Involvement of Protein Kinases on the Upregulation of Endothelin Receptors in Rat Basilar and Mesenteric Arteries

Abstract

Endothelin_B (ET_B) receptors are upregulated in experimental stroke or after 24 hrs of organ culture. This upregulation is manifested both as stronger contraction and as an increase in ET_B receptor messenger RNA (mRNA) levels. The present study was designed to evaluate the importance of protein kinases (c-Jun N-terminal kinase [JNK], protein kinase C [PKC], and extracellular signal-regulated kinase [ERK1/2]) in ET_B receptor upregulation after organ culture. Rat basilar and mesenteric arteries were incubated for 24 hrs in Dulbecco's modified Eagle's medium (DMEM) with or without the PKC inhibitor, RO-31–7549; the ERK1/2 inhibitor, SB386023; or the JNK inhibitor, SP600125, added 3, 6, or 12 hrs after initiation of incubation. Subsequently, vessel segments were mounted in myographs and the contractile responses to ET-1 and sarafotoxin 6c were studied. The ET_B and ET_A receptor mRNA levels were determined with a real-time polymerase chain reaction (PCR). The cellular localization and protein level of ET_B receptors were evaluated by immunohistochemistry. The PKC and ERK1/2 inhibitors attenuated the contraction induced by S6c in the basilar arteries more than in the mesenteric arteries. The efficiency of the inhibitors was proportional to the incubation time. Real-time PCR showed a decrease in the ET_B receptor mRNA levels in arteries treated with PKC or ERK inhibitors. The JNK inhibitor had a significant inhibitory effect on ET_B receptor upregulation in the basilar arteries. Immunohistochemistry revealed that the ET_B receptor upregulation occured in the smooth-muscle cells and that it had the same pattern as in the quantitative PCR. Our results show that the PKC, ERK1/2, and JNK are more important for the upregulation of contractile ET_B receptors in cerebral arteries compared with mesenteric arteries. ERK1/2 seems to be more important for the ET_B receptor upregulation, as compared with PKC and JNK. The evaluation of the time dependency suggests that the phenomenon can be reversed even after its initiation.