Pertussis Toxin-Treated Dog: A Whole Animal Model of Impaired Inhibitory Regulation of Adenyiate Cyciase
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992
Pertussis Toxin-Treated Dog: A Whole Animal
Model of Impaired Inhibitory Regulation of
Adenyiate Cyciase
John W. Fleming, Timothy D. Hodges, and August M. Watanabe
We have shown previously that stimulation of high-affinity GTP hydrolysis and inhibition of adenyiate
cyciase activity by muscarinic agonists are mediated by pertussis toxin (IAP) substrates (G, and GJ
in canine cardiac sarcolemma. We have now used the pertussis toxin-treated dog as a whole animal
model in which G,- and G.-mediated biochemical mechanisms can be studied. Mongrel dogs were
injected intravenously with IAP 48 hours prior to death and isolation of left ventricular sarcolemma.
Treatment of the animal in vivo with the toxin prevented subsequent in vitro IAP-catalyzed
["PJADP-ribosylation of substrates in cardiac, erythrocytic, and renal cortical plasma membranes,
suggesting that ADP-ribosylation occurred in vivo from endogenous substrate. Consistent with our
previous results obtained by treating sarcolemma in vitro with IAP, muscarinic receptor-mediated
stimulation of high-affinity GTP hydrolysis and inhibition of GTP-activated adenyiate cyciase activity
were attenuated in sarcolemma purified from the toxin-treated animals. Proximal to adenyiate cyciase,
guanine nucleotide regulation of muscarinic receptor affinity for agonists was also abolished in
membranes from the toxin-treated animals. In addition, the ability of oxotremorine to attenuate GTP
regulation of stimulation of adenyiate cyciase activity by magnesium ions was abolished in sarcolemma
from the IAP-treated dogs. Thus, cardiac sarcolemma isolated from the IAP-treated animals displayed
biochemical characteristics of an adenyiate cyciase system in which inhibitory regulatory pathways '
had been attenuated. The cardiac biochemical studies and the in vivo ADP-ribosylation of noncardiac
IAP substrates also suggests considerable potential use of this model in the physiological and
biochemical study of regulatory mechanisms mediated by GTP-binding proteins in other systems.
(Circulation Research 1988;62:992-1000)
T he mammalian heart is functionally regulated by
the opposing influences of the two limbs of the
autonomic nervous system.1 Parasympathetic
activation of cardiac muscarinic receptors suppresses
ventricular function, and this inhibitory effect is greater
toxin (islet-activating protein, IAP) has been an im-
portant biochemical probe for the selective identifica-
tion and functional modification of G, in several
tissues.7 Using nicotinamide adenine dinucleotide
(NAD) as substrate, IAP catalyzes the covalent ADP-
when (3-adrenergic receptors are activated (e.g., in the ribosylation of the a-subunit of G, (G^,), which causes
presence of sympathetic stimulation).'-2 The biochem- a loss in the ability of G; to mediate the effects of
ical mechanisms that mediate muscarinic effects are inhibitory hormones.8
varied and include regulation of both cyclic AMP-de- G, and G( interpose between receptors and adenyiate
pendent and cyclic AMP-independent processes.13"* cyciase, and therefore, the functional integrity of the
Because the intracellular effects of catecholamines are regulatory proteins affects processes both at the level
largely mediated by increased synthesis of cyclic AMP of the autonomic receptors and at the level of adenyiate
by adenyiate cyciase [ATP pyrophosphate-ryase (cy- cyciase. We originally reported that muscarinic agon-
clizing) E.C. 4.6.1.1], considerable experimental ef- ists antagonized the ability of guanine nucleoside
fort has been directed toward understanding the mech- 5'-triphosphates to regulate p-adrenergic receptor af-
anism^) by which muscarinic agonists attenuate the finity for catecholamines in cardiac sarcolemma.9 We
increase in cyclic AMP produced by 3-adrenergic have also demonstrated that muscarinic agonists at-
agonists. It is now well established that stimulation and tenuate GTP-activated adenyiate cyciase activity by a
inhibition of adenyiate cyciase activity are mediated at mechanism that is closely linked with GTP hydrolysis
the level of two similar yet unique GTP-binding (GTPase activity).9"" In those studies, muscarinic
proteins designated G, and G,, respectively.7 Pertussis stimulation of specific GTPase activity was inversely
related to muscarinic inhibition of GTP-activated
From the Department of Pharmacology and Toxicology (J.W.F., adenyiate cyciase activity, and the muscarinic effects
T.D.H., A.M.W.), the Department of Medicine (J.W.F., A.M.W.), on both processes were dependent on functional IAP
and the Krannert Institute of Cardiology (J.W.F., A.M.W.), Indiana substrates. We have now further extended these studjes
University School of Medicine, Indianapolis, Indiana.
