Download Is Receptor Cross-Regulation in Human Heart

Clinical Science (1993)
85, 393-399
393
(Printed in Great Britain)
Is receptor cross-regulation in human heart caused by
alterations in cardiac guanine nucleotidebinding proteins?
A. FERRO, C. PLUMPTON and M. J. BROWN
Clinical Pharmacology Unit, University of Cambridge, Addenbrooke’s Hospital, Cambridge, U.K.
(Received 18 September 1992/1 June 1993; accepted 23 June 1993)
1. Guanine nucleotide-binding proteins (G-proteins)
play a central role in signal transduction between a
wide variety of cell-surface receptors and intracellular
second messenger systems. Recently, we and others
have demonstrated that cross-regulation can occur
between a variety of G-protein-linked receptors in
human heart. Chronic /I,-adrenoceptor blockade gives
rise to sensitization of /I,-adrenoceptor and of
5HT,-receptor responses, both of which are mediated
via stimulation of adenylate cyclase through stimulatory G-proteins (Gs), and also gives rise to desensitization of muscarinic M,-receptor responses, which
inhibit adenylate cyclase through inhibitory
Gproteins (Gi).
2. In order to investigate whether these effects are
due to quantitative changes in cardiac G-protein
isoforms, we measured their abundance in right atrial
appendage from patients taking or not taking p,adrenoceptor antagonists, by immunoblotting.
3. Samples of right atrial appendage homogenate
were subjected to SDS/PAGE, and proteins were
electroblotted on to nitrocellulose membranes. These
were then probed with specific anti-G protein antisera, and binding was revealed by means of a secondary antibody labelled with alkaline phosphatase and
using a chromogenic substrate. The resulting bands
were quantified by laser densitometry.
4. No quantitative differences were detected, between
these two groups of patients, in the amounts of asubunit of ‘long’ or ‘short’ G , isoforms (G,aL and
G&), or in the amounts of Gi1+2 a-subunit
(Gial+2). Nor was any difference found in the
abundance of the p-subunit of G-proteins. No ‘other’
G-protein (Go) was detectable in these samples by
immunoblotting.
5. We conclude that the phenomenon of receptor
cross-regulation which we have previously observed in
human right atrial appendage is unlikely to be explained by quantitative changes at the G-protein level.
~~
INTRODUCTION
Guanine nucleotide-binding proteins (G-proteins)
are ubiquitous molecules which are responsible for
coupling a large variety of cell-surface receptors to
second messenger systems and ion channels [l].
They are heterotrimeric proteins, composed of subunits designated a, B and y; whereas the fl and y
subunits are fairly constant between different Gproteins, variations in the a-subunit determine the
unique type and characteristic of each G-protein.
All G-protein-coupled receptors share a common
structure, with seven hydrophobic transmembrane
domains, the third intracytoplasmic loop being
responsible for interacting with the G-protein [Z].
In the human heart B1- and B,-adrenoceptors (AR),
when activated, interact with stimulatory G-proteins
(G,), which in turn stimulate adenylate cyclase. The
same appears to be true of the recently discovered
cardiac SHT,-receptor [3]. The cardiac muscarinic
M,-receptor, by contrast, couples with inhibitory
G-proteins (Gi),which inhibit adenylate cyclase.
We and others have found that strips of right
atrial appendage taken from patients being treated
long-term with B,-AR-selective antagonists exhibit
sensitization of B,-AR and of 5-hydroxytryptaminemediated responses, and desensitization of
M,-receptor-mediated responses, in uitro [4-61.
Recently, we have also demonstrated cardiac B2-AR
sensitization in viuo, both in patients with organic
heart disease and prospectively in healthy subjects
[7, 81. The mechanism of such cross-regulation
between receptors is not clear. Evidence to date
shows no increase in B2-AR density or affinity for
ligands in B,-AR-blocked right atrial appendage,
nor any change in the cellular sensitivity to exogenously applied cyclic AMP analogues [4, 9, lo]. The
most probable explanation, therefore is an alteration
in the coupling efficiency of different receptors to
adenylate cyclase by means of changes at the Gprotein level.
