Vol. 78, No. 2, 1977
BIOCHEMICAL
ELEVATED BETA-ADRENERGIC
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RECEPTOR NUMBER AFTER CHRONIC
PROPRANOLOL TREATMENI George Glaubiger and Robert J. Lefkowitz Departments of Medicine and Biochemistry Uuke University Medical Center Durham, North Carolina 27710
Received
August
9,1977
SUMMAKY: Treatment of rats with the beta-adrenergic antagonist propranolol for two weeks leads to a 100% increase in the number of beta-adrenergic receptors ((-J[3H1dihydroalprenolol binding sites) in cardiac particulate fractions. No change in receptor affinity was observed. These findings may 1) represent the converse of agonist-induced desensitization which is associated with decreases in beta-receptor number, 2) provide a potential explanation for the clinically observed "propranolol withdrawal syndrome". A variety cell the
of pharmacologically
function cell
have been shown
at specific
receptor
Combination
of agonist
in motion
an amplification
cyclase
activation
active
capable
of altering
primary
interaction
with
to have their sites
molecules
on the
with
outer
these
system which
and results
molecules
membrane
receptor
often
in significant
begins
surface(l).
sites
sets
with
adenyl
changes
in physiologic
activity(l).
It has recently changes
in the occupancy
receptor of the
number
et al.,
erythrocyte
receptors
by agonists.
sites
number
by the
antagonist
chronic
Pre.s.s. Inc. form resewed.
in the
occupancy
responsiveness
of frog
such as isoproterenol
erythrocyte
binding.
This
of beta-receptor
cell
membranes
phenomenon mediated
In the same system
720
in
For example,
as well.
in the
propranolol
that
to alterations
alterations
(2).
0 1977 by Acadrmic~ of rrproductiorl it1 any
of systems
by agonists
of receptors
by desensitization
activation
receptor
receptor
number
leads
system
h ave shown that
by (-)[3Hldihydroalprenolol
was accompanied
Copyrigllt All rights
amplification (2)
in a number sites
and in some cases
to a decreased
cyclase
of receptor
beta-adrenergic
as assayed
the
(2)
receptor-coupled
Mukherjee
led
been demonstrated
adenyl
, occupancy
failed
to alter
of
Vol.
78,
No.
2, 1977
In
rat
brain
blockade led
of
to
an
however,
number
of
led
to
between
receptor
in
is
reports
have
cardiac
events
occurring
of
tissue
cardiac
and
the
data
and
receptor
Male eight
normal
similar
dose
the
beta-receptor
hours
assayed
in
methods
(10).
of
were
ventricular
of normal
form
of
from saturation
Dissociation
Scatchard
of
were
Control
saline.
tight
to
determined
of
721
that
myocardial sensitivity
this
information
receptor
occupancy
chronic,
high-dose
injected
intraperitoneally 4 mg/ml
hours
and
Figs.
in
were after
injected the
number
previously
are in
chronic,
suggested
animals
from
experiment
untoward
increased
beta-receptor
(Kg)
Recently
hearts,
mg/kg.
plots
of
(+)propranolol,
constants
a representative
view
rat
according
were
and
in
and
tissue
In
effects
clinically
angina,
between
of
brain.
(5).
been
an
River)
sacrificed
(-)13H]dihydroalprenolol Data
10
has
from
(9).
is
of
unstable
result
a solution
a dose
volumes animals
a number
include
(Charles
rat relationship
angina
It
number
with at
notably
(6,1/,8).
the
in
which
discontinuation
we studied
diminished
a dynamic
acute
beta-agonists
rats
be
that
number.
a relationship
strain
saline,
with
to
number
may
most
may
(4)
6-hydroxydopamine
antagonist
which death,
shown
by
documenting
therapy
number,
C-D
receptor
after
sudden
on
and
appeared
supporting
propranolol
there
by
consequent
beta-receptor
that
haloperidol
have with
COMMUNICATIONS
chronic
measured
(produced
states,
phenomena" and
every
in
propranolol
infarction
al.,
increase
disease
"rebound
Sporn
-- et
that
antagonist
receptors
a beta-adrenergic
several
high-dose
the
dopamine
agonist
RESEARCH
demonstrated
by
by
occupancy
several
been
BIOPHYSICAL
norepinephrine
suggest
Propranolol
these
tissue
an
observations
useful
(3).
occupancy
treatment)
has
of
binding
beta-receptor
AND
receptors
increased
depletion
These
it
dopaminergic
13H]haloperidol chronic
BIOCHEMICAL
last
was
published
maximum Scatchard
binding
for
plots
(11).
presented 1H and
in
B.
the These
Vol.