Supported in part by grant R01 HL-29208 from the National using sarcolemma purified from the ventricles of
Institutes of Health and by a grant-in-aid from the American Heart control and IAP-treated dogs. Several inhibitory bio-
Association, Indiana Affiliate. chemical regulatory processes mediated by muscarinic
Address for correspondence: Dr. John W. Fleming, Department receptors were attenuated or abolished in sarcolemma
of Pharmacology and Toxicology, MS-A402, 635 Barnhill Drive, isolated from animals treated in vivo with LAP. In
Indianapolis, IN 46223.
Received February 5, 1987; accepted November 12, 1987. addition to further supporting our original hypotheses,
Downloaded from http://circres.ahajournals.org/ by guest on May 18, 2015Fleming et al Pertussis Toxin-TVeated Dog 993
the present report also suggests that the IAP-treated was washed with 105 ml of 100 mM potassium
whole dog is an excellent candidate for studies in phosphate (pH 7.0) and 0.5 M NaCl. Pertussis toxin
noncardiac tissues as a model system in which regu- was eluted with 100 ml of 100 mM potassium
lation of biochemical processes by inhibitory receptors phosphate (pH 7.0) and 0.5 M NaCl containing 3 M
linked to IAP substrates has been inactivated. KSCN. The purified toxin was dialyzed and concen-
trated on an Amicon YM-10 ultrafiltration membrane
Table of Abbreviations
(43 mm) (Danvers, Massachusetts) using 100 mM
sodium phosphate (pH 7.0) and 0.5 M NaCl. Pertussis
G, and G,, stimulatory and inhibitory guanine nucleotide binding toxin was stable for at least 1 year when stored in
and regulatory proteins of adenylate cyclase, respectively frozen aliquots at - 2 0 ° C.
G,_, M-45,000 GTP-binding subunit of G, that is ADP-
ribosylatea by cholera toxin
Gfa, M,-41,000 GTP-binding subunit of G, that is ADP- Experimental Design
ribosylated by IAP
G^, inhibitory dimeric complex of M, = 35,000 (f}) and Conditioned mongrel dogs (22-30 kg) were injected
M,~ 10,000 (y) subunits of G, and G, with either 100 mM sodium phosphate (pH 7.0) and 0.5
Gq, GTP-binding protein that regulates muscarinic receptor M NaCl (control) or pertussis toxin (30 jig/kg i.v.) in
affinity for agonists in bovine brain the same buffer. The animals showed no obvious signs
G
«» H ~ 39,000 GTP-binding subunit of Go that is ADP-
ribosylated by IAP of discomfort or disease. Left ventricular sarcolemma
LAP, islet-activating protein {Bordetella pertussis toxin) was purified 48 hours postinjection of toxin and
Gpp(NH)p, guanosine 5'-(P»7-iniino)-tnphosphate subjected to the indicated biochemical assays.
GTP7S, guanosine 5'-(0-tnio)-triphosphate
ARF, ADP-ribosylation factor, a protein cofactor necessary for
cholera toxin-catalyzed ADP-ribosylation of G,,, Preparation of Membranes
QNB, (± )-quinuclidinylbenzilate Purified cardiac left ventricular sarcolemma was
prepared according to Jones et al17 and Jones18 from
Materials and Methods Procedure II membrane vesicles with the exception that
[a-32P]ATP, [7-32P]GTP, [adenylate-32P]NAD, and 0.75 M NaCl and 10 mM /-histidine were substituted
[3H]QNB were obtained from New England Nuclear, for 0.75 M KC1 and 5 mM /-histidine in the starting
Boston, Massachusetts. The purity of the radionu- buffer. The total ouabain-sensitive Na + ,K + -ATPase
cleotides was verified using ascending thin-layer activity of the purified sarcolemma was routinely
chromatography on polyethylenimine cellulose 70-90 ^mol P/hr/mg protein.
