Irreversible binding of a carbostyril-based agonist and antagonist to the β-adrenoceptor in DDT1 MF-2 cells and rat aorta

Article abstract of DOI:10.1038/sj.bjp.0701801

1. The chemoreactive ligands 5(2-(((1'-(4'-isothiocyanatophenylamino)thiocarbonyl)-amino)-2 -methylpropyl)amino-2-hydroxypropoxy)-3,4-dihydrocarbostyril (DCITC) and 8-hydroxy-5(2-(((1'-(4'-isothiocyanatophenylamino)thiocarbonyl)amino)-2 -methylprop-2-yl)a

Full text of DOI:10.1038/sj.bjp.0701801

British Journal of Phar
m
acology (1998) 124, 165 ± 175
1998 Stockton Press All rights reserved 0007 ± 1188/98 $12.00
http://www.stockton-press.co.uk/bjp
Irreversible binding of a carbostyril-based agonist and antagonist to
the b-adrenoceptor in DDT1 MF-2 cells and rat aorta
1Malgorzata D. Deyrup, 2Phillip G. Greco, 1Deborah H. Otero, 1,3Donn M. Dennis, 1,2Craig H. Gelband
and 1,4Stephen P. Baker
2
3
1Departments of Pharmacology and Therapeutics, Physiology and Anesthesiology, University of Florida, College of Medicine,
Gainesville, Florida, 32610, U.S.A.
1
The chemoreactive ligands 5(2-(((1'-(4'-isothiocyanatophenylamino)thiocarbonyl)-amino)-2-methyl-
propyl)amino-2-hydroxypropoxy)-3,4-dihydrocarbostyril (DCITC) and 8-hydroxy-5(2-(((1'-(4'-isothiocya-
natophenylamino)thiocarbonyl)amino)-2-methylprop-2-yl)amino-1-hydroxyethyl)-carbostyril (HCITC)
were synthesized and shown to be potent irreversible antagonist and agonist ligands, respectively, for
the b-adrenoceptor in DDT1 MF-2 (DDT) cells and the rat isolated aorta.
2
In DDT cell membranes DCITC and HCITC inhibited (7)[125I]-iodocyanopindolol (CYP) binding to
the b-adrenoceptor with IC50 values of 1.1 and 18 nM, respectively. (7)-Isoprenaline inhibited [125I]-CYP
binding with an IC50 of 355 nM. Pretreatment of membranes with either chemoreactive ligand produced
a time- and concentration-dependent decrease in the b-adrenoceptor content, indicating irreversible
receptor binding. DCITC at concentrations up to 10 mM did not stimulate cyclic AMP accumulation in
DDT cells nor did it amplify forskolin-stimulated cyclic AMP accumulation.
3
In the rat isolated aorta, DCITC (0.1 mM) did not a€ect either the phenylephrine-mediated tissue
contraction or the acetylcholine-mediated relaxation. DCITC attenuated the maximal (7)-isoprenaline-
mediated relaxation of a phenylephrine contracted aorta in a concentration-dependent manner and
shifted the dose-response curves for (7)-isoprenaline to the right. The DCITC-induced decrease in
maximal response was not reversed by extensive tissue washing. By use of the operational model of
agonism, the calculated dissociation constant for (7)-isoprenaline ws 286 nM and the estimated receptor
reserve for this agonist was 23% at the maximal response.
4
HCITC and (7)-isoprenaline stimulated cyclic AMP accumulation in DDT cells with pD2 values
(negative logarithm to base 10 of EC50) of 7.95 and 7.97, respectively, and both mediated the same
maximal stimulation. In the rat isolated aorta, HCITC produced a concentration-dependent relaxation
of the tissue with a pD2 value of 6.62, whereas the pD2 for (7)-isoprenaline was 7.03. However, HCITC
produced a greater maximal relaxation of the tissue than (7)-isoprenaline. The HCITC-mediated
stimulation of cyclic AMP accumulation and relaxation of the isolated tissue were blocked when the b-
antagonist propranolol was added concurrently. In contrast, once the HCITC-mediated responses were
established, the addition of propranolol did not result in any attenuation indicating that HCITC is an
irreversible b-agonist.
Keywords: b-Adrenoceptor; DDT1 MF-2 cells; isolated aorta; chemoreactive ligands; (7)-isoprenaline; cyclic AMP;
propranolol; 5(2 - (((1'- (4'- isothiocyanatophenylamino) - thiocarbonyl)amino)-2-methylpropyl)amino-2-hydroxypro-
poxy)-3,4-dihydrocarbostyril (DCITC); 8-hydroxy-5(2-(((1'-(4'-isothiocyanatophenylamino)thiocarbonyl)amino)-2-
methylprop-2-yl)amino-1-hydroxyethyl)-carbostyril (HCITC)
Introduction
Chemoreactive ligands are useful probes to study the structure
and function of receptors. These ligands are usually composed
the agonist binding site in the receptor (Baker & Deyrup,
1994).
of
a
pharmacophore based upon known agonists or
A number of chemoreactive ligands for the b-adrenoceptor
have been synthesized and shown to act as irreversible ligands
including those based upon the antagonist pharmacophores
propranolol (NHNP-NBE, Atlas & Levitzki, 1976), alpreno-
lol (BAAM, Pitha et al., 1980; Baker & Pitha, 1982) and a
benzofuranyl derivative (Ro03-7894, Nicholson & Broadley,
1978; Rankin & Broadley, 1982). However, in subsequent
studies irreversible inactivation of the b-adrenoceptor by
NHNP-NBE and Ro03-7894 has been questioned (Krstew et
al., 1984; Minneman & Mowry, 1986). Furthermore, in some
but not all isolated tissue studies, Ro03-7894 and BAAM
appeared to produce nonspeci®c tissue depression either in
the absence or presence of irreversible receptor blockade,
limiting their usefulness in these studies (Krstew et al., 1984;
Posner et al., 1984; May et al., 1985; Ng & Malta, 1989;
Doggrell, 1990). This nonspeci®c e€ect may be related to the
relatively high concentration of chemoreactive ligand
required for receptor blockade which also allows the reactive
antagonists, which provides anity and selectivity for the
receptor and a chemoreactive moiety through which covalent
attachment to the receptor occurs preventing ligand dissocia-
tion. Antagonist based chemoreactive ligands have been used
to determine agonist dissociation constants (KA) and receptor
reserve (Furchgott, 1996; Dennis et al., 1992), basal receptor
turnover (Mahan et al., 1987), mapping of the antagonist
binding site (Dohlman et al., 1988; Curtis et al., 1989) and to
discriminate receptor subtypes (Han et al., 1987; Salles et al.,
1993). Although fewer agonist based irreversible ligands have
been described, they have additional potential in studies on the
mechanism of receptor activation, desensitization and de®ning
4 Author for correspondence at: Department of Pharmacology,
University of Florida, College of Medicine, Box 100267,
Gainesville, Florida, 32610, U.S.A.

