Proc. Nati. Acad. Sci. USA Vol. 74, No. 12, pp. 5290-5294, December 1977
Biochemistry
Probing of fl-adrenergic receptors by novel fluorescent fl-adrenergic blockers (membrane adenylate cyclase/tluorescence microscopy/9-aminoacridylpropranolol/dansyl analogi of promranolol)
DAPHNE ATLAS AND ALEXANDER LEVITZKI Department of Biological Chemistry, The Hebrew University of Jerusalem, 20 Mamilla Road, Jerusalem, Israel
Communicated by Daniel E. Koshland, Jr., August 31, 1977
ABSTRACT
The synthesis of two high-affinity fluorescent
(3-adrenergic blockers is described: dI-N-2-hydroxy-3 (1naphthyloxy)propyl-N2-9-acridyl)-1,2-propanediamine (9aminoacridylpropanolol, 9-AAP) and di-N[2-hydroxy-3-(1naphthyloxy)propyl-N'l-dansylethylenediamine (dansyl analogue of pro ranolol, DAPN). Both 9-AAP and DAPN inhibit competitively the 1-epinephrine-dependent adenylate cyclase activity [ATP pyrophos hate-lyase (cyclizing), EC 4.6.1.11 in turkey erythrocyte membranes without affecting the fluoridestimulated adenylate cyclase activity. Similarly, MAAP and DAPN inhibit in a competitive manner the binding of [12%IJ iodohydroxybenzylpindolol to these fl-adrenergic receptors. The two fluorescent P-adrenergic blockers 9-AAP and DAPN probe specifically ft-adrenergic receptors in the central nervous system
The use of these (3-blockers is to probe postsynaptic (3-adrenergic receptors. This approach is complementary to the well-established fluorescent-histochemical technique (16, 17) used to detect adrenergic and noradrenergic presynaptic pathways. MATERIALS AND METHODS [a-32P]ATP and 3H-labeled cyclic AMP ([3H]cAMP) were purchased from the Radiochemical Centre, Amersham, England. Creatine kinase, creatine phosphate, theophylline, and l-epinephrine were purchased from Sigma. Protein was determined according to Lowry et al. (18) with bovine serum albumin as the standard. Turkey erythrocyte membranes were prepared according to the method described by Steer and Levitzki (19). The activity of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was assayed according to the method of Salomon et al. (20). 125I-HYP binding was measured as described earlier (8). Fluorescence measurements were conducted on a Hitachi-Perkin-Elmer MPF-4 spectrofluorimeter. Front face fluorescence at 300 was measured as described by Gabel et al. (21). A special cell holder for the measurement of front-face fluorescence for either 3-ml or 0.5-ml fluorescence cells was constructed. All fluorescence measurements were conducted at 250. The injection of 9-AAP into rats and mice and the preparation of cryostat sections were performed as described earlier (11-15). Identical procedures were used for the in vivo studies on DAPN.
as well as in other organs when injected into rats. The fluorescence pattern can be monitored by fluorescence microscopy performed on cryostat slices of these organs. The appearance of the characteristic fluorescence pattern can be blocked in a stereospecific fashion by a prior injection of 1-propranolol and not by a prior injection of d-propranolol. These compounds therefore offer a powerful means to map -adrenergic receptors in vivo. The stereospecific displacement of AP from the ,Badrenergic receptors of turkey erythrocyte membranes by Ipropranolol and by 1-epinephrine can be detected in vitro using front-face fluorescence. The potential use of these compounds to probe (-receptors in vitro and in vivo is discussed.
,B-Adrenergic receptors can be monitored using radioactively labeled high-affinity (3-blockers such as [3H]propranolol (1-3), [3H]alprenolol (4, 5), and [125I]iodohydroxybenzylpindolol (125I-HYP) (6-8). Recently irreversible (3-receptor-directed affinity labels have also been prepared (9, 10). These compounds enable one to measure directly the content of (3-receptors in a variety of cell types. The 3H affinity label is currently employed to characterize the molecular nature of the (3-adrenergic receptor (D. Atlas and A. Levitzki, unpublished data). In this communication we report the synthesis and properties of two potent fluorescent ,B-adrenergic blockers: dl-9-aminoacridylpropranolol (9-AAP)* and a dansyl analog of propranolol (DAPN).* These two compounds bind stereospecifically to f-adrenergic receptors and their binding can be probed in vivo using fluorescence microscopy and in vitro using fluorescence spectroscopy. One of the compounds, 9-AAP, has already been used to map f(-adrenergic receptors in vivo (11-14). In these studies fluorescence was found in regions known to possess (3-adrenergic receptors, subsequent to the injection of 9-AAP into rats and mice (15). DAPN, as shown in this communication, was found to be as efficient for probing f(-adrenergic receptors in vivo as 9-AAP. 9-AAP, due to its fluorescence properties, was found to be suitable also for monitoring (3-adrenergic receptors in vitro.
9-Aminoacridylpropranolol (9-AAP). 1,2-Epoxy-3-(1naphthyloxy)propane was synthesized as described elsewhere (10). 1,2-Epoxy-3-(l-naphthyloxy)propane (2.0 mmol) dissolved in 2.0 ml of dioxane is condensed with a mixture of two N-isopropylamino-9-aminoacridine isomers (22) (2.0 mmol) (see Fig.