(Brinkmann, Westbury, New York) in 0.75 M potas- Canine renal cortical plasma membranes were pre-
sium phosphate (pH 3.4), followed by autoradiogra- pared using differential centrifugation essentially ac-
phy. Cyanogen bromide-activated Sepharose was cording to the method of Marx et al," exactly as
purchased from Pharmacia, Piscataway, New Jersey, described by Queener et al.20 Canine erythrocytic plas-
and fetuin was from Sigma Chemical, St. Louis, ma membranes were prepared by hypotonic lysis of
Missouri. Hydroxylapatite (fast flow) was from Cal- erythrocytes obtained at the time of death and removal
biochem, San Diego, California. All other reagents of heart and kidneys. Blood collection, lysis, and
were from Sigma or Fisher Scientific, Pittsburgh, washing of membranes by centrifugation were exactly
Pennsylvania. as described by Fleming and Ross.21 All membranes
were quick-frozen using a dry ice-acetone bath and
Preparation of IAP stored at - 8 0 ° C.
Bordetella pertussis strain 165 was grown in
modified Stainer-Sholte12 medium as described by Adenylate Cyclase Assay
Hewlett et al.13 Pertussis toxin was purified from the Adenylate cyclase activity was assessed by measur-
culture supernatants using hydroxylapatite and fetuin- ing the conversion of [a-32P]ATP to cyclic [32P]AMP,
agarose chromatography using a modification of exactly as described previously.10 Cyclic AMP formed
procedures described elsewhere14"16 as communicated during the reactions was isolated by the method of
by Dr. Erik Hewlett of the University of Virginia, Salomon et al.22 Results are expressed as the mean
Charlottesville. Briefly, 7 1 culture supernatant were adenylate cyclase activity ±SEM of several different
passed through a 200-ml column of hydroxylapatite determinations, each using preparations of sarco-
that had been preequilibrated with 10 mM potassium lemma from individual hearts.
phosphate (pH 9.0). The eluate from the pH-9 column
was adjusted to pH 6.0 with HC1 and applied to a GTPase Assay
200-ml column of hydroxylapatite that had been GTP hydrolysis was determined by measuring the
preequilibrated with 10 mM potassium phosphate (pH release of 32P( from fr-^PJGTP,23 exactly as recently
6.0). This column was then washed sequentially with described by us.10 Specific GTPase activity of each
500 ml of 100 mM potassium phosphate (pH 6.0) and membrane preparation was calculated as the difference
500 ml of 100 mM potassium phosphate (pH 7.0). between the mean total and nonspecific activities in
Pertussis toxin was eluted from the column with 400 duplicate determinations. Results are expressed as the
ml of 100 mM potassium phosphate (pH 7.0) con- mean specific GTPase activity ±SEM of several dif-
taining 0.5 M NaCl. The resulting eluate was applied ferent determinations, each using preparations of
to a 35-ml column of fetuin-Sepharose. The column sarcolemma from individual hearts.