166
M.D. Deyrup et al
Irreversible b-adrenoceptor agonist and antagonist
group to modify covalently nonreceptor tissue components.
To overcome this problem, chemoreactive ligands with a high
anity pharmacophore should produce irreversible receptor
binding at low ligand concentrations with minimal non-
speci®c reactions. Several chemoreactive ligands based on the
high anity pharmacophores pindolol (Br-AAM-pindolol)
and cyanopindolol (BIM) have been shown to bind
irreversibly to the b-adrenoceptor, with little or no
nonspeci®c e€ects in depressing cell or isolated tissue
responses (Homburger et al., 1985; Kusiak & Pitha, 1987;
Jasper et al., 1988; Molenaar et al., 1988). In terms of
chemoreactive agonists for the b-adrenoceptor, BAAM, BIM
and a bromoacetylaminomenthyl derivative of nonadrenaline
(BAAN) have been shown to have varying degrees of partial
agonist activity (Baker et al., 1985; Jasper et al., 1991). In
contrast, a carbostyril-based chemo-reactive ligand (carbo-Br)
was shown to be an irreversible full b-agonist for the
stimulation of adenylyl cyclase activity in an isolated
membrane preparation (Standifer et al., 1989). The e€ects
of this compound on b-adrenoceptor function in cultured
cells and isolated tissues have not been described. None-
theless, continual re®nement in the chemical and pharmaco-
logical properties of antagonist and agonist based
chemoreactive ligands for the b-adrenoceptor will enhance
their usefulness in receptor studies.
microscale puri®cation (silica gel GF, 20620, 2 mm,
Analtech). Silica gel (grade 60, 230 ± 400 mesh, Merck Co.)
was used for column chromatography.
Synthesis of DCITC
5-(2,3-Epoxy)propoxy-3,4-dihydrocarbostyril (0.43 g, 2 mmol)
was reacted with monoprotected N-t-butoxycarbonyl-2-ami-
no-2-methyl propylamine (0.565 g, 3 mmol) in boiling n-
butanol (15 ml) for 6 h. The solvent was removed under
vacuum and the reaction mixture puri®ed by column
chromatography on silical gel with methylene chloride
methanol (98 : 2, 95 : 5, 90 : 10) to elute the product. The yield
1
of puri®ed material was 0.408 g, (50%). H n.m.r. (DMSO):
1.05 (6H, s), 1.41 (9H, s), 2.45 ± 2.55 (2H, t), 2.70 ± 285 (2H, m),
2.95 (3H, t), 3.05 (2H, s), 4.00 (2H, s), 6.50 (1H, d), 6.65 (1H,
d), 7.10 (1H, t). 13C n.m.r. (DMSO): 23.63, 27.37, 29.59, 44.33,
53.46, 69.03, 70.66, 78.69, 106.32, 106.41, 108.36, 111.86,
127.60, 138.49, 155.92, 157.31, 172.37. h.r.m.s. 408.2497
(M+H). C21H34N3O5 requires 408.2498.
The product was deprotected by use of HCl saturated
solution in 1,4-dioxane at room temperature overnight. The
corresponding hydrochloride was very hygroscopic and used
without further puri®cation. A small amount of the hydro-
chloride (20 mg) was neutralized with sodium bicarbonate and
analysed by mass spectrometry. H.r.m.s. 308.1994 (M+H).
C16H26N3O3 requires 308.1974.
To 98 mg (0.285 mmol) of 5-[3-(2-amino-2-methylpropyla-
mino)-2-hydroxypropoxy]-3,4-dihydrocarbostyril hydrochlor-
ide suspended in 1 ml of dry DMF triethylamine (0.079 ml,
0.285 mmol) was added, followed by an excess of 1,4-
phenyldiisothiocyanate (238 mg, 1.24 mmol). This mixture
was stirred at room temperature for 5 h. The solvent was
removed under vacuum, and the crude material puri®ed on
preparative t.l.c. with silica gel and 15% methanol in
methylene chloride as eluent. The stationary phase was
extracted with the same solvent mixture yielding 54 mg
(38%) of the pure product. 1H n.m.r. (DMSO): 1.15 (6H,
two s), 2.45 ± 2.52 (2H, m), 2.65 ± 2.90 (4H, m), 3.53 (1H,
broad signal), 3.90 ± 4.00 (3H, m), 5.50 ± 5.55 (1H, m), 6.45
(1H, t), 6.55 ± 6.62 (1H, m), 7.00 ± 7.10 (1H, m), 7.15 ± 7.35
(3H, m), 7.45 ± 7.50 (1H, m). h.r.m.s. 500.1798 (M+H).
C24H30N5O3S2 requires 500.1790.
In this study, the synthesis and pharmacological
characterization of the b-antagonist 5(2-(((1'-(4'-isothiocya-
natophenylamino)thiocarbonyl)amino) - 2 - methylpropyl) ami-
no-2-hydroxypropoxy)-3,4-dihydrocarbostyril (DCITC) and
the b-agonist 8-hydroxy-5(2-(((1'-(4'-isothiocyanatophenyla-
mino)thiocarbonyl)amino)-2-methylprop-2-yl)amino-1-hydro-
xyethyl)-carbostyril (HCITC) are presented. The pharmaco-
phore for these chemoreactive ligands is based on
a
carbostyril nucleus for which previous derivatives have been
shown to be potent agonists and antagonists for the b-
adrenoceptor (Nakagawa et al., 1974; Yoshizaki et al.,
1976). In the synthesis of DCITC and HCITC, the
chemoreactive moiety selected for incorporation into the
pharmacophore is an isothiocyanate which can readily react
with nucleophilic amino acids (Baker & Deyrup, 1994). This
reactive moiety has been used in chemoreactive ligands for
several other receptors (Jacobson et al., 1989; Kim et al.,
1989; Newman et al., 1990) but has not been employed
previously in irreversible ligands for the b-adrenoceptor. By
use of DDT1 MF-2 (DDT) cells and the rat isolated aorta
preparation, DCITC was shown to be a potent, irreversible
antagonist for the b-adrenoceptor and to be a suitable
pharmacological probe for determining the KA for agonists
and the receptor reserve. On the other hand, HCITC was
shown to be a potent b-agonist that produces sustained,
antagonist-insensitive responses in DDT cells and the
isolated tissue.
Synthesis of HCITC
8-(Benzyloxy)-5-oxiranyl-2(1H)-quinolinone (prepared by use
of a modi®cation of the procedure described by Milecki et al.
(1987) (1.10 g, 3.82 mmol) and N-t-butoxycarbonyl-2-amino-
2-methylpropylamine (1.2 g, 4.80 mmol) were suspended in n-
butanol (6 ml) and heated for 4 h. The solvent was removed
under vacuum, the residue dissolved in methylene chloride
(30 ml) and extracted with HCl solution (15 ml, 1%). The
organic layer was washed with water (15 ml), dried (MgSO4)
and the solvent removed. The residue was then chromato-
graphed on a silica gel column with methylene chloride,
followed by methylene chloride/methanol mixture from 2 ±
5%. Yield was 0.956 g (54%). For C27H35N3O5 found: C 67.11,
Methods
Analytical procedures
1
H 7.39, N 8.59. Required: C 67.34, H 7.33, N 8.72. H n.m.r.
