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(Circulation. 1995;92:2526-2539.)
© 1995 American Heart Association, Inc.

Articles

Sensitization of Human Atrial 5-HT₄ Receptors by Chronic ß-Blocker Treatment

Louise Sanders, MA; James A. Lynham, BSC; Brian Bond, MSC; Federica del Monte, MD; Sian E. Harding, PhD; Alberto J. Kaumann, MD, PhD

From the Human Pharmacology Laboratory, The Babraham Institute (L.S., J.A.L., A.J.K.), Babraham, Cambridge; the Clinical Pharmacology Unit, University of Cambridge, Addenbrooke's Hospital (L.S., A.J.K.), Cambridge; SmithKline Beecham Pharmaceuticals (B.B.), Welwyn; and the Department of Cardiac Medicine, National Heart and Lung Institute (F.d.M., S.E.H.), London, UK.

Correspondence to Dr A.J. Kaumann, Human Pharmacology Laboratory, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK.

	Abstract

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Background Chronic treatment of patients with ß-blockers induces ß₂-adrenergic receptor hyperresponsiveness in atrium and sinoatrial node. To investigate whether other atrial G_s protein–coupled receptors also become hyperresponsive after chronic treatment with ß-blockers, we investigated 5-HT₄ receptors in tissues and myocytes, which mediate serotonin-evoked increases of both contractile force and cAMP levels.

Methods and Results Isolated right atrial strips from patients who had been chronically treated or not treated with a ß-blocker were set up to contract. In tissues from ß-blocker–treated patients (n=27), the maximum inotropic response to serotonin was 56±3% (mean±SEM) of the effect elicited by (-)-isoproterenol (200 µmol/L) compared with a response of 19±6% in tissues from non–ß-blocker–treated patients (n=13) (P<.001). The responsiveness of the tissues to Ca²⁺ was unchanged by chronic ß-blocker treatment. Serotonin (1 and 10 µmol/L) increased tissue cAMP levels, the increase with 10 µmol/L being significantly greater (P<.05) in tissues from ß-blocker–treated (n=9) than in non–ß-blocker–treated (n=7) patients. In paced atrial myocytes, serotonin caused concentration-dependent increases in contraction. Myocytes obtained from atria of ß-blocker–treated patients were more sensitive (P<.01) to the effects of serotonin (-log EC₅₀, 7.9±0.2 mol/L; n=12) than myocytes obtained from non–ß-blocker–treated patients (-log EC₅₀, 7.3±0.2 mol/L, n=12). Chronic ß-blocker treatment had no effect on forskolin-evoked myocyte responses. Carbachol (1 µmol/L) suppressed the effects of both serotonin (n=6) and (-)-isoproterenol (n=6) in the same atrial myocyte.

Conclusions Chronic treatment of patients with ß-blockers causes atrial 5-HT₄ receptor inotropic hyperresponsiveness and enhanced serotonin-evoked increases in cAMP levels. This may be due to modified cross talk between 5-HT₄ receptors, ß-adrenergic receptors, and muscarinic receptors.

Key Words: atrium • receptors, serotonin₄ • receptors, adrenergic, beta • contractility • cAMP • receptors, serotonergic

	Introduction

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5-Hydroxytryptamine₄ receptors^{1 2} have been identified in isolated human atrium.^{3 4 5 6 7 8} Through 5-HT₄ receptors, serotonin (5-HT) has been shown to cause positive inotropic and lusitropic effects,^{3 4 5 6 7} increases in cAMP levels,^{3 4 5} and activation of cAMP-dependent protein kinase.^{3 4 5} In addition, in isolated human atrial cardiomyocytes, 5-HT₄ receptors have been shown to mediate serotonin-induced myocyte shortening⁹ and, as predicted,^{3 4} a marked serotonin-evoked increase in L-type calcium channel permeability^{10 11} that is protein kinase–dependent.¹⁰