Downloaded from http://circres.ahajournals.org/ by guest on May 18, 2015994 Circulation Research Vol 62, No 5, May 1988
[32P]ADP-Ribosylation of Gia and Gm Membrane CARDIAC ERYTHROCYTIC
RENAL
CORTICAL
Covalent modification of the a-subunits of the LAP
substrates was by modification of well-established
In Vivo IAP —
+ —
+ —
+
procedures using [32P]NAD as substrate.24-23 Purified
In Vitro IAP
- + "I*
sarcolemmal membranes (60 (ig) were incubated in 1.44
ml containing potassium phosphate (pH 8.0) 61 mM,
NaCl 54 mM, dithiothreitol (DTT) 2.7 mM, MgCl2 5
mM, thymidine 10 mM, GTP 0.1 mM, ATP 1 mM,
phosphocreatine 3.3 mM, creatine phosphokinase 0.3
units/ml, [32P]NAD (-10,000 cpm/pmol) 50 \iM, and ; #
14.4 [Lg IAP (preactivated with 20 mM DTT, 30 min-
utes at 30° C). The reactions were initiated by the L _ „*
addition of PPJNAD, and intoxication proceeded for
30 minutes at 30° C. Intoxication was terminated by the
addition of 4 ml ice-cold 0.25 M sucrose and 10 mM
/-histidine, and the sarcolemmal membranes were sed-
imented at 40,000 rpm for 20 minutes in a Beckman 40
rotor (HSyOOOg^). Pelleted membranes were resus-
pended to a final concentration of 0.6 mg/ml and boiled
for 5 minutes in gel dissociation medium26 [Tris (pH 6.8)
62.5 mM, sodium dodecyl sulfate (SDS) 1.25%
(vol/vol), P-mercaptoethanol 0.25% (vol/vol); grycerol FIGURE 1. [nP]ADP-ribosylation of GTP-binding proteins in
10% (vol/vol), and bromphenol blue 0.05% (vol/vol)]. canine cardiac, erythrocytic, and renal cortical plasma mem-
branes (see "Materials and Methods"). Membranes purified
SDS Polyacrylamide Gel Electrophoresis of IAP- from control animals and from animals pretreated with islet
Treated Sarcolemma activating protein (LAP) were incubated in vitro with [32P]NAD
After intoxication with IAP, membranes were sub- in the absence and presence of IAP. Following intoxication, the
jected to SDS polyacrylamide gel electrophoresis as membranes were subjected to polyacrylamide gel electropho-
described by Laemmli. M Samples containing 60 (xg resis; the resulting autoradiogram is shown.
protein were loaded onto each lane of a 12% resolving
gel. The gels were run at 20 ma per gel for 30 minutes Results
after the bromphenol blue dye front exited from the Pertussis toxin treatment of the animals in vivo
bottom of the gel. The gels were stained with Coo- altered the ability of the toxin to catalyze the [WP]ADP-
massie blue, destained, dried, and subjected to auto- ribosylation of G^ during subsequent in vitro exposure
radiography using Du Pont Cronex Pelicula x-ray film to the toxin and [32P]NAD. IAP catalyzed the [32P]ADP-
and Du Pont Lightning Plus intensifying screens ribosylation of G,,, in plasma membranes purified from
(Wilmington, Delaware). canine cardiac left ventricles, erythrocytes, and renal
cortex of the control animals (Figure 1). As we have
Muscarinic Receptor Assays recently reported," two substrates for IAP were
The ability of guanine nucleotides to regulate identified in the cardiac tissue: G, (M, = 41,000) and
muscarinic receptor affinity for agonists was deter- another protein corresponding to Go described in
mined in sarcolemmal membranes purified from con- brain29-30 and heart31-32 (Mr = 39,000). In contrast, the
trol and toxin-treated animals in equilibrium bind- ability of IAP to label G^ was nearly abolished in all
ing assays using the antagonist ligand [3H]quinu- three membrane types examined from the animals
clidinylbenzilate ([ 3 H]QNB) n essentially as we have treated with the toxin in vivo (Figure 1). Go was not
previously reported.'-28 Briefly, the 0.1-ml assays con- identified in the erythrocytic and renal cortical plasma
tained Tris/HCl (pH 7.5) 40 mM, MgCl2 10 mM, membranes (Figure 1). [32P]ADP-ribosylated IAP sub-
ascorbic acid 100 |xM, [3H]QNB 5.5 nM, purified strates in cardiac sarcolemma were isolated from
sarcolemma 6 \ig, and the indicated concentrations polyacrylamide gels and quantitated using liquid scin-
of GTP7S and oxotremorine. Binding was allowed tillation spectrometry. Whereas IAP catalyzed the
to proceed to equilibrium for 10 minutes at 37° C. incorporation of 212.8 ± 38 fmol ADP-ribose/mg sar-
[3H]QNB binding to membranes was quantitated colemmal protein in membranes from the hearts (n = 5)
by filtration on Whatman GF/C glass fiber filters of control animals, only 23.8 ± 8 . 8 fmol ADP-ribose/
using an M-24R cell harvester (Brandel, Gaithersburg, mg sarcolemmal protein could be incorporated in
Maryland). membranes from the hearts (n = 7) of the animals that
received IAP in vivo. Thus, intravenous injection
Statistical Analysis of IAP effectively catalyzed the endogenous ADP-
Unless otherwise indicated, reported probability (p) ribosylation of cardiac G; and Go (pFleming et al Pertussis Toxin-TVeated Dog 995
80 I -
O SL from Control Dogs
• SL from IAP Dogi
FIGURE 2. Concentration-effect curves for
oxotremorine stimulation of specific GT-
60
Pase activity in sarcolemma (SL) purified
O from control and islet-activating pro-
<
tein (lAP)-treated dogs. Oxotremorine-
stimulated GTPase activity was assessed in
o «0
membranes purified from control and toxin-
u.
