Naunyn-Schmiedeberg's Arch Pharmacol (1996) 353:592-595 © Springer-Verlag 1996

Alberto J. Kaumann • James A. Lynham Anthony M. Brown

Comparison of the densities of 5-HT4 receptors, ill" and P2-adrenoceptors in human atrium: functional implications

Received: 20 November 1995/Accepted: 20 December 1995

Abstract We measured in human atrium the density of 5- HT4 receptors, labelled with [125I]-SB 207710 (1-butyl-4= piperidinyl) methyl 8-amino-7-iodo-1, 4-benzodioxan-5- carboxylate), and compared it with the density of ill- and fl2-adrenoceptors, labelled with (-)-[~25I]-cyanopindolol. [t25I]-SB 207710 (5-1200 pmol/1) labelled a small popula- tion of saturable binding sites (Bma x ~ 4 fmol/mg protein) with a pKD of 9.7 and with 5-HT4 receptor characteristics, as assessed with competing ligands. The density of atrial binding sites with 5-HT4 receptor characteristics was 10 and 5 times lower, respectively, than the density of/~a- and fl2-adrenoceptors. We suggest that the small 5-HT4 receptor population may in part explain why the positive inotropic effects of 5-HT are smaller than those of catecholamines mediated through ill- and fi2-adrenoceptors.

Key words Human fight atrium • 5-HT4 receptor density • [125I]-SB 207710 • ill- and fi2-adrenoceptor densities

Introduction

5-HT4 receptors mediate increases in contractile force through a pathway involving cyclic AMP-dependent pro- tein kinase in human atrial preparations (Kaumann et al. 1990, 1991; Sanders and Kaumann 1992). The maximum inotropic effects of 5-hydroxytryptamine (5-HT) are smal- ler than those of (-)-isoprenaline (Kaumann et al. 1990, 1991), suggesting that 5-HT4 receptors are less tightly coupled to effectors or exist in lower density than fl-adre-

A.J. Kaumann ([~) • J.A. Lynham Human Pharmacology Laboratory, The Babraham Institute, Babraham, Cambridge CB2 4AR, UK

A.M. Brown SmithKline Beecham Pharmaceuticals, Department of Psychiatry Research, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK

noceptors. Both ill- and fl2-adrenoceptors can mediate greater maximal positive inotropic effects than 5-HT4 re- ceptors in human atrium because (-)-noradrenaline (through fl~-adrenoceptors) and (-),adrenaline (through fl2- adrenoceptors) can produce matching maximal effects (Lemoine et al. 1988; Hall et al. 1990). To assess and compare the density of the three receptor populations we have used [125I]-SB 207710 (Brown et al. 1993; Kaumann et al. 1995) to label 5-HT 4 receptors and (-)-[125I]-cyano- pindolol (Hoyer et al. 1982) to label ill- and fi2-adrenocep- tors.

Methods

Patients. Right atrial appendages were obtained from 38 male patients (Age 61_+10, mean_+SD, range 39-79 years) undergoing surgery at the regional hospital for coronary artery bypass grafts. All patients had re- ceived fl-adrenoceptor blocking agents chronically until the day of surgery. The r-blockers used were (number of patients between par- entheses): atenolol (26), metoprolol (4), bisoprolol (3), oxprenolol (2), pindolol (1), nadolol (1) and propranolol (1). Some of these patients had also been prescribed some of the following drugs: aspirin, warfar- in, isosorbide mononitrate, glyceryl trinitrate, diltiazem, nicardipine, nifedipine, lisinopril, enalapril, captopril, fenofibrate, benzafibrate, simvastatin, omeprazole, cimetidine, ranitidine, furosemide, hydro- chlorothiazide and antibotics. Premedication was with papaveretum and hyoscine. Anaesthesia was induced with alfentanil, ketamine, propofol, fentanyl, thiopentone, imidazolam or O2/N20 and main- tained with fentanyl, ketamine, methoxitone, propofol or trichloro- ethylene. Pancuronium, atracurium or vecuronium was used as mus- cle relaxant.