Evidence is accumulating that chronic treatment of patients with ß-blockers selective for ß₁AR causes enhancement of ß₂AR-mediated atrial inotropic^{12 13 14} and chronotropic¹⁵ responses, whereas ß₁AR-mediated inotropic responses^{12 13 14 15} and responses to dibutyryl cAMP¹³ remain unaffected. Chronic treatment with a ß-blocker also appears to decrease atrial responses to carbachol.¹⁴ The ß₂AR-mediated inotropic hyperresponsiveness^{12 13 14} appears to be unrelated to changes in receptor density, because atrial ß₂AR density has been reported to be unchanged by chronic treatment with ß-blockers selective for ß₁AR (ß₁-selective blockers),¹⁶ whereas ß₁AR density has been reported to be increased.¹⁶ Instead, we have proposed^{17 18} that chronic ß₁-selective blocker treatment may facilitate receptor–effector coupling of receptors other than ß₁AR, eg, ß₂AR and 5-HT₄ receptors, that also stimulate adenylyl cyclase via the stimulatory GTP-binding coupling protein (G_s protein). [Such cross talk between receptor populations probably occurs within a single myocardial cell, since recent evidence indicates, for example, that ß₁AR and ß₂AR functionally coexist in the same ventricular myocyte, both mediating positive inotropic and lusitropic effects of (-)-epinephrine in the same ventricular cell.¹⁹ ] We therefore compared serotonin-evoked positive inotropic responses and associated cAMP levels in paced, isolated human atrial tissues and myocytes obtained from ßB or non-ßB patients, chronically treated (ßB) or not treated (non-ßB) with a ß-blocker, either selective or nonselective for ß₁-adrenergic receptors. We also investigated the influence of chronic treatment with an L-type calcium channel blocker (tissues and myocytes) and of patient age, degree of heart failure, and type of cardiac disease (tissues) on atrial inotropic responses to serotonin. To check whether chronic ß-blocker treatment has any effects on the contractile proteins of the cell, we studied tissue inotropic responses to Ca²⁺. In addition, to distinguish between receptor-related and adenylyl cyclase–related phenomena, we studied the effects of chronic ß-blocker treatment on atrial myocyte responses to forskolin, which activates the catalytic unit of adenylyl cyclase independently of G protein–coupled receptors.²⁰

The presence of cross talk between different receptor populations and its modification by chronic ßAR blockade assumes that these populations coexist and function in the same cell. We therefore looked, in a single atrial myocyte, at the effects of serotonin, (-)-isoproterenol, and carbachol mediated through 5-HT₄ receptors, ßARs, and muscarinic receptors, respectively. Our results show that chronic ß-blocker treatment induces human atrial inotropic 5-HT₄ receptor–mediated hyperresponsiveness and enhanced serotonin-evoked increases in tissue cAMP levels, whereas myocyte and tissue responses to forskolin and calcium, respectively, are unchanged. We also show that the inotropic responses of serotonin are unaffected by chronic calcium channel blocker treatment or the patient-dependent variables investigated. In addition, we have demonstrated that ßARs, muscarinic receptors, and 5-HT₄ receptors function in the same atrial myocyte. It is therefore possible that the 5-HT₄ receptor hyperresponsiveness seen in atrial tissues and myocytes obtained from ßB patients could result from a change in the intracellular cross talk between receptor populations induced by chronic ß₁AR blockade.

	Methods

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Atrial Tissues
Patients. Right atrial appendages were obtained from 89 patients (ßB, n=53, age 60±1 years, mean±SEM; non-ßB, n=36, age 63±1 years) undergoing open-heart surgery for coronary artery disease, aortic valve disease, mitral valve disease, or a combination of these. None of the patients had terminal heart failure. Full details of the patient characteristics and medication are given in Tables 1 through 3. The ßB patients had been taking a ß-blocker on a daily basis for more than 2 months, up to and including the day of operation. Most of the patients were taking a ß₁-selective blocker; 11 patients (see Table 2 and patients 16, 20, and 29 of Table 3) were taking a ß-blocker that was not selective for ß₁AR. All medication was administered up to and including the day of operation except for warfarin and aspirin, which were stopped 1 to 6 days before the day of operation. Premedication was with papaveretum and hyoscine. Anesthesia was induced with alfentanil, ketamine, propofol, fentanyl, thiopentone, midazolam, or O₂/N₂O and maintained with fentanyl, ketamine, methohexitone, propofol, or trichloroethylene; atracurium, pancuronium, or vecuronium was used as muscle relaxant.

