Departments of
Pharmacology (J.Z., L.B., D.H.O., S.P.B) and
Medicine (L.B.), University of Florida, Gainesville, Florida 32610, and
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
Maryland 20892 (K.A.J.).
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Introduction |
Adenosine
is a ubiquitous modulator of physiological processes that acts to both
increase tissue oxygen supply and reduce metabolic demand. The actions
of adenosine are mediated by cell surface receptors and at least four
subtypes, designated A1,
A2A, A2B, and
A3, have been identified, with each subtype
mediating cellular effects by coupling to G proteins (1-4). The AdoR
regulates the activity of several cellular components, including
inhibition of adenylyl cyclase activity, stimulation of phospholipases
A2 and C, and guanylyl cyclase, and regulates the
ion permeability of calcium and potassium channels (2-4). The
A2-AdoRs, on the other hand, mediate stimulation
of adenylyl cyclase activity (1, 2). Although less is known about the
A3-AdoR, it has been reported that this AdoR
subtype mediates an inhibition of adenylyl cyclase and a stimulation of
phospholipase C, resulting in an elevation of inositol phosphates (5,
6). The characterization, function, and regulation of the AdoRs have
been greatly facilitated by the development of potent and selective
agonists and antagonists (1, 7, 8).
Chemoreactive ligands are useful pharmacological probes in structure
and function studies of receptors (9). These ligands are usually
composed of two components: a pharmacophore and a reactive moiety. The
pharmacophore is usually derived from known agonists or antagonists,
which provide affinity and selectivity for the receptor. The reactive
moiety allows covalent incorporation of the ligand into the receptor,
which prevents ligand dissociation (9). These ligands have been used in
a variety of studies, including determination of receptor reserve (10,
11), receptor subtype discrimination (12, 13), basal receptor turnover
(14, 15), and ligand binding site mapping (16, 17).
Jacobson et al. (18) have synthesized a series of potential
irreversible ligands for the adenosine receptor by modifying the high
affinity agonist pharmacophore ADAC with substituents containing
reactive electrophilic groups. Coupling of ADAC with 1,3- or
1,4-phenylene DITC produced p- and m-DITC-ADAC,
which were shown by radioligand binding assays to be relatively
selective and irreversible ligands for the rat brain
A1-AdoR. In the current study, the agonist
properties of p- and m-DITC-ADAC for the
A1-AdoR were determined and contrasted with an
established agonist. In addition, as potential irreversible agonists,
under the appropriate experimental conditions these compounds occupy
and activate a fixed fraction of the total receptor population. From
the fixed fraction receptor occupancy-response relationship, the
receptor reserve for the irreversible agonist can be determined. Toward these goals, the p- and m-DITC-ADAC binding
properties and cAMP accumulation effects mediated by the
A1-AdoR in DDT cells and the
A1-AdoR-induced slowing of AV nodal conduction in
the guinea pig isolated heart were investigated.
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Experimental Procedures |
Materials.
The radioligands [3H]cAMP
(28.4 Ci/mmol) and [3H]CPX (88.2-120 Ci/mmol)
were purchased from New England Nuclear (Boston, MA). CPX and CPT were
from Research Biochemicals (Natick, MA). Dulbecco's modified Eagle's
medium, HBSS, horse serum, and fetal bovine serum were from GIBCO
(Grand Island, NY). DDT cells were purchased from American Type Culture
Collection (Rockville, MD). All other reagents were from Sigma Chemical
(St. Louis, MO) or Fisher Scientific (Orlando, FL). Rolipram was a gift
of Berlex Laboratories (Cedar Knolls, NJ).
Drug preparations.
The synthesis and chemical
characterization of p- and m-DITC-ADAC have been
previously reported (18). The structures of these compounds are shown
in Fig. 1. Stock solutions of
p- and m-DITC-ADAC, CPA, and CPT (10 mM) were prepared in dimethylsulfoxide, stored at 
20°,
and diluted with incubation buffer immediately before use. Stock
solutions of NECA were made in 5 mM HCl, stored at 20°,
and diluted with buffer before use.
Cell culture.
DDT cells were grown as monolayers in 150-mm
culture dishes with Dulbecco's modified Eagle's medium containing 5%
fetal bovine serum, 100 units/ml penicillin G, 0.1 mg/ml streptomycin,
and 2.5 µg/ml amphotericin B in a water-humidified 5%
CO2/95% air mixture at 37°. Cells were seeded
at 0.2-1 × 104
cells/cm2 and subcultured twice weekly after
detachment using 1 mM EDTA in HBSS without the divalent
cations. Experiments were performed on cells 1 day before confluency
unless otherwise indicated.
