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 re- sponse 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 bio- logical 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 main- tained 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-dimethylphen- ethylamine 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. with 10-ml portions of 1.5% methanol in chloroform. Eight Typical displacement curves (Fig. 2) showed that 8 nM ami- 10-ml fractions were collected, and each was analyzed for nobenzylpropranolol blocked half of the fl-receptors in 40 min. product by thin-layer chromatography on silica gel developed (This value was corrected for competition by [125I]iodohy- with chloroform/methanol/ammonium hydroxide, 50:50:1 droxybenzylpindolol.) (vol/vol). Aminobenzylpropranolol migrated at RF 0.82 A unique property of the was its to whereas the major amine-containing contaminant (as detected compound ability cause by fluorescamine staining) migrated at RF 0.29 (presumably 1 O0r * A 100 r B this is unreacted starting amine). Those fractions containing 0: aminobenzylpropranolol (usually fractions 2-5) were pooled 0\0 I 80'F 80F and evaporated to dryness at room temperature. The residue was taken up in 10 ml of chloroform, applied to a second silica 0 60F gel column, and rechromatographed as described above. The fractions containing pure product spot o7D)c (single by thin-layer > 0 40 40F chromatography) were pooled, evaporated, dissolved in methanol, and stored at -16 C in the dark. Overall yield was 0 25% as determined by recovery of 14C radioactivity in the 1 u 20- 20F position of the naphthalene residue. Ultraviolet spectra showed en 9 a maximum at 290 nm (E = 4900). - 10-9 lo 10-' 10-6 10-'p10p 10- 10-7 The product was judged to be pure by a number of criteria. Aminobenzylpropranolol, M Propranolol, M It migrated as a single ninhydrin- or fluorescamine-staining spot FIG. 2. Displacement of 125I-HYP from f3-adrenergic receptors in the following silica-gel thin-layer systems (proportions given of cultured glioma cells by aminobenzylpropranolol and (-)-pro- by volume): n-propanol/cyclohexane/acetic acid, 3:1:1, RF pranolol. Cells were treated for 40 min (370C) with aminobenzyl- 0.21; n-butanol/acetic acid/H20, 24:6:10, RF 0.42; n-buta- propranolol (A) or (-)-propranolol (B) and then specific 125I-HYP binding (@) was measured. In other plates, the cells were pretreated nol/HCl, 10:1, RF 0.60; 0.5 M ammonium formate, pH 8.5/ with the blocking agent and then washed repeatedly for 3 hr (370C) methanol, 1:1, RF 0.62; chloroform/methanol/HCl, 66:33:1, before assessment of 1251-HYP binding (3). For all points, n = 3. RF 0.63; methanol/HCl, 10:1, RF 0.66; diethylamine/ethyl There was no significant difference between the ability of amino- acetate, 1:1, RF 0.72; 95% ethanol/NH40H, 4:1, RF 0.80; benzylpropranolol to block (3-adrenergic receptors when assayed with chloroform/methanol/NH40H, 50:50:1, RF 0.81; and meth- or without washing. The apparent Kd for propranolol (1 nM) was anol/i M sodium acetate, pH 4.8,3:1, RF 0.82. By high-pressure increased 50-fold after washing, suggesting a 98% washout of (-)- it a propranolol. The slow rate of propranolol washout may result from liquid chromatography, appeared as single ultraviolet ab- its hydrophobic nature. The apparent Kd for aminobenzylpropranolol, sorbing peak (Partisil SCX column eluted with 10% metha- after correction for competition with the radioligand (12 pM), was 8 nol/0.1 M ammonium phosphate, pH 4.3). Chemical ionization nM. Downloaded by guest on September 27, 2021 Cell Biology: Terasaki et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6403 a lasting blockade of /3-adrenergic receptors. Inhibition of specific 125I-HYP binding by low concentrations of amino- benzylpropranolol persisted even after several hours of washing 6 *. _Z _-o the cells at 37'C.t The prolonged action of aminobenzyl- propranolol was not shared by (-)-propranolol; 98% of the