Role of Mitogen-Activated Protein Kinase in the Contractions of Rabbit Basilar Artery Induced by Hemolysate, Oxyhemoglob
It has been suggested that tyrosine kinases are involved in smooth muscle contraction [1 ] induced by hemolysate or other spasmogens and might be involved in cerebral vasospasm. Mitogen-activated protein kinase (MAPK), a tyrosine kinase substrate, was act
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Role of Mitogen-Activated Protein Kinase in the Contractions of Rabbit Basilar Artery Induced by Hemolysate, Oxyhemoglobin, and Bloody CSF A. Y. Zubkov, K. Shadon Rollins, B. McGehee, A. D. Parent, and J. H. Zhang Introduction
MAPKAssay
It has been suggested that tyrosine kinases are involved in smooth muscle contraction [1] induced by hemolysate or other spasmogens and might be involved in cerebral vasospasm. Mitogen-activated protein kinase (MAPK), a tyrosine kinase substrate, was activated by oxyhemoglobin (OxyHb) [5] and hemolysate [9] and contributed to cerebral arterial contraction. MAPK inhibitor PD98059 reduced the contraction of a rabbit basilar artery by hemolysate [9] and endothe1in-l [8]. In this study, three spasmogens, hemolysate, OxyHb, and CSF, were used in the contraction and MAPK immunoprecipitation, and the inhibitory effect of a novel and potent MAPK inhibitor U0126 was tested.
The basilar arteries were removed from the brain and were exposed to hemolysate (10%), oxyhemoglobin (10- 4 M), or bloody CSF (30%) for 5 minutes. In another group arteries were pretreated with UOl26 (30 ~M) for 30 minutes and then were exposed to hemolysate (10%), oxyhemoglobin (10- 4 M), or bloody CSF (30%) for 5 minutes. The MAPK activity was studied according to the method described in the MAPK assay (New England Biolabs, Inc., Beverly MA). The density of the bands were quantified with Quantity One software (Biorad, Hercules, CA)
Keywords: Mitogen-activated protein kinase; oxyhemoglobin; vasospasm.
Results
Materials and Methods Materials
UOl26 was purchased from Promega (Madison, WI). AntiMAPK (ERKI/ERK2) antibodies were purchased from Zymed Laboratories (San Francisco, CA). Other chemicals were purchased from Sigma (St. Louis, MO). Hemolysate, Oxyhemoglobin, and Bloody CSF Preparation
Hemolysate [4], OxyHb [3] and CSF [7] were prepared as previously described. Isometric Tension
The rabbit basilar artery rings were preincubated with UOl26 (30 ~M) for 30 minutes, and then dose dependent responses to hemolysate (0.1-10%), OxyHb (0.1-100 ~M), or CSF (30%) were studied. In the second group, the arterial samples were contracted with hemolysate (10%), OxyHb (10 ~M), or CSF (30%) and then a dose dependent relaxation was initiated with UOl26 (1-100 ~M). Each ring was used with only one agonist to avoid cross-reaction.
Data Analysis
Data are expressed as the mean ± the standard error of the mean. Statistical differences between the control and other groups were compared using a one-way analysis of variance (ANOYA) and then the Tukey-Kramer multiple comparison procedure if significant variance was found. A probability value (P) of less than 0.05 was considered statistically significant.
Effects of U0126 on the Arterial Contraction
Pre-incubation of the basilar artery rings with U0126 (30 11M) for 30 minutes significantly reduced the contractions to hemolysate and OxyHb. At a higher concentration, U0126 (100 11M) completely abolished the contraction induced by hemolysate (Fig. lA, P < 0.05-0.01, ANOVA) and significan
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