Determination of cAMP accumulation.
DDT cells were detached
using a cell lifter in 5 ml of HBSS and pelleted by centrifugation at
500 × g for 5 min. Cells (0.2-0.3 mg of protein) were
then incubated in microfuge tubes with 500 µl of HBSS containing 100 µM rolipram and the indicated concentrations of drugs.
Incubations were performed at 37° for 10 min unless otherwise
indicated. Reactions were terminated by placing the tubes in a boiling
water bath for 5 min. After cooling to room temperature, the tubes were
centrifuged for 2 min at 10,000 × g, and the
supernatants were saved for assays.
The cAMP content was determined by a modified competitive protein
binding assay (19). Briefly, an aliquot of the supernatant (50 µl)
was incubated in a total volume of 0.2 ml of 25 mM
Tris·HCl buffer, pH 7.0, containing 8 mM theophylline,
0.8 pmol of [3H]cAMP, and 24 µg of bovine
heart cAMP-dependent protein kinase at 4° for 60 min. At the end of
the incubation, 70 µl of 50% (v/v) hydroxyapatite/water suspension
was added, followed by 4 ml of ice-cold 10 mM Tris·HCl
buffer, pH 7.0. The suspension was then filtered through Whatman GF/B
glass-fiber filters under reduced pressure using a Brandel Cell
Harvester (Montreal, Quebec, Canada); the filters were washed twice
with 3 ml of ice-cold 10 mM Tris·HCl, pH 7.0, and placed
in a scintillation vial with 3 ml of Scinti-Verse BD. The radioactivity
retained on the filters was determined in a liquid scintillation
counter. The amount of cAMP in the assay was calculated from a standard
curve determined with known concentrations of unlabeled cAMP.
Membrane preparation.
Monolayers of DDT cells were washed
two times with 10 ml of ice-cold HBSS, and the cells were scraped free
in 5 ml of ice-cold 50 mM Tris·HCl buffer, pH 7.4, containing 5 mM MgCl2. The
suspensions were then homogenized with a SDT-100EN homogenizer (Tekmar,
Cincinnati, OH) at the lowest speed for 5 sec, diluted to 35 ml with
homogenization buffer, and centrifuged at 27,000 × g
for 10 min in a Sorvall RC-5B centrifuge. The supernatants were
discarded, and the pellets were washed two additional times by
resuspension and centrifugation as above. The final pellets were
resuspended in one volume of homogenization buffer for assays.
Guinea pig ventricular membranes were prepared as previously described
(20). Briefly, guinea pig hearts were perfused for 10 min with
oxygenated Krebs-Henseleit solution to remove blood. Ventricles were
then isolated, minced, and homogenized in 10 volumes of ice-cold 50 mM Tris·HCl buffer, pH 7.4. The homogenate was centrifuged at 48,000 × g for 15 min to pellet the
membranes. The pellet was resuspended in 30 ml of homogenization buffer
and centrifuged as above, and the membranes were washed twice more by
resuspension and centrifugation. The final pellet was resuspended in
one volume of appropriate buffer for assays. The protein content of
membrane preparations was determine according to Bradford (21) method,
with a BioRad (Richmond, CA) assay and bovine serum albumin as
standard.
Radioligand binding assays..
A1-AdoRs
in DDT cell and cardiac membranes were determined according to the
specific binding of [3H]CPX as described by
Scammells et al. (22) and Belardinelli et al.
(20). Briefly, membrane protein (0.1-0.2 mg) was incubated in a volume
of 0.25 ml containing 50 mM Tris·HCl buffer, pH 7.4, 5 mM MgCl2, 2 units/ml adenosine
deaminase, and 0.06-4 nM [3H]CPX
for 90 min at 25°. Nonspecific binding was determined using CPX (10 µM) to displace the specific binding. At the end of the incubation, the suspensions were diluted with 3 ml of ice-cold incubation buffer, and the membranes with bound radioligand were collected by filtration through GF/B glass-fiber filters under reduced
pressure. The filters were washed with an additional 6 ml of ice-cold
buffer and placed in scintillation vials with 3 ml of Scinti-Verse BD,
and the radioactivity was determined using a liquid scintillation
counter. Specific [3H]CPX binding to the
A1-AdoR was calculated as the difference between
total binding in the absence of CPX and the nonspecific binding
determined in the presence of CPX.
The functional A1 response in guinea pig isolated
hearts.