latter 0)5 , - drug was lost during a 3-hr washout period (Fig. 2). E E _ ~ /a 0 It would be predicted that the occupation of a receptor by / / 0 a specific ligand should be seen as an effective loss of that re- ceptor, provided that the ligand dissociates sufficiently.slowly or not at all. In another experiment it was observed that glioma cells treated with 10 nM aminobenzylpropranolol and then IL)I, , washed displayed a marked decrease in maximal receptor binding capacity (Fig. 3). This apparent loss of receptors was not accompanied by a change in the affinity of the remaining receptors for 125I-HYP. 13/ Aminobenzylpropranolol Inhibition of Isoproterenol- Stimulated Cyclic AMP Production in Cultured Glioma C-A0a, 0' 0 a Cells. Aminobenzylpropranolol inhibited increases in intra- 109 10-8 107 10-6 1o- cellular cyclic AMP in isoproterenol-treated glioma cells (Fig. Isoproterenol, M 4). This inhibition was seen only at submaximal concentrations FIG. 4. Isoproterenol dose-response curves for intracellular cyclic of the catecholamine, and isoproterenol dose-response curves AMP accumulation in cultured glioma cells pretreated with amino- were shifted to the right. Whereas 0.1 and 1 ,uM aminoben- benzylpropranolol. Cells were treated for 40 min with 0 (*), 0.1 ,M zylpropranolol increased the apparent activation constants for (&), or 1 ,M (o) aminobenzylpropranolol and then washed for 3 hr 1.6 orders of (37°C). At the end bfthe wash period, the cells were treated for 20 min isoproterenol by 0.9 and magnitude, respectively, with (-)-isoproterenol and extracted with HOC to determine intra- there was no change in the maximal attainable level of intra- cellular AMP For all n = 3. cellular cyclic AMP. A concentration of 1 ,uM aminobenzyl- cyclic (1). points, propranolol was sufficient to eliminate >95% of the fl-adren- ergic receptors (Fig. 2). bition was dose- and time-dependent. Guinea pig and rat atria Aminobenzylpropranolol Antagonism of Isoproterenol- treated with aminobenzylpropranolol and then washed five Stimulated Contractility in Isolated Atria. Aminobenzyl- times to remove free inhibitort displayed decreased sensitivity propranolol inhibited isoproterenol-stimulated tension devel- toward isoproterenol. The degree of inhibition was similar to opment in atria isolated from rats and guinea pigs. This inhi- that observed in glioma cells. In rat and guinea pig atria, 0.1 and 10 ,M aminobenzylpropranolol (for i hr) caused shifts of 0.5 t Washing experiments of cultured C6 cells could not be carried out and 2 orders of magnitude, respectively, in the cumulative beyond 5-6 hr because specific 125I-HYP binding appeared to di- isoproterenol dose-response curves (Fig. 5). Although the po- minish markedly after this time. tency of isoproterenol was decreased in aminobenzylpropra- nolol-treated atria, the efficacy§ (maximal response) was un- changed. By analogy with the glioma cell experiments, 10 AM 0.06 aminobenzylpropranolol would be expected to have eliminated >99% of the tissue 3-adrenergic receptors. The positive ino- 0.05 tropic response of guinea pig atria to histamine was unaltered by aminobenzylpropranolol. The onset of the inhibitory action of aminobenzylpropranolol 0.04 \ was slow. Increases in the apparent Kact. for isoproterenol were 1.2-, 5.2-, and 13.5-fold (1.1 order of magnitude) in rat atria 0.03 pretreated with 1 AM aminobenzylpropranolol for 5, 20, and 0 50 min prior to washing. Persistent Blockade of Atria by Aminobenzylpropranolol. o.c Rat atria treated with aminobenzylpropranolol remained blocked even when washed for extended periods of time at 0 * Transfer of the medium bathing an atrium that had been treated with aminobenzylpropranol (and subsequently washed) to a fresh atrium revealed no inhibitory activity in the medium, suggesting that washing removes essentially all of the free drug. Bound '251-HYP, fmol § Efficacy was assessed in two ways. (i) Some atria were pretreated with FIG. 3. Scatchard plot of 1251-HYP binding in cultured glioma cells. 