78,
No.
00
2, 1977
BIOCHEMICAL
IO 20 30 [(-)[3H]D,hydroalpre”olol]
AND
BIOPHYSICAL
Fig.
COMMUNICATIONS
40 nM
(-)~H]DHA
Fig.lA
RESEARCH
Bound
(fm/mg
prorem)
Specific (-)L3H]dihydroalprenolo1 binding to ventricular membranes from control and propranolol treated rats as a function of (-)[3H]dihydroalprenolol concentration. Groups of four control and treated rats were sacrificed by cervical dislocation and the ventricles rapidly extirpated and minced with a scissors to a particle size of 1-3 mn in 20 ml of ice-cold sucrose 0.25 M, Tris-HCI 5 mM, pH 7.4, MgCl 1 mM. The ventricular pieces were allowed to sett P e and the supernatant decanted and replaced with 30 ml of fresh ice-cold buffer. ihe suspension was homogenized at 0 C using 20 strokes of a motor-driven grooved leflon-glass homogenizer . After cheese cloth filtration, the material was centrifuged at 4OC for 10 m&n at 480 xg and the resulting supernatant centrifuged at 4 C for 10 minutes at 30,000 xg. The pellet was resuspended in 40 ml of Tris HCl 50 mM, pH 7.5, MgC12 10 mM, and centrifuged at 4'C for 10 min at 30,000 xg. This step was repeated and the final pellet was suspended in 4-6 ml of Tris HCl 75 mM , pH 7.65, MgC12 25 mM. The binding assay was initiated by dding 100 ~1 of the membrane preparation !l!HJdihydroalprenolol to 50 ~1 of (-)[ with and without (+)propranolol 3 X 10 M. The mixtures were incubated fEr 10 min at 37OC with shaking. One hundred ul aliquots were then withdrawn and immediately dispersed in 5 ml of ice-cold Tris HCl 75 mM, pH 7.65, MgC12 25 mM and filtered by suction through a 25mM Schleicher and Schuell IVO. 25 filter disc. The filters were washed four times with 5 ml of buffer, dried and counted. Specific binding was defined as the difference betwgen binding in the absence and presence of (+)propranolol IO M expressed as fmol Specific binding was usually 50-70x bound/mg protein.of total binding. A representative saturation curve with a membrane preparation derived from control and treated groups of animals is shown. Oata.points are means of closely agreeing duplicate observations. 16
Scatchard ventricular rats. details
plot of (-)[3H]dihydroalprenolol membranes from control Data of Figure 1H have been are as described in legend
722
binding to and propranolol treated replotted. Experimental to Figure 1A.
Vol. 78, No. 2, 1977
A
BIOCHEMICAL
AND BIOPHYSICAL
T p 0.5)
of membrane
experiments
synthesis
receptor rats
was not significantly
100% increase
to compensate
of binding.
in Figs.
treatment
of beta-receptors
of beta-adrenergic propranolol-treated
affinity
(-)[3H]dihydroalprenolol
cnronic
with
I lTROL
C
A) Dissociation constants (Kd) and 6) ?laximum specific binding of (-)[3H]dihydroalprenolol in ventricular membranes from control and gropranolol treated rats. Membranes were prepared and (-)] HJdihydroalprenolol binding assays performed as described in sigure 1 legend. Dissociation constants and maximum (-)[ Hldihydroalprenolol binding were determined from least square fits to Scatchard plots of the data. The results represent the mean + S.E. of seven experiments. Statistical analysis was performed by Student's t test (unpaired).
2
indicate
for
RESEARCH COMMUNICATIONS
in an attempt
occupancy
associated
2) a decreased
Vol. 78, No. 2, 1977
rate
of degradation
3) reversal effect
of receptors
of a chronic
of endogenous Whether
the
demonstrated
chronic,
results
therapy
observed
sensitivity represents
after
reflected
been determined.
provide
It
is possible
of agonist
to agonist
and the
part
of this
occupancy increased
change
explanation
discontinuation
receptors
in altered
blockade
a possible acute
lowering
beta-adrenergic
is
antagonist
of beta-receptors
phenomena"
in sensitivity.
