TY - JOUR
T1 - Alkylation of alpha-1 receptors with a chemically reactive analog of prazosin reveals low affinity sites for norepinephrine in rabbit aorta
AU - Piascik, M. T.
AU - Kusiak, J. W.
AU - Pitha, J.
AU - Butler, B. T.
AU - Le, H. T.
AU - Babich, M.
PY - 1988
Y1 - 1988
N2 - The effect of a newly synthesized irreversible blocker of the alpha-1 receptor {1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2- bicyclo[2,2,2]octa-2,5-dienylcarbonyl)-piperazine; SZL-49} has been evaluated in contractile studies in rabbit aorta and binding studies in aorta and brain. SZL-49 produced long lasting inhibition of norepinephrine-induced contractions which was apparent 21 hr after drug washout. The inhibition, which was dose and time dependent, was characterized by progressive shift to the right in the norepinephrine dose-response curve. The ED50 for norepinephrine was shifted from 10-7 M to 8 x 10-7, 3 x 10-6, 1 x 10-5 and 5 x 10-4 M after incubation (30 min) and washout of increasing concentrations of SZL-49. Surprisingly, SZL-49, irrespective of the dose or incubation time, did not decrease the maximal response of aortic rings to norepinephrine. This resulted in a norepinephrine dose-response curve after SZL-treatment that is parallel to the control. SZL-49 had no effect on the spasmogenic actions of histamine, serotonin, KCl or CaCl2. In contrast to the inhibitory pattern seen with SZL-49, incubation with 10-7 M phenoxybenzamine shifted the norepinephrine dose-response curve to the right in a nonparallel manner and significantly depressed the maximal response obtainable with norepinephrine. Incubation with 10-6 M phenoxybenzamine for 30 min virtually abolished the response to norepinephrine. Phenoxybenzamine (10-7 M) was without effect on aortic rings treated with a maximally effective dose of SZL-49. Prazosin weakly antagonized the contractile actions of norepinephrine observed after SZL-49 treatment, whereas yohimbine was without effect on these norepinephrine-induced contractions. Inn control binding studies [3H]prazosin bound to two classes of sites in both aorta and brain preparations. Affinities and densities for these sites were K1 = 67.5 pM, K2 = 309 pM; R1 = 38.2 fmol/mg, R2 = 46.47 fmol/mg in aorta and K1 = 29.6 pM, K2 = 182 pM; R1 = 6.6 fmol/mg and R2 = 30.4 fmol/mg in brain. Treatment with increasing amounts of SZL-49 (10-10 to 10-8 M) progressively reduced the number of [3H]prazosin sites without altering the affinity of the sites remaining. At 10-7 M, SZL-49 eliminated completely all specific [3H]prazosin binding. Our results indicate that the site mediating norepinephrine contraction after treatment with SZL-49 does not possess the characteristics of an alpha-1 receptor and supports the hypothesis that a low affinity site for norepinephrine and prazosin exists in vascular smooth muscle. The fact that our control binding studies reveal two high affinity [3H]prazosin sites suggests that there could be three binding sites for norepinephrine and prazosin in rabbit aorta.
AB - The effect of a newly synthesized irreversible blocker of the alpha-1 receptor {1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2- bicyclo[2,2,2]octa-2,5-dienylcarbonyl)-piperazine; SZL-49} has been evaluated in contractile studies in rabbit aorta and binding studies in aorta and brain. SZL-49 produced long lasting inhibition of norepinephrine-induced contractions which was apparent 21 hr after drug washout. The inhibition, which was dose and time dependent, was characterized by progressive shift to the right in the norepinephrine dose-response curve. The ED50 for norepinephrine was shifted from 10-7 M to 8 x 10-7, 3 x 10-6, 1 x 10-5 and 5 x 10-4 M after incubation (30 min) and washout of increasing concentrations of SZL-49. Surprisingly, SZL-49, irrespective of the dose or incubation time, did not decrease the maximal response of aortic rings to norepinephrine. This resulted in a norepinephrine dose-response curve after SZL-treatment that is parallel to the control. SZL-49 had no effect on the spasmogenic actions of histamine, serotonin, KCl or CaCl2. In contrast to the inhibitory pattern seen with SZL-49, incubation with 10-7 M phenoxybenzamine shifted the norepinephrine dose-response curve to the right in a nonparallel manner and significantly depressed the maximal response obtainable with norepinephrine. Incubation with 10-6 M phenoxybenzamine for 30 min virtually abolished the response to norepinephrine. Phenoxybenzamine (10-7 M) was without effect on aortic rings treated with a maximally effective dose of SZL-49. Prazosin weakly antagonized the contractile actions of norepinephrine observed after SZL-49 treatment, whereas yohimbine was without effect on these norepinephrine-induced contractions. Inn control binding studies [3H]prazosin bound to two classes of sites in both aorta and brain preparations. Affinities and densities for these sites were K1 = 67.5 pM, K2 = 309 pM; R1 = 38.2 fmol/mg, R2 = 46.47 fmol/mg in aorta and K1 = 29.6 pM, K2 = 182 pM; R1 = 6.6 fmol/mg and R2 = 30.4 fmol/mg in brain. Treatment with increasing amounts of SZL-49 (10-10 to 10-8 M) progressively reduced the number of [3H]prazosin sites without altering the affinity of the sites remaining. At 10-7 M, SZL-49 eliminated completely all specific [3H]prazosin binding. Our results indicate that the site mediating norepinephrine contraction after treatment with SZL-49 does not possess the characteristics of an alpha-1 receptor and supports the hypothesis that a low affinity site for norepinephrine and prazosin exists in vascular smooth muscle. The fact that our control binding studies reveal two high affinity [3H]prazosin sites suggests that there could be three binding sites for norepinephrine and prazosin in rabbit aorta.
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M3 - Article
C2 - 2843622
AN - SCOPUS:0023697090
VL - 246
SP - 1001
EP - 1011
IS - 3
ER -