Membrane preparation. After excision the appendages were immedi- ately placed into modified oxygenated Krebs' solution, kept on ice, containing (mmol/1): Na + 125, K + 5, Ca 2+ 2.25, Mg 2+, C1-98,5, SO ]- 0.5, HCO~ 29, HPO42- 1, EDTA 0.04 and equilibrated with 95% 02/5% CO2. All further procedures were on ice. After washing free of blood in ice-cold solution the appendages were carefully dis- sected and freed of fat and connective tissue, blotted, quickly weighed and freeze-clamped in liquid nitrogen, and stored at -70°C until use. Further procedures were those of Kaumann et al. (1995) with minor modifications. Atrial tissue (0.5 g) was reduced to a pow- der under liquid nitrogen and homogenised for 10 s in 0.5 ml of 50 retool/1 Tris HCI and 1 retool/1 EDTA, pH 7.0 at 20°C, with a Polytron PT7 probe at speed setting 8 and diluted with further 4.5 ml

of the same buffer. The diluted homogenate was filtered through gauze and centrifuged for 10 rain at 14000 g, 4°C. The pellet was re- suspended in 5 ml and centrifuged as before, then finally resuspeuded in binding buffer containing (mmol/1): Tris HC1 50, EDTA 1, EGTA 5, MgC12 2, ascorbate 1, phenyl methyl sulphonyl fluoride 0.5, and 0.5% bovine serum albumin, pH 7.4 at 37°C.

Binding assays. 5-HT4 receptor binding assays were carried out in tubes containing 100 or 200 gl binding buffer with 200-500 lag mem- brane protein. Saturation binding was carried out with [~25I]- SB 207710, 5-1200 pmol/1, in the absence or presence of 5- HT 1 mmol/1. Specific binding was defined as the difference between total binding and binding in the presence of 5-HT 1 retool/1. For com- petitive binding studies, around 40-50 pmol/l[12sI]-SB 207710 was used in the absence and presence of 8-10 concentrations of compet- ing ligand; the medium also contained 100 lamol/1 GTR All assays were carried out in duplicate. After an incubation period of 60 rain at 37°C, the contents were filtered on Whatman GF/C filters presoaked in 0.3% polyethylenediamine and 1 lamol/1 SB 207710 using a Bran- del harvester and washed 3 times with ice-cold solution containing 50 mmol/1 Tris HC1 and 2 mmol/1 MgC12, pH 7.0. Filters were counted in a Packard ?,-counter at 79% efficiency.

The densities of ill- and fi2-adrenoceptors were assessed with (-)- [~25I]-cyanopindolol 1-300 pmol/1 for saturation binding and around 50 pmol/1 for inhibition of binding by the fll-adrenoceptor-selective antagonist CGP 20712A (Dooley et al. 1986) as used on human heart (300 nmol/1) (Kanmann and Lemoine 1987). Binding removed by 200 gmol/1 (-)-isoprenaline was considered specific. After 2 h incuba- tion at 37°C, 200 lal samples were diluted with ice-cold buffer, fil- tered and counted as described above.

Protein was measured by the method of Bradford (1976) with bo- vine serum albumin as standard.

Data from saturation binding and inhibition of binding by compet- ing ligands were analysed by non-linear regression using GRAFIT (Leatherbarrow 1992).

593

Drugs and materials. [125I]-SB 207710 was obtained from Amersham (Little Chalfont, Buckinghamshire, UK) and (-)-[125I]-cyanopindolol from NEN (Stevenage, UK). Bovine serum albumin (fraction V), GTP disodium salt, 5-hydroxytryptamine hydrochloride (5-HT), and (-)-isoprenaline hydrochloride were purchased from Sigma (Poole, Dorset). Tropisetron was purchased from RBI (Natick, MA, USA). SB 207710 (1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1, 4-benzo- dioxan-5-carboxylate), renzapride, 5-carboxamidotryptamine (5-CT) and SB 203186{(1-piperidinyl)ethyl 1H-indole 3 carboxylate} were synthesized at SmithKline Beecham. Cisapride was a gift from Jans- sen (Beerse, Belgium). CGP 20712A 1-[2-{(carbamoyl 4-hydroxy) phenoxy } ethylamino]-3- { 4-(1-methyl-4-trifluoromethyl-2-imidazolyl)- phenoxy}-propanol methanesuffonate was a gift from CIBA-Geigy (Basle, Switzerland).

Stock solutions were made up in twice distilled water except tro- pisetron and cisapride (dimethylsulphoxide). 5-HT and (-)-isoprena- line were in 0.2 mmol/1 ascorbate.

