Immediate early gene expression in vascular smooth-muscle cells synergistically induced by hemolysate components.
OBJECTIVE
Vasospasm after subarachnoid hemorrhage is associated with changes in modulators of vascular tone in the arterial wall and is related to the presence of erythrocyte hemolysate in the subarachnoid space. The purpose of this study was to determine the compounds in erythrocyte hemolysate that are responsible for changing smooth-muscle cell gene expression.


METHODS
Rat aorta smooth-muscle cells were exposed to erythrocyte hemolysate in vitro and the effects on immediate early gene messenger (m)RNA levels were determined by competitive reverse transcriptase-polymerase chain reaction. Message levels for c-fos, jun B, and c-jun were increased in the presence of hemolysate, reaching maximum expression between 30 and 60 minutes, whereas the level of jun D mRNA was unaffected. Increasing doses of hemolysate caused greater expression of c-fos and jun B, but not c-jun. Adenosine triphosphate and hemoglobin, possible spasmogens present in hemolysate, caused much smaller and more rapid increases in c-fos expression than whole hemolysate. Size fractionation showed that all of the c-fos mRNA-inducing activity of hemolysate was recovered with molecules greater than 6 kD. Following separation of hemolysate proteins by hydrophobic interaction chromatography, only one of the three fractions had partial activity. Recombining the three fractions, however, yielded greater c-fos activation than any combination of two.


CONCLUSIONS
Multiple high-molecular-weight components present in erythrocytes have synergistic effects on gene expression in smooth-muscle cells. The differences in patterns of gene induction suggest that multiple signaling pathways are activated.
Wang
X
X
Department of Surgery, Pritzker School of Medicine, University of Chicago, Illinois 60637, USA.
Marton
L S
LS
Weir
B K
BK
Macdonald
R L
RL
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D005591:Chemical Fractionation; D005786:Gene Expression Regulation / Q000502:physiology*; D017781:Genes, Immediate-Early / Q000235:genetics*; D016762:Genes, fos / Q000235:genetics; D006461:Hemolysis / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation
D012333:RNA, Messenger