for
the
"rebound
of chronic
propranolol
in man. In favor
haloperidol
curve
of this
view
administration
receptor
number
(3)
of apomorphine
of rats have
has not
would
of cardiac
experiments
high-level,
Such a finding
or receptor agonist.
number
present
in an increased
number
by the antagonist;
"desensitizing"
increased
function
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
occupied
catecholamine
in the
physiologic that
BIOCHEMICAL
treated
observed
in basal
with (4)
is the
observation
to rats
caused
and a leftward
shift
- stimulated
motor
intraventricular
an increase
and isoproterenol et -- al.,
(13)
adenyl
cyclase
to norepinephrine
denervation
in dogs.
decreased agonist
agonist
occupancy
sensitivity
activity
response
[12).
In brains
several
Sporn
number
sensitivity
of receptors
Further,
of ventricular
chronic
systems
al -et -1
and an increase
CAMP accumulation.
after
as measured
in dopamine
in the dose
an increased
Thus,
an increase
in beta-receptor
Palmer
chronic
b-hydroxydopamine
stimulated
found
that
surgical exist
cardiac
where
results
chronic
in increased
by biochemical
and pharmacological
responses. By contrast
it
sensitivity
to agonist
chronotropy
after
administration experiments
has been reported
that
as measured
by changes
acute both
discontinuation
in rats
is
however
724
not an increased
in inotropy
of chronic
and man (14).
is open to criticism
there
propranolol
Interpretation since
it
and
is difficult
of these
1
Vol. 78, No. 2, 1977
BIOCHEMICAL
to exclude
of residual
the
preparations
presence
used
In conclusion
for
these
it
number.
regulatory
agonist
catecholamines
clinical
implications.
RESEARCH COMMUNICATIONS
in the physiological
studies.
antagonist,
"up-regulation" This
propranolol
has been demonstrated
of a beta-adrenergic significant
AND BIOPHYSICAL
that
propranolol, of cardiac
effect, in other
chronic to rats
administratlon is associated
beta-adrenergic
which
is opposite
systems
may have
with
receptor to that
important
exerted
by
physiological
Acknowledgement This worK was supported by N.I.H. grant number HL 16037 and HL 20339 and by a grant-in-aid from The American Heart Association with funds Dr. Lefkowitz contributed in part by The North Carolina Heart Association. is a Howard Hughes Medical Investigator. REFERENCES 1. 2. ;: 5.
;: 8. 9. 10. 11. 12. 13. 14.
Ariens, E.J. and Simonis, A.M. (1974) Beta-Adrenoceptor Blocking Agents. (tds. Saxena, P.R. and Forsyth, K.P.), page 3, (North Holland Publishing Co., Amsterdam). Mukherjee, C. and Lefkowitz, R.J. (1976) Proc. Natl. Acad. Sci. USA 73: 1494-1498,. Burt, D.R., Creese, I. and Snyder, S. Science, In Press. Sporn, J.K., Harden, T.K., Wolfe, B.B. and Molinoff, P.B. (1976) Science 194: 624-626. Dollery, C.T. and George, C. (lY75) in: Cardiovascular Clinics, (Ed. Brest, A.N.) Vol. 6, #2, page 255-268, (P.A. Davis Co., Philadelphia). Slome, P. (1973) Lancet 1: 156. Alderman, E.L., Coltart, D.J., Weittach, G.E. and Harrison, D.C. (1974) Ann. Int. Med. 81: 625. Uiaz, R.G., Somberg, J.C. and Freman, E. (1973) Lancet 1: 1068. Harrison, D.c. and Alderman, E.L. (19/6) Chest 69: 1. Williams, L.T., Lefkowitz, R.J., Watanabe, A.M., Hathaway, D.R. and Besch, H.R., Jr. (1977) J. Biol. Chem., 252: 2787-2789. Scatchard, G. Ann. N.Y. Acad. Sci. 51: 660-676. Moore, K.E. and Ihornberg, J.E. (1975) in : Advances in Neurology (Eds. Calne, D.B., Chase, T.N. and Barbeau, A.) page 93-104, (Raven Press, New York). Palmer, G.C., Spurgeon, H.A. and Priola, D.V. (1975) J. Cyclic Nucleotide Research 1: 89-95. Faulkner, S.L., Hopkins, J.I., Boerth, R.C., Young, J.L., Jr., Jellett, L.B., Nies, U.S., Bender, H.W. and Shand, D.G. (1973) New England Journal of Medicine 289: 607-609.
