F3-Adrenergic Antagonist (Receptors/Cyclic AMP/Aminobenzylpropranolol/Iodohydroxybenzylpindolol/Isoproterenol) WESLEY L

Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6401-6405, December 1979 Cell Biology Quantitative relationship between 3-adrenergic receptor number and physiologic responses as studied with a long-lasting f3-adrenergic antagonist (receptors/cyclic AMP/aminobenzylpropranolol/iodohydroxybenzylpindolol/isoproterenol) WESLEY L. TERASAKI, JOEL LINDEN, AND GARY BROOKER* Department of Pharmacology, School of Medicine, University of Virginia, Charlottesville, Virginia 22908 Communicated by Paul Greengard, August 20, 1979 ABSTRACT The aminobenzyl analog of propranolol, atrium when f3-adrenergic receptor number is selectively de- 1- (p-amino-a,a-dimethylphenethylamino)-3(1-naphthoxy)2- creased. To investigate this problem, a new propanol, was synthesized and found tobe a potent B-adrenergic f3-adrenergic blocking agent. The fl-adrenergic receptors of cultured rat C6 blocking agent, 1-(p-amino-a,a-dimethylphenethylamino- glioma cells (2B clone) as assessed by [' 51]iodohydroxybenzyl- 3-(1-naphthoxy)-2-propanol (aminobenzylpropranolol), was pindolol binding were decreased 50 and >95% afterpretreat- synthesized. It was found to be a selective agent for the elimi- ment with 8 nM and 1 ,M aminobenzylpropranolol, respec- nation of cellular /3-adrenergic receptors. tively. Unlike propranolol, aminobenzylpropranolol displayed a prolonged blockade of receptors that was maintained during several hours of washing. [121s]Iodohydroxybenzylpindolol MATERIALS AND METHODS saturation binding experiments in cells exposed to aminoben- Tissue Culture. C6 rat glioma cells, 2B subclone (7), were zylpropranolol and subsequently washed indicated that the compound effectively diminished receptor number with no grown in monolayer cultures as described (1) in 16-mm plastic change in the affinity of the remaining receptors for iodohy- cluster dishes. In all experiments, the cells were changed from droxybenzylpindolol. Aminobenzylpropranool inhibited cat- growth medium (Ham's F-10 containing 10% fetal bovine echolamine-stimulated intracellular cyclic AMP accumulation; serum) to a simple salt medium containing 130 mM NaCl, 4 with increasing blockade, isoproterenol dose-response curves mM KCI, 0.6 mM MgSO4, 0.3 mM CaCI2, and 5 mM sodium became progressively shifted to the right but the maximal response was unaltered. Aminobenzylpropranolol inhibited the phosphate buffer (pH 7.4). All glioma cell washing experiments fl-adrenergic contractile response in atria isolated from rats and utilized this salt solution as the wash medium. guinea pigs. Treatment with 0.1 and 10 AM aminobenzyl- Binding of [i25I]Iodohydroxybenzylpindolol (125I-HYP) to propranolol produced decreases of 0.5 and 2 orders of magnitude Intact Glioma Cells. Specific (propranolol-displaceable) in the contractile potency of isoproterenol. As in glioma cells, binding of 125I-HYP (8) to C6 rat glioma cells was performed aminobenzylpropranolol failed to decrease the maximal response to isoproterenol. The effects of aminobenzylpropranolol by the method developed by Terasaki and Brooker (1), with the persisted during extensive washing of atria (up to 17 hr). Re- exception that phentolamine was omitted from all solutions and, peated exposures to isoproterenol at concentrations sufficient at the termination of the radioligand binding reaction, 0.1 mM to produce maximal tension development also failed to alleviate (d)-propranolol was included in the cell wash solution. the blockade. The inotropic potency of histamine in guinea pig Isolated Atrial Preparations. Hearts were excised from male atria was not affected by aminobenzylpropranolol. These data Wistar rats (200-250 g) or male Hartley guinea pigs (300-400 suggest that catecholamines are capa le of eliciting full biological responses in glioma cells and isolated atria even though g). All animals were pretreated with reserpine (5 mg/kg') ad- the great majority of fl-adrenergic receptors are persistently ministered intraperitoneally 15-18 hr before the animals were blocked. killed by a blow to the back of the neck. Left atria were maintained at 30'C (pH 7.4) in buffer containing 118-mM NaCl, The nature of the coupling interaction between /3-adrenergic 4.75 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM receptors and adenylate cyclase and the effect of this interaction dextrose, 25 mM NaHCO3, and 1 mM CaCl2. The buffer was upon subsequent biochemical events have yet to be understood vigorously gassed with 95% 02/5% CO2. Atria hooked to plat- in molecular detail. Receptor binding studies performed on inum/iridium electrodes were stimulated at 1-sec intervals with intact living cells (1, 2) reveal considerable complexity in the 10-msec monophasic pulses at twice the threshold voltage for mechanism(s) by which the receptors regulate adenylate cyclase contractions. Developed tension was recorded on Beckman activity (3). Because cells appear to be capable of changing their oscillographic recorders (Dynographs) and the signals were receptor