View this table: [in this window] [in a new window]   Table 1. Patient Details, Drug Treatment, and Inotropic Data for Serotonin Concentration-Effect Curves on Atrial Tissues From non-ßB Patients

View this table: [in this window] [in a new window]   Table 2. Patient Details, Drug Treatment, and Inotropic Data for Serotonin Concentration-Effect Curves on Atrial Tissues From ßB Patients

View this table: [in this window] [in a new window]   Table 3. Patient Details and Drug Treatment for cAMP Determinations and CaCl2 Concentration-Effect Curves in Atrial Tissues

Tissues. After excision, the right atrial appendages were dissected into two to six strips 1 mm thick and set up to contract as previously described.^{3 4} Each atrial strip was cut in such a way as to include endocardium and usually epicardium. Each strip was paced at either 1-second (1-Hz) or 2-second (0.5-Hz) intervals as described.^{3 4}

Concentration–effect curves. The inotropic efficacy and potency of serotonin were estimated from cumulative concentration–effect curves determined at a pacing frequency of 0.5 Hz as described.^{3 4} To exclude the possibility that serotonin might be exerting its inotropic effects through activation of the atrial ß₁AR or ß₂AR by release of norepinephrine, serotonin concentration–effect curves were also determined in the presence of a ß-blocker not selective for ß₁AR, either (-)-pindolol (1 µmol/L) or (±)-propranolol (400 nmol/L), preincubated for at least 45 minutes. To investigate whether tissue capture of serotonin was affected by chronic ß-blocker treatment, tissues from each group of patients (non-ßB and ßB) were exposed to 6 µmol/L cocaine [in the presence of 1 µmol/L (-)-pindolol].³

Only one serotonin concentration–effect curve was determined per atrial strip. On occasion, parallel atrial strips from a patient were used to investigate the effects of serotonin in both the absence and the presence of a ß-blocker or cocaine in the organ bath. The type of experiments carried out using tissue from any one individual is indicated in Tables 1 and 2.

Sensitivity to calcium. To see whether chronic ß-blocker treatment has nonspecific effects rather than a receptor-specific modifying effect on atrial inotropic responses, atrial strips were exposed to graded CaCl₂ concentrations. A cumulative concentration–effect curve to CaCl₂ (0.2 to 11.2 mmol/L) was carried out at a pacing frequency of 1 Hz on atrial strips obtained from non-ßB and ßB patients (see Table 3 for patient details).

cAMP levels. Tissue levels of cAMP were determined in freeze-clamped atrial strips that had been exposed or not exposed for 5 minutes to 1 or 10 µmol/L serotonin^{3 4} (see Table 3 for patient details). Modifications of previous methods^{3 4} consisted of pacing the strips at 1 Hz, exposing all the strips for at least 60 minutes to 300 nmol/L CGP 20712A to block ß₁AR-mediated responses,²¹ and using an enzyme immunoassay kit rather than a radioimmunoassay kit for the determination of the cAMP levels. The protein content was determined by the method of Bradford²² using BSA as the standard.

Atrial Myocytes
Patients. Right atrial appendages were obtained from 37 patients (ßB, n=18, age 60±2 years; non-ßB, n=19, age 60±2 years). Full details of the patient characteristics are given in Table 4.

View this table: [in this window] [in a new window]   Table 4. Patient Details and Drug Treatment for Experiments Using Atrial Myocytes

Preparation of myocytes. Pieces of right atrial appendage were transported in cold cardioplegic solution; average transit time to the laboratory was 10 minutes. Myocytes were isolated from the tissues as described.^{23 24}

Contraction studies. Single myocytes were superfused at 32°C and electrically stimulated with biphasic pulses at 0.5 Hz. Contractile amplitude was monitored with a video length-detection system, as described.^{24 25} Cumulative concentration–effect curves to serotonin, renzapride, or forskolin were constructed. Only myocytes that responded to serotonin, renzapride, or forskolin with graded and reversible increases in contraction amplitude were used (54 myocytes from 37 patients). (-)-Isoproterenol was added at a concentration (0.2 µmol/L) sufficient to stimulate atrial ßARs maximally after maximum responses to serotonin had been reversed by washout. Carbachol, when present, was added at a concentration (1 µmol/L) sufficient to activate atrial muscarinic receptors maximally in the presence of a maximally effective concentration of either serotonin or (-)-isoproterenol.