Guinea pig isolated hearts were perfused at a constant
flow (10 ml/min) and instruments for measurement of AV nodal conduction time were attached as described previously (23). The hearts were
perfused with oxygenated Krebs-Henseleit solution (pH 7.4, 35°)
containing the drugs as indicated. The atrium of the heart was
stimulated at a constant cycle length of 300 msec throughout the
experiment. The A1-AdoR-mediated prolongation of
the SH interval, an index of the delay in AV nodal conduction caused by
adenosine and other compounds, was measured on a beat-by-beat
throughout the experiment.
Data analysis.
The receptor concentrations
(Bmax) and dissociation constants
(Kd) for the radiolabeled ligands
were determined from nonlinear regression analysis of Rosenthal (24)
plots. The concentration of drugs that inhibited cAMP accumulation by
50% (IC50) was determined using a
concentration-effect analysis with a nonlinear regression algorithm
(Marquardt-Levenberg). The occupancy-response relationship was fitted
to a sigmoid equation using nonlinear regression (Table Curve V2.0;
Jandel Scientific, San Rafel, CA) to estimate the receptor reserve for
the maximal response defined as >90%. Statistical analysis of the
experimental data was performed using the Student's t test
and were considered significant if p < 0.05.
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Results |
Effect of p- and m-DITC-ADAC on
A1-AdoRs in DDT cells.
The effect of p- and
m-DITC-ADAC to decrease (A1-AdoR
effect) cAMP accumulation was examined using DDT cells. For comparison, the well established A1-AdoR agonist CPA was also
tested. Fig. 2 illustrates the effects of
p- and m-DITC-ADAC and CPA to attenuate ()-isoproterenol-stimulated cAMP accumulation in DDT cells.
()-Isoproterenol (5 µM) alone increased cAMP from 5 to
60 pmol/mg/min, a 12-fold increase above basal. This increase in
cellular cAMP accumulation was inhibited by both m- and
p-DITC-ADAC and CPA in a concentration-dependent manner with
the same maximal attenuation (85%). Under the incubation conditions
used (10 min, 37°), the IC50 values for
p- and m-DITC-ADAC and CPA to inhibit
()-isoproterenol-stimulated cAMP accumulation were 19.4 ± 0.2, 11.0 ± 2.0, and 1.4 ± 0.1 nM, respectively. The inhibitory effect of the three agonists at 0.1 µM was
prevented by coincubation with the selective A1
antagonist CPX (10 µM, data not shown), indicating that
this inhibitory response is mediated by the
A1-AdoR.
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Fig. 2.
Effects of p- and m-DITC-ADAC
on inhibition of ()-isoproterenol-stimulated cAMP accumulation in DDT
cells. Intact DDT cells were incubated with 1 µM
()-isoproterenol, 50 µM rolipram, and the indicated
concentrations of CPA or p- or m- DITC-ADAC for 10 min at 37°. At the end of the incubation, the cAMP accumulated was
determined as described in Experimental Procedures. Values are the
mean ± standard error of four separate experiments, each performed in duplicate. The basal cAMP was 5.0 ± 1.7 pmol/mg of protein/min, and the cAMP accumulated in the presence of 5 µM ()-isoproterenol alone was 60.0 ± 9.0 pmol/mg
of protein/min.
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To examine the reversibility of the p-DITC-ADAC and CPA
effects in DDT cells, the selective A1 antagonist
CPX was used to attempt to displace the agonists bound at the
A1-AdoR. As shown in Fig.
3, ()-isoproterenol (1 µM) increased cAMP accumulation in a time-dependent
manner from 20 (basal) to 605 pmol/mg of protein over a 12-min
incubation period. This increase of cAMP accumulation was attenuated by
70% and 80% after a 5-min incubation in the presence of 1 µM p-DITC-ADAC or 1 µM CPA,
respectively, and by 80% and 81%, respectively, after 12 min of
incubation. When CPX (1 µM) was added after incubation
with p-DITC-ADAC (1 µM) and ()-isoproterenol
(1 µM), no reversal of the
p-DITC-ADAC-mediated attenuation of
()-isoproterenol-stimulated cAMP accumulation was observed. This
demonstrated that once receptor activation had been established, the
inhibition by p-DITC-ADAC was insensitive to CPX, a finding
consistent with an irreversible interaction between
p-DITC-ADAC and the A1-AdoR. In
contrast, the addition of CPX after a 4-min incubation with
()-isoproterenol and CPA resulted in a time-dependent increase in
cAMP accumulation such that at the end of the 12-min incubation period,
the attenuation of cAMP accumulation by CPA was reduced from 81% in
the absence of CPX to 36% in its presence. This indicates that
CPA-mediated attenuation of cAMP accumulation and the interaction of
CPA with the A1-AdoR are reversible.