Such changes in the G-proteins could take several
forms, for example an increase in total G, or a
decrease in total Gi or both. Alternatively, there
may be a selective change in expression of one of
the G, or Gi isoforms only, which affects its activity.
Or there may be a change in activity of one of these
Key words fl1-adrenoceptor blockade, flradrenocepton. guanine nucleotide-binding proteins, human right atrial appendage, receptor cross-regulation.
Abbreviations AR, adrenoceptor; G-protein. guanine-nucleotide-binding protein; Gi, inhibitory guanine-nucleotide-binding protein; Go. ‘other’ guaninwucleotide-binding
protein; G,, stirnulatory guanine-nucleotide-binding protein; TBS, Tris-buffered saline (see the text for compition); TBS-Tween, Tris-buffered saline containing 0.1% (v/v)
Tween-20.
Correspondence Dr A Ferro. Clinical Pharmacology Unit, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CBZ ZQQ,U.K.
394
A. Ferro et al.
isoforms with no change in quantity; this could
occur because of differences in post-translational
modification of the expressed proteins.
In order to investigate whether any quantitative
changes occur in G, or G i in response to P,-AR
blockade, we examined the levels of G, and G i
isoforms in right atrial appendage from P,-ARblocked and from non-/3-AR-blocked patients, using
the technique of immunoblotting. We had two
possible hypotheses: the first, prompted by our
finding of preferential expression of G,aL (the 'long'
form of G p ) in human right atrium, was that p,-AR
blockade induces a further switch in the ratio of
G,aL/G,aS (the 'short' form of G,a); the second was
that P,-AR blockade blocks the well-documented
increase in G i a expression by P-AR stimulation
[ll], so that the P,-AR-blocked patients would
have reduced levels of Gia.
METH0DS
Patients
Right atrial appendage was obtained from 26
patients undergoing cardiac surgery, either coronary
artery bypass grafting or mitral or aortic valve
surgery, at the time of institution of cardiopulmonary bypass. Premedication was with papaveretum and hyoscine; anaesthesia was induced with
midazolam, fentanyl and propofol, with pancuronium as muscle relaxant. Propofol infusion was
used for maintenance of anaesthesia. Sixteen of
these patients were on long-term treatment with
P,-AR-selective blockers (atenolol, metoprolol or
bisoprolol), and ten were not treated with P-AR
blockers. All B,-AR-blocked patients and five of the
ten non-P-AR-blocked patients suffered from ischaemic heart disease and were undergoing coronary
artery bypass surgery. Of the remaining five non-pAR-blocked patients, four were undergoing aortic
valve replacement and one was undergoing mitral
valve repair. Patients with ischaemic heart disease
were also taking other drugs, including aspirin,
nitrates, calcium-channel antagonists, diuretics and
angiotensin-converting enzyme inhibitors. The
b,-AR-blocked and non-b-AR-blocked patients with
ischaemic heart disease were evenly matched both
for these other drugs and for age, sex and clinical
assessment of myocardial function. Two of the
patients undergoing aortic valve replacement were
receiving diuretic therapy, and another was receiving sulphasalazine and non-steroidal anti-inflammatory analgesic therapy for rheumatoid arthritis.
Materials
Anti-G-protein antisera were a gift from Dr G.
Milligan, Department of Biochemistry, Glasgow
University. Goat anti-rabbit immunoglobulins were
from Dako Ltd. Acrylamide/bisacrylamide, ammonium persulphate, glycine, SDS, N,N,N',N'-
tetramethylethylenediamine, Tris and Tween-20
were from Bio-Rad Laboratories Ltd. Dithiothreitol
was from Boehringer Mannheim U.K. All other
chemicals used were from Sigma Chemical
Company Ltd.