Data Analysis and Statistics
Atrial Tissues
The magnitude of the positive inotropic response to serotonin at each concentration was calculated as a percentage of the inotropic effect induced by 200 µmol/L (-)-isoproterenol in the same atrial strip and in terms of the maximum developed force (in millinewtons) achieved by each patient's tissue in the presence of serotonin, regardless of the concentration of serotonin eliciting the maximum force.

The potency of serotonin was evaluated by determination of the EC₅₀ value (mol/L) of serotonin (ie, the concentration of serotonin causing a half-maximal response) for each concentration–effect curve by use of a logistic response curve fitted to the serotonin-induced responses for each tissue (by a modified Newton method within the Genstat computer software package). The data were used in the multiple regression analysis (see below). The effects of serotonin on cAMP levels were assessed by paired t test and the Welch test²⁶ for unpaired samples, as appropriate.

Results are given as mean±SEM. The significance of differences between results with inotropic data was assessed by paired or unpaired t test, as appropriate. For all the statistical analyses, results were considered significant at a value of P.05. Values of n indicate the number of patients.

Multiple Regression Analysis
To determine whether certain independent variables might be influencing the magnitude and/or sensitivity of the atrial tissue responses to serotonin, we carried out two separate multivariate regression analyses. These analyses examined the influence of the independent variables on either the absolute magnitude (in millinewtons) of the maximum force developed in the presence of serotonin (ie, the serotonin-induced force plus the basal force, regardless of the concentration of serotonin producing the maximal force) or the EC₅₀ values (in mol/L) of the serotonin concentration–effect curves, respectively. The independent variables entered into the multivariate regression analyses were the basal contractile force of the paced tissue (millinewtons), the total contractile force (millinewtons) achieved in the presence of 200 µmol/L (-)-isoproterenol, the ß-blocker status of the patient (ie, whether or not the patient was treated with a ß-blocker), the calcium channel blocker status of the patient (ie, whether or not the patient was treated with an L-type calcium channel blocker), the ß-blocker status of the experiment (ie, whether or not a ß-blocker had been added to the organ bath), the status of the experiment with respect to cocaine (ie, whether or not cocaine had been added to the organ bath), the demographic data of patient age and degree of heart failure (graded according to the NYHA classification of heart failure), and the nature of the cardiac disease (coronary artery disease, aortic valve disease, mitral valve disease, or some combination of these) suffered by the patient.

Before the regression analyses were carried out, the base 10 logarithm of the maximal force achieved in the presence of serotonin (millinewtons), the basal contractile force (millinewtons), the total force achieved in the presence of 200 µmol/L (-)-isoproterenol (millinewtons), and the EC₅₀ value (mol/L) for serotonin were determined for each atrial strip. Logarithm values were used (1) to linearize the relation between the force achieved in the presence of serotonin and the basal contractile force and force achieved in the presence of (-)-isoproterenol and (2) to stabilize the variation in responses across the whole range of measurements. To explain the variation of the serotonin-evoked inotropic responses, an optimum model was chosen through a stepwise regression procedure. The optimum model derived for the analysis of the variables affecting the responses to serotonin was

where log(5-HT force) is the log of the maximum total force (millinewtons) achieved by the tissue in the presence of serotonin (ie, basal force plus serotonin-evoked response); K is a constant; B_i is the effect of chronic ß-blocker treatment (i=1 or 2, with 1=ßB patient and 2=non-ßB patient); _i is the slope of the log(basal force) relation for ßB versus non-ßB tissue; log(basal force) is the log of the basal contractile force (millinewtons) shown by the tissue before the addition of serotonin; _i is the slope of the log(ISO force) relation for ßB versus non-ßB tissue; log(ISO force) is the log of the total force (millinewtons) achieved by the tissue in the presence of 200 µmol/L (-)-isoproterenol; _ij is the residual error of the jth observation for ßB versus non-ßB treatment; and 0<basal force 5-HT forceISO force.