O treated animals as described in "Materials
o and Methods. " Basal specific GTPase ac-
tivity was 21.9 ±3.1 pmol P/min/mg pro-
20
3 tein (control) and 10.7±2.2pmol PJminl
mg protein (IAP treated), (n = 3, control;
n=3, IAP treated.)
-5
10 10*7 10"6 10 10
[OXOTREMORINE] (M)
high-affinity GTP hydrolysis displayed by canine the IAP-treated animals, muscarinic stimulation was
cardiac sarcolemma.1011 In the present study, in vivo significantly reduced in membranes from the IAP-
pretreatment of dogs with IAP markedly reduced treated animals at essentially all concentrations tested
oxotremorine stimulation of specific GTPase activity in (j>996 Circulation Research Vol 62, No 5, May 1988
FIGURE 4. Effects of in vivo islet-activating protein
OSL from Control Dog (IAP) treatment on the ability of GTPyS to regulate
1.50
• Control SL + 100 pM CTPTS
muscarinic receptor affinity for agonist in purified
3 A SL from IAP Dog ogS>
1.25 sarcolemma (SL). Specific muscarinic receptor bind-
A I A P SL • 100 M" CTPTS
»o ing was determined in sarcolemma purified from
1.00
K
j y hearts taken from control (control SL) and IAP-
Q / S
Jv -r
Z 0.75 - treated dogs (IAP SL) as described in "Materials and
o
/ Methods." The assays contained 5.6 nM f'HJQNB and
m 0.50 the indicated concentrations of oxotremorine. Where
z
at indicated, 100 UJW GTPyS was also present in the
O 0.25
assays. The amount of oxotremorine bound was
3
or determined from the amount of /3H]QNB displaced
o 0.00
X from the membranes at each concentration tested.
o -0.25
* • • * •
• Lines, nonlinear least-squares fit of the data to either
| | 1 I I i |
a two-site (control) or one-site (IAP) mass action
10"' 10"» 10"' 10" 6 10" s to-* 10-5
binding model as described in the text.
[OXOTREMORINE] (U)
(p< 0.001). Maximal muscarinic inhibition of GTP- affinity for agonists in subsequently isolated sarco-
stimulated adenylate cyclase activity of 84.3% in the lemma. Binding of [3H]QNB to sarcolemma from the
control membranes was reduced to 20.4% in mem- hearts of both control and IAP-treated animals Was to
branes from the hearts of the toxin-treated animals. a single class of receptors (Kd = 10-15 pM, n = 5).
