Mechanisms underlying a decrease in KCl-induced contraction after long-term serum-free organ culture of rat isolated mesenteric artery.
Organ culture of blood vessel is a better technique to investigate the long-term effects of drugs. However, some functional changes may occur from freshly isolated vessel (Fresh). Mammalian/mechanistic target of rapamycin (mTOR) regulates smooth muscle differentiation and Ca(2+) mobilization. We thus investigated mechanisms of alteration in smooth muscle contractility after serum-free organ culture focusing on mTOR. Rat isolated mesenteric arteries were cultured for 5 days without (0% serum) or with rapamycin. In 0% serum, absolute contraction by KCl significantly decreased from Fresh, which was significantly rescued by rapamycin. In 0% serum, mTOR expression significantly increased from Fresh, which was significantly rescued by rapamycin. In 0% serum, expression of myocardin, a key regulator of smooth muscle differentiation markers, significantly decreased from Fresh, which was significantly rescued by rapamycin. However, the decrease in expression of contractile proteins, including SM22α and calponin, was not changed by rapamycin. Basal phosphorylation of calmodulin-dependent protein kinase II significantly increased in 0% serum, which was significantly rescued by rapamycin. In 0% serum, absolute contraction by caffeine significantly decreased from Fresh, which was significantly rescued by rapamycin. In conclusion, expression of mTOR increased during serum-free organ culture of rat isolated mesenteric artery for 5 days, which may be at least partly responsible for the decreased smooth muscle contractility perhaps due to the decrease in the stored Ca(2+) in smooth muscle.
Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Aomori 034-8628, Japan.
Tomoka
T
Morita
Muneyoshi
M
Okada
Hideyuki
H
Yamawaki
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D008297:Male; D008638:Mesenteric Arteries / Q000502:physiology*; D009924:Organ Culture Techniques; D011189:Potassium Chloride / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D020123:Sirolimus / Q000494:pharmacology; D014661:Vasoconstriction / Q000187:drug effects*
D016895:Culture Media, Serum-Free; D002110:Caffeine; D011189:Potassium Chloride; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D020123:Sirolimus