1) by heating the mixture at 500 for 24 hr. The solution is cooled and acidified with concentrated hydrochloric acid (6.0 mmol). The aqueous phase is concentrated under vacuum. The mixture of hydrochlorides separates as an oil. The oil is treated with traces of methanol to complete dissolution and then precipitated by the addition of ether. The compound 9-AAP is separated on thin-layer plates 1.0 mm thick, made of Kieselgel H type 60, Merck. 1-Butanol/acetic acid/water (4:1:4 by volume) is used as the solvent system (RF = 0.4). The outline of synthesis is shown in Fig. 1. The absorption spectrum, fluorescence excitation spectrum, and fluorescence emission spectrum of the compound are given in Fig. 2. Analysis: % N = 9.2 (calculated Abbreviations: 9-AAP, dl-N1-[2-hydroxy-3-(1-naphthyloxy)propyl]N2-(9-acridyl)-1,2-propanediamine, or dl-9-aminoacridylpropranolol; DAPN, dl-N-[2-hydroxy-3-(1-naphthyloxy)propyl]-N'-dansylethylenediamine, or dansyl analog of propranolol; 125I-HYP, [125I]iodohydroxybenzylpindolol; cAMP, cyclic AMP. * Now available commercially from Yissum Research Development Co., Hebrew University, Jerusalem, Israel.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5290
Proc. Natl. Acad. Sc. USA 74 (1977)
Biochemistry: Atlas and Levitzki
OH
CH3
OCH2CH-CH2 NH CH 2-CH2 NH2
NH2CHCH2NH
OCH2 CH - CH2
5291
ct
Cl SO2
NH
NH2CH2CH
N CH3 CH3
CH3 T2.HCI
OH OCH2 CH-CH2-NHCH2CH2NH SO2
CH3
OH
OCH2 CHCH2NHCH CH2 - NH t
X
~~~~~~A
2
Hydrochloride
SalIt
OH and
N
CH3 CH3
FIG. 3. Outline of synthesis of DAPN.
OCH2CHCH2NHCH2CH- NH
CH3
cm-1 in water. The absorption spectrum, the fluorescence excitation spectrum, and the fluorescence emission spectrum are shown in Fig. 4.
B
FIG. 1. Outline of synthesis of 9-AAP.
RESULTS Spectroscopic Properties of the Ligand. The absorption, fluorescence excitation, and fluorescence emission spectra of 9-AAP and DAPN are shown in Figs. 2 and 4, respectively. The quantum yield of 9-AAP decreases as a function of hydrophobicity, as can be seen from Fig. 5, in which the effect of dioxane on the intensity of the fluorescence emission is demonstrated. Binding of 9-AAP and of DAPN to the fl-Adrenergic Receptor. The compounds 9-AAP and DAPN inhibit competitively the epinephrine-dependent adenylate cyclase activity from turkey erythrocyte membranes (Fig. 6). Both 9-AAP and DAPN exert no effect on the fluoride-stimulated activity. The dissociation constants for 9-AAP and DAPN calculated from the inhibition of the l-epinephrine-dependent adenylate cyclase activity are summarized in Table 1. The inhibition of 125I-HYP binding by 9-AAP and DAPN is shown in Fig. 7. Both 9-AAP
= 9.31). The molar extinction coefficient of 9-AAP at 260 nm was found to be 200,000 M-1 cm-' in water. Dansyl Analogue of Propranolol (DAPN). dl-N-[2-Hy-
droxy-3-(1-naphthyloxy)propyl]ethylenediamine (10) (2.0 mmol) is partially dissolved in dioxane (3.0 ml), and triethylamine (6.0 mmol) is added for neutralization. Ten percent molar excess of dansyl chloride (2.2 mmol) is added to the reaction mixture, and the reaction is allowed to proceed for 1.0 hr at room temperature. Then the mixture is heated up to 50° and kept at this temperature for 0.5 hr. After evaporation under reduced pressure the oily residue is dissolved in traces of isopropanol and methanol and precipitated with ether. The white flocculent precipitate (DAPN) is collected, washed with ether, water, and ether once again, and dried in a desiccator over KOH and CaC12. Analysis: % N = 6.21 (calculated = 6.96). The outline of synthesis of DAPN is shown in Fig. 3. The molar extinction coefficient at 295 nm was found to be 3060 M-1
WAELNGH nmm420nmc WAVELENGTH (nm) D WAVeLENTH(n) 4..
z 410
co) < 0.6 20
z W
z
260 32
WU 0 z202020
e
Q4
o30303
9
o4
260
320 380
440 50
WAVELENGTH (nm)
0
D
250
8
2
480
520
Z w 0
~~~~~~~~~~~~~~~~~~~~~~C/)
Cl)
200
4
0
o wUzo-
0.2,
zFO -0 w
w
270
290
310
330
350 370
390
WAVELENGTH (nm)
410
430
0
D
400
440
WAVELENGTH (nm)
FIG. 2. The absorption spectrum, fluorescence excitation spectrum, and fluorescence emission spectrum of 9-AAP. The absorption spectrum of 9-AAP was measured on a 5 AM solution. The molar extinction coefficient of 9-AAP at 260 nm can be calculated to be 200,000 M-1 cm-1. A concentration of 2.4 MM 9-AAP in water was used for the measurement of the fluorescence excitation spectrum and the fluorescence emission spectrum in the MPF-4 spectrofluorimeter at 250. When the excitation spectrum was monitored, the emission wavelengths (Aem) used were 420 and 445 nm. When the fluorescence emission spectrum was monitored, the excitation wavelength used was 385 nm. The slight difference in the excitation spectra at 420 and 445 nm may reveal the higher contribution to the fluorescence of the naphthyloxy moiety in the 9-AAP molecule. This finding may also be taken as an indication for the relative inefficiency in the energy transfer between the naphthyloxy moiety and the acridine nucleus.
Biochemistry: Atlas and Levitzki
5292
Proc. Natl. Acad. Sci. USA 74 (1977) I-
t~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.