Although oxotremorine somewhat inhibited GTP- Although in vivo IAP treatment of the animals had no
stimulated adenylate cyclase activity in sarcolemma effect on the affinity of muscarinic receptors for
from the IAP-treated animals, muscarinic inhibition [3H]QNB, toxin pretreatment of the animals abolished
was significantly reduced in membranes from the the ability of GTP7S to regulate muscarinic receptor
IAP-treated animals at all concentrations tested affinity for agonist (Figure 4). In sarcolemma from the
(p< 0.001). In summary, IAP-cataryzed ADP-ribo- control animals, specific oxotremorine binding to
sylation of sarcolemmal substrates in vivo attenuated muscarinic receptors indicated complex binding kinet-
the ability of oxotremorine to both stimulate specific ics (pseudo-Hill coefficient, nFleming et al Pertussis Toxin-Treated Dog 997
TABLE 2. Effect of In Vivo IAP Treatment on Gnanine Nu- (LKd = 117.9 nM), and the addition of 100 LIM GTP7S
cleotide Inhibition of Forskolin-Stimulated Adenyiate Cyciase resulted in no significant change in affinity (LKd = 140.6
Activity
nM). Furthermore, the pseudo-Hill coefficient for
Adenyiate cyciase activity oxotremorine binding to muscarinic receptors in sar-
(pmol cAMP/min/mg protein)
colemma from the IAP-treated animals was approxi-
Additions Control LAP treated mately 1.0, indicating simple binding to a homoge-
None 3,038 + 301 2,151 ±322 neous population of receptors. ADP-ribosylation of the
0.1 /xM cardiac IAP substrates thus totally abolished GTP7S
GTPyS 2,578±220 (-14.5%) 2,057±315 (-4,3%) regulation of muscarinic receptor affinity for oxotre-
Adenyiate cyciase activity was determined as described in "Ma- morine.
terials and Methods" in the presence of 0.25 mM ATP, 1 mM Nonhydroryzable GTP analogues characteristically
MgCl2> Md 10 fiM forskolin. Sarcolemma purified from the hearts inhibit forskolin-stimulated adenyiate cyciase activity
of control and IAP-treated.animals was assayed in the absence and 15-30% in membranes that display functional G;,35 and
presence of 0.1 pM GTP7S. The percent inhibition due to GTPyS is
indicated in parentheses. we have shown previously that Gpp(NH)p inhibits the
IAP, islet-activating protein. forskolin-stimulated canine cardiac enzyme.10 In sar-
colemma from the control animals, 0.1 \LM GTP7S
where R is the concentration of receptor molecules, i inhibited adenyiate cyciase activity in the presence of
is an index referring to each of the m different classes 10 u,M forskolin by 14.5% (Table 2). In contrast,
(affinities) of receptors, nx is the number of receptors in GTP-yS inhibited the forskolin-stimulated enzyme by
each of the i affinity states, and Kdi is the corresponding only 4.3%. The ability of GTP7S to inhibit the
dissociation constant for each of the affinity states of forskolin-stimulated enzyme was significantly reduced
the receptors.34 Sarcolemma purified from the hearts of (p < 0.05) in membranes from the toxin-treated animals
control animals displayed two populations of mus- (Table 2).
carinic receptors with high- and low-affinity states Finally, muscarinic effects on the ability of GTP and
differing approximately 100-fold in relative affinity for magnesium ions to regulate adenyiate cyciase activity
oxotremorine (Table 1). In the absence of regulation by were examined in sarcolemma from the hearts of
guanine nucleotide, 6 1 % of the receptors displayed control and IAP-treated animals. In sarcolemma from
high-affinity binding ( H K d = 1.4 nM), while 39% were both control and IAP-treated animals, MgCl2 aug-
in the low-affinity state (LKd = 76.0 nM). The presence mented GTP-stimulated adenyiate cyciase activity in a
of GTP7S shifted the majority (67%) of the receptors concentration-dependent manner (p< 0.001, Figure
into the low-affinity state (LKd = 135.9 nM), with 33% 5). Although 10 LIM oxotremorine markedly inhibited
of the receptors remaining in a relatively high-affinity GTP-stimulated adenyiate cyciase activity at all con-
state (HKd = 20.3 nM). Therefore, GTP7S converted centrations of MgCl2 tested in the control sarcolemma
the majority of the high-affinity receptors to the (/?< 0.001), muscarinic inhibition of the enzyme was
low-affinity state in sarcolemma from the hearts of essentially abolished in sarcolemma from the IAP-
control animals. In contrast, guanine nucleotide reg- treated animals (Figure 5).
