title: Alkylation of alpha-1 receptors with a chemically reactive analog of prazosin reveals low affinity sites for norepinephrine in rabbit aorta.
abstract: The effect of a newly synthesized irreversible blocker of the alpha-1 receptor [1-(4-amino-6,7-dimethoxy-2-quinazolnyl)-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. In 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.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology, University of Kentucky, College of Medicine, Lexington.
authors.forename: M T
authors.initials: MT
authors.lastname: Piascik
authors.forename: J W
authors.initials: JW
authors.lastname: Kusiak
authors.forename: J
authors.initials: J
authors.lastname: Pitha
authors.forename: B T
authors.initials: BT
authors.lastname: Butler
authors.forename: H T
authors.initials: HT
authors.lastname: Le
authors.forename: M
authors.initials: M
authors.lastname: Babich
mesh_terms: D000478:Alkylation; D000818:Animals; D004305:Dose-Response Relationship, Drug; D008861:Microsomes / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects; D009638:Norepinephrine / Q000378:metabolism*; D010643:Phenoxybenzamine / Q000494:pharmacology; D011224:Prazosin / Q000031:analogs & derivatives / Q000494:pharmacology*; D011817:Rabbits; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D015016:Yohimbine / Q000494:pharmacology
chemical_list: D011942:Receptors, Adrenergic, alpha; D010643:Phenoxybenzamine; C051307:SZL 49; D015016:Yohimbine; D009638:Norepinephrine; D011224:Prazosin