0.6
Z ~~~~~~AgS2Smm
A1.d5e
I.-
A.-4S0Mm
OA
240
260
320
360
200
400
240
280
320
360
440
400
480
WAELENGTH (nm)
WANELENGTH (nm)
520
600
560
640
700
MVELENGTH (nm)
FIG. 4. The absorption spectrum, fluorescence excitation spectrum, and fluorescence emission spectrum of DAPN. The absorption spectrum of 0.14 mM DAPN was measured in water. The concentration of DAPN for the fluorescence measurements was 1.0 ,uM in p-dioxane. When the excitation spectrum was monitored, the emission wavelengths used were 440, 480, and 525 nm. When the fluorescence emission spectrum was monitored, the excitation wavelength was 350 nm.
and DAPN compete with the fl-adrenergic blocker 125I-HYP for binding to the Preceptor. The dissociation constants for 9-AAP and DAPN were calculated from the Scatchard plots presented in Fig. 7, as explained in the legend to the figure. Fluorescence Mapping of f-Receptors In Vivo by DAPN. DAPN (1.0 mg/ml) in 0.5 ml of saline was administered by slow intravenous injection into the tail veins of albino rats (200-220 g, 2.5 mg of DAPN per kg of body weight). Fifteen to thirty minutes later the animals were sacrificed by decapitation as described earlier (11-14). The brains were removed immediately and frozen in liquid nitrogen. Sections of 8-16 ,.m were cut in a cryostat at -20°. The frozen sections were mounted on glass slides, dried in air, and visualized under phase contrast and transmitted ultraviolet illumination on a Zeiss Universal fluorescent microscope (11-13). Prior injection of dl-propranolol (5.0 mg/kg) inhibited formation of the fluorescence pattern. Only slight reduction in the fluorescence was observed after injection of d-propranolol. The fluorescence pattern observed in the kidney, with 9-AAP, and the fluorescence pattern observed in the pyramidal cells of the hippocampus, with DAPN, are shown in Fig. 8.
Fluorescence Changes upon 9-AAP Binding to f-Receptors In Vitro. In Figs. 9 and 10, the front-face fluorescence spectrum of a mixture of turkey erythrocyte membranes at 10 mg/ml and 0.10 ,M 9-AAP is shown. Upon addition of either I-propranolol
but measurable in(Fig. 9) or l-epinephrine (Fig. 10) a crease in the fluorescence intensity can be detected. This flufor the I-antagonist and 1orescence change is
small
stereospecific
agonist, respectively, because d-propranolol and d-epinephrine do not display any effect on the spectrum when added to the mixture of membranes with 9-AAP (Figs. 9 and 10). The displacement of 9-AAP from the receptor results in an increase of the 9-AAP fluorescence intensity. This is probably due to the fact that the 9-AAP is displaced from the hydrophobic environment in the f-receptor to the more hydrophilic environment of the buffer. The fluorescence of the acridine derivative in-
when transferred from a hydrophobic medium to a hydrophilic medium (Fig. 5). The fluorescence peak at 540 nm is an intrinsic fluorescence
creases
E
W C
''o
A
oo
-.
C0
FIGX
cyclase
% DIOXANE
60
60
40
40
40
2 -
civiy lt y l g'N mdn amod
~
a
measured as a function of I-epinephrine concentration in the presence and in the absence of 9-AAP or DAPN. (A)
zoct inwa 420
44
6
8
0
0.20MM 9-AAP. (B)
*, No 9-AAP present;
*, No DAPN present; ,0.15M DAPN present;
0, 1.5 MM DAPN present. The data in both cases are plotted according to Eadie:
20
W-) 00 -Pa FIG 5.Tefurseceitniyo
ucino
v/[E]
=
Vmax
-J
420
440
460
480
WAVELENGTH,
500
520
nm
The fluorescence intensity of 9-AAP as a function of FIG. 5. p-dioxane concentration. The fluorescence emission spectrum of 9-AAP in water in the presence of increasing dioxane concentrations was measured at 250 on the MPF-4 spectrofluorimeter.
in which v is the measured adenylate cyclase activity,
[El
is the epi-
nephrine concentration, KE is the epinephrine-receptor dissociation constant,
[I]
is the concentration of the
,8-blocker, Vma,, is
blocker-receptor dissociation constant, and
specific activity. for
The values calculated for the
K1
dissociation
9-AAP and DAPN are summarized in Table
1.
is the
(3-
the maximal constants
Biochemistry:
Atlas and Levitzki
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. The dissociation constants of 9-AAP and DAPN towards the ,-receptor Dissociation constant, nM From inhibition of 125I-HYP From inhibition kinetics Compound binding 9-AAP 34.0 + 3.0 21.3 2.0 DAPN 34.5 + 3.0 28.3 + 2.0 The values of the dissociation constants of 9-AAP and DAPN to the f3-adrenergic receptors were calculated from Figs. 6 and 7, as explained in the legends to the figures.
peak of the membrane and may represent membrane-bound flavins. This fluorescence peak remains unchanged upon addition of 9-AAP, propranolol, or epinephrine. From the practical point of view, the constant value of this peak during the experiment may be taken as an internal reference. Although the compound DAPN is shown to bind with high affinity to the (-receptor (Table 1) it was found that its nonspecific solubility in the membrane causes a large fluorescence enhancement that does not allow us, at the present time, to resolve the fluorescence enhancement due to the binding to the (-receptor. Furthermore, the fluorescence peak of the bound DAPN overlaps the intrinsic, fluorescence peak of the turkey membranes, which further complicates the use of this compound in studies in vitro. However, in studies in vivo, DAPN was found to be as effective as 9-AAP (Fig. 8 and ref. 23). DISCUSSION The fluorescent (3-blockers 9-AAP and DAPN bind specifically to the (3-adrenergic receptors of turkey erythrocyte membrane fragments. The specificity of the binding is established by three criteria: (i) the inhibition of the catecholamine-dependent adenylate cyclase activity (Fig. 6); (ii) the inhibition of 125IHYP binding to the (3-receptor (Fig. 7); and (iii) the ability of i-propranolol and I-epinephrine to displace the fluorescent (3-blocker and the inability of d-propranolol and d-epinephrine to cause its displacement (Figs. 9 and 10). The stereospecific displacement of the fluorescent (3-blocker by i-agonist or I-antagonist from the receptor is accompanied by an increase of the ligand fluorescence (Figs. 9 and 10). An increase in the fluorescence upon its displacement from a hydrophobic environment to a hydrophilic one is in accordance 2.4
24.
