Mitogen-activated protein kinase mediation of hemolysate-induced contraction in rabbit basilar artery

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Object. Mitogen-activated protein kinase (MAPK) is an important signaling factor in vascular proliferation and contraction, which are the two features of cerebral vasospasm that follow subarachnoid hemorrhage. The authors studied the possible involvement of MAPK in hemolysate-induced signal transduction and contraction in rabbit basilar artery (BA).

Methods. Isometric tension was used to record the contractile response of rabbit BA to hemolysate, and Western blots were obtained using antibodies for MAPK.

The following results are reported. 1) Hemolysate produced a concentration-dependent contraction of rabbit BA; however, preincubation of arteries with the MAPK kinase (MEK) inhibitor PD-98059 markedly reduced this contraction. The administration of PD-98059 also relaxed, in a concentration-dependent fashion, the sustained contraction induced by 10% hemolysate. 2) The Janus tyrosine kinase 2 inhibitor AG-490, preincubated with arterial rings, reduced the contractile response to hemolysate but failed to relax the sustained contraction induced by this agent. The Src-tyrosine kinase inhibitor damnacanthal and the phosphatidylinositol 3—kinase inhibitor wortmannin failed to reduce hemolysate-induced contraction. 3) Hemolysate produced a time-dependent elevation of MAPK immunoreactivity as seen on Western blots of rabbit BA. The MAPK was enhanced 1 minute after hemolysate exposure and the effect reached maximum levels at 5 minutes. The immunoreactivity of MAPK decayed slowly over time, but the level of this kinase was still higher than the basal level, even at 2 hours after exposure to hemolysate. Preincubation of arteries with the MEK inhibitor PD-98059 abolished the effect of hemolysate on MAPK immunoreactivity.

Conclusions. Hemolysate produced contraction of rabbit BA, possibly by activation of MAPK, and therefore MAPK inhibitors may be useful in the treatment of cerebral vasospasm.

Article Information

Address reprint requests to: Alexander Y. Zubkov, M.D., Department of Neurosurgery, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216–4505. email: azubkov@neurosurgery.umsmed.edu.

© AANS, except where prohibited by US copyright law.

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Figures

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    Graphs displaying the inhibitory effects of the PD-98059, AG-490, wortmannin, and damnacanthal. Arterial rings were contracted by exposure to hemolysate in the absence of PD-98059, AG-490, wortmannin, and damnacanthal in control rings. In the treated groups, 30 µM each of PD-98059, AG-490, wortmannin, and damnacanthal were preincubated in separate chambers with arterial rings for 30 minutes before the rings were contracted with hemolysate. A: Preincubation with PD-98059 significantly inhibited contraction to 1% hemolysate (0.60 g and 0.53 g, control compared with 1% hemolysate; p = 0.018) and to 10% of hemolysate (0.89 g and 0.60 g, control compared with 10% hemolysate; p = 0.002). B: Preincubation with AG-490 abolished the contraction induced by 1% hemolysate (p < 0.05) but did not reduce the contraction from 10% hemolysate (C and D). The concentration-dependent contraction on addition of hemolysate to the arterial rings inhibited with wortmannin or damnacanthal were not different in the control group. The “n” in parentheses indicates the number of arterial rings studied. *p < 0.05; **p < 0.01 (ANOVA).

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    Graphs displaying the relaxant effects of PD-98059, AG-490, wortmannin, and damnacanthal on sustained contraction induced by 10% hemolysate. When a stable contraction was obtained, PD-98059, AG-490, wortmannin, and damnacanthal were applied to relax the contracted arterial rings. Only one antagonist was used with each arterial ring to prevent cross-reaction. A: Application of PD-98059 significantly relaxed the contracted artery in a dose-dependent manner (p = 0.036). B–D: Application of wortmannin, AG-490, and damnacanthal failed to reverse contraction induced by hemolysate. The “n” in parentheses indicates the number of arterial rings studied. *p < 0.05 (ANOVA).

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    Time course of activation of MAPK after stimulation with 10% hemolysate. Upper: Western blot showing MAPK activation within 1 minute, peak by 5 to 10 minutes, and sustained activity above the baseline for at least 120 minutes. Lower: Graphs of the laser density of the protein bands showing a quantified amount of activation of MAPK on different time points after stimulation. The peak of activation was at 5 minutes and activity of MAPK was maintained above the baseline level for at least 120 minutes. 42 = p42ERK; 44 = p44ERK.

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    Upper: Western blot demonstrating the effect of PD-98059 on activation of MAPK after stimulation with 10% hemolysate. Incubation of the control vessel with PD-98059 did not significantly decrease the level of basal activity of MAPK. The effect of 10% hemolysate on MAPK was abolished in arterial rings preincubated with PD-98059 for 30 minutes. Lower: Graph displaying the laser density of the protein band after preincubation of arterial rings with PD-98059. A nonsignificant decrease in the basal level of MAPK activity appeared after the preincubation. Preincubation with PD-98059 for 30 minutes abolished the effect of hemolysate on MAPK (p = 0.041). 42 = p42ERK; 44 = p44ERK; H = vessels treated with 10% hemolysate for 5 minutes; H + PD = vessels preincubated with PD-98059 for 30 minutes, and then treated with 10% hemolysate for 5 minutes; S = saline-treated group of arteries; S + PD = vessels preincubated with PD-98059 for 30 minutes and then treated with saline for 5 minutes.

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    Diagram showing possible pathways of MAPK activation. Activation of growth factor receptors (receptor tyrosine kinases) by EGF or PDGF leads to Src activation, assisted with adapter proteins Grb2 or Shc and exchange factor son of sevenless (SOS). The Src stimulates Ras, which activates Raf-1. Activation of G protein—coupled receptors leads to activation of PLC (PLCβ), generating IP3 and diacylglycerol (DAG), which are involved in intracellular Ca++ mobilization and PKC activation, respectively. The Ca++ activates Pyk2, which is involved in the pathogenesis of cerebral vasospasm, and tyrosine kinase may activate MAPK through Src or Ras protein phosphorylation. Activation of PKC may phosphorylate either Raf-1 or Pyk2. Phosphatidylinositol 3—kinase (PI 3K) may be involved in activation of the Ras protein through the Src tyrosine kinase protein family. Activation of Src leads to the activation of Janus tyrosine kinase 2 (JAK2), which is important in growth hormone activation. Activation of the Ras protein leads to activation of Raf-1, which phosphorylates MEK. The function of MEK is to phosphorylate both threonine and tyrosine regulatory sites in MAPK (ERK 1/2). The MAPK plays an important role in cell proliferation through activation of transcription factors and target genes and may phosphorylate caldesmon, which plays an important role in prolonged smooth-muscle contraction. Activation of MEK by Raf-1 is inhibited by PD-98059 and thus inhibits MAPK activation. Wortmannin, AG-490, damnacanthal, genistein, and staurosporine inhibit phosphatidylinositol 3—kinase, Janus tyrosine kinase 2, the Src family, Pyk2, and PKC, respectively. ATP = adenosine triphosphate; G = GTP-binding protein; Grb2 = growth factor receptor—bound protein 2; OxyHb = oxyhemoglobin; R = receptor; Ras = p21Ras.

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