Preparation of homogenates of right atrial appendage
Samples of right atrial appendage were collected
into modified Krebs' solution (composition in
mmol/l: Na', 125; K + , 5; C a 2 + , 2.25; Mg2+, 0.5;
CI-, 98.5; SO:-, 0.5; HCO,, 32; H P O i - , 1; EDTA,
0.04) on ice. Connective tissue and fat were
removed, and the remaining tissue was placed into
1 mmol/l KHCO, (10pl/mg of tissue). This was
homogenized in a Polytron homogenizer, at maximum speed (setting 10) for 40s. To this homogenate
was added an equal volume of modified Laemmli
sample buffer [composition: 16% (v/v) glycerol, 3.2%
(w/v) SDS, 64mmol/l dithiothreitol, 0.1 mol/l TrisHCI, p H 6 . Q and this was heated at 100°C for
10min. The mixture was clarified by centrifugation
at 11 OOOg for lOmin, and the resulting supernatant
was stored at -70°C before use in immunoblotting
assays.
lmmunoblotting
Samples were subjected to SDS/PAGE [lo%
(w/v) polyacrylamide, 8.5 cm total gel length], as
described by Laemmli [12]. For each sample, l00pg
of total protein was loaded on to the gel. Protein
was measured in 96-well plates using the Bio-Rad
protein assay (Bio-Rad Laboratories Ltd). Molecular
mass markers (Bio-Rad Prestained SDS-PAGE
Standards, low range, 18.5-106 kDa, Bio-Rad
Laboratories Ltd) were also loaded on to each gel.
After electrophoresis, proteins were transferred to
a nitrocellulose membrane (pore size 0.45 pm;
0.8 mA/cm2 for 1 h; LKB 21 17-250 Novablot apparatus), soaked in transfer buffer of the following
composition: glycine, 39 mmol/l; Tris, 48 mmol/l;
SDS, 0.0375% (w/v) methanol, 20% (v/v). The membranes were then washed briefly in Tris-buffered
saline (TBS, composition 200 mmol/l NaCI,
50 mmol/l Tris-HC1, pH 7.4), and remaining protein
binding sites were subsequently blocked by overnight incubation in 5% (w/v) non-fat dried milk
(Marvel) made up in TBS, at 4°C.
After the blocking step, the membranes were
incubated with specific rabbit anti-G-protein antisera, diluted 1: 500 in 5% (w/v) non-fat dried milk
made up in TBS containing 0.1% (v/v) Tween-20
(TBS-Tween), for 4 h at room temperature with
gentle agitation. The anti-G-protein antisera used
were as follows: CS-1 (anti-G, a-subunit), SG-1
(anti-G,1 & 2 mubunit), BN-3 (anti-/%subunit) and
IM- 1 (anti-Go mubunit). Their characterization has
been described previously [13-1 51. After these incubations, the membranes were washed three times
(10 min per wash) with TBS-Tween. They were then
Guanine nucleotidebinding proteins in human heart
incubated with goat anti-rabbit immunoglobulins
conjugated to alkaline phosphatase [diluted 1:500
in 5% (w/v) non-fat dried milk made up in TBSTween] for 2 h at room temperature, and subsequently washed six times (10 min per wash) with
TBS-Tween. Bound primary antibodies were
revealed by incubating the membranes with the
following mixture: Nitro Blue Tetrazolium, 15mg/l;
MgC12, 4 mmol/l; 5-bromo-4-chloro-3-indolyl phosphate, 60 mg/l; ethanolamine, 0.1 mol/l, pH 9.6. Colour development was stopped after approximately
5min by rinsing with water, and the membranes
were dried between two sheets of filter paper and
stored in darkness.
