A new signaling paradigm for serotonin: use of Crk-associated substrate in arterial contraction.
Crk-associated substrate (CAS), a 130-kDa adaptor protein, was discovered as a tyrosine kinase substrate of Src that was important to cellular motility and actin filament formation. As the tyrosine kinase Src is utilized by the 5-hydroxytryptamine (5-HT)(2A) receptor in arterial contraction, we tested the hypothesis that CAS was integral to 5-HT(2A) receptor-mediated vasoconstriction. Rat thoracic aorta was used as a model of the arterial 5-HT(2A) receptor. Western and immunohistochemistry analyses validated the presence of CAS in the aorta, and tissue bath experiments demonstrated reduction of contraction to 5-HT (13.5 +/- 5% control maximum) and the 5-HT(2) receptor agonist alpha-methyl-5-HT (6 +/- 2% maximum) by latrunculin B (10(-6) mol/l), an actin disruptor. In aorta contracted with 5-HT (10(-5) mol/l), tyrosine phosphorylation (Tyr410) of CAS was significantly increased (approximately 225%), and both contraction and CAS phosphorylation were reduced by the 5-HT(2A/2C) receptor antagonist ketanserin (3 x 10(-8) mol/l). Src is one candidate for 5-HT-stimulated CAS tyrosyl-phosphorylation as 5-HT promoted interaction of Src and CAS in coimmunoprecipitation experiments, and the Src tyrosine kinase inhibitor PP1 (10(-5) mol/l) abolished 5-HT-induced tyrosyl-phosphorylation of CAS and reduced 5-HT- and alpha-methyl-5-HT-induced contraction. Antisense oligodeoxynucleotides delivered to the aorta reduced CAS expression (33% control) and arterial contraction to alpha-methyl-5-HT (45% of control), independent of changes in myosin light chain phosphorylation. These data are the first to implicate CAS in the signal transduction of 5-HT.
Ogden
Kevin
K
Dept. of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824, USA.
Thompson
Janice M
JM
Hickner
Zachary
Z
Huang
Taoying
T
Tang
Dale D
DD
Watts
Stephanie W
SW
D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D050719:Crk-Associated Substrate Protein / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology; D007650:Ketanserin / Q000494:pharmacology; D008297:Male; D011189:Potassium Chloride / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D044402:Receptor, Serotonin, 5-HT2A / Q000502:physiology; D012701:Serotonin / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D053778:Thiazolidines / Q000494:pharmacology; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology*
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D019086:Bridged Bicyclo Compounds, Heterocyclic; D050719:Crk-Associated Substrate Protein; D004791:Enzyme Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D012333:RNA, Messenger; D044402:Receptor, Serotonin, 5-HT2A; D017366:Serotonin Receptor Agonists; D053778:Thiazolidines; C042390:alpha-methylserotonin; D012701:Serotonin; D011189:Potassium Chloride; D007650:Ketanserin; C037068:latrunculin B