1H and 13C nuclear magnetic resonance (n.m.r.) spectra were
measured on QE 300 MHz instrument with TMS as an
internal standard in deuterated solvents as indicated. High
resolution mass spectra (h.r.m.s.) were obtained by means of
FAB ionization mode with CH5+. Precoated silica gel sheets
(60F-254, 0.2 mm, EM-Reagents) were used for thin layer
chromatography (t.l.c.). Preparative layer plates were used for
(DMSO): 1.26 (6H, s) 1.38 (9H, s), 2.90 ± 3.32 (4H, m), 3.44
(1H, broad signal), 5.35 (2H, s), 5.52 (1H, d), 6.58 (1H, d),
7.18 ± 742 (5H, m), 7.60 (2H, d), 8.41 (1H, d), 10.85 (1H, broad
signal). 13C.m.r. (DMSO): 21.69, 21.79, 28.58, 46.10, 48.62,
59.77, 65.74, 70.12, 78.72, 112.58, 116.96, 119.98, 122.85,
128.29, 128.75, 129.81, 130.84, 136.87, 137.04, 144.16, 156.58,
161.31.

M.D. Deyrup et al
Irreversible b-adrenoceptor agonist and antagonist
167
The protected aminoalcohol (2.6 g, 5.4 mmol) was dis-
solved in methanol (30 ml) and ammonium formate (500 mg),
followed by palladium on charcoal (10%, 100 mg) was added
at once under nitrogen. The reaction was re¯uxed and
monitored by t.l.c. After no more starting material was
detected the mixture was cooled to room temperature, ®ltered
through ®ne ®lter and the ®lter concentrated under vacuum.
The residue was puri®ed by column chromatography on silica
gel and 10% methanol in methylene chloride as eluent. Yield
of the debenzylated product was 1.5 g (71%). For C20H29N3O5
found: C 60.96, H 7.80, N 10.65. Required: C 61.30, H 7.47, N
Membrane preparations and pretreatments
Cell monolayers were washed twice with HBSS (2610 ml),
scraped free of the plate with the aid of a cell scraper in 4 ml of
50 mM Tris-HCl bu€er pH 7.4 at 48C, and the suspension
homogenized with a Tekmar homogenizer at setting 4 for 10 s.
The suspension was then centrifuged at 15,0006g for 10 min.
The pellet was resuspended in 50 mM Tris-HCl bu€er pH 7.4
by vortexing and centrifuged as before. The ®nal pellet was
resuspended in 50 mM Tris-HCl bu€er pH 7.4 containing
5 mM MgCl2 for assays. The protein content was determined
by the method of Bradford (1976), with bovine serum albumin
as the standard.
In pretreatment experiments, membranes (2 mg pro-
tein ml71) were incubated in 50 mM Tris-HCl at pH 7.4
containing 5 mM MgCl2, 100 mM 5'-guanylylimidodiphosphate
(Gpp(NH)p) and without or with varying concentrations of
DCITC or HCITC for 30 min at 368C. At the end of the
incubation, the suspensions were diluted to 25 ml with ice-cold
incubation bu€er and centrifuged at 48,0006g for 10 min. The
pellet was then washed a further 9 or 5 times for membranes
treated with DCITC or HCITC, respectively, by resuspension
in 25 ml of bu€er and centrifugation. The ®nal membrane
pellets were resuspended in 1 ml of bu€er for assays.
1
10.43. H n.m.r. (DMSO): 0.916 (6H, s), 1.384 (9H, s), 2.624
(2H, d), 2.851 (2H, d), 4.900 (1H, t), 6.489 (1H, d), 6.917 (1H,
d), 7.087 (1H, d), 8.182 (1H, d).
To remove the t-butoxycarbonyl group the protected
aminoalcohol (1.8 g, 4.6 mmol) was suspended in dioxane
solution of hydrogen chloride (15 ml, 4 M) and the mixture
was stirred overnight. The solvent was removed under vacuum
and the product, a white solid was dried over phosphorous
1
pentoxide. Yield was 1.34 g (89%). H n.m.r. (D2O): 1.5 (6H,
d), 3.28 ± 3.41 (2H, m), 3.60 ± 3.64 (2H, m), 5.30 ± 5.35 (1H, m),
6.40 (1H, d), 6.75 (1H, d), 7.12 (1H, d), 8.05 (1H, d). 13C n.m.r.
(D2O+dioxane): 20.88, 21.08, 45.08, 47.42, 58.23, 65.26,
114.65, 117.90, 119.21, 121.69, 121.73, 126.72, 138.09, 143.38,
162.35. H.r.m.s. 292.1590 (M+H). C15H22N3O3 requires
292.1661.
Receptor binding assay
8-Hydroxy-5-[2-(2-amino-2-methylpropylamino)-1-hydro-
xyethyl]carbostyril hydrochloride (0.312 g, 0.952 mmol) was
dissolved in 5 ml of dry DMF, then triethylamine (0.193 g,
0.266 ml, 1.9 mmol) was added followed by 1,4-phenyldii-
sothiocyanate (1 g, 5 mmol). The mixture was stirred at room
temperature for 4 h and then DMF was removed under
reduced pressure. The residue was puri®ed on preparative t.l.c.
plates (silica gel) by use of 10% methanol in methylene
chloride as developing mixture. The silica gel was then scraped
and extracted with the same mixture of solvents. Yield of the
isothiocyanate was 0.21 g (47%). H.r.m.s. 484.1483 (M+H).
C23H26N5O3S2 requires 484.1477. 1H n.m.r. (MeOD): 1.85 (6H,
s), 3.55 ± 3.70 (2H, m), 4.25 ± 4.31 (2H, broad signal), 5.70 ±
5.75 (1H, m), 7.27 (1H, d), 7.65 (1H, d), 7.85 ± 7.95 (3H, m),
8.17 (2H, m), 8.90 ± 9.02 (1H, m).
b-Adrenoceptors in cell membranes were determined by
speci®c (7)[125I]-iodocyanopindolol (CYP) binding (Standifer
et al., 1989). Brie¯y, membrane protein (15 mg) was incubated
in a total volume of 0.25 ml containing 50 mM Tris-HCl bu€er
at pH 7.4, 5 mM MgCl2, [125I]-CYP (6 ± 100 pM), with or
without 3 mM (+)-alprenolol for 60 min at 368C. At the end of
incubation, the cell suspensions were diluted with 3 ml of ice-
cold incubation bu€er and the suspension ®ltered through
glass ®bre ®lters under vacuum by use of a Brandel cell
harvester. The ®lters were rinsed with a further 6 ml of ice-cold
bu€er and the retained radioactivity was determined with a
gamma counter. Speci®c
[
125I]-CYP binding to the b-
adrenoceptor was calculated as a di€erence between the total
binding in the absence of (+)-alprenolol and the nonspeci®c
binding determined in the presence of (+)-alprenolol.