ulation of muscarinic receptor affinity for oxotremorine
was totally absent in sarcolemma from the hearts of Discussion
IAP-treated animals (Figure 4 and Table 1). Least- Treatment of intact dogs with IAP resulted in
squares analysis revealed that 100% of the receptors alterations in the interactions of the components of the
(~2 pmol/mg protein) were in the low-affinity state hormone-regulated adenyiate cyciase system in cardiac
1 AHA
/I
»- o SL from Control Dog•
u
< • Control SL • 10>jM OXO
FIGURE 5. Effects of in vivo islet activating
CTP-S1r IMULATE0 AOENYI.ATE CYCLASf
c A SL from IAP Dog. O .
1000 protein (IAP) treatment on regulation of
1
1.
A IAF SL * 1«>IM OXO
a adenyiate cyciase activity by oxotremorine
(OXO) and MgCl2 in sarcolemma (SL) puri-
I 750
fied from control and IAP-treated dogs.
c
ol cAWP/n
Adenyiate cyciase activity was assessed as
500 - - described in "Materials and Methods. " The
assays contained the indicated concentra-
250
I- I1
V ^A tions of oxotremorine (OXO) and MgCl2. The
increment in adenyiate cyciase activity due to
the presence of 10 \xM GTP is shown, (n = 3,
- ^
control; n = 2, IAP treated,)
1 1 | i i 1 i i 1 _J
0 2 1 6 t 10 12 1« 16 11 20
1 MgCI J 1 (mM)
Downloaded from http://circres.ahajournals.org/ by guest on May 18, 2015998 Circulation Research Vol62, No 5, May 1988
sarcolemma purified from the hearts taken from these chick cardiac muscarinic receptor. In addition, treat-
dogs. We have previously shown that muscarinic ment of chicks with IAP 48 hours before preparation
agonists modulate both ^-adrene^gic receptor affinity of cardiac membranes resulted in elimination of the
for catecholamines9 and GTP-activation of adenylate highest affinity state of the muscarinic receptors and
cyclase. 9 "" It has been shown that the biochemical increases in the proportions of receptors in the inter-
effect of IAP is the ADP-ribosylation of the a-subunit mediate- and low-affinity states.40 The Kd of the
of G, (and possibly Go), and this modification by the high-affinity state of the canine cardiac receptor (1.4
toxin results in the inactivation of the ability of the IAP nM) was similar to that reported in other cardiac
substrates to mediate inhibition of adenylate cyclase tissue,40 but lower-affinity states did not differ to the
activity.7-8 The present report describes the attenuation degree reported in other systems. For example, the
of biochemical mechanisms mediated by IAP sub- highest and lowest affinity states described in the
strates in cardiac sarcolemma isolated from the hearts mammalian neuronal and chick cardiac membranes
of animals treated in vivo with the toxin. differed by two to three orders of magnitude and
In vivo ADP-ribosylation of Gfa and GM from required fitting of the data to a three-site binding
endogenous NAD was confirmed by the inability of the model. In contrast, the high- and low-affinity states of
substrates to be [wP]ADP-ribosylated on subsequent the muscarinic receptors in the present study differed
exposure to the toxin and [32P]NAD in vitro (Figure 1). by two orders of magnitude or less and did not require
The ability of oxotremorine to stimulate high-affinity the hypothesis of a third affinity state for adequate fit
GTPase activity and inhibit GTP-activated adenylate of the data. It can be seen in Table 1 that the confidence
cyclase activity was markedly attenuated or abolished interval for the intermediate affinity state of the receptor
in membranes from the toxin-treated animals (Figures in the presence of GTP7S(HKd = 11.SM0.9 nM) over-
2 and 3). In addition, the ability of GTP7S to regulate lapped with that of the low-affinity state seen in the
muscarinic receptor affinity for agonists was abolished absence of guanine nucleotide (LKd = 38.0-152.9 nM).