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Fig. 3.
Time course effect of p-DITC-ADAC on the
inhibition of ()-isoproterenol-stimulated cAMP accumulation in DDT
cells. DDT cells were incubated with 50 µM rolipram and 1 µM ()-isoproterenol and without ( ) or with 1 µM p-DITC-ADAC ( ) or 1 µM CPA
( ) at 37°. After 4 min of incubation with p-DITC-ADAC
( ) or CPA ( ), 1 µM CPX was added, and the
accumulated cAMP was determined at the indicated time points.
X, Basal cAMP levels. Points, mean ± standard error of three separate determinations assayed in duplicate; arrow, addition of CPX.
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To further investigate the interaction of p-DITC-ADAC with
the A1-AdoR, radioligand binding experiments were
performed. DDT cells were incubated with 1 µM
p-DITC-ADAC at 37° for 1 hr and washed six times with
HBSS, and the Bmax and
Kd values for
[3H]CPX binding to cell membranes were
determined on the basis of Rosenthal (24) plots. As shown in Table
1, preincubation of DDT cells with
p-DITC-ADAC (1 µM) followed by
extensive cell washing reduced the Bmax
value of [3H]CPX binding to the
A1-AdoR by 44%, with no change in the
Kd value for the remaining receptors.
In contrast, pretreatment with CPA (1 µM),
followed by cell washing, had no effect on the
Bmax or
Kd value of
[3H]CPX binding. A representative Rosenthal
plot of [3H]CPA binding to DDT cell membranes
is shown in Fig. 4. These results further
indicate that the interaction of p-DITC-ADAC with the
A1-AdoR of DDT cells is irreversible. Similar to
p-DITC-ADAC, pretreatment of DDT cells with 1 µM m-DITC-ADAC resulted in a reduction of A1-AdoRs (data not shown). As
illustrated in Fig. 5, the
p-DITC-ADAC-induced loss of [3H]CPX
binding sites (i.e., A1-AdoRs) in DDT cells was
time dependent. After incubation of DDT cells with 1 µM p-DITC-ADAC for 5, 15, 30, and 60 min, there was a 16%, 29%, 39%, and 47% reduction, respectively, of
specific [3H]CPX binding to cell membranes.
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TABLE 1
Effects of pretreatment of DDT cells with p-DITC-ADAC on
radioligand binding to cell membranes
DDT cells were incubated with 1 µM p-DITC-ADAC
or 1 µM CPA for 1 hr at 37° followed by six washes with
HBSS. Cell membranes were then prepared, and the A1-AdoRs
were determined by [3H]CPX (0.125-4 nM)
binding to DDT cell membranes. The Kd and
Bmax values were derived from Rosenthal plots.
Data are the mean ± standard error of four or five separate
determinations.
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Fig. 4.
Representative Rosenthal plots of
[3H]CPX binding to DDT cell membranes after pretreatment
of intact cells with p-DITC-ADAC. DDT cells were incubated
with 1 µM p-DITC-ADAC or 1 µM
CPA at 37° for 1 hr followed by six washes with HBSS. Cell membranes were then prepared, and the A1-AdoRs were determined by
[3H]CPX (0.06-4 nM) binding in DDT cell
membranes. Points, mean of triplicate determinations.
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Fig. 5.
Time course of p-DITC-ADAC-induced loss
of specific [3H]CPX binding to DDT cells. DDT cells were
incubated without () or with () 1 µM
p-DITC-ADAC for the indicated times followed by six washes with HBSS. The cells were then homogenized in 50 mM
Tris·HCl buffer, pH 7.4, containing 5 mM
MgCl2, and the suspension was centrifuged at 48,000 × g for 15 min. The membranes were resuspended in buffer, and
the A1-AdoR content was determined with 4 nM
[3H]CPX as described in Experimental Prcedures.
Points, mean ± standard error of three separate
experiments assayed in triplicate. The control specific
[3H]CPX binding was 212 ± 30 fmol/mg of protein.
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The relationship between A1-AdoR occupancy by
p-DITC-ADAC and responsiveness in DDT cells.
The
relationship between p-DITC-ADAC-mediated inhibition of
()-isoproterenol-stimulated cAMP accumulation and
A1-AdoR occupancy in DDT cells is shown in Fig.
