Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Hemolysate Preparation

Hemolysate was prepared as described previously.(22) Briefly, heparinized dog arterial blood was centrifuged at 2500 xg for 15 minutes at 4 °C and the supernatant aspirated. The erythrocyte rich precipitate was washed three times with sterile saline (saline/erythrocyte fraction ratio, 1:3) and lysed by ultrasonic waves. The particulate material was centrifuged at 15,000 xg for 90 minutes at 4 °C, and the supernatant (erythrocyte lysate) was collected and stored at -80 °. Concentration of oxyhemoglobin in the preparation of 100% hemolysate was 12.20 ± 0.93 mM (n = 5).

New Zealand white rabbits (n = 45) of either sex, 2-3 pound weight, were anesthetized with an intravenous injection of thiopental (20 mg/kg) and euthanitized by exsanguination. The basilar arteries were removed and cut into 3 mm rings in a dissecting chamber filled with modified Krebs-Henseleit bicarbonate solution, bubbled with 95% O² and 5% CO². No attempt was made to remove endothelial cells. The modified Krebs-Henseleit solution contained (mM): NaCl 120, KCl 4.5, MgSO4 1, NaHCO3 27, KH2PO4 1, CaCl2 2.5 and dextrose 10. All procedures were approved by the Animal Care and Use Committee at the University of Mississippi Medical Center.

The rings were suspended at 500 mg tension (Radnoti transducer, Radnoti Glass, Monrovia, CA) between stainless steel hooks in 10 ml water-jacketed tissue baths (Radnoti Glass, Monrovia, CA) filled with modified Krebs-Henseleit buffer bubbled with 95% O²/5% CO² at pH 7.4 and 37 °C. Rings were equilibrated for 90 minutes and bath solution was changed every 20 minutes. Tension was recorded continuously with a force-displacement transducer as described previously (15).

The tissues were divided into five groups and the following studies were performed. In the control group, concentration-dependent contractions were induced with hemolysate (0.01, 0.1, 1 and 10%) in the absence of inhibitors. In the treated groups, the rings were incubated for 30 min with MAPK kinase inhibitor PD 98059 (30 µM). Concentration-dependent contractions were produced with hemolysate in the presence of this inhibitor.

In another series studies, the rings were first contracted with hemolysate (10%) and, when a stable plateau contraction was maintained, PD 98059 (1-100 µM) was applied on the sustained contraction induced by hemolysate. Relaxation was calculated as a percentage of the maximum contraction to hemolysate.

The basilar arteries were removed from the brainstem and incubated with 10% hemolysate diluted in Krebs-Henseleit buffer. The arteries were treated for 1, 3, 5, 10, 30, 60 and 120 minutes, and then immediately frozen in the liquid nitrogen. The arteries were homogenized in (mM) 50 Tris-HCl pH 7.5, 100 NaCl, 5 EDTA, 1 PMSF and 100 µlof IGEPAL CA-630 for 20 minutes at 4 °C. The insoluble materials were removed by centrifugation at 13,000 xG for 10 minutes at 4°C. The samples (20 µg protein) were applied to 12% SDS-PAGE. After electrophoretic transfer of the separated polypeptides to nitrocellulose membrane, the membranes were blocked using 5% non-fat milk in Tween-PBS (PBS containing 0.1% Tween 20) for 1hour. The membranes were washed with Tween-PBS and incubated at room temperature for 2 hours in a 1:5000 dilution of mouse anti-MAPK antibodies (ERK1+2, monoclonal mouse antibody, Zymed Laboratories, San Francisco, CA)(13). Nitrocellulose membranes were later washed with Tween-PBS and incubated with 1:5000 dilution of sheep anti-mouse IgG antibody, linked with horseradish peroxidase. The enhanced chemiluminescence (ECL) system (Amersham, Buckinghamshire, England) was used for visualization of protein bands. The results were quantified by laser densitometry of the films and integrated whole band analysis (Molecular Dynamics, Image QuantTM, Sunnyvale, CA, USA).

PD-98059 was purchased from BIOMOL (Plymouth Meeting, PA). Other chemicals were purchased from Sigma (Sigma, St. Louis, MO).

Data are expressed as mean ± standard error of the mean. Statistical differences between the control and other groups were compared using one-way analysis of variance (ANOVA) and then Turkey-Kramer multiple comparison procedure (95% lower and upper confidence interval) if significant variance was found. A value of p < 0.05 was considered statistically significant.

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