725
and
BIOCHEMICAL
ELEVATED BETA-ADRENERGIC
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RECEPTOR NUMBER AFTER CHRONIC
PROPRANOLOL TREATMENI George Glaubiger and Robert J. Lefkowitz Departments of Medicine and Biochemistry Uuke University Medical Center Durham, North Carolina 27710
Received
August
9,1977
SUMMAKY: Treatment of rats with the beta-adrenergic antagonist propranolol for two weeks leads to a 100% increase in the number of beta-adrenergic receptors ((-J[3H1dihydroalprenolol binding sites) in cardiac particulate fractions. No change in receptor affinity was observed. These findings may 1) represent the converse of agonist-induced desensitization which is associated with decreases in beta-receptor number, 2) provide a potential explanation for the clinically observed "propranolol withdrawal syndrome". A variety cell the
of pharmacologically
function cell
have been shown
at specific
receptor
Combination
of agonist
in motion
an amplification
cyclase
activation
active
capable
of altering
primary
interaction
with
to have their sites
molecules
on the
with
outer
these
system which
and results
molecules
membrane
receptor
often
in significant
begins
surface(l).
sites
sets
with
adenyl
changes
in physiologic
activity(l).
It has recently changes
in the occupancy
receptor of the
number
et al.,
erythrocyte
receptors
by agonists.
sites
number
by the
antagonist
chronic
Pre.s.s. Inc. form resewed.
in the
occupancy
responsiveness
of frog
such as isoproterenol
erythrocyte
binding.
This
of beta-receptor
cell
membranes
phenomenon mediated
In the same system
720
in
For example,
as well.
in the
propranolol
that
to alterations
alterations
(2).
0 1977 by Acadrmic~ of rrproductiorl it1 any
of systems
by agonists
of receptors
by desensitization
activation
receptor
receptor
number
leads
system
h ave shown that
by (-)[3Hldihydroalprenolol
was accompanied
Copyrigllt All rights
amplification (2)
in a number sites
and in some cases
to a decreased
cyclase
of receptor
beta-adrenergic
as assayed
the
(2)
receptor-coupled
Mukherjee
led
been demonstrated
adenyl
, occupancy
failed
to alter
of
Vol.
78,
No.
2, 1977
In
rat
brain
blockade led
of
to
an
however,
number
of
led
to
between
receptor
in
is
reports
have
cardiac
events
occurring
of
tissue
cardiac
and
the
data
and
receptor
Male eight
normal
similar
dose
the
beta-receptor
hours
assayed
in
methods
(10).
of
were
ventricular
of normal
form
of
from saturation
Dissociation
Scatchard
of
were
Control
saline.
tight
to
determined
of
721
that
myocardial sensitivity
this
information
receptor
occupancy
chronic,
high-dose
injected
intraperitoneally 4 mg/ml
hours
and
Figs.
in
were after
injected the
number
previously
are in
chronic,
suggested
animals
from
experiment
untoward
increased
beta-receptor
(Kg)
Recently
hearts,
mg/kg.
plots
of
(+)propranolol,
constants
a representative
view
rat
according
were
and
in
and
tissue
In
effects
clinically
angina,
between
of
brain.
(5).
been
an
River)
sacrificed
(-)13H]dihydroalprenolol Data
10
has
from
(9).
is
of
unstable
result
a solution
a dose
volumes animals
a number
include
(Charles
rat relationship
angina
It
number
with at
notably
(6,1/,8).
the
in
which
discontinuation
we studied
diminished
a dynamic
acute
beta-agonists
rats
be
that
number.
a relationship
strain
saline,
with
to
number
may
most
may
(4)
6-hydroxydopamine
antagonist
which death,
shown
by
documenting
therapy
number,
C-D
receptor
after
sudden
on
and
appeared
supporting
propranolol
there
by
consequent
beta-receptor
that
haloperidol
have with
COMMUNICATIONS
chronic
measured
(produced
states,
phenomena" and
every
in
propranolol
infarction
al.,
increase
disease
"rebound
Sporn
-- et
that
antagonist
receptors
a beta-adrenergic
several
high-dose
the
dopamine
agonist
RESEARCH
demonstrated
by
by
occupancy
several
been
BIOPHYSICAL
norepinephrine
suggest
Propranolol
these
tissue
an
observations
useful
(3).
occupancy
treatment)
has
of
binding
beta-receptor
AND
receptors
increased
depletion
These
it
dopaminergic
13H]haloperidol chronic
BIOCHEMICAL
last
was
published
maximum Scatchard
binding
for
plots
(11).
presented 1H and
in
B.
the These
Vol.