6. After preincubation of cells with
increasing concentrations of p-DITC-ADAC (0.1-2
µM) at 37°C for 1 hr followed by six cell washes,
()-isoproterenol (5 µM)-stimulated cAMP accumulation
was determined in the presence of 1 µM CPX. This A1-AdoR
antagonist was added to prevent any response from residual
p-DITC-ADAC present in the cells after washing. In parallel samples, the A1-AdoR occupancy by
p-DITC-ADAC was determined by the loss of specific
[3H]CPX binding. At receptor occupancy levels
of 16%, 29%, 34%, and 46%, there was a 32%, 58%, 73%, and 78%
inhibition of ()-isoproterenol-stimulated cAMP accumulation,
respectively (Fig. 6). Using nonlinear regression analysis, the
calculated receptor reserve for p-DITC-ADAC in DDT cells was
~64% because the maximal inhibition of ()-isoproterenol-stimulated cAMP accumulation (defined as >90%) was obtained at 36% receptor occupancy. This level of receptor reserve for p-DITC-ADAC is
based on the assumption that all A1-AdoRs that
were measured are coupled to the inhibition of cAMP accumulation.
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Fig. 6.
The relationship between the inhibition of
()-isoproterenol-stimulated cAMP accumulation and receptor occupancy
by p-DITC-ADAC in DDT cells. Cells were pretreated with 0.1, 0.5, 1.0, or 2.0 µM p-DITC-ADAC for 1 hr at
37° followed by six washes with HBSS. The inhibition of cAMP
accumulation by the A1-AdoRs occupied by pretreatment with
p-DITC-ADAC was determined by incubating the cells with 50 µM rolipram, 1 µM CPX, and 5 µM ()-isoproterenol at 37° for 10 min. The
accumulated cAMP was determined as described in Experimental
Procedures. The A1-AdoR occupancy by p-DITC-ADAC was determined as the percent loss of specific [3H]CPX (5 nM) binding to cell membranes compared with untreated controls. Values are mean ± standard error of three or four
separate experiments, each performed in duplicate or triplicate. The
control ()-isoproterenol (5 µM)-stimulated cAMP
formation was 50 ± 5 pmol/mg of protein/min, and the
control-specific [3H]CPX binding was 195 ± 4 fmol/mg of protein. The maximal inhibition of
()-isoproterenol-stimulated cAMP accumulation in the presence of 5 µM p-DITC-ADAC was 82 ± 3% (three
experiments).
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Effect of m-DITC-ADAC on A1-AdoR-mediated
functional response in guinea pig isolated hearts.
As shown in
Fig. 7A, CPA (50 nM)
prolonged the SH interval of isolated guinea pig hearts from 35 to 70 msec. This effect of CPA was rapidly and nearly completely reversed on
washout or the addition of the antagonist CPT (5 µM).
Similar to CPA, m-DITC-ADAC (5 µM) prolonged
the SH interval from 37 to 79 msec, which was sustained during a 40-min
washout (Fig. 7B). Furthermore, perfusion of the heart with a
supramaximal concentration of CPT (5 µM) caused only a
small attenuation of the m-DITC-ADAC effect. In contrast, when the hearts were pretreated with the antagonist CPT (5 µM) before exposure to m-DITC-ADAC, the
negative dromotropic effect (i.e., SH interval prolongation) of
m-DITC-ADAC could no longer be elicited (Fig.
8). On washout of CPT but still in the
presence of 5 µM m-DITC-ADAC, the SH interval
rapidly lengthened from 40 to 82 msec and remained prolonged during a
subsequent 45-min washout period. Thus, the functional effect of
m-DITC-ADAC could be prevented by preincubation with the
antagonist CPT, but once the effect was established, it persisted and
could not be reversed.
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Fig. 7.
Time course of the effects of CPA,
m-DITC-ADAC, and A1-AdoR antagonist CPT on SH
interval of guinea pig isolated, perfused hearts. Hearts were paced at
an atrial cycle length of 300 msec throughout an experiment. A, CPA (50 nM) maximally and reversibly prolonged the SH interval.
This effect was rapidly and nearly completely reversed by its washout
or by the antagonist CPT (5 µM). Points,
averages of SH interval determinations from two hearts. B, Lack of
reversibility of m-DITC-ADAC (5 µM, 20 min)-induced SH interval prolongation. m-DITC-ADAC (5 µM) added to the perfusate increased the SH interval and
caused second-degree AV block. After a 40-min washout, 1:1 AV
conduction resumed, but the SH interval remained prolonged (79 ± 2 msec). A supramaximal concentration of CPT (5 µM)
caused only a small (p > 0.05) reversal of the
effect of m-DITC-ADAC. Points, mean ± standard error of responses of four hearts.
