Background and Purpose--Previous studies have shown that contractile endothelin type B (ET_B) receptors are upregulated in cerebral arteries after experimental focal cerebral ischemia. The aim of this study was to examine the upregulation of contractile ET_B receptors in cerebral arteries after organ culture and to elucidate the intracellular pathways involved.

Methods--Rat middle cerebral arteries (MCAs) were incubated with or without inhibitors. The vessels were mounted in myographs, and the contractile responses to endothelin-1 (ET-1) (ET_A and ET_B receptor agonist) and sarafotoxin 6c (ET_B receptor agonist) were measured. Levels of ET_B receptor mRNA were measured with real-time polymerase chain reaction.

Results--In fresh MCA, sarafotoxin 6c had no contractile effect. However, after organ culture, a strong concentration-dependent contraction was induced. ET-1 produced a strong contraction, in which the E_max was unaffected by organ culture but the EC₅₀ was decreased with time. The sarafotoxin 6c-induced contraction after 24 hours of organ culture was attenuated by the transcriptional inhibitor actinomycin D and the translational inhibitor cycloheximide as well as the protein kinase C inhibitor Ro-31-8220. Real-time polymerase chain reaction revealed that the mRNA levels of the ET_B receptor were increased after organ culture compared with fresh vessels. Actinomycin D and Ro-31-8220 diminished the enhanced mRNA levels considerably.

Conclusions--The results suggest that, in fresh MCA, the ET_A receptor is the most prominent subtype, while after organ culture ET_B receptors also contribute to the contraction. This upregulation is due to de novo transcription of receptors. Protein kinase C is involved in the upregulation as Ro-31-8220 attenuates the contraction and the mRNA increase.