Impaired agonist-dependent myosin phosphorylation and decreased RhoA in rat portal hypertensive mesenteric vasculature.
The purpose of the present study was to examine the effects of portal hypertension on agonist-induced myosin phosphorylation and RhoA expression in vascular smooth muscle. A possible link to cAMP-dependent events was also examined. Portal hypertension was produced by stenosis of the portal vein. Vessel segments were treated with or without 50 microM of the PKA inhibitor Rp-cAMPS for 30 min and subsequently stimulated with 10(-4) M phenylephrine. Myosin regulatory light-chain phosphorylation was detected by immunoblotting. Total RNA from first-order mesenteric arteries and portal veins was isolated and amplified by RT-PCR using RhoA and GAPDH primers. RhoA protein expression was also measured in first-order mesenteric arteries using Western blot analysis. Myosin phosphorylation in maximally stimulated first-order mesenteric arteries was significantly lower in portal hypertensive animals (19.9 +/- 2.86%) when compared with sham-operated control (43.8 +/- 3.53%). Inhibition of PKA selectively increased myosin phosphorylation to 34.7 +/- 4.18%. Rp-cAMPS did not affect the phosphorylation of the portal veins or superior mesenteric arteries. RhoA mRNA and membrane-associated RhoA protein expression in portal hypertensive first-order mesenteric arteries were significantly lower when compared with controls. Acute inhibition of PKA had no effect on RhoA mRNA expression. However, it restored membrane-associated RhoA protein expression in portal hypertensive vessels to control levels. The results suggest that reductions in membrane-associated RhoA expression, which appear to be regulated by cAMP-dependent events, lead to reduced myosin phosphorylation and may underlie the reduced vasoconstrictor effectiveness in the resistance vasculature of portal hypertensive intestine.
Zhang
Hai-Ying
HY
Dean, Graduate School Professor of Pharmacology, Physiology & Therapeutics, Univ. of North Dakota, 414 Twamley Hall, Grand Forks, ND 58202, USA.
Shirasawa
Yuichi
Y
Chen
Xuesong
X
Yu
Hong
H
Benoit
Joseph N
JN
D000818:Animals; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D006975:Hypertension, Portal / Q000378:metabolism*; D008297:Male; D008638:Mesenteric Arteries / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013873:Thionucleotides / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D018994:Myosin Light Chains; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D020742:rhoA GTP-Binding Protein