Nonspeci®c binding was similar if the (+)-alprenolol was
replaced with 100 mM (7)-isoprenaline and speci®c binding
was 90 ± 95% of the total bound.
In some experiments, the ability of several compounds to
inhibit speci®c [125I]-CYP binding was determined. The assays
were the same as above except the [125I]-CYP concentration
was 30 pM and 100 mM Gpp(NH)p was included.
Drug preparations
Stock solutions (10 mM) of synthesized compounds were made
in DMSO and diluted with HBSS before use. The dilution of
vehicle (DMSO) was a minimum of 100 and 1000 fold for the
isolated tissue and cultured cell experiments, respectively, and
had no e€ect on responses in either preparation. All other
drugs were made fresh at the time of use.
Cyclic AMP accumulation
Cell culture
Monolayers of DDT cells were rinsed with HBSS (2610 ml),
detached into HBSS (5 ml) using a cell lifter and the resulting
suspension was centrifuged at 50006g for 5 min. Aliquots of
the cell suspension (0.5 mg protein) were then incubated in
HBSS (500 ml total volume) for 5 min at 378C, before addition
of rolipram to ®nal concentration of 50 mM and b-
adrenoceptor ligands as indicated in text. The resulting
suspensions were incubated for a further 10 min, placed in a
boiling bath for 5 min, cooled to room temperature and then
centrifuged at 13,0006g for 2 min. The adenosine 3' : 5'-cyclic
monophosphate (cyclic AMP) content of the supernatant was
determined by a modi®cation of a radioimmunoassay (Harper
& Brooker, 1975). Brie¯y, an aliquot of the supernatant (50 ml)
was added to 50 ml HBSS containing adenosine 3',5'-cyclic
DDT cells were grown as monolayers in 57 cm2 petri dishes
in Dulbecco's modi®ed Eagle's medium (DMEM) supple-
mented with 5% foetal bovine serum, 2.75 mg ml71
amphotericin B, 100 u ml71 penicillin G, and 0.1 mg ml71
streptomycin sulphate in a humidi®ed atmosphere of 95%
air and 5% CO2. Cells were routinely subcultured from
stocks once or twice a week, with 1 mM EDTA in Hank's
balanced salt solution (HBSS) without divalent cations to
detach the cells and HBSS to disperse them. The cells were
initially seeded at 1.26105 cells/plate and experiments were
performed 4 days later at 50 ± 60% con¯uence (46106 cells/
plate). Cell viability was determined by exclusion of trypan
blue.

168
M.D. Deyrup et al
Irreversible b-adrenoceptor agonist and antagonist
phosphoric acid 2'-O-succinyl[125I]-iodotyrosine methyl ester
([125I]-ScAMP, 10,000 c.p.m.). After the addition of anti-cyclic
AMP antibody (50 ml of 1 : 2000 dilution with 0.1% BSA in
water), the samples were incubated at 48C for 18 h. At the end
of incubation 70 ml of a 50% (v/v) hydroxyapatite suspension
in water was added to each tube followed by incubation for
10 min at 48C. The antibody/radioligand complex adsorbed to
the hydroxyapatite was retained on glass ®bre ®lters during
rapid ®ltration with the Brandel cell harvester. The ®lters were
washed with 6 ml of ice-cold 10 mM Tris-HCl bu€er at pH 7.0.
Nonspeci®c binding of [125I]-ScAMP was determined in
parallel assays that contained unlabelled cyclic AMP (0.1 mM),
and was subtracted from the total binding. The amount of
cyclic AMP was calculated from a standard curve constructed
with known amounts of unlabelled cyclic AMP.
at submaximal levels of response decreases proportionally with
the remaining fraction of non-inactivated receptors (t=t'q),
was allowed to vary between individual concentration-
response relations during the curve ®tting process. Unless
equation 1 gave a statistically improved ®t (P50.05) compared
to equation 2, the latter was used in the analysis.
Materials
DDT cells,
a smooth muscle cell line derived from
lieomyosarcoma of the ductus deferens of the syrian hamster,
was obtained from American Type Culture Collection (Rock-
ville, MD). (7)[125I]-CYP (2000 ± 2200 Ci mmol71
)
was
purchased from Amersham Corp (Arlington Heights, IL).
125I]-ScAMP was prepared by the method described by (Patel
[
& Linden, 1988) and was generously provided by Dr John
Shryrock from the Department of Medicine at the University
of Florida. All other chemicals, including cyclic AMP,
Gpp(NH)p, (7)-isoprenaline, (+)-alprenolol, 2'-O-monosuc-
cinyladenosine 3',5'-cyclic monophosphate tyrosyl methyl ester
and hydroxyapatite were purchased from Sigma Chemical Co.
(St. Louis, MO). Glass ®bre ®lters were from Schleicher and
Schuell (Keene, NH).
Tension measurements
Male rats were killed and the aorta was rapidly excised and
placed in cold, oxygenated (95% O2/5% CO2) Krebs-Henseleit
Bu€er (KHB), containing in mM: NaCl 120, NaHCO3 20, KCl
4.2, KH2PO4 1.2, MgCl2 0.5, CaCl2 1.8 and glucose 11 at
pH 7.4. A 3 ± 4 cm segment of the aorta was dissected free and
cleaned of fat and adventitia. Ring segments (3 ± 5 mm long)
were mounted onto two triangular tungsten wires and placed
into a 10 ml isolated organ chamber. The bottom triangle was
mounted to a stable hook while the top triangle was attached to
a Gould strain gage. The bath was maintained at 378C and a
resting force of 1.5 g was applied to the aortic rings. Vessel
segments were initially equilibrated for 2 h with alternating
5 min exposures to KCl (80 mM) and phenylephrine (1 mM). All
concentration-response curves were performed cumulatively.
Results
Synthesis of DCITC and HCITC
The multistep synthesis of DCITC was carried out by use of
previously published procedures (Tamura et al., 1970; Dubas-
Sluyter et al., 1972; Shono et al., 1981). 5-(2,3-Epoxy)propoxy-
3,4-dihydrocarbostyril, prepared in a good yield, was reacted
with N - t - butoxycarbonyl - 2 - amino - 2 - methylpropylamine
(Wang et al., 1986) to yield the corresponding adduct, which
was subsequently cleaved with hydrogen chloride etheral
solution to give the amine hydrochloride. The amine
hydrochloride was then reacted with phenyl 1,4-diisothiocya-
nate to give the chemoreactive antagonist. Similarly, HCITC
was synthesized starting with the corresponding 8-(benzyloxy)-
5-oxiranylcarbostyril (Milecki et al., 1987). This epoxide was
treated with N-t-butoxycarbonyl-2-amino-2-methylpropyla-
mine to give the protected adduct which was hydrogenated
(to remove benzyl group) and reacted with hydrogen chloride
to deprotect the amino group. The amine hydrochloride was
coupled with phenyl 1,4-diisothiocyanate to render the
corresponding chemoreactive agonist. The structures of the
two chemoreactive compounds are shown in Figure 1.