-AA
-
o-
xz
a
co
B
20 20
2.0
I.6 0
1.2
126
0.8
0.8
0.4
0.4 02
04
06
08
51 -HYP
1.0
1.2
-
02
04
06
08
1.0
1.2
BOUND, pmol / mg
FIG. 7. The displacement of 125I-HYP from ,8-receptors by 9-AAP and DAPN. The binding of 125I-HYP to turkey erythrocyte membranes was measured as described by Maguire et al. (8). The binding data are presented in the form of Scatchard plots. The values for the dissociation constants for 9-AAP and DAPN are given in Table 1. (A) 0, Binding of 125I-HYP in the absence of 9-AAP; &, binding of 1251-HYP in the presence of 0.10MM 9-AAP; 0, binding of 125I-HYP in the presence of 0.50MM 9-AAP. (B) 0, Binding of 125I-HYP in the absence of DAPN; 0, binding of 125I-HYP in the presence of 0.50 gM DAPN.
5293
FIG. 8. (A) Distribution of DAPN fluorescence within the pyramidal cell layer of the rat hippocampus. (B) Localization of 9-AAP fluorescence in the region corresponding to the afferent arteriole and its juxtaglomerular cells in rat kidney (G = glomeruli). The fluorescence pattern is observed by using an UV-illuminated phase microscope of cryostat slices (8-16 Am) of rat brain (A) and rat kidney (B) 15 min after intravenous injection of DAPN (A) or 9-AAP (B).
with the fluorescence properties of the compound (Fig. 5). Under the experimental conditions used (Figs. 9 and 10) 11.5 nM of the total concentration of 100 nM 9-AAP are bound to the receptor. The fluorescence change measured amounts to 8.5-10%. This finding may be taken as an indication that the receptor binding site is highly hydrophobic. The absolute magnitude of the fluorescence change is, however, at present too small to be used as a probe for the quantitative titration of (-receptors in vitro. It is hoped that this goal will be achieved when higher concentrations of receptor will be accessible experimentally. At present the highest (-adrenergic receptor concentration accessible is about 10-20 nM. The availability of fluorescent (3-blockers allows one to localize (-receptors in a variety of tissues. Indeed, 9-AAP has already been used successfully to map (3-adrenergic receptors (11-15). The specific fluorescence staining is inhibited by a prior injection of i-propranolol and not by a prior injection of d-propranolol (14). DAPN was also found to bind in a stereospecific fashion to (3-adrenergic receptors in vivo both in the brain and in the periphery (Fig. 8). Attempts, however, to monitor DAPN binding in vitro to the (3-adrenergic receptors of turkey erythrocyte membranes have thus far failed. This is probably due to the fact that the intrinsic fluorescence peak of the membranes coincides with that of the bound DAPN. The much simpler synthetic procedure for the preparation of DAPN as compared to the procedure to prepare 9-AAP and the fact that DAPN is at least as effective as 9-AAP in in vivo studies make DAPN the ligand of choice for the task of mapping (3-adrenergic receptors. The availability of specific postsynaptic blockers such as 9-AAP or DAPN for the (3-adrenergic receptor enables the researcher to selectively identify adrenergic and noradrenergic nerve terminals. It would also be useful to synthesize fluorescent a-adrenergic blockers as well as fluorescent blockers for dopamine receptors.' This work was supported by a grant from the U.S.-Israel Binational Research Foundation (BSF), Jerusalem, Israel. D.A. was supported by the Lady Davis Fellowship Trust and by a Research Advancement of Science and Technology grant from the Bat-Sheva de Rothschild Fund. 1. Levitzki, A., Atlas, D. & Steer, M. L. (1974) Proc. Natl. Acad. Sci. USA, 71, 2773-2776. 2. Atlas, D., Steer, M. L. & Levitzki, A. (1974) Proc. Natl. Acad. Sci. USA, 71, 4246-4248. 3. Levitzki, A., Sevilla, N., Atlas, D. & Steer, M. L. (1975) J. Mol. Biol. 97, 35-53. 4. Lefkowitz, R. J., Mukherjee, C., Coverstone, M. & Caron, M. G. (1974) Biochem. Biophys. Res. Commun. 60,703-709.
Biochemistry: Atlas and Levitzki
5294
Proc. Natl. Acad. Sci. USA 74 (1977)
Iz
~~~ZU ~~~~~~_
U
~ 42
WVLEGH n 60
50
A B
WAEEGH nm 4
50
40 4 40 52c6
WAVELENGTH, nm WAVELENGTH, nm FIG. 9. The fluorescence change upon displacement of 9-AAP from the receptor by I-propranolol. A suspension of turkey erythrocyte membranes (10 mg of protein per ml, 15 nM receptor) was mixed with 0.1 gM 9-AAP in 50 mM Tris-HCl containing 2 mM MgCl2 and 1 mM EDTA, pH 7.4 at 250. Curves A: membranes and 9-AAP; curves B: membranes, 9-AAP, and the stereoisomer of propranolol. The propranolol concentrations used for the displacement experiment were 1 AM for the I or the d stereoisomers. The excitation wavelength was 385 nm. The fluorescence emission was monitored in the front-face mode at 30° as described by Gabel et al. (21). One can calculate on the basis of the dissociation constant of the j3-receptor-9-AAP complex (Table 1) that 11.5 nM of the receptor is bound with 9-AAP. 5. Romero, J. A., Zats, M., Kebabian, J. W. & Axelrod, J. (1976) Nature 258,435-436. 6. Aurbach, G. D., Fedak, S. A., Woodward, C. J., Palmer, J. S., Hauser, D. & Troxler, F. (1974) Science 186, 1223-1224. 7. Brown, E. M., Fedak, S. M., Woodward, C. J., Aurbach, G. D. & Rodbard, D. (1976) J. Biol. Chem. 251, 1239-1246.
