Nitric oxide-induced biphasic mechanism of vascular relaxation via dephosphorylation of CPI-17 and MYPT1.
Nitric oxide (NO) from endothelium is a major mediator of vasodilatation through cGMP/PKG signals that lead to a decrease in Ca(2+) concentration. In addition, NO-mediated signals trigger an increase in myosin light chain phosphatase (MLCP) activity. To evaluate the mechanism of NO-induced relaxation through MLCP deinhibition, we compared time-dependent changes in Ca(2+), myosin light chain (MLC) phosphorylation and contraction to changes in phosphorylation levels of CPI-17 at Thr38, RhoA at Ser188, and MYPT1 at Ser695, Thr696 and Thr853 in response to sodium nitroprusside (SNP)-induced relaxation in denuded rabbit femoral artery. During phenylephrine (PE)-induced contraction, SNP reduced CPI-17 phosphorylation to a minimal value within 15 s, in parallel with decreases in Ca(2+) and MLC phosphorylation, followed by a reduction of contractile force having a latency period of about 15 s. MYPT1 phosphorylation at Ser695, the PKG-target site, increased concurrently with relaxation. Phosphorylation of RhoA, MYPT1 Thr696 and Thr853 differed significantly at 5 min but not within 1 min of SNP exposure. Inhibition of Ca(2+) release delayed SNP-induced relaxation while inhibition of Ca(2+) channel, BK(Ca) channel or phosphodiesterase-5 did not. Pretreatment of resting artery with SNP suppressed an increase in Ca(2+), contractile force and phosphorylation of MLC, CPI-17, MYPT1 Thr696 and Thr853 at 10 s after PE stimulation, but had no effect on phorbol ester-induced CPI-17 phosphorylation. Together, these results suggest that NO production suppresses Ca(2+) release, which causes an inactivation of PKC and rapid CPI-17 dephosphorylation as well as MLCK inactivation, resulting in rapid MLC dephosphorylation and relaxation.
Kitazawa
Toshio
T
Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA. Kitazawa@bbri.org
Semba
Shingo
S
Huh
Yang Hoon
YH
Kitazawa
Kazuyo
K
Eto
Masumi
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D005246:Feedback / Q000502:physiology; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D009119:Muscle Contraction / Q000502:physiology*; D009124:Muscle Proteins; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D014664:Vasodilation / Q000502:physiology
D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; C107507:PPP1R14A protein, human; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human; D006152:Cyclic GMP; D002118:Calcium