Data analysis
The IC50 values from radioligand binding assays and EC50
values from functional assays were determined from nonlinear
regression analysis of the curves by use of the GraphPad Inplot
programme (Graphpad Software, San Diego, CA). The
dissociation constant (Kd) and maximal [125I]-CYP binding
was determined from nonlinear regression analysis (GraphPad
Inplot). Statistical analysis of paired data was performed by
use of Student's t-test.
The KA and receptor reserve for (7)-isoprenaline in the
isolated aorta was determined by simultaneously curve ®tting
the concentration-response curves using the operational model
of agonism as described by Black and Le€ (1983) with a
multiple function nonlinear regression technique which used
the Marquardt-Levenberg algorithm (SigmaPlot 4.0, SPSS
Inc., Chicago, IL). The experimental data were ®t to two forms
of the operational model. Equations 1 and 2 predict the
behaviour of nonhyperbolic (slope factor ≠1) and a rectangular
hyperbolic (slope factor=1) shaped concentration-response
curves, respectively,
E€ect of DCITC and HCITC on the b-adrenoceptor in
DDT cells
The e€ect of DCITC, HCITC and the b-agonist (7)-
isoprenaline on [125I]-CYP binding to the b-adrenoceptor in
DDT cell membranes is shown in Figure 2a. These competition
assays were performed in the presence of 100 mM Gpp(NH)p to
maintain the receptor in the agonist low anity state
(Lefkowitz et al., 1976; Kent et al., 1980). The concentration
of (7)-isoprenaline that inhibited speci®c [125I]-CYP binding
by 50% (IC50) was 355+25 nM (n=5). Both DCITC and
HCITC were relatively potent as inhibiting [125I]-CYP binding
with IC50 values of 1.1+0.5 and 18+3 nM, respectively. The
agonist e€ects of the three compounds were examined by the
stimulation of cyclic AMP accumulation in DDT cells. As
depicted in Figure 2b, (7)-isoprenaline and HCITC increased
n
Emaxꢀn‰AŠ
E ˆ
ꢀ1†
ꢀ2†
n
n
ꢀ‰AŠ ‡ KA† ‡ ꢀn‰AŠ
Emaxꢀ‰AŠ
E ˆ
ˆ KA ‡ ꢀ1 ‡ ꢀ†‰AŠ
where E is the observed response, Emax is the theoretical
maximal response. A is agonist concentration, t is the
operational ecacy of the agonist, n is the slope factor and
KA is the agonist equilibrium dissociation constant. The family
of concentration-response curves were ®tted assuming a
common value of Emax, KA and n (Equation 1). Only t, which
cyclic AMP accumulation in
a concentration-dependent

M.D. Deyrup et al
Irreversible b-adrenoceptor agonist and antagonist
169
manner producing the same maximal stimulation above basal
levels [(7)-isoprenaline, 11.2+2 fold, and HCITC, 11.5+1.5
fold, n=6]. The pD2 values (negative logarithm to base 10 of
the EC50) was 7.97+0.08 (mean+s.e., n=6) for (7)-isoprena-
line and 7.95+0.04 (mean+s.e., n=6) for HCITC. In
contrast, DCITC did not increase cyclic AMP accumulation
at concentrations up to 10 mM (Figure 2b). Jasper et al. (1990,
1991) showed that the weak partial agonist activity for several
b-adrenoceptor ligands can be readily observed by ampli®ca-
tion of forskolin-stimulated cyclic AMP accumulation in
several cell lines. This experimental approach was used to test
further for DCITC agonist activity. Forskolin (1 mM) and (7)-
isoprenaline (0.1 mM) increased cyclic AMP accumulation in
DDT cells 7.9+0.6 (n=3) and 15.8+0.3 (n=3) fold above
basal levels, respectively. In the presence of both forskolin and
(7)-isoprenaline, there was an increase in the stimulation fold
to 28.9+2.4 (n=3, P50.005 vs forskolin alone). However,
when DCITC (0.1 mM) was incubated with forskolin, there was
no change in the fold stimulation of cyclic AMP accumulation
(7.8+1.0, n=3) as compared to forskolin alone.
To investigate further the interaction of HCITC and DCITC
with the b-adrenoceptor, DDT cell membranes were incubated
with these compounds in the presence of 100 mM Gpp(NH)p,
followed by membrane washing and nonlinear regression
analysis of speci®c [125I]-CYP binding. As shown in Table 1,
preincubation of DDT cell membranes with 0.5, 1 and 5 nM
DCITC for 30 min at 368C followed by 10 membrane wash
cycles resulted in a 30, 47 and 75% decrease in [125I]-CYP
binding, respectively, with no change in the Kd value of the
radioligand for the receptors remaining. HCITC also produced
a concentration-dependent decrease of b-adrenoceptors. In-
cubation of DDT cell membranes for 30 min at 368C with 5 nM
HCITC followed by 6 membrane wash cycles reduced speci®c
Figure 1 Structure of DCITC and HCITC.
Iso
DCITC
HCITC
a
[
125I]-CYP binding by 43%. Incubation with 10 nM HCITC
reduced speci®c binding by 60% and with 100 nM HCITC the
reduction was 84%. For each concentration of HCITC used
there was no change in the Kd value for [125I]-CYP binding to
the receptors remaining (Table 1). The DCITC and HCITC-
induced loss of [125I]-CYP binding to the b-adrenoceptor was
time-dependent (Figure 3). Incubation of DDT cell membranes
with 10 nM DCITC or 100 nM HCITC in the presence of
100 mM Gpp(NH)p reduced [125I]-CYP binding by 54 and 30%
b
Table 1 E€ect of DCITC and HCITC on b-adrenoceptors
in DDT1 MF-2 cell membranes
DCITC
pretreatment
[
125I]-CYP Bmax
Kd
(nM)
(fmol mg71 protein)
Control
0.5 nM
1.0 nM
5.0 nM
173+11 (3)
126+17 (3)*
96+3 (3)**
47+4 (3)***
26+2
27+5
28+3
28+1
HCITC
pretreatment
Control
5 nM
10 nM
100 nM
228+7 (4)
134+13 (3)***
91+5 (4)***
30+5 (3)***
43+4
40+3
42+4
34+2
Figure 2 Concentration-dependent e€ect of (7)-isoprenaline (Iso),
DCITC and HCITC to (a) inhibit [125I]-CYP binding in DDT cell
membranes and (b) stimulate cyclic AMP accumulation in DDT cells.
(a) DDT cell membranes were incubated in the presence of 100 mM
Gpp(NH)p, 30 pM [125I]-CYP and the indicated concentration of
ligand for 60 min at 368C. The control [125I]-CYP binding was
63+3 fmol mg71 protein. (b) DDT cells were incubated with 50 mM
rolipram and the indicated concentration of ligand for 10 min at
378C. The basal and maximal (7)-isoprenaline-stimulated cyclic
AMP accumulation were 45+2 and 515+68 pmol mg71 protein 10 -
min71, respectively. Data from both series of experiments are the
mean with vertical lines showing s.e.mean; n=3 ± 4.
