Degradation of leucine zipper-positive isoform of MYPT1 may contribute to development of nitrate tolerance.
OBJECTIVE
A depressed cGMP-dependent protein kinase (PKG) activity is implicated in nitrate tolerance. The present study determines whether the leucine zipper-positive (LZ+) isoform of myosin phosphatase target subunit 1 (MYPT1), a key target protein for PKG actions, is involved in the development of nitrate tolerance.


RESULTS
Nitrate tolerance in in vitro preparations was obtained by a 24 h incubation with nitroglycerin (NTG). Nitrate tolerance in in vivo preparations was obtained by subcutaneous injection of mice with NTG, and the aortas were used. Protein levels of total MYPT1, MYPT1 (LZ+), PP1Cdelta, myosin light chain (MLC), and phosphorylated MLC were determined by Western blot analysis. Isometric vessel tension was determined by an organ chamber technique. Protein levels of MYPT1 (LZ+), but not of PP1Cdelta, were significantly reduced in in vitro and in vivo nitrate-tolerant arteries. The decrease in the MYPT1 (LZ+) protein level of coronary artery was also induced by a nitric oxide donor and a cGMP analogue, which was prevented by the inhibitors of soluble guanylyl cyclase and PKG. The decrease in MYPT1 (LZ+) protein levels was not affected by the inhibitor of protein synthesis, but was prevented by the inhibitors of proteasomes. The diminished inhibition of dephosphorylation of MLC as well as the attenuated relaxation of porcine coronary artery and mouse aorta to NTG was improved by proteasome inhibitors.


CONCLUSIONS
This study demonstrates that a reduction in the protein level of MYPT1 (LZ+) is involved in nitrate tolerance. This may result in part from a proteasome-dependent degradation of MYPT1 (LZ+).
Dou
Dou
D
Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100191, People's Republic of China.
Ma
Huijuan
H
Zheng
Xiaoxu
X
Ying
Lei
L
Guo
Yixuan
Y
Yu
Xiaoxing
X
Gao
Yuansheng
Y
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D003331:Coronary Vessels / Q000187:drug effects / Q000502:physiology; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004361:Drug Tolerance / Q000502:physiology; D016350:Leucine Zippers / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D005996:Nitroglycerin / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034421:Sus scrofa; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology; D014665:Vasodilator Agents / Q000494:pharmacology*
D011500:Protein Synthesis Inhibitors; D014665:Vasodilator Agents; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; C510645:Ppp1r12a protein, mouse; D046988:Proteasome Endopeptidase Complex; D005996:Nitroglycerin; D006152:Cyclic GMP