in membranes from the toxin-treated animals (Figure Thus, it cannot be stated unequivocally whether the
4 and Table 1). Thus, several biochemical regulatory canine receptors actually exist in three affinity states
systems related to adenylate cyclase were interrupted that differ by a factor of 10 or less or whether the
by in vivo ADP-ribosylation of cardiac sarcolemma receptors exist in only two affinity states. Regardless of
with IAP: muscarinic stimulation of GTPase activity, the strict interpretation of the oxotremorine displace-
muscarinic inhibition of adenylate cyclase activity, ment curves, the important conclusion remains that the
inhibition of forskolin-stimulated adenylate cyclase highest affinity state of the receptors was eliminated by
activity by GTP7S, and, proximal to adenylate cyclase, either the presence of GTP-yS or by complete uncou-
guanine nucleotide regulation of muscarinic receptor pling of the receptors from LAP substrate(s) by admin-
affinity for agonists. istration of the toxin to the animals in vivo.
Apart from the effects of in vivo IAP administration In addition to G^, we have shown that IAP catalyzes
to uncouple guanine nucleotide regulation of oxotrem- the [32P]ADP-ribosylation of an additional substrate in
orine binding to subsequently isolated cardiac musca- canine cardiac sarcolemma (Figure 1 and J. W. Fleming
rinic receptors, it was of interest to us that GTP-yS did and A.M. Watanabe"). The additional IAP substrate is
not shift 100% of the receptors from the high-affinity analogous to G^, described previously in bovine
state to the low-affinity state in the sarcolemma from brain,2930 chick heart,31 and rabbit heart.32 Although GM
the control animals (Figure 4 and Table 1). Rather, 33% has not been directly demonstrated to affect adenylate
of the receptors remained in a relatively high, inter- cyclase activity,42 Go has been shown to regulate
mediate affinity state ("Kd = 20.3 nM) in the presence muscarinic receptor affinity for agonists.43 We have
of GTP-yS. Because the data were obtained in the recently shown that the most significant mechanism for
presence of MgCl2 and a high concentration of GTP7S muscarinic effects on adenylate cyclase activity in
(100 M-M), it seemed unlikely that the intermediate state canine cardiac sarcolemma is mediated indirectly by
of receptor affinity resulted simply from an incomplete inhibition of activated G,,, (GM-GTP).'°The mechanism
effect of GTP-yS. We have consistently observed that of inhibition is closely linked to the hydrolysis of
approximately one third of the receptors do not shift GTP9"" and may be related to the inhibitory receptor-
completely to the low-affinity state but rather shift to catalyzed release of functionally similar, if not iden-
an affinity intermediate between the affinities displayed tical, G^-subunits from G, and Go in the presence of
in the absence of guanine nucleotide. Furthermore, this GTP.7 Ivatada and coworkers8 have shown that ADP-
observation was true regardless of whether the guanine ribosylation of IAP substrates increases the affinity of
nucleotide was GTP7S or Gpp(NH)p (data not shown). the a-subunits for the p-y-subunits, thus reducing the
Although there is substantial precedent in the liter- ability of inhibitory P7-subunits to dissociate from the
ature for muscarinic receptors existing in three affinity G proteins in response to inhibitory receptor agonists.
states,36^1 the present data did not allow strict differ- It appears likely that the biochemical alterations seen
entiation between the possibilities of two or three in sarcolemma purified from the hearts of IAP-treated
affinity states of the canine cardiac receptors. Birdsall animals result from ADP-ribosylation of the a-subunits
et a P first demonstrated mammalian neuronal musca- of both G, and Go. Present experiments are directed
rinic receptors existing in three affinity states, and toward selective definition of the relative roles of G,
Hosey et al40 have observed three affinity states of the and Go in the mechanism of inhibitory regulation of
Downloaded from http://circres.ahajournals.org/ by guest on May 18, 2015Fleming el al Pertussis Toxin-Treated Dog 999
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Downloaded from http://circres.ahajournals.org/ by guest on May 18, 2015Pertussis toxin-treated dog: a whole animal model of impaired inhibitory regulation of
adenylate cyclase.
J W Fleming, T D Hodges and A M Watanabe
Circ Res. 1988;62:992-1000
doi: 10.1161/01.RES.62.5.992
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