Characterisation of 5-HT receptors in human coronary arteries by molecular and pharmacological techniques

Abstract

5-Hydroxytryptamine (5-HT) can produce both vasoconstrictor and vasorelaxant effects in human coronary arteries and the response to 5-HT can be influenced by the presence of disease. The aim of the present study was to elucidate the 5-HT receptor subtypes responsible for mediating 5-HT-evoked contraction of human coronary arteries using pharmacological, molecular and immunocytochemical approaches. Normal human coronary arteries, with intact endothelium, were mounted in tissue baths, and the vascular responses to 5-HT and 5-HT receptor agonists were studied. The effects of 5-HT 1 and 5-HT 2 receptor antagonists on these responses were also studied. Expression of messenger ribonucleic acid (mRNA) encoding different 5-HT receptors in human coronary arteries, atrium, ventricle wall and epicardium was determined using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis. The expression of 5-HT 1B or 5-HT 1D receptor protein was studied using subtype selective antibodies and standard immunocytochemical techniques. The rank order of 5-HT receptor agonist potency in causing vasoconstriction was 5-carboxamido tryptamine, (5-CT)>zolmitriptan=BW183C91 ( N 10-desmethyl zolmitriptan)=α-methyl-5-hydroxytryptamine (α-CH 3-5-HT)=5-HT=sumatriptan>2-methyl-5-hydroxytryptamine (2-CH 3-5-HT)=8-hydroxy-DPAT (8-OH-DPAT). α-CH 3-5-HT, 5-CT, 5-HT, zolmitriptan and BW 183C91 were significantly more potent (approximately 3-fold) than sumatriptan and 2-CH 3-5-HT, which in turn were more potent than 8-OH-DPAT. Ketanserin and methiothepin (5-HT 2 and 5-HT 1 receptor antagonists, respectively) caused parallel rightward shifts of the concentration–effect curves to α-CH 3-5-HT or 5-CT, respectively, without changing the maximum contractile response. In human coronary arteries, atrium, ventricle and epicardium. RT-PCR products corresponding to the human 5-HT 2A, 5-HT 1B and 5-HT 1F receptors were expressed in high levels, mRNAs coding for 5-HT 7, 5-HT 1A and 5-HT 1D receptors were only weakly expressed. No 5-HT 1E receptor mRNA was detected. In coronary arteries there was a differential expression of 5-HT 1B versus 5-HT 1D receptor mRNAs, with 5-HT 1B mRNAs being found in greater abundance. Dense 5-HT 1B-immunoreactivity was detected on smooth muscle layer within coronary artery, however, 5-HT 1D-immunoreactivity was not detected. It is concluded that 5-HT-evoked contraction of human coronary arteries is most probably mediated via the activation of both 5-HT 1B and 5-HT 2A receptors.