Importance of ERK1/2 in upregulation of endothelin type B receptors in cerebral arteries

Abstract

This study examines the importance of mitogen-activated protein kinases (MAPKs) in upregulation of endothelin type B (ETB) receptors. Rat middle cerebral arteries (MCAs) were incubated for 24 h with or without kinase inhibitors. Vessel segments were mounted in myographs and the contractile responses to endothelin-1 (ET-1; ETA and ETB receptor agonist) and sarafotoxin 6c (S6c; ETB receptor agonist) were studied. We used real-time PCR to measure the receptor mRNA levels. An ELISA assay showed the activation of ERK1/2 kinases after 3 h. Immunohistochemistry revealed the presence of ETB receptors on the vessels. After organ culture, S6c induced vasoconstriction. Incubation with the MEK/ERK inhibitors U0126 and SB386023 diminished the contractile response to S6c. The p38 MAPK inhibitor SB239063 did not affect the S6c-induced contraction. The ET-1-induced vasoconstriction was increased after incubation with SB386023 or SB239063, while unaffected by U0126. The ETB receptor mRNA levels were diminished by SB386023 and U0126. The ETA receptor mRNA levels were unaffected. The levels of activated ERK1/2 kinases were significantly higher after 3 h of organ culture as compared to fresh vessels. The level of ETB receptor protein on the smooth muscle cells of the MCA, visualised by immunohistochemistry, was somewhat diminished by SB386023. Our results show that the ERK1/2 MAPK is important in the upregulation of contractile ETB receptors in MCA after organ culture. Since there is a similar upregulation in models of focal ischaemia and subarachnoid haemorrhage, this may be an important pathophysiological event.

Figure 1

Contractile responses elicited by cumulative application of S6c in rat MCA incubated for 24 h (control) and rat MCA incubated for 24 h with the kinase inhibitors U0126 (10 μM) and SB386023 (10 μM). Each point represents mean values±s.e.m., n=11–15. For statistical analysis, see Table 1.

Figure 2

Effect of the kinase inhibitors U0126 (10 μM), SB386023 (10 μM) and SB239063 (10 μM) on the mRNA levels of the ET_A and ET_B receptors after 24 h of organ culture. Data were obtained by real-time PCR and the results are expressed as mean values±s.e.m. relative to EF-1 mRNA levels, n=3–7. ^**P<0.01.

Figure 3

Levels of activated ERK1/2 in fresh vessels and in vessels incubated for 3 h. Data are expressed as per cent of control, n=4. ^*P<0.05.

Figure 4

Sections from the rat MCA showing ET_B immunoreactivity in the smooth muscle cell layer in (a) fresh segments, (b) after organ culture and (c) after organ culture in the presence of the raf inhibitor SB386023 (10 μM).