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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. 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