Membranes were incubated with 100 mM Gpp(NH)p and the
indicated concentrations of DCITC or HCITC for 30 min at
368C. At the end of the incubation, the membranes were
washed (DCITC, 10 times; HCITC, 6 times) and the Bmax
and Kd for [125I]-CYP binding to the b-adrenoceptor were
determined. Data are the means+s.e. Values in parentheses
are the number of experiments. *P50.05, **P50.01,
***P50.0005 compared to respective control.

170
M.D. Deyrup et al
Irreversible b-adrenoceptor agonist and antagonist
DCITC
HCITC
Iso
Iso + prop (4 m in)
HCITC
HCITC + prop (4 m in)
HCITC + prop
Basal
Figure 3 Time course of DCITC and HCITC-induced loss of
speci®c [125I]-CYP binding. DDT cell membranes were incubated
with 10 nM DCITC or 100 nM HCITC in the presence of 100 mM
Gpp(NH)p. At the times indicated, the membranes were washed and
the speci®c binding of 100 pM [125I]-CYP determined. Data are the
mean with vertical lines showing s.e.mean; n=3 ± 4. The control
Figure 4 Time course of (7)-isoprenaline and HCITC mediated
stimulation of cyclic AMP accumulation. DDT cells were incubated
with 50 mM rolipram and 1 mM (7)-isoprenaline (Iso) or 1 mM
HCITC and at the times indicated the cyclic AMP content was
determined. Propranolol (Prop; 20 mM) was added after 4 min of
incubation with (7)-isoprenaline (Iso+prop, 4 min) or HCITC
(+prop, 4 min). HCITC and propranolol added together (HCITC+-
prop) and basal cyclic AMP. Data are the mean with vertical lines
showing s.d. of triplicate determinations and is representative of 3
experiments.
[
125I]-CYP binding at time 0 was 133+12 fmol mg71 protein.
after 0.5 min and reached a maximal reduction of 81 and 70%,
respectively, by 10 min of incubation.
The b-antagonist propranolol was used to investigate the
reversibility of (7)-isoprenaline and HCITC-mediated stimu-
lation of cyclic AMP accumulation in DDT cells. As shown in
Figure 4, both (7)-isoprenaline (1 mM) and HCITC (1 mM)
increased cyclic AMP accumulation in
a
similar time-
the (7)-isoprenaline pD2s were 6.95+0.08 (n=5) and
6.68+0.06 (n=6, P50.005 vs control) and the maximal
relaxation was reduced to 61+13 and 31.8+1.5%, respec-
tively. Pretreatment of the tissue with 100 nM DCITC further
attenuated the maximal (7)-isoprenaline-induced relaxation
to 9.0+2.8% (n=3) with a pD2 of 6.39+0.04 (n=3, P50.005
vs control).
The reversibility of the DCITC e€ect on (7)-isoprenaline-
induced relaxation was investigated by tissue washout
experiments. As depicted in Figure 5b, the maximal (7)-
isoprenaline-mediated relaxation of the control was
75.8+1.3% (n=5) and this relaxation was decreased to
34.1+1.9% (n=5) after pre-exposure of the tissue for 30 min
to 10 nM DCITC. After a 1 h washout period no recovery of
the maximal relaxation was observed (38.8+0.6%, n=5).
To determine if DCITC produced non-b-adrenoceptor-
mediated tissue e€ects, the response to phenylephrine and
acetylcholine in the presence of DCITC was determined.
Phenylephrine (500 nM) alone increased aortic contraction and
this contraction was transiently attenuated after the addition
of acetylcholine (1 mM). Neither the phenylephrine contraction
nor the acetylcholine relaxation was a€ected in the presence of
0.1 mM DCITC (Figure 6).
dependent manner with 256+26 (mean+s.d.) pmol cyclic
AMP formed after 1 min of incubation and this was increased
to 467+57 after 12 min. When an excess of propranolol
(20 mM) was added after a 4 min incubation with (7)-
isoprenaline alone, there was a subsequent time-dependent
decrease in cyclic AMP accumulation to 33% of the maximal
stimulated level at the end of the 12 min incubation period.
This indicates that the (7)-isoprenaline-mediated increase in
cyclic AMP accumulation, and the interaction of (7)-
isoprenaline with the b-adrenoceptor is reversible. In contrast,
when propranolol was added after a 4 min incubation with
HCITC alone, the rate of cyclic AMP accumulation was the
same as that observed in the absence of the antagonist. When
propranolol was added concurrently with HCITC, the
stimulation of cyclic AMP accumulation was blocked over
the 12 min incubation (Figure 4). This demonstrated that once
bound to the b-adrenoceptor HCITC produced an antagonist-
insensitive response, consistent with this compound binding to
the receptor in an irreversible manner.
E€ect of DCITC on the rat isolated aorta
The e€ect of DCITC on (7)-isoprenaline-mediated relaxation
of the rat isolated aorta contracted with 500 nM phenylephrine
is shown in Figure 5a. In these experiments, the tissue was
incubated with DCITC for 30 min before the e€ects of (7)-
isoprenaline were determined. This preincubation period
(30 min) was used because the maximal DCITC-induced
receptor loss in DDT cells occured before this time (Figure
3). (7)-Isoprenaline alone produced a concentration-depen-
dent relaxation of the smooth muscle preparation with an pD2
of 7.02+0.09 and a maximal relaxation of 71+4.4% (n=4).
After exposure of the tissue to 1 or 10 nM DCITC for 30 min,
KA and receptor reserve for (7)-isoprenaline in rat
isolated aorta
Compared to the hyperbolic form of the operational model of
agonism, the more complex form (i.e., nonhyperbolic form)
did not statistically improve the ®t of the concentration-
response data depicted in Figure 5a (P=0.42). By use of the
hyperbolic form, the estimated KA and operational ecacy (t)
of (7)-isoprenaline were 286+92 nM and 1.63+0.97, respec-
tively. Pretreatment of the aortic tissue with 1, 10 or 100 nM

M.D. Deyrup et al
Irreversible b-adrenoceptor agonist and antagonist
171
DCITC, not only cause a concentration-dependent reduction
in the magnitude of the maximal relaxation (Figure 5a), but
also in the fraction of non-inactivated receptors (q) to
0.71+0.07, 0.22+0.06 and 0.05+0.018, respectively. A plot
of q (%) against maximal relaxation at each concentration of
DCITC pretreatment revealed an occupancy-response rela-
tionship that was slightly hyperbolic (Figure 7). The receptor
reserve for the (7)-isoprenaline-mediated maximal relaxation,
de®ned as 90% of the maximum, is 23%.