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Fig. 8.
Ado-R blockade prevents the negative dromotropic
effect of m-DITC-ADAC on the guinea pig isolated, perfused
heart. The heart was paced at a constant atrial cycle length of 300 msec. CPT (5 µM) prevented the prolongation by
m-DITC-ADAC (5 µM) of the SH interval. After
washout of CPT in the presence of m-DITC-ADAC, the SH
interval rapidly lengthened and remained prolonged despite washout of
m-DITC-ADAC. Identical results were obtained in two additional hearts.
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The effect of m-DITC-ADAC on [3H]CPX
binding to the A1-AdoR in guinea pig ventricle
membranes is illustrated in Fig. 9. In control membranes, maximal [3H]CPX binding was
38.5 ± 3.9 fmol/mg of protein with a
Kd value of 4.3 ± 0.5 nM. Preincubation of the membranes with
m-DITC-ADAC (1 µM) for 20 min
followed by four wash cycles caused a 35% reduction in the
Bmax value with a
Kd value of 1.4 ± 0.1 nM for [3H]CPX binding to
the remaining receptors.
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Fig. 9.
Rosenthal plot of specific [3H]CPX
binding to guinea pig ventricular membranes after pretreatment with
m-DITC-ADAC. Membranes were incubated with 1 µM m-DITC-ADAC for 20 min and then washed four
times by centrifugation and resuspension. The A1-AdoR
density was then determined by the specific binding of
[3H]CPX (0.6-10 nM) as described in
Experimental Procedures. Points, mean of four determinations
assayed in triplicate. The Kd values are 4.3 ± 0.5 and 1.4 ± 0.1 nM for control
and m-DITC-ADAC-treated membranes, respectively.
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The relationship between the SH interval prolongation and
A1-AdoR occupancy with m-DITC-ADAC in
guinea pig isolated, perfused hearts is shown in Fig.
10. After a 5-, 10-, 15-, or 30-min
perfusion with 5 µM m-DITC-ADAC, the hearts
were perfused with 5 µM CPT, and the SH response was
recorded. In the same perfused hearts, the
A1-AdoR occupancy at each time point was
determined by [3H]CPX binding after preparation
of ventricular membranes. A nearly linear relationship between receptor
occupancy by m-DITC-ADAC and prolongation of the SH interval
was observed, with the maximal response requiring ~80-90% receptor
occupancy. With the assumption that all of the
A1-AdoRs are coupled to the prolongation of the SH interval, the receptor reserve for m-DITC-ADAC to produce
the maximal response is 10-20%.
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Fig. 10.
Correlation between SH interval prolongation and
A1-AdoR occupancy by m-DITC-ADAC in guinea pig
isolated, perfused hearts. Hearts paced at a constant atrial cycle
length of 300 msec were perfused with Krebs-Henseleit solution
containing 5 µM m-DITC-ADAC for 5, 10, 15, and
30 min. At the end of each perfusion period, the tissue was perfused
with 5 µM CPT, and the irreversible component of SH
interval prolongation was recorded. After washout for 1 hr, the
ventricles were harvested, and membranes were prepared for
determination of the number (Bmax) and
affinity (Kd) of A1-AdoRs by measuring specific binding of [3H]CPX. The
irreversible component of SH interval prolongation caused by
m-DITC-ADAC is plotted as a function of percent of total A1-AdoRs occupied [Bmax (fmol/mg of
protein of untreated control)/Bmax of
m-DITC-ADAC-treated hearts × 100). The
Bmax and Kd
values of four untreated hearts were 33 ± 4 fmol/mg of protein
and 3.2 ± 0.2 nM, respectively. There was no
significant difference between the Kd
values of [3H]CPX binding to membranes of untreated and
m-DITC-ADAC-treated hearts.
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Discussion |
Agonist effects and irreversible binding of p- and
m-DITC-ADAC to the A1-AdoR.
The
results from the current study show that p- and
m-DITC-ADAC are potent and full agonists at the
A1-AdoR in DDT cells. Under the study incubation
conditions, the IC50 values for both compounds to
inhibit ()-isoproterenol-stimulated cAMP accumulation were in the low
nanomolar range, whereas the maximal inhibition (85%) was the same as
for the full A1-AdoR agonist CPA. As discussed below, p- and m-DITC-ADAC are irreversible
ligands for the A1-AdoR; therefore, the
IC50 values obtained will depend on the
incubation time. The observation that p- and
m-DITC-ADAC- and CPA-induced inhibition of cAMP accumulation
was prevented by coincubation with the
A1-AdoR-selective antagonist CPX indicates that
the inhibitory effect elicited by all three agonists was mediated by
the A1-AdoR.