78,
No.
00
2, 1977
BIOCHEMICAL
IO 20 30 [(-)[3H]D,hydroalpre”olol]
AND
BIOPHYSICAL
Fig.
COMMUNICATIONS
40 nM
(-)~H]DHA
Fig.lA
RESEARCH
Bound
(fm/mg
prorem)
Specific (-)L3H]dihydroalprenolo1 binding to ventricular membranes from control and propranolol treated rats as a function of (-)[3H]dihydroalprenolol concentration. Groups of four control and treated rats were sacrificed by cervical dislocation and the ventricles rapidly extirpated and minced with a scissors to a particle size of 1-3 mn in 20 ml of ice-cold sucrose 0.25 M, Tris-HCI 5 mM, pH 7.4, MgCl 1 mM. The ventricular pieces were allowed to sett P e and the supernatant decanted and replaced with 30 ml of fresh ice-cold buffer. ihe suspension was homogenized at 0 C using 20 strokes of a motor-driven grooved leflon-glass homogenizer . After cheese cloth filtration, the material was centrifuged at 4OC for 10 m&n at 480 xg and the resulting supernatant centrifuged at 4 C for 10 minutes at 30,000 xg. The pellet was resuspended in 40 ml of Tris HCl 50 mM, pH 7.5, MgC12 10 mM, and centrifuged at 4'C for 10 min at 30,000 xg. This step was repeated and the final pellet was suspended in 4-6 ml of Tris HCl 75 mM , pH 7.65, MgC12 25 mM. The binding assay was initiated by dding 100 ~1 of the membrane preparation !l!HJdihydroalprenolol to 50 ~1 of (-)[ with and without (+)propranolol 3 X 10 M. The mixtures were incubated fEr 10 min at 37OC with shaking. One hundred ul aliquots were then withdrawn and immediately dispersed in 5 ml of ice-cold Tris HCl 75 mM, pH 7.65, MgC12 25 mM and filtered by suction through a 25mM Schleicher and Schuell IVO. 25 filter disc. The filters were washed four times with 5 ml of buffer, dried and counted. Specific binding was defined as the difference betwgen binding in the absence and presence of (+)propranolol IO M expressed as fmol Specific binding was usually 50-70x bound/mg protein.of total binding. A representative saturation curve with a membrane preparation derived from control and treated groups of animals is shown. Oata.points are means of closely agreeing duplicate observations. 16
Scatchard ventricular rats. details
plot of (-)[3H]dihydroalprenolol membranes from control Data of Figure 1H have been are as described in legend
722
binding to and propranolol treated replotted. Experimental to Figure 1A.
Vol. 78, No. 2, 1977
A
BIOCHEMICAL
AND BIOPHYSICAL
T p 0.5)
of membrane
experiments
synthesis
receptor rats
was not significantly
100% increase
to compensate
of binding.
in Figs.
treatment
of beta-receptors
of beta-adrenergic propranolol-treated
affinity
(-)[3H]dihydroalprenolol
cnronic
with
I lTROL
C
A) Dissociation constants (Kd) and 6) ?laximum specific binding of (-)[3H]dihydroalprenolol in ventricular membranes from control and gropranolol treated rats. Membranes were prepared and (-)] HJdihydroalprenolol binding assays performed as described in sigure 1 legend. Dissociation constants and maximum (-)[ Hldihydroalprenolol binding were determined from least square fits to Scatchard plots of the data. The results represent the mean + S.E. of seven experiments. Statistical analysis was performed by Student's t test (unpaired).
2
indicate
for
RESEARCH COMMUNICATIONS
in an attempt
occupancy
associated
2) a decreased
Vol. 78, No. 2, 1977
rate
of degradation
3) reversal effect
of receptors
of a chronic
of endogenous Whether
the
demonstrated
chronic,
results
therapy
observed
sensitivity represents
after
reflected
been determined.
provide
It
is possible
of agonist
to agonist
and the
part
of this
occupancy increased
change
explanation
discontinuation
receptors
in altered
blockade
a possible acute
lowering
beta-adrenergic
is
antagonist
of beta-receptors
phenomena"
in sensitivity.