Atrial Myocytes
Data are expressed as mean±SEM throughout. -Log EC₅₀ values (-log EC₅₀=pD₂) for concentration–effect curves of serotonin were calculated with an iterative curve-fitting program. If more than one cell from an atrial appendage was used, the results were combined so that n values always apply to patients. Statistical comparisons were made with paired and unpaired t tests. Values of P.05 were considered significant.

Drugs and Materials
The following drugs and materials were purchased: Biotrak cAMP enzyme immunoassay kits from Amersham; carbachol, IBMX (3-isobutyl-1-methylxanthine), and forskolin from Sigma Chemical Co; trioctylamine and freon (1,1,2-trichlorotrifluoroethane) from Aldrich; and collagenase for the isolation of atrial myocytes from Boehringer Mannheim. CGP 20712A {1-[2(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluoromethyl 2-imidazolyl)phenoxy]-2-propanol methane sulfonate} was a gift from Dr Maître, CIBA Geigy (Basel, Switzerland). The sources of all other drugs used have previously been reported.^{3 4 24 25 27}

	Results

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Atrial Tissues
Enhanced Inotropic Responsiveness to Serotonin Induced by Chronic ß-Blockade
We have previously shown that serotonin causes a potent positive inotropic effect in tissues obtained from ßB patients.^{3 4} We now demonstrate that serotonin caused positive inotropic effects in tissues obtained from non-ßB patients as well as ßB patients (Fig 1). However, the maximum responses to serotonin of tissues from non-ßB patients were significantly smaller than those of tissues from ßB patients (Fig 1, top; Table 5). Expressed as a percentage of the response to 200 µmol/L (-)-isoproterenol, the maximum inotropic response to serotonin was 19±6% (n=13; range, 0% to 71%) in tissues from non-ßB patients (who had a variety of diseases) compared with 56±3% (n=27; range, 9% to 86%) in tissues from ßB patients (who primarily had coronary artery disease) (P<.001) (see Tables 1 and 2). The same phenomenon was observed when only patients with coronary artery disease were compared. In this case, the maximum inotropic response to serotonin was 33±13% in tissues from non-ßB patients (n=5; range, 0% to 71%) compared with 57±4% in tissues from ßB patients (n=24; range, 9% to 86%) relative to the response elicited by (-)-isoproterenol (P=.002) (see Tables 1 and 2; Fig 1, bottom; and below). The effect of chronic ß-blocker treatment on atrial inotropic responses was also seen when the developed force achieved by the tissues was expressed in terms of millinewtons. The maximum contractile force evoked by serotonin in the atrial tissues was significantly greater for ßB patients than for non-ßB patients (Fig 1), whereas there was no significant difference between the two groups of patients (unpaired t test) in the basal contractile force or the force achieved in the presence of 200 µmol/L (-)-isoproterenol. However, although the efficacy of serotonin was more than twofold greater in ßB tissues than in non-ßB tissues, its potency, as judged by EC₅₀ values, was unchanged by chronic ß-blocker treatment (Figs 1 and 2, Table 6).

View larger version (23K): [in this window] [in a new window]   Figure 1. Graphs showing effect of chronic ß-blocker treatment on the inotropic responsiveness to serotonin of paced human right atrial strips. Non-ßB indicates tissues obtained from non-ßB patients; ßB, tissues obtained from ßB patients. Top, Serotonin concentration–effect curves determined in the absence of any ß-blocker in the organ bath. The non-ßB patients (, n=13) had a variety of diseases (Table 1); the ßB patients (, n=27, of whom 24 received a ß1-selective blocker, see Table 2) primarily had coronary artery disease. Bottom, Comparison of patients who had only coronary artery disease. , n=9 strips from 8 patients; , n=36 strips from 29 patients. Columns show the absolute developed force (mN) achieved by the same strips. Basal indicates developed force before the addition of serotonin; Serotonin, maximal developed force reached in the presence of serotonin, regardless of the concentration of serotonin causing maximal activation; and Isoproterenol, developed force reached after the addition of 200 µmol/L (-)-isoproterenol at the end of the serotonin concentration–effect curve. Statistics within the columns show absolute force developed in the presence of a maximally activating concentration of serotonin or in the presence of (-)-isoproterenol vs the corresponding basal force (paired t test). Statistics between the columns show comparison between tissues from non-ßB and ßB patients of the inotropic force achieved in the presence of a maximally active concentration of serotonin. When no statistics are shown, there was no significant difference between tissues from ßB patients compared with non-ßB patients, ie, either in the basal developed force or in the total developed force achieved after the addition of 200 µmol/L (-)-isoproterenol.