zF , zD
i
UJ W a:
CC
Biochemistry
Probing of fl-adrenergic receptors by novel fluorescent fl-adrenergic blockers (membrane adenylate cyclase/tluorescence microscopy/9-aminoacridylpropranolol/dansyl analogi of promranolol)
DAPHNE ATLAS AND ALEXANDER LEVITZKI Department of Biological Chemistry, The Hebrew University of Jerusalem, 20 Mamilla Road, Jerusalem, Israel
Communicated by Daniel E. Koshland, Jr., August 31, 1977
ABSTRACT
The synthesis of two high-affinity fluorescent
(3-adrenergic blockers is described: dI-N-2-hydroxy-3 (1naphthyloxy)propyl-N2-9-acridyl)-1,2-propanediamine (9aminoacridylpropanolol, 9-AAP) and di-N[2-hydroxy-3-(1naphthyloxy)propyl-N'l-dansylethylenediamine (dansyl analogue of pro ranolol, DAPN). Both 9-AAP and DAPN inhibit competitively the 1-epinephrine-dependent adenylate cyclase activity [ATP pyrophos hate-lyase (cyclizing), EC 4.6.1.11 in turkey erythrocyte membranes without affecting the fluoridestimulated adenylate cyclase activity. Similarly, MAAP and DAPN inhibit in a competitive manner the binding of [12%IJ iodohydroxybenzylpindolol to these fl-adrenergic receptors. The two fluorescent P-adrenergic blockers 9-AAP and DAPN probe specifically ft-adrenergic receptors in the central nervous system
The use of these (3-blockers is to probe postsynaptic (3-adrenergic receptors. This approach is complementary to the well-established fluorescent-histochemical technique (16, 17) used to detect adrenergic and noradrenergic presynaptic pathways. MATERIALS AND METHODS [a-32P]ATP and 3H-labeled cyclic AMP ([3H]cAMP) were purchased from the Radiochemical Centre, Amersham, England. Creatine kinase, creatine phosphate, theophylline, and l-epinephrine were purchased from Sigma. Protein was determined according to Lowry et al. (18) with bovine serum albumin as the standard. Turkey erythrocyte membranes were prepared according to the method described by Steer and Levitzki (19). The activity of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was assayed according to the method of Salomon et al. (20). 125I-HYP binding was measured as described earlier (8). Fluorescence measurements were conducted on a Hitachi-Perkin-Elmer MPF-4 spectrofluorimeter. Front face fluorescence at 300 was measured as described by Gabel et al. (21). A special cell holder for the measurement of front-face fluorescence for either 3-ml or 0.5-ml fluorescence cells was constructed. All fluorescence measurements were conducted at 250. The injection of 9-AAP into rats and mice and the preparation of cryostat sections were performed as described earlier (11-15). Identical procedures were used for the in vivo studies on DAPN.
as well as in other organs when injected into rats. The fluorescence pattern can be monitored by fluorescence microscopy performed on cryostat slices of these organs. The appearance of the characteristic fluorescence pattern can be blocked in a stereospecific fashion by a prior injection of 1-propranolol and not by a prior injection of d-propranolol. These compounds therefore offer a powerful means to map -adrenergic receptors in vivo. The stereospecific displacement of AP from the ,Badrenergic receptors of turkey erythrocyte membranes by Ipropranolol and by 1-epinephrine can be detected in vitro using front-face fluorescence. The potential use of these compounds to probe (-receptors in vitro and in vivo is discussed.
,B-Adrenergic receptors can be monitored using radioactively labeled high-affinity (3-blockers such as [3H]propranolol (1-3), [3H]alprenolol (4, 5), and [125I]iodohydroxybenzylpindolol (125I-HYP) (6-8). Recently irreversible (3-receptor-directed affinity labels have also been prepared (9, 10). These compounds enable one to measure directly the content of (3-receptors in a variety of cell types. The 3H affinity label is currently employed to characterize the molecular nature of the (3-adrenergic receptor (D. Atlas and A. Levitzki, unpublished data). In this communication we report the synthesis and properties of two potent fluorescent ,B-adrenergic blockers: dl-9-aminoacridylpropranolol (9-AAP)* and a dansyl analog of propranolol (DAPN).* These two compounds bind stereospecifically to f-adrenergic receptors and their binding can be probed in vivo using fluorescence microscopy and in vitro using fluorescence spectroscopy. One of the compounds, 9-AAP, has already been used to map f(-adrenergic receptors in vivo (11-14). In these studies fluorescence was found in regions known to possess (3-adrenergic receptors, subsequent to the injection of 9-AAP into rats and mice (15). DAPN, as shown in this communication, was found to be as efficient for probing f(-adrenergic receptors in vivo as 9-AAP. 9-AAP, due to its fluorescence properties, was found to be suitable also for monitoring (3-adrenergic receptors in vitro.
9-Aminoacridylpropranolol (9-AAP). 1,2-Epoxy-3-(1naphthyloxy)propane was synthesized as described elsewhere (10). 1,2-Epoxy-3-(l-naphthyloxy)propane (2.0 mmol) dissolved in 2.0 ml of dioxane is condensed with a mixture of two N-isopropylamino-9-aminoacridine isomers (22) (2.0 mmol) (see Fig.