Several criteria using radioligand binding assays are commonly used to
determine whether a chemoreactive ligand binds in an irreversible
manner to a receptor: (i) a decrease in radioligand binding capacity,
(ii) lack of receptor recovery after extensive washing to remove the
free chemoreactive ligand, and (iii) a time-dependent loss of the
receptor (9). In the current study, incubation of intact DDT cells with
p- or m-DITC-ADAC or of guinea pig ventricular membranes with m-DITC-ADAC reduced the
Bmax value for
[3H]CPX binding to the
A1-AdoR with little or no change in the
Kd value for this radioligand to the
receptors remaining. The p- and
m-DITC-ADAC-induced decreases in
A1-AdoRs in DDT cells and guinea pig isolated
heart were time dependent and not reversed by extensive washing to
remove the free ligand. These findings are consistent with the two
agonists binding to the A1-AdoR in an
irreversible manner and confirm the findings with these two compounds
as reported by Jacobson et al. (18), who used rat brain
membranes as the receptor source. Because the preincubation protocol
involved intact DDT cells, the p- and
m-DITC-ADAC-induced A1-AdoR decreases
could be explained by a combination of receptor acylation and
agonist-induced receptor down-regulation. However, the contribution of
agonist-induced down-regulation is likely to be minor because
pretreatment of the cells with the higher potency reversible agonist
CPA for the same time period (1 hr) did not result in a decrease in
specific [3H]CPX binding to the
A1-AdoR. Furthermore, the half-life of
agonist-induced down-regulation of the A1-AdoR in
DDT cells has been reported to be ~8 hr (25). This relatively slow
rate of agonist-induced down-regulation suggests that only a small
fraction of the receptors would have been lost during the 1-hr
preincubation protocol of the current study.
The apparent irreversible binding of p- and
m-DITC-ADAC to the A1-AdoR is likely
due to an addition reaction leading to covalent bond formation between
the electrophilic isothiocyanate moiety of the compounds and a
nucleophilic amino acid located within or in close proximity to the
ligand binding site of the receptor. Whether both p- and
m-isomers of DITC-ADAC react with the same or different
nucleophiles in the A1-AdoR remains to be
determined.
In several experiments, the interaction of p-DITC-ADAC with
the A2A-AdoR in PC12 cells was investigated using
[3H]NECA, a radioligand previously used to
label A2A-AdoRs in PC12 cells (26, 27).
Pretreatment of PC12 cells with p-DITC-ADAC (1 µM) for 60 min, conditions that reduced the
A1-AdoR content of DDT cells, did not affect
[3H]NECA binding to the
A2A-AdoR (data not shown). This suggests that
p-DITC-ADAC did not irreversibly bind to the
A2A-AdoR. However, an irreversible interaction of
p-DITC-ADAC with the A2A-AdoR may be
possible if higher concentrations of the chemoreactive ligand or longer
incubation periods are used.
Sustained A1-AdoR activation by p- and
m-DITC-ADAC.
The results from the time course of cAMP
accumulation in DDT cells and prolongation of the SH interval in guinea
pig isolated hearts show that p- and m-DITC-ADAC
elicit sustained, antagonist-insensitive A1-AdoR-mediated responses. This was shown by the
observation that the addition of a 20-fold excess of CPX after a 4-min
incubation with p-DITC-ADAC did not reverse the inhibition
of ()-isoproterenol-stimulated cAMP accumulation in DDT cells (Fig.
3). As expected, when CPX was added after a 4-min incubation with CPA
alone, the inhibition of cAMP accumulation was largely reversed, which
is consistent with CPA being a reversible A1-AdoR
agonist. Similar to the effects in DDT cells, the
m-DITC-ADAC-induced prolongation of the SH interval in the
guinea pig isolated heart, an A1-AdoR-mediated
response (4), was not reversed by the addition of an excess of the
antagonist CPT or by extensive washout of m-DITC-ADAC (Fig.
7). This contrasts with the predicable behavior of the reversible
agonist CPA; that is, the CPA-induced prolongation of the SH interval
was completely reversed by washout of this agonist or addition of the
antagonist CPT. Thus, the antagonist-insensitive effects of
p- and m-DITC-ADAC to mediate cell and tissue
responses are consistent with the radioligand binding data indicating
these compounds bind irreversibly to the A1-AdoR.