0.1 AM isoproterenol, which was maximally effective in the absence Specific binding [the difference between total binding and the non- of the inhibitor. After washing and treatment with 10 MM amino- specific binding seen in the presence of 1 ,M (-)-propranololl was benzylpropranolol, the maximal level of tension development re- measured as a function of radioactive ligand concentration in washed mained the same. (ii) Efficacy was also assessed by using the phos- control cells (-, Kd = 20 pM) and in cells treated with 10 nM ami- phodiesterase inhibitor isobutylmethylxanthine, which is able to elicit nobenzylpropranolol (o, Kd = 22 pM) for 40 min (37°C) and washed a maximal inotropic response by itself. Addition of 100 AM iso- for 3 hr. For all points, n = 3. Maximal binding capacity was dimin- butylmethylxanthine to atria treated with 10 MM aminobenzyl- ished 37% in aminobenzylpropranolol-treated cells, even after pro- propranolol plus 10 MM isoproterenol produced <5% increase in longed washing, but the equilibrium dissociation constant (Kd) for developed tension. By both criteria, concentrations of the blocker 1251-HYP (20-22 pM) was not different from controls. The straight sufficient to produce a 2-order-of-magnitude shift in the potency of lines are the least squares linear regression fits of the data (r = 0.89 isoproterenol did not decrease the efficacy of the catecholamine in for control and 0.92 for aminobenzylpropranolol-treated cells). atria. Downloaded by guest on September 27, 2021 6404 Cell Biology: Terasaki et al. Proc. Nati. Acad. Sci. USA 76 (1979) 1 OOr measuring biological responses in two dissimilar /3-adrenergi9 systems in which receptor numbers have been diminished by 801 selective chemical means. In both cultured glioma cells and isolated atrial tissues, maximal cyclic AMP or contractile re- 601 sponses were elicited despite a substantial (>95%) decrease in

AX% the number of hormone receptors by aminobenzylpropranolol. - 4U F The molecular mechanism by which persistent blockade by this m agent is achieved is unknown, but this is not essential to the 20 argument that physiologic function may be controlled by only E a minority of cellular receptors. In both glioma cells and atria, ' OL isoproterenol dose-response curves were shifted rightward as x receptor numbers were artificially decreased. These shifts ap- E 100 parently reflect an increased interaction'of the remaining re- c 0 ceptors with adenylate cyclase molecules or other secondary c 80 IF 0 A~~~~~~~~~~~. sites that mediate the f3-adrenergic response. ' The finding that cells that have lost most of their receptors --I60 ./ - can still manifest full biological responses is not surprising. Takayanagi et al. (12) attempted to decrease f3-adrenergic 401- receptors of guinea pig taenia by photolysis with isoproterenol or 2-(2-hydroxy-3-isopropylaminopropoxy)iodobenzene. When 201 20p such photolyzed tissues were treated with isoproterenol, potency was decreased but maximal relaxation could still be elicited. 0O I 10-9I I 10-84Z I 10-7 10-6 I 1o-, Isoproterenol had decreased potency but evoked maximal in- Isoproterenol or histamine, M otropic and cyclic AMP responses in cat papillary muscle when FIG. 5. Effect of aminobenzylpropranolol on the contractile re- the receptors had been irreversibly blocked by N-[2-hydroxy- sponse of atria to isoproterenol and histamine. Guinea pig (Upper) 3- (1 - naphthoxy) - propyl]-N-bromoacetylethylenediamine or rat (Lower) atria were pretreated with 0 (0), 0.1 AiM (-), or 10 ,gM (13). (A) aminobenzylpropranolol and then washed five times. Subse- By contrast, cultured murine S49 lymphoma cells (2) and quently, cumulative dose-response curves for (-)-isoproterenol (solid human VA2 fibroblasts (13) show decreases in their symbols) or histamine (open symbols) were determined (only one maximal curve was obtained from each atrium, n = 2 or 3). At the termination