1) by heating the mixture at 500 for 24 hr. The solution is cooled and acidified with concentrated hydrochloric acid (6.0 mmol). The aqueous phase is concentrated under vacuum. The mixture of hydrochlorides separates as an oil. The oil is treated with traces of methanol to complete dissolution and then precipitated by the addition of ether. The compound 9-AAP is separated on thin-layer plates 1.0 mm thick, made of Kieselgel H type 60, Merck. 1-Butanol/acetic acid/water (4:1:4 by volume) is used as the solvent system (RF = 0.4). The outline of synthesis is shown in Fig. 1. The absorption spectrum, fluorescence excitation spectrum, and fluorescence emission spectrum of the compound are given in Fig. 2. Analysis: % N = 9.2 (calculated Abbreviations: 9-AAP, dl-N1-[2-hydroxy-3-(1-naphthyloxy)propyl]N2-(9-acridyl)-1,2-propanediamine, or dl-9-aminoacridylpropranolol; DAPN, dl-N-[2-hydroxy-3-(1-naphthyloxy)propyl]-N'-dansylethylenediamine, or dansyl analog of propranolol; 125I-HYP, [125I]iodohydroxybenzylpindolol; cAMP, cyclic AMP. * Now available commercially from Yissum Research Development Co., Hebrew University, Jerusalem, Israel.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5290
Proc. Natl. Acad. Sc. USA 74 (1977)
Biochemistry: Atlas and Levitzki
OH
CH3
OCH2CH-CH2 NH CH 2-CH2 NH2
NH2CHCH2NH
OCH2 CH - CH2
5291
ct
Cl SO2
NH
NH2CH2CH
N CH3 CH3
CH3 T2.HCI
OH OCH2 CH-CH2-NHCH2CH2NH SO2
CH3
OH
OCH2 CHCH2NHCH CH2 - NH t
X
~~~~~~A
2
Hydrochloride
SalIt
OH and
N
CH3 CH3
FIG. 3. Outline of synthesis of DAPN.
OCH2CHCH2NHCH2CH- NH
CH3
cm-1 in water. The absorption spectrum, the fluorescence excitation spectrum, and the fluorescence emission spectrum are shown in Fig. 4.
B
FIG. 1. Outline of synthesis of 9-AAP.
RESULTS Spectroscopic Properties of the Ligand. The absorption, fluorescence excitation, and fluorescence emission spectra of 9-AAP and DAPN are shown in Figs. 2 and 4, respectively. The quantum yield of 9-AAP decreases as a function of hydrophobicity, as can be seen from Fig. 5, in which the effect of dioxane on the intensity of the fluorescence emission is demonstrated. Binding of 9-AAP and of DAPN to the fl-Adrenergic Receptor. The compounds 9-AAP and DAPN inhibit competitively the epinephrine-dependent adenylate cyclase activity from turkey erythrocyte membranes (Fig. 6). Both 9-AAP and DAPN exert no effect on the fluoride-stimulated activity. The dissociation constants for 9-AAP and DAPN calculated from the inhibition of the l-epinephrine-dependent adenylate cyclase activity are summarized in Table 1. The inhibition of 125I-HYP binding by 9-AAP and DAPN is shown in Fig. 7. Both 9-AAP
= 9.31). The molar extinction coefficient of 9-AAP at 260 nm was found to be 200,000 M-1 cm-' in water. Dansyl Analogue of Propranolol (DAPN). dl-N-[2-Hy-
droxy-3-(1-naphthyloxy)propyl]ethylenediamine (10) (2.0 mmol) is partially dissolved in dioxane (3.0 ml), and triethylamine (6.0 mmol) is added for neutralization. Ten percent molar excess of dansyl chloride (2.2 mmol) is added to the reaction mixture, and the reaction is allowed to proceed for 1.0 hr at room temperature. Then the mixture is heated up to 50° and kept at this temperature for 0.5 hr. After evaporation under reduced pressure the oily residue is dissolved in traces of isopropanol and methanol and precipitated with ether. The white flocculent precipitate (DAPN) is collected, washed with ether, water, and ether once again, and dried in a desiccator over KOH and CaC12. Analysis: % N = 6.21 (calculated = 6.96). The outline of synthesis of DAPN is shown in Fig. 3. The molar extinction coefficient at 295 nm was found to be 3060 M-1
WAELNGH nmm420nmc WAVELENGTH (nm) D WAVeLENTH(n) 4..
z 410
co) < 0.6 20
z W
z
260 32
WU 0 z202020
e
Q4
o30303
9
o4
260
320 380
440 50
WAVELENGTH (nm)
0
D
250
8
2
480
520
Z w 0
~~~~~~~~~~~~~~~~~~~~~~C/)
Cl)
200
4
0
o wUzo-
0.2,
zFO -0 w
w
270
290
310
330
350 370
390
WAVELENGTH (nm)
410
430
0
D
400
440
WAVELENGTH (nm)
FIG. 2. The absorption spectrum, fluorescence excitation spectrum, and fluorescence emission spectrum of 9-AAP. The absorption spectrum of 9-AAP was measured on a 5 AM solution. The molar extinction coefficient of 9-AAP at 260 nm can be calculated to be 200,000 M-1 cm-1. A concentration of 2.4 MM 9-AAP in water was used for the measurement of the fluorescence excitation spectrum and the fluorescence emission spectrum in the MPF-4 spectrofluorimeter at 250. When the excitation spectrum was monitored, the emission wavelengths (Aem) used were 420 and 445 nm. When the fluorescence emission spectrum was monitored, the excitation wavelength used was 385 nm. The slight difference in the excitation spectra at 420 and 445 nm may reveal the higher contribution to the fluorescence of the naphthyloxy moiety in the 9-AAP molecule. This finding may also be taken as an indication for the relative inefficiency in the energy transfer between the naphthyloxy moiety and the acridine nucleus.
Biochemistry: Atlas and Levitzki
5292
Proc. Natl. Acad. Sci. USA 74 (1977) I-
t~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.