Irreversible activation of the A1-AdoR by
m-DITC-ADAC would explain the sustained decrease in heart
rate caused by this agonist in the rabbit isolated heart (28). Other
irreversible agonists for AdoRs have been reported. Lohse et
al. (29) showed that the photoaffinity ligand
(R)-2-azido-N6-p-hydroxyphenylisopropyladenosine
produced irreversible activation of the A1-AdoR
in fat cells. This compound, however, caused an irreversible blockade
of the A2-AdoR in human platelets (30). More
recently, Niiya et al. (31) studied an arylisothiocyanate derivative of a functionalized congener of adenosine,
2-[(2-aminoethyl-aminocarbonylethyl)phenylethylamino]-5
-N-ethylcarboxamidoadenosine, and this compound produced sustained, antagonist insensitive coronary vasodilation in the guinea pig heart, indicating irreversible activation of the A2A-AdoR. It is not known
whether this latter compound acts as an irreversible agonist at the
A1-AdoR.
Receptor reserve for p- and
m-DITC-ADAC.
The concept of receptor reserve, in which
a maximal response can be achieved at submaximal receptor occupancy,
was introduced by Stephenson in 1956 (32). Typically, the receptor
reserve is estimated with the use of irreversible antagonists (9-11,
33, 34). The persistent activation of the A1-AdoR
by p- and m-DITC-ADAC allowed us to determine the
relationship between fixed fraction receptor occupancy and response and
hence the receptor reserve for these irreversible agonists. In DDT
cells, the maximal inhibition of ()-isoproterenol-stimulated cAMP
accumulation was achieved when ~36% of the
A1-AdoRs were irreversibly activated by
p-DITC-ADAC (Fig. 6), which indicates that there is a
~64% receptor reserve for the maximal response produced by
p-DITC-ADAC in DDT cells. The presence of a relatively high
receptor reserve for the A1-AdoR-mediated inhibition of isoproterenol-stimulated cAMP accumulation in fat cells
has also been reported for a photoaffinity A1
adenosine agonist (29). It should be pointed out that the magnitude of the receptor reserve, as determined in the current study, may be
affected by several cell-dependent factors, including the presence of
internalized receptors and the possibility that not all of the
receptors measured are coupled to G proteins. Furthermore, in addition
to the inhibition of cAMP accumulation, the
A1-AdoR in DDT cells has been shown to mediate
inositol phosphate accumulation (35). Thus, the receptor reserve for an
agonist eliciting more than one response in a cell by the same receptor
may be different, as has been shown for the muscarinic receptor in
chick heart cells (36).
In contrast to DDT cells, the occupancy-response relationship for
m-DITC-ADAC-mediated prolongation of the SH interval showed that 80-90% of the A1-AdoRs must be
irreversibly activated to achieve the maximal response, indicating a
10-20% receptor reserve (Fig. 10). This small receptor reserve is
similar to that determined, with an irreversible antagonist, for
A1-AdoR activation of an inwardly rectifying
K+ current by adenosine in guinea pig atrial
myocytes (37). On the other hand, Dennis et al. (11), using
an irreversible antagonist in combination with the method developed by
Furchgott (38), calculated a 54% receptor reserve for the maximal
CPA-mediated increase in the SH interval of the guinea pig heart.
Because the receptor reserve is dependent on both the tissue and
agonist (36), the difference in receptor reserve for
m-DITC-ADAC and CPA to maximally increase the SH interval
may in part be due to CPA having a higher intrinsic efficacy than
m-DITC-ADAC. Alternatively, the difference in receptor
reserve for these two agonists may be related to CPA being a reversible
agonist, whereas m-DITC-ADAC irreversibly activates the
A1-AdoR.
In summary, the data from the current study show that p- and
m-DITC-ADAC are relatively potent, full agonists at the
A1-AdoR. The irreversible binding and persistent
agonist properties of these compounds suggest that they or related
derivatives may be useful probes for further studies on the mechanism
of receptor activation and effector coupling, desensitization and, in
radiolabeled form, for elucidation of the structure of the
A1-AdoR agonist binding site.
We thank Dr. Malgorzata Deyrup for helpful discussions during
preparation of the manuscript.
This work was supported by a Grant-in-Aid from the American
Heart Association, Florida Affiliate, and National Institutes for
Health Grant HL35272.
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Summary
Introduction
Summary