Confirmation of specificity of G-protein bands
In order to confirm the specificity of the bands
detected at the appropriate molecular masses of the
respective G-proteins, affinity purification experiments were performed using immobilized GTP,
since preimmune sera were no longer available for
use as negative controls. A 3g sample of human
right ventricle (from an explanted heart obtained
from a patient undergoing cardiac transplantation
for end-stage cardiac failure secondary to ischaemic
heart disease) was homogenized as described above
and incubated with 1 mmol/l isoprenaline at 4°C for
10min. Debris was pelleted at 5000g for lOmin,
and the supernatant was centrifuged at 50000g
for 45min, all at 4°C. The pellet was resuspended
in 60ml of solubilization buffer [10mmol/l 4(2-hydroxyethy1)-1r pipemine-ethanesulphonic acid
(sodium salt), 20 mmol/l 2-mercaptoethanol, 1mmol/
1 EDTA, pH8.01 containing 1% (w/v) sodium cholate, with constant stirring for 1 h at 4"C, and the
suspension was then ultracentrifuged at 100OOOgfor
Wmin at 4°C. The resulting supernatant was passed
through a 1cmx5cm GTP-agarose column at
0.3 ml/min at room temperature and, after washing
with three column volumes of solubilization buffer
containing 0.5% sodium cholate, specifically bound
proteins were competitively eluted with 0.15 mmol/l
GTP dissolved in the same buffer. The pass-through
fraction from the first run was then applied to the
column, which was then washed and eluted as
above. Minity-isolated GTP-binding proteins were
concentrated using five volumes of acetone (- 20°C)
and centrifugation at 10000g. Samples of solubilized protein, pass-through fractions (1 and 2), and
GTP-binding proteins (eluates 1 and 2) were subsequently mixed with an equal volume of modified
Laemmli sample buffer as above, heated at 100°C
for lOmin, and subjected to SDS/PAGE and
immunoblotting as already described, except that
the secondary antibody used was goat anti-rabbit
immunoglobulins conjugated to horseradish peroxidase (1:500 dilution) and immunodetection was by
enhanced chemiluminescence (ECL Western blotting
kit from Amersham U.K.).
395
Quantification of G-proteins
The bands obtained on immunoblots were
scanned by one-dimensional laser densitometry
(LKB Ultroscan XL), measuring the absorbance at
633 nm. The areas under the peaks thus generated
were measured (LKB 2400 Gelscan XL software
package). Statistical comparison of areas under
peaks between B,-AR-blocked and non-B-ARblocked samples was performed by means of
unpaired Student's t test, with P < 0.05 being considered significant.
RESULTS
Homogenate preparations of human right atrial
appendage were subjected to SDS/PAGE, and
electroblotted on to nitrocellulose membranes, using
specific anti-G-protein antisera for immunodetection. Gels were loaded with right atrial appendage homogenate samples from both B1-ARblocked and non-/3-AR-blocked patients; each gel
was loaded with samples from 13 patients, eight of
whom were B,-AR-blocked and five of whom were
non-b-AR-blocked, In all, samples from 26 patients
were analysed (16 B,-AR-blocked and 10 non-B-ARblocked). Thus, for each anti-G-protein antiserum,
two immunoblots (13 patients each) were obtained
and analysed.
Typical immunoblots for G,a, Gial + 2 and G ,
are shown in Figs. 1-3. For all samples, antiserum
CS-1 stained two bands which ran at 52 kDa (in the
position of G,aL) and 45kDa (in the position of
G,aS); SG-1 produced one band at 40kDa
(Gial+2), BN-3 produced a band at 36 kDa (G,)
and IM-1 gave no bands (G,,a).
In addition to the bands described, other apparently non-specific bands were also seen in the
CS-1 and SG-1 immunoblots. In order to confirm
the specificity of the bands at the appropriate
molecular masses of the G, and Gi a-subunits,
solubilized proteins from a sample of human right
ventricular myocardium were affinity purified by
passage through a GTP-agarose column. The eluate
and pass-through fractions were subjected to SDS/
PAGE and immunoblotting as before. Fig. 4 shows
the result of such an experiment using the antiserum
SG-1 for immunoblotting. Using both antisera SG-1
and CS-1, the appropriate bands were abolished in
the second pass-through fraction, but were present
in both eluate fractions.
Blots were scanned by laser densitometry, measuring the absorbance of the bands at 633nm in
each lane; a typical tracing is shown in Fig. 5, where
a lane on a G,a blot has been scanned. The area
under each peak was taken to represent the relative
amount of G-protein (a- or /3-subunit) in each band.