View this table: [in this window] [in a new window]   Table 5. Probability of No Relation of Independent Factors and Demographic Variables With log(5-HT Force)=log of Maximum Force (mN) Achieved by Tissue in Presence of Serotonin, Regardless of Concentration of Serotonin Producing Maximal Activation

View larger version (14K): [in this window] [in a new window]   Figure 2. Scatterplot showing inverse relation for human right atrial tissues between basal contractile force (mN), expressed as log(basal force), and the inotropic potency of serotonin (mol/L), expressed as -log EC50 values. Tissues were obtained from non-ßB (open symbols) and ßB (solid symbols) patients. The enhancing effect of cocaine on the inotropic potency of serotonin is also shown. Each symbol represents data from an individual atrial strip, regardless of whether concentration–effect curves were determined in both the absence and the presence of (-)-pindolol or (±)-propranolol for a particular patient, so that in a few cases more than one data point for a patient is shown. Circles show data from curves determined in the absence of cocaine; squares show data for curves determined in the presence of 6 µmol/L cocaine. Dashed line indicates average values for data obtained in the absence of cocaine; solid line, average values for data obtained in the presence of 6 µmol/L cocaine.

View this table: [in this window] [in a new window]   Table 6. Probability of No Relation of Independent Factors and Demographic Variables With log(EC50) for Serotonin

Lack of Effect of In Vitro ß-Adrenergic Receptor Blockade
Neither (-)-pindolol (6 non-ßB patients, 7 ßB patients) nor (±)-propranolol (5 non-ßB patients, 5 ßB patients) in the organ bath had any effect on the magnitude of the serotonin-evoked inotropic responses of either non-ßB or ßB tissues (Tables 1, 2, and 5), excluding an indirect contribution of atrial ßAR to the action of serotonin. The potency of serotonin was unaffected by the presence of either of the ß-blockers in the organ bath for both groups of tissues (Table 6). There were no significant differences (unpaired t test) between the two groups of tissues in either the basal contractile force or the force achieved in the presence of 200 µmol/L (-)-isoproterenol in the presence of either of the ß-blockers in the organ bath. Since neither (-)-pindolol nor (±)-propranolol had any influence on the inotropic effects of serotonin, we included data obtained in the presence of these ß-blockers for our comparison of the inotropic responses of patients suffering from coronary artery disease only (see above, Fig 1, bottom).

Comparison of Chronic Treatment With ß-Blockers Selective and Not Selective for ß₁AR
Although most of the ßB patients in our sample had been taking a ß₁-selective blocker (see Table 2), some of the patients had been chronically treated with a ß-blocker that is not selective for ß₁AR, ie, that blocks both ß₁AR and ß₂AR. We were interested to know whether chronic blockade of the ß₂AR in addition to blockade of the ß₁AR would give rise to additional or different effects on the inotropic efficacy of serotonin compared with those caused by blockade of the ß₁AR alone. To obtain data from a reasonable number of patients treated with ß-blockers not selective for ß₁AR, we included in the analysis all the serotonin concentration–effect curve data we obtained for these patients in the presence of (-)-pindolol or (±)-propranolol in the organ bath, since we had shown that these agents do not affect the inotropic responses of the tissues to serotonin. The inotropic efficacy and potency of serotonin were unchanged in tissues from patients taking a ß-blocker not selective for ß₁AR compared with tissues from patients taking a ß₁-selective blocker. Expressed as a percentage of the response to 200 µmol/L (-)-isoproterenol, the serotonin-evoked inotropic responses were 61±12% (n=6; range, 9% to 89%) for tissues from patients treated with a ß-blocker not selective for ß₁AR and 56±3% (n=26; range, 16% to 86%) for tissues from patients treated with a ß₁-selective blocker (see Table 2). The potency of serotonin, as shown by -log EC₅₀ values (mol/L), was 6.72±0.16 (n=8) and 6.60 ± 0.12 (n=26) for the two groups of tissues, respectively.