0.6
Z ~~~~~~AgS2Smm
A1.d5e
I.-
A.-4S0Mm
OA
240
260
320
360
200
400
240
280
320
360
440
400
480
WAELENGTH (nm)
WANELENGTH (nm)
520
600
560
640
700
MVELENGTH (nm)
FIG. 4. The absorption spectrum, fluorescence excitation spectrum, and fluorescence emission spectrum of DAPN. The absorption spectrum of 0.14 mM DAPN was measured in water. The concentration of DAPN for the fluorescence measurements was 1.0 ,uM in p-dioxane. When the excitation spectrum was monitored, the emission wavelengths used were 440, 480, and 525 nm. When the fluorescence emission spectrum was monitored, the excitation wavelength was 350 nm.
and DAPN compete with the fl-adrenergic blocker 125I-HYP for binding to the Preceptor. The dissociation constants for 9-AAP and DAPN were calculated from the Scatchard plots presented in Fig. 7, as explained in the legend to the figure. Fluorescence Mapping of f-Receptors In Vivo by DAPN. DAPN (1.0 mg/ml) in 0.5 ml of saline was administered by slow intravenous injection into the tail veins of albino rats (200-220 g, 2.5 mg of DAPN per kg of body weight). Fifteen to thirty minutes later the animals were sacrificed by decapitation as described earlier (11-14). The brains were removed immediately and frozen in liquid nitrogen. Sections of 8-16 ,.m were cut in a cryostat at -20°. The frozen sections were mounted on glass slides, dried in air, and visualized under phase contrast and transmitted ultraviolet illumination on a Zeiss Universal fluorescent microscope (11-13). Prior injection of dl-propranolol (5.0 mg/kg) inhibited formation of the fluorescence pattern. Only slight reduction in the fluorescence was observed after injection of d-propranolol. The fluorescence pattern observed in the kidney, with 9-AAP, and the fluorescence pattern observed in the pyramidal cells of the hippocampus, with DAPN, are shown in Fig. 8.
Fluorescence Changes upon 9-AAP Binding to f-Receptors In Vitro. In Figs. 9 and 10, the front-face fluorescence spectrum of a mixture of turkey erythrocyte membranes at 10 mg/ml and 0.10 ,M 9-AAP is shown. Upon addition of either I-propranolol
but measurable in(Fig. 9) or l-epinephrine (Fig. 10) a crease in the fluorescence intensity can be detected. This flufor the I-antagonist and 1orescence change is
small
stereospecific
agonist, respectively, because d-propranolol and d-epinephrine do not display any effect on the spectrum when added to the mixture of membranes with 9-AAP (Figs. 9 and 10). The displacement of 9-AAP from the receptor results in an increase of the 9-AAP fluorescence intensity. This is probably due to the fact that the 9-AAP is displaced from the hydrophobic environment in the f-receptor to the more hydrophilic environment of the buffer. The fluorescence of the acridine derivative in-
when transferred from a hydrophobic medium to a hydrophilic medium (Fig. 5). The fluorescence peak at 540 nm is an intrinsic fluorescence
creases
E
W C
''o
A
oo
-.
C0
FIGX
cyclase
% DIOXANE
60
60
40
40
40
2 -
civiy lt y l g'N mdn amod
~
a
measured as a function of I-epinephrine concentration in the presence and in the absence of 9-AAP or DAPN. (A)
zoct inwa 420
44
6
8
0
0.20MM 9-AAP. (B)
*, No 9-AAP present;
*, No DAPN present; ,0.15M DAPN present;
0, 1.5 MM DAPN present. The data in both cases are plotted according to Eadie:
20
W-) 00 -Pa FIG 5.Tefurseceitniyo
ucino
v/[E]
=
Vmax
-J
420
440
460
480
WAVELENGTH,
500
520
nm
The fluorescence intensity of 9-AAP as a function of FIG. 5. p-dioxane concentration. The fluorescence emission spectrum of 9-AAP in water in the presence of increasing dioxane concentrations was measured at 250 on the MPF-4 spectrofluorimeter.
in which v is the measured adenylate cyclase activity,
[El
is the epi-
nephrine concentration, KE is the epinephrine-receptor dissociation constant,
[I]
is the concentration of the
,8-blocker, Vma,, is
blocker-receptor dissociation constant, and
specific activity. for
The values calculated for the
K1
dissociation
9-AAP and DAPN are summarized in Table
1.
is the
(3-
the maximal constants
Biochemistry:
Atlas and Levitzki
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. The dissociation constants of 9-AAP and DAPN towards the ,-receptor Dissociation constant, nM From inhibition of 125I-HYP From inhibition kinetics Compound binding 9-AAP 34.0 + 3.0 21.3 2.0 DAPN 34.5 + 3.0 28.3 + 2.0 The values of the dissociation constants of 9-AAP and DAPN to the f3-adrenergic receptors were calculated from Figs. 6 and 7, as explained in the legends to the figures.
peak of the membrane and may represent membrane-bound flavins. This fluorescence peak remains unchanged upon addition of 9-AAP, propranolol, or epinephrine. From the practical point of view, the constant value of this peak during the experiment may be taken as an internal reference. Although the compound DAPN is shown to bind with high affinity to the (-receptor (Table 1) it was found that its nonspecific solubility in the membrane causes a large fluorescence enhancement that does not allow us, at the present time, to resolve the fluorescence enhancement due to the binding to the (-receptor. Furthermore, the fluorescence peak of the bound DAPN overlaps the intrinsic, fluorescence peak of the turkey membranes, which further complicates the use of this compound in studies in vitro. However, in studies in vivo, DAPN was found to be as effective as 9-AAP (Fig. 8 and ref. 23). DISCUSSION The fluorescent (3-blockers 9-AAP and DAPN bind specifically to the (3-adrenergic receptors of turkey erythrocyte membrane fragments. The specificity of the binding is established by three criteria: (i) the inhibition of the catecholamine-dependent adenylate cyclase activity (Fig. 6); (ii) the inhibition of 125IHYP binding to the (3-receptor (Fig. 7); and (iii) the ability of i-propranolol and I-epinephrine to displace the fluorescent (3-blocker and the inability of d-propranolol and d-epinephrine to cause its displacement (Figs. 9 and 10). The stereospecific displacement of the fluorescent (3-blocker by i-agonist or I-antagonist from the receptor is accompanied by an increase of the ligand fluorescence (Figs. 9 and 10). An increase in the fluorescence upon its displacement from a hydrophobic environment to a hydrophilic one is in accordance 2.4
24.