Results

Binding of [125I]-SB 207710

Figure 1A shows in a representative experiment that [~25I]- SB 207710 labelled a population of saturable binding sites as defined with 1 mmol/1 5-HT. Resuks from 5 independent experiments yielded a Bmax of 3.7 fmol/mg protein with a KD of 190 pmol/1 (Table 1). Non-specific binding was 51+8% (mean_+SD, range=39-67%, n=29) with approxi- mately 50 pmol/l[125I]-SB 207710. Binding of [125I]-SB

Fig. 1 Specific binding of [125I]- A SB 207710 (A) and inhibition of binding by antagonists (B), partial agonists (C) and agonists (D). Data points of (A) are duplicates a

of 1 experiment; Bmax=3.1 fmol/ .~ mg and KD=126 pmol/1. Data £ points of B, C and D are mean ~- 2 values from n experiments shown < in Table 1 -s

, I 1 I I T i I [ I 1 I

2 0 0 4 0 0 . 6 0 0 8 0 0 1 0 0 0 1 2 0 0

[["~1] - SB207710] pmol/I

B

I ~ I i I l " I I

1 0 0 ~ i s e =~ so o Iron

i~ 60

4o

0

-12 -11 -10 -9 -8 -7 -6 -5 -4 Log [drug] mol/I

53 ~5 o

& u)

C

1 0 0

80

60

40

2 0

0

0 ~ ~ 0 I Cisaprid e •

R e n z a p r i d e ' / ~

" % 9 o I I J { , 1 ~ I

-8 -7 -6 -5 -4 Log[drug] rnol,I

-3

D

1 0 0 ~,

80

ifi 60

o. 40

2 0

0 -8

- • •

- • O -CT

4

-7 -6 -5 -4 -: Log[drug] mol/I

594

Table 1 Characteristics of 5-HT4 receptors. Comparison with ill- and fl2-adrenoceptors (Data are mean_+SEM)

5-HT4 receptors n

Density (fmol/m~)25 3.74_+0.45 5 KD (pmol/1) for [ ]-SB207710 190 _+83 5

Competing ligand

pKi (mol/1)

Functional evidence

pKB pKp pECso

SB 207710 9.6 _+0.2 6 SB 203186 8.0 +0.1 8 Tropisetron 6.1 _+0.2 7 Renzapride 6.4 _+0.2 3 Cisapride 6.0 _+0.2 3 5-HT 5.8 _+0.2 7 5-CT 4.9 _+0.2 3

10.1 a 8.7 b 6.7 c

6.7 d 6.2 a

6.3 cl <6.1 d

7.4--7.9 °-e 4.7 d

fl-adrenocepto rs Density (fmol/mg) 57.5 +5.0 9 (/31- and flz-adrenoceptors) fll-adrenoceptors (%) 65 _+1.2 9 flz-adrenoeeptors (%) 35 _+1.2 9

n= number of patients. On occasion more than one assay, using different ligands, was carried out on atrial membranes from the same patient. a From Kaumann et al. (1994) b From Parker et al. (1995) c From Kaumann et al. (1990) a From Kaumann et al. (1991) e From Sanders et al. (1995)

207710 was inhibited by antagonists (Fig. 1B), partial ago- nists (Fig. 1C) and agonists (Fig. 1D). pKi values (pKi = - l og Ki M) for competing ligands are listed in Table 1.

Binding of (-)-[125I]-cyanopindolol

Saturation binding with (-)-[125I]-cyanopindolol revealed a density of 57.5 fmol/mg fl-adrenoceptors of which 65% were fll-adrenoceptors and 35% fla-adrenoceptors, as esti- mated from a biphasic binding inhibition curve of CGP 20712A (data summarised in Table 1).

Discussion

We have identified in human right atrium a small popula- tion of saturable binding sites for [lzsI]-SB 207710 with the following 5-HT 4 receptor characteristics: 1. The pKD of 9.7 of the radioligand agrees with the pKi of 9.6 ob- tained from competition with its non-radioactive analogue and is also in line with pKB of 10.1 for unlabelled SB 207710 as competitive antagonist of the positive ino- tropic effects of 5-HT in human atrium (Kaumann et al. 1994). 2. The estimated pKi values for the binding of the antagonists SB 203186 and tropisetron as well as the par- tial agonists renzapride and cisapride are also in line with the corresponding pKB (Parker et al. 1995; Kaumann et al. 1990) or pKp values (and pECso values) (Kaumann et al. 1991). 3. As expected for 5-HT4 receptors, including those

of human atrium (Kaumann et al. 1991), the agonist 5-HT has higher affinity than the agonist 5-CT. However, the agonist potency of 5-HT is at least 40 times greater than its affinity (pECs0-pKi>l.6), suggesting the existence of a high 5-HT4 receptor reserve for 5-HT in human right atrial appendage.