In Vitro Potentiation by Cocaine
Since we had shown that (-)-pindolol has no effect on the serotonin-evoked inotropic responses of atrial tissues, the effects of the neuronal amine uptake blocker cocaine²⁸ on the serotonin-evoked inotropic responses of tissues from non-ßB compared with ßB patients were assessed in the presence of (-)-pindolol so that inotropic effects of any neuronally released norepinephrine (effects potentiated by cocaine) would be blocked. Cocaine (6 µmol/L) had no effect on the maximum inotropic response to serotonin of tissues from either group of patients (non-ßB or ßB) (Fig 3; Tables 1, 2, and 5). Cocaine did, however, cause a significant shift to the left of the serotonin concentration–effect curves in tissues from both ßB and non-ßB patients (compare Figs 1 and 3), enhancing the potency of serotonin (Fig 2, Table 6). It can be seen from Fig 2 that the potentiating effect of cocaine tended to be greater in tissues from ßB patients than in tissues from non-ßB patients.

View larger version (20K): [in this window] [in a new window]   Figure 3. Graphs showing effects of cocaine on the inotropic responsiveness to serotonin of human right atrial tissues obtained from non-ßB (, n=8) and ßB patients (, n=8, of whom 6 had received a ß1-selective blocker; see Table 2). Curves are cumulative concentration–effect curves for serotonin carried out in the presence of 6 µmol/L cocaine and 1 µmol/L (-)-pindolol. Other details as in Fig 1.

Relations Between the Response to Serotonin and Basal Contractile Force, the Response in the Presence of (-)-Isoproterenol, and Patient-Dependent Variables
To exclude the effects of confounding independent variables, such as the age and disease state of the patients (see "Methods"), on the enhancement of the inotropic efficacy of serotonin by chronic ß-blocker treatment and to examine closely whether any of these variables affected the potency of serotonin (ie, EC₅₀ values), multivariate regression analysis was used. In particular, it had become apparent that basal contractile force and the response of the tissues to (-)-isoproterenol showed large variability (Figs 1 and 3), so these variables were included in the analyses.

The probability of there not being a relation between the independent variables and the inotropic efficacy of serotonin, expressed as log(5-HT force), is presented in Table 5. The results of the multivariate regression analysis suggest that the inotropic efficacy of serotonin was not influenced by the age or degree of heart failure of the patients, by the nature of the cardiac disease suffered by the patients, or by chronic treatment of the patients with an L-type calcium channel blocker. The variability seen in the inotropic efficacy of serotonin is largely explained by chronic ß-blocker treatment and by variations in the basal contractile force of the tissues [log(basal force)] and the inherent responsiveness of the tissues as shown by their responses to a ßAR-saturating concentration of (-)-isoproterenol [log(ISO force)] (87.8% of the variability is explained by these factors).

The interactions between these three variables and their contribution to the variability in the inotropic efficacy of serotonin were investigated further by use of the model described by the Equation. This analysis revealed that the interaction between ß-blocker treatment and log(basal force) and between ß-blocker treatment and log(ISO force) contributed a further small (1.4%) but significant (P=.001 and P=.012, respectively) proportion to the variability. Overall, multivariate regression analysis revealed that in the region in which the model described in the Equation is valid, ie, where 0<basal force<5-HT forceISO force, the effect of chronic ß-blocker treatment is to increase log(5-HT force), ie, the inotropic efficacy of serotonin, but that the magnitude of the effect depends on the magnitude of log(basal force) and log(ISO force), ie, on the basal contractile force of the tissue and the maximum achievable force in the presence of maximal ßAR activation. In tissues in which there is a high basal contractile force and a high force in response to (-)-isoproterenol, the effect of chronic ß-blocker treatment on the inotropic efficacy of serotonin is small, whereas in tissues in which both the basal contractile force and the force in response to (-)-isoproterenol are low, the inotropic efficacy of serotonin in tissue from ßB patients is greater than twice that in tissue from non-ßB patients. Predicted and experimental values illustrating this phenomenon are shown in Tables 7 and 8.

