Signal transduction pathways involved in U46619- and vasopressin-mediated enhancement of 2 adrenoceptor-induced vasoconstriction in the porcine ear artery Bhattacharya B. & Roberts R. E. Institute of Cell Signalling, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH.

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We have previously shown that the thromboxane-mimetic U46619, and vasopressin enhance the ₂ adrenoceptor-mediated vasoconstriction in the porcine ear artery through ERK-dependent (Bhattacharya &Roberts 2003a, in press) and independent pathways (Bhattacharya & Roberts 2003b, in press) respectively. The aim of this study was to elucidate the different signalling components involved in these two apparently different pathways.

Ears from pigs of both sexes were obtained from a local abattoir. The ear arteries were dissected into 5mm ring segments and suspended in an isolated organ bath containing Krebs-Henseleit buffer maintained at 37°C and gassed with 95%O₂ and 5% CO₂. Contractions were measured using an isometric force transducer linked to PCLab Data acquisition system. The selective ₂ adrenoceptor agonist UK14304 (1µM) was added in the presence or absence of U46619 or vasopressin (10-20% of 60mM KCl response). The Rho kinase inhibitor Y27632 (10µM in 0.1 % ethanol; Davies et al., 2000) or the phosphatidylinositol 3- kinase (PI 3-K) inhibitor LY294002 (50µM in 0.1 % DMSO; Vlahos et al., 1994) were added for 1h prior to addition of agonists. Alternatively, the cytosolic phospholipase A₂ (cPLA₂) inhibitor AACOCF3 (50µM in 0.1 % DMSO; Street et al., 1993) was added for 3 h.

Y27632 (Rho-kinase inhibitor) or LY294002 (PI 3-K inhibitor) inhibited the response to UK14304 alone by 100% or 96 ± 1.6% (mean ± s.e.mean) respectively (both p<0.05, Student's 2-tailed,unpaired t-test, n=11-13). On the other hand, AACOCF3 (cPLA₂ inhibitor) had no effect on the response to UK14304 alone. Y27632 caused only a 25 ± 4% inhibition (mean ± s.e.mean) of the enhanced response to UK14304 in the presence of U46619, but a 70 ± 3.5 % (n=5) inhibition of the enhanced response to UK14304 in the presence of vasopressin (both p<0.05, Student's 2-tailed, unpaired t-test). Similarly, LY294002 caused a 21 ± 3 % (n=7) inhibition of the enhanced response to UK14304 in the presence of U46619, but a 79 ± 2% (n=6) inhibition of the response to UK14304 in the presence of vasopressin (both p<0.05, Student's 2-tailed, unpaired t-test). In contrast, AACOCF3 caused a 54 ± 6 % inhibition of the enhanced response to UK14304 in the presence of U46619 (n=5, p<0.05, Student's 2-tailed, unpaired t-test), but had no significant effect on the enhanced response to UK14304 in the presence of vasopressin.

These data suggest that Rho-kinase and PI 3-K play a greater role in the vasopressin-enhanced ₂-adrenoceptor-mediated vasoconstriction than in the U46619-enhanced response. On the other hand, cPLA₂ appears to be important in the U46619-enhanced response, but plays no role in either the direct, or vasopressin-enhanced vasoconstriction.

Supported by The Wellcome Trust.

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Bhattacharya B. & Roberts R. E. (2003b), Br. J. Pharmacol., 138, 159P.
Davies S. P., Reddy H., Caivano M. et al., (2000). Biochem J., 351, 95-105.
Street I.P., Lin H.K., Laliberte F. et al., Biochemistry, 32, 5935-5940.
Vlahos C. J., Mather W. F., Hui K. Y. et al. (1994) J. Biol. Chem. 269, 5241-5248.