Both the potency and efficacy of renzapride and cisa- pride, with respect to 5-HT, are considerably lower in hu- man atrium than in mammalian brain cells (Dumuis et al. 1989). Therefore human atrial 5-HT receptors were consid- ered similar, ie 5-HT4_like , but not identical, to cerebral 5- HT 4 receptors (Kaumann et al. 1991). More recent work has provided further evidence for a higher affinity of cisa- pride for cerebral 5-HT4 receptors (Grossman et al. 1993; Brown et al. 1993), including those of man (Domenech et al. 1993) than for 5-HT4 receptors of porcine right atrium (Kaumann 1990; Kaumann et al. 1995) and human right at- rial appendage (present work). This situation suggests the more likely existence of tissue-dependent distinct popula- tions of 5-HT4 receptor subtypes than mere species homolo- gues of 5-HT4 receptors. Two splice variants of the 5-HT4 receptor that differ in the length and sequence of their C-ter- mini, long (5-HT4L) and short (5-HT4s), have been cloned for rat and man (Gerald et al. 1994, 1995). In rat atrium only the S isoform was detected while transcripts in rat brain were mostly of the L form, with the exception of stria- turn which was of the S form (Gerald et al. 1995). Future work is required to establish whether the isoform of 5-HT4 receptors of human atrial myocytes differs from the 5-HT4 receptor isoform in certain areas of human brain.

595

We assume that the 5-HT4 receptor population labelled by [125I]-SB 207710 in human right atrial tissues is mostly located in atrial myocytes because in these contractility is increased by 5-HT through 5-HT4 receptors (Harding et al. 1994; Sanders et al. 1995). Recently some m R N A for the 5-HT4 receptor has also been detected in isolated human endothelial cells (Ullmer et al. 1995) but we are not aware of functional evidence. Right atrial appendages contain small coronary blood vessels, whose endothelial cells could conceivably contain some 5-HT4 receptors labelled by [125I]-SB 207710. A low density of fl2-adrenoceptors has also been detected in human atrial endothelial cells (Bux- ton et al. 1987) although the density is lower than in myo- cytes. Our estimated 5-HT 4 receptor density and fl2-adreno- ceptor density therefore represent upper limits for the corre- sponding receptor populations in human atrial myocytes.

The 5-HT4 receptor population, detected in human right atrial appendage, was 10 times smaller than that of fll- adrenoceptors and 5 times smaller than that of fi2-adreno- ceptors. This low atrial 5-HT4 receptor density could be related, at least in part, to the smaller maximal positive in- otropic effects of 5-HT observed in tissues (Kaumann et al. 1990, 1991) compared to those of catecholamines mediated through i l l- and fl2-adrenoceptors (Lemoine et al. 1988; Hall et al. 1990). At first sight this interpretation ap- pears to be inconsistent with another finding in the present work that 5-HT4 receptors of human atrium appear to be tightly coupled to effectors, as estimated indirectly by the demonstration of a large receptor reserve for 5-HT. This implies that the smaller 5-HT-evoked effects cannot reflect low 5-HT4 receptor density in individual myocytes. Furthermore, we have shown recently that in single human atrial myocytes the maximum contractile responses to 5- HT and (-)- isoprenal ine are not different (Sanders et al. 1995). These observations may be reconciled when it is noted that our assessment of atrial 5-HT 4 receptor density refers to the whole tissue and not necessarily to individual myocytes. A possible explanation is then that not all myo- cytes expressing fl-adrenoceptors also express 5-HT4 re- ceptors and that in the whole tissue, the fraction of myo- cytes responsive to 5-HT is insufficient to generate maxi- mum contractions as large as those caused by a catechol- amine through fl-adrenoceptor activation.

We conclude that human right atrial appendages pos- sess a small population of 5-HT 4 receptors compared to /71- and fla-adrenoceptors.

Acknowledgements AJK wishes to thank the surgical staff of Pap- worth Everard Hospital for the reliable supply of atrial tissues.

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