isoproterenol responses when /3-adrenergic receptors are per- of the experiment, maximal tension was determined by exposing atria sistently blocked. Furthermore, mutant clones of S49 cells iso- to 100jM isobutylmethylxanthine. lated by selecting for those cells having diminished cyclic AMP responsiveness have apparently undergone corresponding losses 30'C. Rat atria washed for 17 hr after treatment with the amine in f3-adrenergic receptors (14). The resolution of these con- (3 ,uM for 1 hr) showed no detectable decrease in the inhibition flicting reports probably lies in the existence of real differences of the isoproterenol response compared to parallel washed between the cell systems examined to date. If one were to pick control tissues: dose response curves remained shifted rightward some cell types over others as being more "physiological," one by 1.5 orders of magnitude. would tend to choose the isolated muscle preparations rather When high concentrations of isoproterenol were added to than the cultured cell lines. It may be more appropriate, how- atria pretreated with aminobenzylpropranolol, the catechol- ever, to consider differences among the systems to be more amine did not reverse the blockade, even after multiple washes quantitative than qualitative. and rechallenges with concentrations of agonist that evoked It has been hypothesized that adenylate cyclase activation repeated maximal inotropic responses. In other words, succes- may persist beyond the time when hormone is bound to the sive isoproterenol dose-response curves remained shifted receptor (14). In order to explain systems such as the heart and rightward.1 glioma cells, a more explicit mechanism would be required wherein each receptor molecule would have to have access to DISCUSSION many, if not all, adenylate cyclase molecules. If, after the re- In a previous study, we showed that C6 rat glioma cells appear ceptor-cyclase interaction was terminated, the receptors were to need few f3-adrenergic receptors in order to respond maxi- freed to activate additional enzyme molecules, then full acti- mally to f3-adrenergic agonists (1). This idea was directly in- vation is possible despite receptor loss. Such a mode of action ferred from experiments on intact cells which revealed a gross has been described as a collision coupling model by Levitzki discrepancy between the measured dissociation constant (Kd) (3). The magnitude of the cyclic AMP response (and presum- of pure agonists for 125I-HYP binding sites and their apparent ably functions mediated by cyclic AMP) would be some func- activation constant (Kact.) for cyclic AMP synthesis. In essence, tion of the probability that an activating receptor molecule it was shown that the affinity of agonist-binding was much too would interact with the cyclase molecule(s). Loss of receptors, low to be understood in terms of a simple fixed receptor-cyclase whether induced chemically or genetically, would decrease, coupling model. In the present report, the necessity of having but not necessarily eliminate, the chance that any given cyclase a full complement of receptors has been tested directly by will be activated. In this way, maximal activation of total cel- lular cyclase activity could be dependent on many additional Multiple exposures of control atria (not pretreated with the inhibitor) and perhaps cell-specific factors such as membrane fluidity, to the catecholamine caused a small degree of desensitization (de- the molecular properties of the receptors themselves, and the crease in potency for isoproterenol) with no decrease in efficacy. (For lag time of the decay of the enzyme active state. instance, in rat atria the apparent Kact. of isoproterenol during first Regardless of the f-adrenergic system studied the phrase and second dose-response curves was 0.9 ± 0.2 and 4.7 ± 2.1 nM.) The magnitude of shifts in curves generated from antagonist-treated "spare receptors" is misleading. Even in those cases in which