In the case of the G,a immunoblots, the 52kDa
band was present in quantities approximately eight
times as great as the 45kDa band. There was no
significant difference in the amounts of the two
A. Ferro et al
396
kDa
Fig. I. lmmunoblot demonstrating the presence of G,KL and G,aS in extracts of human right atrial appendage. Proteins
were separated by SDS/PAGE, electroblotted on to a nitrocellulose membrane, probed using antiserum CS-l and bands were revealed
as outlined in the Methods section Lanes 1-8 are samples from /j,-AR-blocked patients. lanes 9-13 are samples from non-P-ARblocked patients G p L runs at 52 kDa and G,IS at 45 kDa (arrows)
kDa
41-
3 s
24I
2
3
4
5
6
7
8
9
1
0
1
1
1
2
1
3
Fig. 2. lmmunoblot demonstrating the presence of Cia1 + 2 in extracts of human right atrial appendage. Detection was as
outlined in legend to Fig I , using antiserum SG-l to probe for G,zI 2 Lanes 1-8 are samples from /I,-AR-blocked patients. lanes
9-13 are samples from non-/i-AR-blocked patients G,al 2 runs at 40 kDa (arrow)
+
bands, nor in their relative ratio, between
fl,-AR-blocked and non-fl-AR-blocked patients. For
G i a l + 2 , and also for GB, there was n o significant
difference in the amounts of the appropriate bands
between P,-AR-blocked and non-fl-AR-blocked
patients. Values of mean areas under each peak are
shown in Table I .
Fig. 6 shows typical dilution curves for a right
atrial sample, for both the CS-I and SG-1 antisera;
over the range of &125pg of protein, there was a
linear relationship between total protein loaded and
the area under each peak. Therefore, at least over
this range, the amount of G-protein (G,aL, G,aS
and G i a l + 2 ) present was proportional to the measured area under the absorbance peaks. Similar
linear relationships were found, over the same range
+
of protein, in experiments on three separate samples
of right atrial appendage, using antisera CS-1, SG-1
and BN-3.
In another experiment, 1oOpg of protein from a
single sample of right atrial appendage was loaded
into 13 different lanes of a gel, and immunoblotting
(with antisera CS-1, SG-I and BN-3) and detection
were performed as before. Coefficients of variation
for the area under the peaks as detected by laser
densitometry were as follows: G,aL, 13.87;; G,aS
1 l.6x;Cia, 11.3%; G,, 11.1%.
DISCUSSION
Our results indicate that human right atrial
appendage possesses both G , (G,aL and G,aS) and
Guanine nucleotidebinding proteins in human heart
397
kDa
I lo-
84-
47-
33-
24-
Fig. 3. lmmunoblot demonstrating the presence of C/?in extracts of human right atrial appendage. Detection was as
outlined in legend t o Fig. I,using antiserum BN-3 to probe for GB. Lanes 1-8 are samples from B,-AR-blocked patients; lanes %I3
are samples from non-SAR-blocked patients. GP runs at 36 kDa (arrow).
t
II
I
'2
3
4
5
6
Fig. 4. lmmunoblot of solubilized proteins from human right
ventricle, before and after affinity purification on a CTP-agarose
column. The blot was probed using antiserum SG-l (for Gial +2). and
subsequent detection was as outlined in the Methodr section. Lane I:before
ultracentrifugation of solubilized myocardial membranes. Lane 2: after
ultracentrifugation but before affinity purification. Lanes 3 and 4 passthrough I and 2, respectively. Lanes 5 and 6 eluates I and 2, respectively.
The arrow indicates 6 kDa.
Gi. No 'other' G-protein (Go)was detected under
these conditions, indicating that it is absent, that it
is present in much smaller amount than G, or Gi,
or that the antiserum has a relatively low activity
against this protein. Staining of G,aL was significantly greater than that of G,aS; the levels of both
of these, as well as the G,aL/G,aS ratio, were not
different between P,-AR-blocked and non-P-ARblocked patients. Similarly, the amounts of G i a l + 2
and GP were not different between the two groups
of patients. It has been demonstrated previously
that Gia2 is the predominant Gi isoform in human
heart, with very little if any Gial being present, at
least at the mRNA level [16]; we may say, therefore,
that the lack of difference demonstrated using the
anti-Gi antiserum represents a lack of difference in
the amount of Gia2 in our samples.
We and others have previously demonstrated that
samples of human right atrial appendage, from
I'
;
{L~,
T
T
Fig. 5. Laser densitometer scan of a lane on an immunoblot probed
with antiserum CS-I. For details of immunoblotting and scanning see the
Methods section. Absorbance units at 633nm is plotted along the vertical
axis, against the position on the lane along the horizontal axis. Two peaks
were detected. corresponding to G,aS (left arrow) and G,aL (right arrow).
