Role of calcium. ERK-MAP kinase, and the Rho-kinase in the thromboxane receptor-mediated vasoconstriction in the porcine isolated ear artery Williams R. & Roberts R. E. Institute of Cell Signalling, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH.

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Vasoconstriction mediated through the thromboxane receptor is dependent upon influx of extracellular calcium (Tosun et al., 1998). However, recent studies in the porcine ear artery have demonstrated that the thromboxane-mimetic U46619 can cause vasoconstriction in the absence of calcium (Bhattacharya & Roberts). The aims of this study were to determine the role of calcium in U46619-mediated vasoconstriction in the porcine ear artery, and to determine the role, if any, of the ERK-MAP kinase pathway, or Rho kinase.

Ears from pigs of both sexes were obtained from a local abattoir. Ear arteries were dissected into 5 mm segments and mounted in a tissue bath containing Krebs-Henseleit buffer maintained at 37^oC, and gassed with 95% O₂/ 5% CO₂. Contractions were measured using an isometric force transducer linked to a Grass-Telefactor polygraph recorder. After reproducible responses to 60 mM KCl were obtained, responses to the thromboxane-mimetic U46619 were obtained. In experiments in the absence of calcium, the Krebs-Henseleit buffer was replaced with calcium-free Krebs-Henseleit in which the calcium was replaced with 2 mM EGTA. In some experiments 50 µM PD98059 (an inhibitor of ERK activation, Alessi et al., 1995), or 10 µM Y27632 (a Rho kinase inhibitor, Davies et al., 2000) were added 1 hr prior to the addition of U46619.

U46619 caused a concentration-dependent contraction of the porcine ear artery, with a maximum response of 78 ± 20 % of the 60 mM KCl response (mean ± s. e. mean, n=5). In the absence of calcium U46619 caused a contraction, but this was reduced to 21 ± 7 % (n=5). In the presence of calcium, 50 µM PD98059 reduced the maximum U46619 response from 78 ± 5 % to 58 ± 5 % (p<0.01, Student's 2-tailed, unpaired t-test, n=8). In the absence of calcium, PD98059 had no effect on the U46619 response (response to 1 µM U46619 8 ± 1 % in the absence of PD98059, compared to 13 ± 3 % in the presence of PD98059, n=6). Pre-incubation w ith the Rho kinase inhibitor Y27632 (10 µM) in the presence of calcium reduced the maximum U46619 response from 105 ± 8 % to 41 ± 12 % (p<0.05, Student's 2-tailed, unpaired t-test, n=9). In the absence of calcium, Y27632 completely inhibited the response to 1 µM U46619. These data demonstrate that U46619-mediated contractions in the porcine ear artery occur through calcium-dependent and independent pathways. The calcium dependent pathway involves both the ERK-MAP kinase pathway to a certain degree, and the Rho kinase pathway to a greater degree. On the other hand, the ERK-MAP kinase pathway is not involved in the calcium-independent pathway, but Rho kinase plays a major role.

Supported by The Wellcome Trust.

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