1 and
CPI-17. Consistent with these data, either forskolin,
isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor,
but not a PKC inhibitor, abolished KCl-induced
di-phosphorylation of MLC. These observations indicate that
cyclic AMP inhibits Ca2+-mediated
activation of the MLCP-regulating signaling pathway
comprising PI3K-C2, Rho, and Rho kinase in a manner
independent of Ca2+,
and point to the novel mechanism of the cyclic AMP actions
in the regulation of vascular smooth muscle contraction.