The area under each peak corresponds t o the relative amount of each
protein present on the blot.
patients on long-term treatment with P,-AR antagonists, show a tenfold increased sensitivity to the
effects of P2-AR agonists, with no change in P,-AR
sensitivity [4, 63. We have also found evidence of
similar cross-sensitization of SHT,-receptor responses in human right atrial appendage after longterm P,-AR blockade [ S ] . The mechanism of this
cross-regulation between different G-protein-linked
receptor systems is not clear. There is known to be
no change in P,-AR density, whereas P,-AR density
is increased after chronic B,-AR antagonism [9, lo];
nor is there any change in the apparent affinity of
salbutamol for the P2-AR [4]. Furthermore, the
inotropic response of right atrial strips to dibutyryl
cyclic AMP is unaltered, indicating no change in the
A. Ferro et al.
398
Table I. Areas under absorbance peak at 633nm for &-AR-blocked
and non-PAR-blocked patients. Values given are in absorbance units
(AU) x mm for each band at the stated molecular mas, and are expressed
as meansf SEM. For each band, there was no significant difference
(P >0.05) between atria from /l,-AR-blocked and from non/l-AR-blocked
patients.
Area under absorbance peak (AU x mm)
Antiserum CS-l (anti-G ,a)
45 kDa
52 kDa
Antiserum SG-l (anti-G,al +2)
40 kDa
Antiserum BN-3 (anti-G/l)
36 kDa
/l,-AR-blocked
patients
(n = 16)
Non-/l-AR-blocked
patients
(n = 10)
0.045
0.006
0.372 0.045
0.0% f0.009
0.438 f0.073
0.046f0.005
0.042 f0.005
'1
s
x
0.30
-m
x
'-I
s
0.10
e
4
0
0.081 fl.008
0.062 fl.016
cellular response to cyclic A M P generated by P-AR
activation [4]. For these reasons, it appears most
likely that the observed receptor cross-regulation is
produced by differential alteration in the coupling of
the various receptors to adenylate cyclase; we have
therefore postulated that changes at the G-protein
level must take place, involving alterations either in
quantity or in function of different G-protein isoforms. If the different G-protein isoforms preferentially couple different receptors to adenylate cyclase,
such alterations could give rise to the phenomenon
observed.
Our results have failed to provide evidence of any
quantitative change in Gi- or in G,-proteins after
long-term p, -AR blockade. We consider that small
quantitative changes below the limits of resolution
of our assays are unlikely to explain the order of
magnitude changes in receptor coupling described
above. We have recently reported a similar negative
result for measurements of the messenger R N A
encoding the G, and Cia-subunits [17]. Therefore it
now seems likely that fl,-AR blockade influences
post-translational modification of the G-proteins.
There is evidence that G,, Gi and transducin (G,)
can be phosphorylated [18-211, Gi, Go and transducin can be N-myristylated, and transducin can
also be N-modified by other fatty acids [22, 231; G,,
and probably other G-proteins, may also undergo
endogenous ADP-ribosylation [24]; and isoprenylation of the y-subunit of G-proteins has recently been
demonstrated to be of functional importance in the
regulation of signal transduction, by enabling membrane association and activation of fl-adrenergic
receptor kinase [25]. Such modifications may be
important in determining G-protein activity, so that
changes in one or more of these processes may give
rise to differential coupling of receptors to adenylate
cyclase.
We have found that, in human right atrial appendage, G,aL staining is considerably greater (by
approximately eight-fold) than that of G,aS. It is
20
40 60 80 loo 120
Amount of protein loaded (pg)
140
-g0.06
8
n
4
;
i
o'02v
0.04
5 0.01
,-I
0.00
0
20
40 60 80 100 120
Amount of protein loaded (pg)
I40
Fig. 6. Relation between area under absorbance peak at 633nm and
amount of protein loaded. Different amounts of protein from a sample of
right atrial appendage were loaded into different lanes on a gel. run,
blotted, probed and analysed by laser densitometry as outlined in the
Methods section. Here we show typical curves obtained from one such
experiment, using antisera ( 0 ) CS-l and (b) SG-I. Area under peak is
expressed as absorbance units (AU) x mm.