for
the
"rebound
of chronic
propranolol
in man. In favor
haloperidol
curve
of this
view
administration
receptor
number
(3)
of apomorphine
of rats have
has not
would
of cardiac
experiments
high-level,
Such a finding
or receptor agonist.
number
present
in an increased
number
by the antagonist;
"desensitizing"
increased
function
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
occupied
catecholamine
in the
physiologic that
BIOCHEMICAL
treated
observed
in basal
with (4)
is the
observation
to rats
caused
and a leftward
shift
- stimulated
motor
intraventricular
an increase
and isoproterenol et -- al.,
(13)
adenyl
cyclase
to norepinephrine
denervation
in dogs.
decreased agonist
agonist
occupancy
sensitivity
activity
response
[12).
In brains
several
Sporn
number
sensitivity
of receptors
Further,
of ventricular
chronic
systems
al -et -1
and an increase
CAMP accumulation.
after
as measured
in dopamine
in the dose
an increased
Thus,
an increase
in beta-receptor
Palmer
chronic
b-hydroxydopamine
stimulated
found
that
surgical exist
cardiac
where
results
chronic
in increased
by biochemical
and pharmacological
responses. By contrast
it
sensitivity
to agonist
chronotropy
after
administration experiments
has been reported
that
as measured
by changes
acute both
discontinuation
in rats
is
however
724
not an increased
in inotropy
of chronic
and man (14).
is open to criticism
there
propranolol
Interpretation since
it
and
is difficult
of these
1
Vol. 78, No. 2, 1977
BIOCHEMICAL
to exclude
of residual
the
preparations
presence
used
In conclusion
for
these
it
number.
regulatory
agonist
catecholamines
clinical
implications.
RESEARCH COMMUNICATIONS
in the physiological
studies.
antagonist,
"up-regulation" This
propranolol
has been demonstrated
of a beta-adrenergic significant
AND BIOPHYSICAL
that
propranolol, of cardiac
effect, in other
chronic to rats
administratlon is associated
beta-adrenergic
which
is opposite
systems
may have
with
receptor to that
important
exerted
by
physiological
Acknowledgement This worK was supported by N.I.H. grant number HL 16037 and HL 20339 and by a grant-in-aid from The American Heart Association with funds Dr. Lefkowitz contributed in part by The North Carolina Heart Association. is a Howard Hughes Medical Investigator. REFERENCES 1. 2. ;: 5.
;: 8. 9. 10. 11. 12. 13. 14.
Ariens, E.J. and Simonis, A.M. (1974) Beta-Adrenoceptor Blocking Agents. (tds. Saxena, P.R. and Forsyth, K.P.), page 3, (North Holland Publishing Co., Amsterdam). Mukherjee, C. and Lefkowitz, R.J. (1976) Proc. Natl. Acad. Sci. USA 73: 1494-1498,. Burt, D.R., Creese, I. and Snyder, S. Science, In Press. Sporn, J.K., Harden, T.K., Wolfe, B.B. and Molinoff, P.B. (1976) Science 194: 624-626. Dollery, C.T. and George, C. (lY75) in: Cardiovascular Clinics, (Ed. Brest, A.N.) Vol. 6, #2, page 255-268, (P.A. Davis Co., Philadelphia). Slome, P. (1973) Lancet 1: 156. Alderman, E.L., Coltart, D.J., Weittach, G.E. and Harrison, D.C. (1974) Ann. Int. Med. 81: 625. Uiaz, R.G., Somberg, J.C. and Freman, E. (1973) Lancet 1: 1068. Harrison, D.c. and Alderman, E.L. (19/6) Chest 69: 1. Williams, L.T., Lefkowitz, R.J., Watanabe, A.M., Hathaway, D.R. and Besch, H.R., Jr. (1977) J. Biol. Chem., 252: 2787-2789. Scatchard, G. Ann. N.Y. Acad. Sci. 51: 660-676. Moore, K.E. and Ihornberg, J.E. (1975) in : Advances in Neurology (Eds. Calne, D.B., Chase, T.N. and Barbeau, A.) page 93-104, (Raven Press, New York). Palmer, G.C., Spurgeon, H.A. and Priola, D.V. (1975) J. Cyclic Nucleotide Research 1: 89-95. Faulkner, S.L., Hopkins, J.I., Boerth, R.C., Young, J.L., Jr., Jellett, L.B., Nies, U.S., Bender, H.W. and Shand, D.G. (1973) New England Journal of Medicine 289: 607-609.
725
and
