The role of the 5-HT1B receptor and the Na:Ca exchanger (NCX) in mediating the contraction to 5-HT in bovine pulmonary arteries.

H Maghoud, AM Shaw. Glasgow Caledonian University, Glasgow, UK

This study investigated the role of the 5-HT1B receptor, the Na:Ca exchanger (NCX), the Na:H exchanger (NHX) and the Na:K ATPase (NKA) in the contractile response to 5-HT in bovine pulmonary arteries (BPA).

Bovine lungs from animals under 20 months were dissected within 50 minutes of slaughter. Ring segments 0.1-0.3cm in diameter from the 3th and 4th arterial generations were dissected out and mounted in 5ml Linton organ baths under a resting tension of 2g in Krebs physiological saline solution gassed with 95/5% O2/CO2 at 37°C. Tissues were allowed to equilibrate for 1 hour before an initial contraction with 60mM KCl. The study investigated the effect of the 5-HT1B receptor antagonist SB216641 (1µM), the selective reverse mode NCX inhibitor KB-R7943 (10µM, Watanabe et al., 2006), the NHX inhibitor 5-(N-Ethyl-N-isopropyl) amiloride (EIPA, 100nM, Horinouchi et al., 2008), the non-selective store-operated/cation channel blocker SKF96365 (100µM, Poburko et al., 2007) and the NKA inhibitor ouabain (Croyle et al., 1997). Inhibitors were pre-incubated for 40mins. Results are expressed as % of the contraction to KCl (60mM) and are presented as means ± s.e.m. Statistical analysis was carried out using Student's t-test with p < 0.05 considered significant.

5-HT induced a concentration-dependent contraction that was shifted to the right to a similar extent by SB216641, KB-R7943 and EIPA but was unaffected by SKF96365 (pEC50 values: 5-HT control, 6.0 ± 0.06; SB216641, 5.4 ± 0.06, p<0.05, n=4; KB-R7943, 5.48±0.07, p<0.05, n=4; EIPA, 5.28±08, p<0.0001, n=5). A single concentration of 5-HT (1µM) produced a contraction that was sustained for more than 40 min. The presence of SB216641 or KB-R7943 changed the sustained contraction to a transient contraction. After 40 min the tone was reduced by 17.0±0.04% (control), 73.3±0.7% (KB-R9743) and 78.5±0.6% (SB216641). EIPA caused a fall in the sustained contraction though less than that observed with SB216641 or KB-R7943. Ouabain did not affect the concentration response curve (CRC) to 5-HT nor the contraction to a single concentration of 5-HT but prevented the SB216641- and KB-R7943-evoked rightward shift of the 5-HT CRC and maintained a sustained contraction in the presence of SB216641 or KB-R7943.

This study indicates that the 5-HT1B receptor and NCX (reverse mode) is particularly important in maintaining the sustained contraction to 5-HT and may indicate that activation of the 5-HT1B receptor stimulates calcium entry via NCX. Na entry via NHX but not via an SKF96365-sensitive cation channel may, at least in part, drive NCX in reverse mode. The loss of sustained tone in the presence of SB216641 or KB-R7943 may be explained by inhibition of the reverse mode of NCX and loss of calcium entry. If however, NCX reverse mode is blocked then the NHX-mediated locally elevated [Na]i could stimulate NKA lowering local [Na]I and this in turn would facilitate NCX forward mode and calcium extrusion, which could explain the ability of ouabain to prevent the loss of tone by SB216641 and KB-R7943.

Watanabe et al., (2006) J. Pharmacol. Sci. 102, 7-16. Horinouchi et al., (2008) J. Pharmacol. Sci. 107, 456-459. Poburko et al., (2007) Circ Res. 101,1030-1038. Croyle et al., (1997) Eur. J. Biochem. 248, 488.