52kDa band (antiserum
CS-I); 0.
45kDa band (antiserum CS-I);
40kDa (antiserum SG-I).
..
+,
likely that this result reflects a true difference in the
relative abundance of G,aL and G,aS in human
atrium, since the antiserum CS-I was raised against
a C-terminal peptide common to both proteins. The
finding of more G,aL in human atrium is in contrast to cardiac tissues from other species such as
rat and dog, where G,aS staining is approximately
equal to that of G,aL ( A . Ferro et al., unpublished
work). Interestingly, human heart contains the
highest /l2-AR/B1-AR ratio (approximately 30: 70) of
all species [26]. It is possible, if these observations
are connected, that the two fl-AR subtypes couple
preferentially to different G,a isoforms, and this
would in turn explain the paradox that most adenylate cyclase in human heart is coupled to the fl,-AR
[27]. However, direct evidence of differential cou-
Guanine nucleotidebinding proteins in human heart
pling of fl-AR subtypes to different G-protein isoforms is not available at present.
In conclusion, our results indicate that the previously observed cross-regulation of G-proteincoupled receptors in response to long-term fl,-AR
blockade does not appear to be the result of
quantitative changes occurring at the G-protein
level. Other processes involving the G-proteins may
well be involved, but their nature remains to be
elucidated.
ACKNOWLEDGMENTS
We thank the Theatre staff at Papworth Hospital
for their assistance in the supply of tissues. The antiG-protein antisera were kindly donated by Dr G.
Milligan, Department of Biochemistry, Glasgow
University. We also thank Dr B. Hazleman and Mr
G. Riley, Rheumatology Research Unit, Cambridge
University, for use of, and assistance with, the laser
densitometer. A.F. is an MRC Training Fellow. C.P.
is funded by the British Heart Foundation.
REFERENCES
I. Simon MI, Strathmann MP, Gautam N. Diversity of G proteins in signal
transduction. Science (Washington DC) 1991; %2: 802-8.
2. Leviaki A. From epinephrine to cyclic AMP. Science (Washington DC) 1988;
MI:
3. Kaumann AJ, Sanders L, Brown AM, Murray KJ, Brown MJ. A SHT,-like
w.
4.
5.
6.
7.
8.
9.
receptor in human right atrium. Naunydchmiedeberg’s Arch Pharmacol
1991; 344: 1%9.
Hall JA. Kaumann AJ, Brown MJ. Selective 8,-adrenoceptor blockade enhances
positive inotropic responses t o endogenous catecholamines mediated through
fi,-adrenoceptors in human atrial myocardium. Circ Res 1990; 6 1610-13.
Kaumann AJ, Sanders L, Brown MJ. Chronic p,-adrenoceptor blockade
enhances positive inotropic responses to Mydroxytryptamine in human
atrium. J Mol Cell Cardiol 1990; 22 (Suppl. Ill):PW6.
Motomura S, Deighton NM, Zerkowski H-R. Doetsch N, Michel MC,
Brodde 04. Chronic P,-adrenoceptor antagonist treatment sensitises
&adrenoceptors, but desensitises M,-murcarinic receptors in the human right
atrium. Br J Pharmacol 1990; 101: 363-9,
Hall JA, Ferro A, Dickerson JEC, Brown MI. ,!-Adrenoceptor subtype cross
regulation in the human heart. Br Heart J 1993; 69: 331-7.
Hall ]A, Petch MC. Brown MI. In vivo demonstration of cardiac
&drenoceptor sensitisation by PI-antagonist treatment. Circ Res 1991; 69:
959-64.
Michel MC, Pingsmann A, Beckeringh JJ. Zerkowski H-R. Doetsch N,
Brodde 04. Selective regulation of PI- and j,-adrenoceptors in the human
399
heart by chronic padrenoceptor antagonist treatment. Br J Pharmacol 1988;
W: 685-91.