tissues were always corrected for this desensitization and are reported the full efficacy of 3-adrenergic agonists is maintained, all the relative to identically treated controls. receptors are probably required for full potency. A corollary Downloaded by guest on September 27, 2021 Cell Biology: Terasaki et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6405 to this idea is that, because many cells display agonist-induced spectrometric analyses. This work was supported by National Institutes receptor losses, certain tissues would be expected to udergo. of Health Grants HL15985, AM22125, and HL19242. G.B. received desensitization (decrease in agonist potency) as opposed to re- Research Career Development Award HL00098 from the National fractoriness (decrease in agonist efficacy). The heart seems to Institutes of Health. be an example of a tissue showing a desensitization phenome- non; it makes physiological sense that this critical organ would 1. Terasaki, W. L. & Brooker, G. (1978) J. Biol. Chem. 253, respond to prolonged catecholamine stimulation with a decrease 5418-5425. in sensitivity to the hormones rather than with a loss of con- 2. Insel, P. A. & Stoolman, L. M. (1978) Mol. Pharmacol. 14, tractility. 549-561. 3. Levitzki, A. (1978) Biochem. Pharmacol. 27,2083-2088. The f3-adrenergic blocking agent aminobenzylpropranolol 4. Lefkowitz, R. J. & Williams, L. T. (1978) Adv. Cyclic Nucleotide promises to be a useful investigational tool in several respects. Res. 9, 1-32. (i) As shown in the present report, its long duration of action 5. Su, U. F., Harden, T. K. & Perkins, J. P. (1979) J. Biol. Chem. 254, makes it an effective agent for decreasing or virtually elimi- 38-41. nating f3-adrenergic receptors from cell surface membranes. 6. Sporn, J. R., Wolfe, B. B., Harden, T. K. & Molinoff, P. B. (1977) It is more potent than other agents that have been used for this Mol. Pharmacol. 13, 1170-1180. purpose (12, 15). (if) The aniline portion of the molecule should 7. de Vellis, J. & Brooker, G. (1973) in Tissue Culture of the Ner- be easily radioiodinated by established procedures (16), and vous System, ed. Sato, G. (Plenum, New York), pp. 231-245. preliminary data in our laboratory indicate that such labeling 8. Maguire, M. E., Wiklund, R. A., Anderson, H. J. & Gilman, A. readily occurs. Alternatively, the blocking agent could be G. (1976) J. Biol. Chem. 251, 1221-1231. synthesized in a 3H-labeled form. Because the effect of the 9. Bayley, H. & Knowles, J. R. (1977) Methods Enzymol. 46,69- compound is essentially irreversible over the time span used in 114. 10. Crowther, A. F. & Smith, L. H. (1968) J. Med. Chem. 11, most binding studies, radiolabeled aminobenzylpropranolol 1009-1013. may prove to be useful as a f-adrenergic receptor ligand. (iii) 11. Howe, R., Crowther, A. F., Stephenson, J. S., Rao, B. S. & Smith, The compound is easily diazotized and converted to the aryl L. H. (1968) J. Med. Chem. 11, 1000-1008. azide, azidobenzylpropranolol, which has obvious potential as 12. Takayanagi, I., Yoshioka, M., Takagi, K. & Tamura, Z. (1976) a true photoaffinity label (9). (iv) Chemical modifications in- Eur. J. Pharmacol. 35, 121-125. volving the terminal primary amine, such as carbodiimide 13. Venter, J. C. (1979) Fed. Proc. Fed. Am. Soc. Exp. Biol. 38,595 coupling or reaction with N-hydroxysuccinimide derivatives, (abstr.). can be used to immobilize aminobenzylpropranolol to solid 14. Maguire, M. E., Ross, E. M. & Gilman, A. G. (1977) in Adv. Cyclic supports, high molecular weight polymers, and electron-dense Nucleotide Res. 8, 1-84. or fluorescently tagged proteins. 15. Atlas, D., Steer, M. L. & Levitzki, A. (1976) Proc. Natl. Acad. Sci. USA 73, 1921-1925. The expert technical assistance of Ms. Celinda Johnson is greatly 16. Hunter, W. M. & Greenwood, F. C. (1962) Nature (London) 194, appreciated. We thank Dr. Don Hunt and Jeff Shabanowitz for mass 495-496. 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