(Figure 8). The pD2 values were 7.03+0.03 (n=6) for (7)-
isoprenaline and 6.62+0.03 (n=6) for HCITC. The maximal
relaxation for (7)-isoprenaline and HCITC was 62+0.3%
and 97+0.6% (P50.05), respectively. The relaxation pro-
duced by both agonists (1 mM) was blocked in the presence of
10 mM propranolol (data not shown). The reversibility of the
agonist-induced relaxation was examined by the use of
propranolol (Figure 9). (7)-Isoprenaline (1 mM) and HCITC
(1 mM) produced a 40 and 80% relaxation, respectively, of the
phenylephrine contracted aorta. After the subsequent addition
of 20 mM propranolol, the (7)-isoprenaline relaxation was
rapidly and completely reversed. In contrast, the addition of
propranolol had no e€ect on the HCITC-mediated relaxation,
demonstrating that this agonist produced an antagonist-
insensitive relaxation.
E€ect of HCITC on the rat isolated aorta
In phenylephrine-precontracted aortae, (7)-isoprenaline and
HCITC produced
a concentration-dependent relaxation
Control
1nM DCITC
10 nM DCITC
100 nM DCITC
Discussion
a
The results from the present study show that DCITC and
HCITC are potent ligands acting as an antagonist and agonist,
respectively for the b-adrenoceptor. Under the incubation
Control
DCITC
Wash
Figure 6 Phenylephrine contraction and acetylcholine relaxation of
rat aorta in the presence of DCITC. Aortae were contracted with
500 mM phenylephrine and at the maximal contraction, acetylcholine
(1 mM) was added. After washing to remove drugs the phenylephrine
contraction and acetylcholine relaxation was repeated in the presence
b
of 0.1 mM DCITC. Tracing is representative of
experiments.
3 separate
Figure 5 E€ect of DCITC on (7)-isoprenaline-mediated relaxation
of rat aorta. In (a): aortae were precontracted with phenylephrine
(500 nM) and the tissue was exposed to solution in the absence
(control) or presence of 1, 10 or 100 nM DCITC for 30 min. The
concentration-response for relaxation by (7)-isoprenaline was then
determined. Data are the means, n=3 ± 6. *P50.05 and **P50.01
compared to the control (7)-isoprenaline values. In (b) aortae were
contracted with 500 nM phenylephrine and the tissue was then
incubated in the absence (control) or presence of 10 nM DCITC for
30 min followed by the concentration-response for (7)-isoprenaline.
The tissue was then washed over a period of 60 min during which the
tissue bath solution was exchanged with drug free solution three
times every ®ve min. At the end of the wash period, the
concentration-response to (7)-isoprenaline was repeated (Wash).
Data are the means, n=5. *P50.01 compared to control. In (a) and
(b), vertical lines show s.e.mean.
Figure
7 Occupancy-response relationship for (7)-isoprenaline-
mediated relaxation of rat isolated aorta. Receptor occupancy (q)
was calculated from (7)-isoprenaline dose-response curves (Figure
5a) after treatment with 1, 10, and 100 nM DCITC by use of the
operational model.

172
M.D. Deyrup et al
Irreversible b-adrenoceptor agonist and antagonist
conditions used in the radioligand binding assays (60 min,
368C), these ligands inhibited [125I]-CYP binding to the b-
adrenoceptor in DDT cell membranes in the low nanomolar
range. In the functional assays, the potency of HCITC to
stimulate cyclic AMP accumulation in DDT cells and to relax
the rat isolated aorta was in the low to mid nanomolar range.
However, for several reasons, the potency of DCITC and
HCITC may be underestimated. First, as discussed below,
DCITC and HCITC are irreversible ligands and as such their
apparent potency will increase as a function of incubation
time. Second, it is well known that the (7)-enantiomer of b-
agonists and antagonists are more potent than the (+)-
enantiomers or their racemic mixtures (Patil et al., 1974). In
the present study, both DCITC and HCITC were used as
racemic mixtures and it is likely that their (7)-enantiomers
will be more potent.
DCITC alone did not increase cyclic AMP accumulation in
DDT cells. Furthermore, DCITC unlike (7)-isoprenaline did
not amplify forskolin-stimulated cyclic AMP accumulation in
DDT cells, an experimental approach previously used to detect
partial agonist activity of b-adrenoceptor ligands (Jasper et al.,
1991). These observations are consistent with DCITC acting as
a b-adrenoceptor antagonist without detectable partial agonist
activity. In DDT cells, HCITC produced the same maximal
stimulation of cyclic AMP accumulation as (7)-isoprenaline
indicating that this chemoreactive ligand is a full b-agonist.
However, in the isolated aorta, HCITC produced a greater
maximal relaxation than (7)-isoprenaline. This di€erence in
the maximal relaxation response for the two agonists may
re¯ect a di€erential ability to activate the cellular transduction
mechanisms coupled to the b-adrenoceptor in the smooth
muscle cells of the isolated tissue (Kenakin, 1996).
Iso
HCITC
A number of criteria, with radioligand binding assays,
have been used to establish the irreversible binding of a
chemoreactive ligand to a receptor. These include: (1) loss of
receptor binding capacity as a function of chemoreactive
ligand concentration and incubation time and (2) lack of
receptor recovery with extensive washing to remove free
ligand (Baker & Deyrup, 1994). In the present study, DCITC
and HCITC reduced maximal [125I]-CYP binding to the b-
adrenoceptor in DDT cell membranes in a concentration-
dependent manner, without a change in the Kd value for the
radioligand to the receptors remaining. Furthermore, the
DCITC and HCITC-induced reduction in receptor content
was time-dependent and not reversed by extensive membrane
washing. In addition, exposure of the isolated aorta to
DCITC reduced in a concentration-dependent manner, the
maximal (7)-isoprenaline-induced relaxation and this reduc-
tion was not reversed by extensive washing of the tissue.
These ®ndings with DDT cells and the isolated aorta are
consistent with DCITC and HCITC binding to the b-
adrenoceptor in an irreversible manner. By use of pharma-
cological criteria, the majority of b-adrenoceptors expressed
in DDT cells and mediating relaxation of the rat aorta are
the b2 subtype (Norris et al., 1983; O'Donnell & Wanstall,
1984; Hadcock & Malbon, 1988). Thus, the present data
indicate that DCITC and HCITC are irreversible ligands for
the b2-adrenoceptor. Further studies will be needed to
determine if they bind in an irreversible manner to the other
b-adrenoceptor subtypes. The previously described irrever-
sible antagonists BAAM (Pitha et al., 1980), Br-AAM-
pindolol (Homburger et al., 1985) and BIM (Kusiak & Pitha,
1987), and the irreversible agonist carbo-Br (Standifer et al.,
1989), use an electrophilic haloacetamide as the reactive
moiety that may form a covalent bond by reaction with
nucleophilies in the receptor by a second order SN2 process.