number and because such changes have been impli- processed by a DEC LSI-1 1 microcomputer. cated in the phenomena of refractoriness (4, 5) and super- Purification of p-Amino-a,a-dimethylphenethylamine. sensitivity (6), the physiological consequences of these changes One gram of commercial p-amino-a,a-dimethylphenethyla- have become an important area of study from the standpoints mine (Aldrich 536658-7) was dissolved in 80 ml of chloroform of both hormonal regulation and enzymology. Our previous and applied to a 180-ml column of silica gel (Bio-Sil A, 100-200 results have implied that full hormone responses in C6-2B mesh) in chloroform. The column was washed with 600 ml of glioma cells may occur when only a minority of the available chloroform and then successively eluted with chloroform receptors are occupied (1). containing 2% methanol (600 ml), 4% methanol (600 ml), 10% In the present study we were concerned with what happens methanol (1600 ml), and 33% methanol (1200 ml). Chroma- to fl-adrenergic responses in the glioma cell and the mammalian tography was monitored by diluting an aliquot of the column The publication costs of this article were defrayed in part by page Abbreviations: aminobenzylpropranolol, 1-(p-amino-a,a-dimethyl- charge payment. This article must therefore be hereby marked "ad- phenethylamino)-3-(1-naphthoxy)-2-propanol; 125I-HYP, [1251]io- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate dohydroxybenzylpindolol. this fact. * To whom correspondence should be addressed. 6401 Downloaded by guest on September 27, 2021 6402 Cell Biology: Terasaki et al. Proc. Natl. Acad. Sci. USA 76 (1979) eluate 1:300 in methanol and measuring ultraviolet absorbance at 240 nm (e = 8750). Purified p-amino-a,a-dimethylphenethylamine was obtained between 900 and 1600 ml of 10% -CH- CH-CH2- NH --C- QCH NH2 methanol and in the first 700 ml of 33% methanol. The pooled solutions were evaporated at 220C in a rotary evaporator, and the residue was dissolved in 10 ml of n-butanol. The golden brown solution represented 62% of the starting material and yielded a single fluorescamine-reactive spot (RF, 0.29) on silica FIG. 1. Aminobenzylpropranolol. gel thin-layer chromatography in chloroform/methanol/ mass spectrometry with methane indicated a molecular weight ammonium hydroxide, 50:50:1 (vol/vol). The compound was of 364, consistent with the structure given in Fig. 1. stored as a 0.38 M solution at -16'C in the dark. The primary amine formed by the present synthetic scheme Synthesis of a-Naphthyloxy-l-propane-2,3-oxide. 1- is clearly the aryl amine and not the alkyl amine. It is readily Naphthol (Sigma, grade III, recrystallized) (432 mg), 1-[1- converted to the aryl azide by conventional procedures (9), and 14C]naphthol (Amersham, 20.1 mCi/mmol; 1 Ci = 3.7 X 1010 the azido derivative shows a shift in absorption maximum to becquerels; 5 ,gCi in 25 ,ul of ethanol), sodium hydroxide (130 shorter wavelengths with a typical increase in extinction coef- mg), and epichlorhydrin (Aldrich, E105-5) (230 1l) were ficient (Emax = 13,000 at 275 nm). In addition, mass spectrom- combined in a glass tube and heated at 60'C for 20 hr. The etry revealed a major ionized fragment of molecular weight 258 cooled reaction mixture was extracted with 2-3 ml of dichlo- which corresponds to the isopropylamine(1-naphthoxy)pro- romethane three times. The pooled extracts were evaporated panol cleavage product. Such a product could not be obtained at 600C, and the residue was dissolved in n-butanol to make a from the alkyl amine. Lastly, phenylamino(l-naphthoxy)pro- 0.38 M solution (concentration was determined from the ul- panol is reported to be devoid of biological activity (10). traviolet absorbance of a methanolic solution, assuming e = 4700 at 290 nm). The product, an orange solution, was stored RESULTS at -16'C in the dark; it was used for synthetic work without further purification. Aminobenzylpropranolol Antagonism of 125I-HYP Binding of to Cultured Glioma Cells. Aminobenzylpropranolol (Fig. 1) Synthesis 1(p-Amino-a,a-dimethylphenethylamino)- is an analog of propranolol and has the features generally as- 3-1(naphthoxy)2-propanol. Purified p-amino-a,a-di- cribed to the most potent 13-adrenergic antagonists: a methoxy methylphenethylamine (380 Mmol) was combined with 1 linkage between the 1 position of the naphthalene equivalent of a-naphthoxy-l-propane-2,3-oxide in 2 ml of moiety and n-butanol and heated for 48 hr at the alkyl side chain, and a secondary-alkyl amino group at- 650C. The solvent was re- tached to a methyl- or dimethyl-substituted a carbon (10, 11). moved by evaporation at 65°C in an open beaker. The residue When this compound was tested for biologic activity on a cul- was dissolved in 10 ml of chloroform and applied to a 4-ml tured glioma cell line, it was found to be a relatively potent column of silica gel (Bio-Sil A, 100-200 mesh) in chloroform. 3-adrenergic antagonist, displacing the specific radioligand The column was washed with 40 ml of chloroform and then 125I-HYP from 3-adrenergic receptors in intact living cells.

Load more