10. Brodde 0 4 Hundhausen H-J, Zerkowski H-R, Michel MC. Lack of effect of
chronic calcium antagonist treatment on 8,-and /?,-adrenoceptors in right
atria from patients with or without heart failure. Br J Clin Pharmacol 1992;
3 3 169-74.
II. Eschenhagen T, Mende U, Nose M, et al. Isoprenalinejnduced increase in
mRNA levels of inhibitory G-protein a-subunits in rat heart.
Naunyn-Schmiedeberg’r Arch Pharmacol 1991; 34% 609-15.
12. Laemmli UK. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature (London) 1970 221: 680-5.
13. Milligan G, Unson CG. Persistent activation of the a-subunit of G, promotes
its removal from the plasma membrane. Biochem J. 1989, 160: 837-41.
14. Mitchell FM, Griffiths SL, Saggerson ED, Houslay MD, Knowler JT. Milligan G.
Guanine-nucleotide-binding proteins expressed in rat white adipose tissue.
Biochem J 1989; 262: 403-6.
15. Green A, Johnson JL, Milligan G. Down-regulation of Gi subtypes by
prolonged incubation of adipocytes with an A, adenosine receptor agonist.
J Biol Chem 1990; U:520610.
16. Eschenhagen T, Mende U, Nose M. et al. Increased messenger RNA level of
the inhibitory G protein a subunit Gia-2 in human end-stage heart failure.
Circ Res 1992; 70: 688-96.
17. Jia H, Monteith S, Brown MJ. Messenger RNA levels of the a subunits of Gi
protein and G, protein in &blocked and non-gblocked human atrium. Br J
Pharmacol 1993; I00 (Suppl.): 117P.
18. Pyne NJ, Freissmuth M, Pyne S. Phosphorylation of the recombinant spliced
variants of the a-subunit of the stirnulatory guanine-nucleotide binding
regulatory protein (G,) by the catalytic subunit of protein kinase A. Biochem
Biophys Res Commun 1992; 1% 10816.
19. Pyne NJ, Freissmuth M. Palmer S. Phosphorylation of the spliced variant
forms of the recombinant stirnulatory guanine-nucleotide-binding regulatory
protein (G,a) by protein kinase C. Biochem J 1992; Z& 333-6.
10. Katada T, Gilman AG, Watanabe Y. Bauer S, Jakobs KH. Protein kinase C
phorphorylates the inhibitory guanine-nucleotide-binding regulatory
component and apparently suppresses its function in hormonal inhibition of
adenylate cyclase. Eur J Biochem 1985; 151: 431-7.
21. Zick Y, Sagi-Eisenberg R, Pines M, Gierschik P, Spiegel AM. Multisite
phosphorylation of the a subunit of transducin by the insulin receptor kinase
and protein kinase C. Proc Natl Acad Sci USA 1986. 8& 92947.
22. Buss JE, Mumby SE, Casey PJ. Gilman AG, Sefton BM. Myristoylated a
subunits of guanine nucleotide-binding regulatory proteins. Proc Natl Acad Sci
USA 1987; M 7493-7.
l3. Kokame K, Fukada Y, Yorhizawa T, Takao T, Shimonishi Y. Lipid modification
at the N terminus of photoreceptor G-protein a-subunit. Nature (London)
1992; 359 749-52.
14. Molina y Vedia L, Nolan RD. Lapetina EG. The effect of iloprost on the
ADP-ribosylation of G,a (the a-subunit of G,). Biochem J 1989; 261: 841-5.
25. lnglese J. Koch WJ, Caron MG, Lefkowitz RJ. lsoprenylation in regulation of
signal transduction by G-proteimoupled receptor kinases. Nature (London)
1992; 359: 147-50.
16. Buxton BF, Jones CR, Molenaar P, Summers RJ. Characterization and
autoradiographic localization of 8-adrenoceptorr in human cardiac tissues. Br J
Pharmacol 1987; 92: 299-310.
17. Kaumann AJ, Hall JA, Murray KJ, Wells FC, Brown MI. A comparison of the
effects of adrenaline and noradrenaline on human heart: the role of /?,- and
/?,-adrenoceptors in the stimulation of adenylate cyclase and contractile force.
Eur Heart J 1989; 10 (Suppl. 6): 19-37.