-AA
-
o-
xz
a
co
B
20 20
2.0
I.6 0
1.2
126
0.8
0.8
0.4
0.4 02
04
06
08
51 -HYP
1.0
1.2
-
02
04
06
08
1.0
1.2
BOUND, pmol / mg
FIG. 7. The displacement of 125I-HYP from ,8-receptors by 9-AAP and DAPN. The binding of 125I-HYP to turkey erythrocyte membranes was measured as described by Maguire et al. (8). The binding data are presented in the form of Scatchard plots. The values for the dissociation constants for 9-AAP and DAPN are given in Table 1. (A) 0, Binding of 125I-HYP in the absence of 9-AAP; &, binding of 1251-HYP in the presence of 0.10MM 9-AAP; 0, binding of 125I-HYP in the presence of 0.50MM 9-AAP. (B) 0, Binding of 125I-HYP in the absence of DAPN; 0, binding of 125I-HYP in the presence of 0.50 gM DAPN.
5293
FIG. 8. (A) Distribution of DAPN fluorescence within the pyramidal cell layer of the rat hippocampus. (B) Localization of 9-AAP fluorescence in the region corresponding to the afferent arteriole and its juxtaglomerular cells in rat kidney (G = glomeruli). The fluorescence pattern is observed by using an UV-illuminated phase microscope of cryostat slices (8-16 Am) of rat brain (A) and rat kidney (B) 15 min after intravenous injection of DAPN (A) or 9-AAP (B).
with the fluorescence properties of the compound (Fig. 5). Under the experimental conditions used (Figs. 9 and 10) 11.5 nM of the total concentration of 100 nM 9-AAP are bound to the receptor. The fluorescence change measured amounts to 8.5-10%. This finding may be taken as an indication that the receptor binding site is highly hydrophobic. The absolute magnitude of the fluorescence change is, however, at present too small to be used as a probe for the quantitative titration of (-receptors in vitro. It is hoped that this goal will be achieved when higher concentrations of receptor will be accessible experimentally. At present the highest (-adrenergic receptor concentration accessible is about 10-20 nM. The availability of fluorescent (3-blockers allows one to localize (-receptors in a variety of tissues. Indeed, 9-AAP has already been used successfully to map (3-adrenergic receptors (11-15). The specific fluorescence staining is inhibited by a prior injection of i-propranolol and not by a prior injection of d-propranolol (14). DAPN was also found to bind in a stereospecific fashion to (3-adrenergic receptors in vivo both in the brain and in the periphery (Fig. 8). Attempts, however, to monitor DAPN binding in vitro to the (3-adrenergic receptors of turkey erythrocyte membranes have thus far failed. This is probably due to the fact that the intrinsic fluorescence peak of the membranes coincides with that of the bound DAPN. The much simpler synthetic procedure for the preparation of DAPN as compared to the procedure to prepare 9-AAP and the fact that DAPN is at least as effective as 9-AAP in in vivo studies make DAPN the ligand of choice for the task of mapping (3-adrenergic receptors. The availability of specific postsynaptic blockers such as 9-AAP or DAPN for the (3-adrenergic receptor enables the researcher to selectively identify adrenergic and noradrenergic nerve terminals. It would also be useful to synthesize fluorescent a-adrenergic blockers as well as fluorescent blockers for dopamine receptors.' This work was supported by a grant from the U.S.-Israel Binational Research Foundation (BSF), Jerusalem, Israel. D.A. was supported by the Lady Davis Fellowship Trust and by a Research Advancement of Science and Technology grant from the Bat-Sheva de Rothschild Fund. 1. Levitzki, A., Atlas, D. & Steer, M. L. (1974) Proc. Natl. Acad. Sci. USA, 71, 2773-2776. 2. Atlas, D., Steer, M. L. & Levitzki, A. (1974) Proc. Natl. Acad. Sci. USA, 71, 4246-4248. 3. Levitzki, A., Sevilla, N., Atlas, D. & Steer, M. L. (1975) J. Mol. Biol. 97, 35-53. 4. Lefkowitz, R. J., Mukherjee, C., Coverstone, M. & Caron, M. G. (1974) Biochem. Biophys. Res. Commun. 60,703-709.
Biochemistry: Atlas and Levitzki
5294
Proc. Natl. Acad. Sci. USA 74 (1977)
Iz
~~~ZU ~~~~~~_
U
~ 42
WVLEGH n 60
50
A B
WAEEGH nm 4
50
40 4 40 52c6
WAVELENGTH, nm WAVELENGTH, nm FIG. 9. The fluorescence change upon displacement of 9-AAP from the receptor by I-propranolol. A suspension of turkey erythrocyte membranes (10 mg of protein per ml, 15 nM receptor) was mixed with 0.1 gM 9-AAP in 50 mM Tris-HCl containing 2 mM MgCl2 and 1 mM EDTA, pH 7.4 at 250. Curves A: membranes and 9-AAP; curves B: membranes, 9-AAP, and the stereoisomer of propranolol. The propranolol concentrations used for the displacement experiment were 1 AM for the I or the d stereoisomers. The excitation wavelength was 385 nm. The fluorescence emission was monitored in the front-face mode at 30° as described by Gabel et al. (21). One can calculate on the basis of the dissociation constant of the j3-receptor-9-AAP complex (Table 1) that 11.5 nM of the receptor is bound with 9-AAP. 5. Romero, J. A., Zats, M., Kebabian, J. W. & Axelrod, J. (1976) Nature 258,435-436. 6. Aurbach, G. D., Fedak, S. A., Woodward, C. J., Palmer, J. S., Hauser, D. & Troxler, F. (1974) Science 186, 1223-1224. 7. Brown, E. M., Fedak, S. M., Woodward, C. J., Aurbach, G. D. & Rodbard, D. (1976) J. Biol. Chem. 251, 1239-1246.
zF , zD
i
UJ W a:
CC