In contrast, DCITC and HCITC contain an electrophilic
isothiocyanate group as the reactive moiety, that can undergo
an addition reaction when a receptor nucleophile attacks the
electrophilic carbon of the ligand leading to the formation of
a stable bond (Baker & Deyrup, 1994). DCITC and HCITC
are the ®rst b-adrenoceptor ligands which contain the
isothiocyanate moiety and the data indicate that this reactive
group is useful in the development of chemoreactive
irreversible ligands for this receptor.
Figure 8 Relaxation e€ect of (7)-isoprenaline and HCITC on the
rat aorta. Aortae were precontracted with 500 nM phenylephrine and
the relaxation determined in the presence of the indicated
concentrations of (7)-isoprenaline (Iso) or HCITC. Data are the
mean and s.e., n=6. *P50.01 and **P50.05 compared to the (7)-
isoprenaline values.
Iso
HCITC
Figure 9 E€ect of propranolol on (7)-isoprenaline and HCITC
mediated relaxation in rat aorta. Aortae were contracted with 500 nM
phenylephrine and then exposed to 1 mM (7)-isoprenaline (Iso) or
1 mM HCITC. After a stable relaxation was established, propranolol
(20 mM) was added. Data are the means, n=3; vertical lines show
s.e.mean.

M.D. Deyrup et al
Irreversible b-adrenoceptor agonist and antagonist
173
Several lines of evidence indicated that DCITC produced
little if any nonspeci®c depression of responses in DDT cells or
the rat isolated aorta at concentrations that irreversibly
blocked the b-adrenoceptor. Forskolin, which activates
adenylyl cyclase through a nonreceptor mechanism (Dar¯er
et al., 1982), stimulated cyclic AMP accumulation in DDT
shown by the ability of propranolol to reverse the established
HCITC-mediated increase in the rate of cyclic AMP
accumulation in DDT cells or the HCITC-mediated relaxation
of the aorta. These antagonist-insensitive responses were due
to activation of the b-adrenoceptor because they were
prevented when propranolol was added concurrently with
HCITC. As expected for a reversible ligand, the (7)-
isoprenaline-mediated increase in cyclic AMP accumulation
and relaxation of the smooth muscle preparation was rapidly
reversed by the addition of propranolol. These observations in
conjuction with the radioligand binding data demonstrate that
HCITC is an irreversible agonist for the b-adrenoceptor. In
comparison, Baker et al. (1985) showed that a bromoacetyla-
minomenthyl derivative of noradrenaline (BAAN) could bind
to the b-adrenoceptor in an irreversible manner. However, this
compound was a weak partial agonist, chemically unstable,
and although it initially stimulated adenylyl cyclase activity in
membrane preparations, after irreversible binding it appeared
to act as an antagonist. The reason for the di€erence between
HCITC acting as an irreversible agonist and the transient
agonist e€ects of BAAN is not clear. However, the di€erence
may be related to BAAN alkylating a critical amino acid
residue required for receptor activation, whereas HCITC may
acylate a noncritical residue in the receptor. Chemoreactive
agonists that produce sustained or transient responses for the
muscarinic receptor have also been described (Ringdahl et al.,
1984; Bolden & Baker, 1990; Crocker & Russell, 1990). A
carbostyril-based b-agonist with a haloacetamide as the
reactive moiety (carbo-Br) has been shown to bind irreversibly
to the b-adrenoceptor and produce antagonist-insensitive
activation of adenylyl cyclase activity in reticulocyte mem-
branes (Standifer et al., 1989). Although HCITC and carbo-Br
both appear to be potent irreversible ligands in isolated
membrane preparations, the latter compound has not been
studied in cultured cells or isolated tissue preparations.
cells. This increase was not a€ected by DCITC at
a
concentration (100 nM) greater than that required for receptor
inactivation (0.5 ± 10 nM). In the isolated aorta preparation,
DCITC did not a€ect the basal tone or the contraction
mediated by the a-adrenoceptor agonist phenylephrine.
Furthermore, DCITC did not a€ect aortic relaxation mediated
by acetylcholine-induced nitric oxide production. However,
because of the high reactivity of the isothiocyanate moiety, it is
possible that DCITC will cause some nonspeci®c tissue
depression at high concentrations by reaction with non-
receptor cellular or tissue components. Nonspeci®c tissue
depression has been shown for the irreversible b-adrenoceptor
antagonist BAAM (Posner et al., 1984; Ng & Malta, 1989).
Nonetheless, the observation that DCITC can irreversibly
block the b-adrenoceptor at concentrations that do not appear
to cause any nonspeci®c e€ects, indicates that this compound
will be useful on this receptor in a variety of biological systems.
By use of the operational model of agonism developed by
Black and Le€ (1983), the calculated KA for the (7)-
isoprenaline-mediated relaxation of aortic smooth muscle was
286+92 nM. This value is similar to the experimentally derived
EC50 values for this agonist obtained after treatment of the
isolated tissue with 10 (218+29 nM) or 100 (409+33 nM)
DCITC. Furthermore, by use of the Furchgott (1966) method,
May et al. (1985) obtained a KA value of 281 ± 330 nM for (7)-
isoprenaline to decrease the twitch response of the rat isolated
vas deferens, a b2-adrenoceptor response. Their KA range is in
excellent agreement with the KA for (7)-isoprenaline
determined in the present study. Based upon the q values
(fraction of non-inactivated receptors) derived from the
operational model, the b-adrenoceptor occupancy-response
relationship for aortic muscle relaxation was slightly hyper-
bolic with a 23% receptor reserve for (7)-isoprenaline at the
maximal response. This receptor reserve at the maximal
response is similar to that obtained (19%) for the selective
b2-agonist procaterol mediating a relaxation of the rat isolated
aorta (Doggrell, 1989), and suggests a tight coupling between
the receptor and its intracellular processes that lead to muscle
relaxation. However, the receptor reserve at 50% of the
maximal response was about 20% for (7)-isoprenaline (Figure
7) and 45% for procaterol (Doggrell, 1989). This di€erence in
receptor reserve for the two agonists at a submaximal response
probably re¯ects di€erences in their intrinsic ecacies
(Kenakin, 1993).
In summary, the data from the present study show that
DCITC and HCITC are potent, irreversible ligands for the b-
adrenoceptor in DDT cells and the rat isolated aorta. DCITC
is an irreversible antagonist which produces no signi®cant
nonspeci®c cell or tissue depressant e€ects at concentrations
that inactivate the receptor. This chemoreactive ligand appears
useful for determining the KA and receptor reserve of agonists.
HCITC, on the other hand, is an irreversible full b-agonist.
The characteristics of these compounds suggest that they are
potentially useful ligands in further studies on the structure
and function of the b-adrenoceptor.
This work was supported by grants from the National Institutes of
Health (HL 52189) and the Council for Tobacco Research to
C.H.G. and a grant from the American Heart Association, Florida
Aliate (to S.P.B.).
The results with DDT cells and the rat isolated aorta
preparation demonstrated that HCITC produced sustained,
antagonist-insensitive b-adrenoceptor responses. This was
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(Received August 4, 1997
Revised January 9, 1998
Accepted January 23, 1998)