Studies On 5-HT2A Receptor Transduction In Bovine Pulmonary Arteries

Ross McCallum, Angus Shaw, Allan MacDonald. Glasgow Caledonian University, Glasgow, UK

In bovine pulmonary arteries (BPA) 5-HT induces constriction through activation of 5-HT2A. 5-HT1B receptors increase the tissue sensitivity to 5-HT (McKenzie et al., 2010). The present study aimed to investigate the receptor transduction for the 5-HT2A receptor in BPA. Concentration response curves for contraction to the 5-HT2A selective agonist TCB-2 (McLean et al., 2006) were constructed alone or in the presence of a PLCβ inhibitor U73122 (Bleasdale et al., 1990), an InsP3 receptor antagonist, 2-APB (Maruyama et al., 1997), a store-operated calcium channel blocker SKF96365, (Merritt et al., 1990), the voltage-gated calcium channel blockers nifedipine (CaV 1.1-1.2) and verapamil (CaV 1.1-1.3, Alexander et al., 2008), the MAP-kinase inhibitors PD98059 and U1026 (Zubkov et al., 2002), a PI3-kinase inhibitor LY294002 (Vlahos et al., 1994) and the PKB inhibitor 10-DEBC (Thimmaiah et al., 2005).

Ring segments of 0.1-0.3cm in diameter from the 4th and 5th pulmonary arterial generations were dissected from animals under 20 months within 50 min of death and mounted in 5ml Linton organ baths under a resting tension of 2g in a modified Krebs PSS gassed with 95/5% O2/CO2 at 37°C. Tissues were equilibrated for 1 hour before the addition of drugs. All tissues were first contracted with 60mM KCl. Results are expressed as % of the KCl contraction and are presented as means±s.e.m. Statistical analysis was carried out using Student's t-test with p < 0.05 considered significant.

TCB-2 (1nM – 10μM) induced a concentration dependent contraction (pEC50 7.38 ±0.12; Rmax 94.36±5.18, n=4). The concentration response curve was unaffected by U73122 (10μM), SKF96365 (10μM), nifedipine (10μM), PD98059 or U1026 (both 10μM) but was shifted to the right and the maximum response reduced by verapamil (10 μM, pEC50 6.00±0.34, p=0.0002; Rmax 92.04±23.56, n=4) and 10-DEBC (10μM, pEC50 5.97±0.17 p=<0.0001; Rmax 31.04±5.35, n=4). LY294002 (10μM) also produced a rightward shifted but with no change in the maximum response (10 μM, pEC50 6.30±0.10, p=<0.0001; Rmax 103.30±8.23, n=4). 2-APB did not change the tissue sensitivity but reduce the maximum response (30 μM, pEC50 7.223±0.20, Rmax 78.48± 7.97, p=0.023, n= 4)

The present study suggests that contraction of BPA by activation of the 5-HT2A receptor does not involve coupling to PLCβ or the MAP-kinase pathways nor does it involve calcium entry via a nifedipine-sensitive L-type- or store-operated- calcium channel. The inhibitory effect of verapamil suggests that calcium entry via CaV 1.3 may contribute to the contraction. The reduced contraction in the presence of 2-APB may also suggest the involvement of calcium release from InsP3 receptors. The fact that the PI3-kinase inhibitor LY294002 and the PKB inhibitor 10-DEBC reduced the tissue sensitivity to TCB-2 suggests that the 5-HT2A receptor is linked to the PI3-kinase and PKB pathway.

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