1 Adrenoceptor subtype mediates noradrenaline induced contraction of the human internal mammary artery: radioligand and functional studies
Authors: M. Bevilacqua, T. Vago, A. Monopoli, G. Baldi, A. Forlani, C. Antona, P. Biglioli, R. Scrofani, G. Norbiato
Journal: Cardiovascular Research (1991)

The aim was to evaluate the characteristics of alpha adrenergic binding sites on human internal mammary arteries and the alpha adrenoceptor mediated vasoconstrictor response to catecholamines.Human internal mammary arteries were cut longitudinally, the intimal layer was scraped, and the arteries homogenised and centrifuged at 50,000 g to obtain a membrane pellet. Saturation isotherms with [3H]-prazosin were done with 50-100 micrograms plasma membranes per tube and increasing concentrations of [3H]-prazosin (non-specific binding: 2.5 mM noradrenaline plus superoxide dismutase and catalase). Kinetic isotherms were done with 100 micrograms plasma membranes and 1-5 nM [3H]-prazosin for time periods ranging from 1 to 90 min; at the equilibrium, dissociation of [3H]-prazosin was achieved by 10 microM prazosin. alpha 2 Adrenoceptor density on internal mammary artery membranes was assessed with [3H]-rauwolscine (non-specific binding: 1 microM yohimbine). Separation of membrane bound radioactivity was achieved by rapid vacuum filtration through Whatman GF/C fibre filters. Saturation isotherms were evaluated by Scatchard plots and kinetic data, and competition isotherms by Enzfitter analysis. Contractility studies were done with helical strips of artery (without adventitial layer) placed in a thermostated perfusion bath. Data were obtained in the presence of different concentrations of agonists and antagonist to obtain Schild plots. Antagonist drugs were employed at only one concentration for each preparation.Mammary arteries were collected from 51 patients (age range 42-65 years) undergoing surgery for coronary grafting.The binding of [3H]-prazosin to arterial plasma membrane was rapid and reversible. The K + 1 was 0.13 (SD 0.03) X 10(9) M.min-1 (n = 5) and the Kd, determined as a ratio between k-1/K + 1, was 0.34(0.01) nM (n = 5). [3H]-Prazosin binding, displaceable by 2.5 mM (-)-noradrenaline, was saturable and disclosed an alpha 1 adrenoceptor density of 30(3) fmol.mg-1 protein with a dissociation constant (Kd) of 215(50) pM (n = 18). The adrenergic agonists competed with [3H]-prazosin in the following order of potency: (-)-adrenaline [Ki = 0.6(0.1) microM; n = 5] greater than (-)-noradrenaline [Ki = 1.05(0.015) microM; n = 12] much greater than (-)-isoprenaline [Ki = 150(10) microM; n = 4]. Specific binding of [3H]-rauwolscine to IMA plasma membranes was negligible (about 2 fmol.mg-1 protein) (n = 15) with an unfavourable ratio of non-specific v specific binding. Catecholamines induced a dose dependent contractile response in arterial strips; (-)-noradrenaline: EC50 = 0.48(0.12) microM, n = 20; (-)-adrenaline: EC50 = 0.15(0.16) microM, n = 10; and methoxamine, a selective alpha 1 adrenergic agonist: EC50 0.67(0.15) microM, n = 10. The alpha 2 adrenoceptor agonists BHT-933, BHT-920, and guanabenz did not contract the arterial strips (up to 10 mM). Prazosin (0.03-0.1 microM) produced concentration dependent right shifts of the (-)-noradrenaline [pA2 = 9.83(0.11), n = 19], (-)-adrenaline [pA2 = 9.50(0.31), n = 10], and methoxamine [pA2 = 8.96(0.18), n = 10] concentration-response curve. CONCLUSIONS - Internal mammary artery plasma membranes possess alpha 1 adrenoceptors which are involved in the vasoconstrictor response to catecholamines. alpha 2 Adrenoceptors seem not to be involved.