The probability of there not being a relation between the independent variables and the potency of serotonin, expressed as -log EC₅₀ values, is presented in Table 6. The multivariate regression analysis suggests that only cocaine and variations in the basal contractile force have any effect on -log EC₅₀ (together, they explain 44.3% of the variability in -log EC₅₀). There was an inverse relation between -log EC₅₀ values and log(basal force) regardless of whether the tissues were from non-ßB or ßB patients (Fig 2). In tissues in which there was a high basal contractile force, the EC₅₀ tended to be reduced (Fig 2).

Responses to Calcium
Tissue sensitivity to Ca²⁺ was unchanged by chronic treatment with ß-blockers. Concentration–effect curves to CaCl₂ were not different for atrial tissues obtained from ßB patients compared with non-ßB patients (Fig 4).

View larger version (16K): [in this window] [in a new window]   Figure 4. Graphs showing lack of effect of chronic ß-blocker treatment on the inotropic responsiveness to CaCl2 of atrial tissues. Curves are cumulative CaCl2 concentration–effect curves. , Non-ßB patients (n=5, age 66±2 years); , ßB patients (n=6, age 59±3 years, 4 of whom were taking a ß1-selective blocker). All the patients had coronary artery disease. Columns show the basal force of the same tissues (mN) in 2.25 mmol/L CaCl2 before the concentration–effect curve to CaCl2 was started and the force achieved (mN) in the presence of a maximally activating concentration of CaCl2 in the same atrial strips. Open columns show data for the non-ßB patients; solid columns, data for the ßB patients.

Increase in Atrial cAMP Levels With Serotonin
Serotonin (1 and 10 µmol/L) increased the cAMP content of atrial strips obtained from non-ßB patients as well as from ßB patients (Fig 5). Serotonin-evoked cAMP levels tended to be higher in tissues from ßB compared with non-ßB patients, becoming significant with 10 µmol/L serotonin (Fig 5).

View larger version (13K): [in this window] [in a new window]   Figure 5. Effects of serotonin (1 and 10 µmol/L) on the cAMP content of human right atrial tissues; comparison between tissues from ßB (open symbols) and non-ßB (filled symbols) patients. Columns show paired basal cAMP levels for atrial strips exposed to 1 (, ) and 10 (, ) µmol/L serotonin. Graph, cAMP levels for atrial strips exposed to 1 and 10 µmol/L serotonin. Both 1 and 10 µmol/L serotonin significantly enhanced the cAMP content of both non-ßB tissues and ßB tissues (statistics on graph [small figures], paired t test comparison with basal levels). The total cAMP content of tissues exposed to serotonin tended to be greater for ßB tissues than for non-ßB tissues (Welch test26 ). This reached significance for 10 µmol/L serotonin (statistics on graph, large figures). Figures in the columns are the number of patients.

Atrial Myocytes
Positive Inotropic Effects of Serotonin
Serotonin caused concentration-dependent increases of contractile amplitude in isolated atrial myocytes (Fig 6), as previously observed.⁹ A maximally effective concentration of serotonin increased contractile amplitude to the same extent as did 0.2 µmol/L (-)-isoproterenol (Figs 7 and 8, Table 9).

View larger version (15K): [in this window] [in a new window]   Figure 6. Representative tracing of a concentration–effect curve to serotonin in a human atrial myocyte (obtained from patient 11, Table 4). An upward deflection indicates cell shortening.

View larger version (21K): [in this window] [in a new window]   Figure 7. Representative tracing of responses to serotonin (1 µmol/L), (-)-isoproterenol (0.2 µmol/L), and (-)-isoproterenol plus carbachol (1 µmol/L) in the same human atrial myocyte (obtained from patient 8, Table 4). The arrows indicate arrhythmic contractions.

View larger version (21K): [in this window] [in a new window]   Figure 8. Bar graph showing contraction amplitude (percent shortening of the cell with each beat) of six human atrial myocytes, each from a different patient (1 ßB), under basal conditions (1 mmol/L Ca2+ only) or in the additional presence of maximally inotropic concentrations of serotonin (0.1 to 1 µmol/L) or (-)-isoproterenol (Iso, 0.2 µmol/L). Carbachol (1 µmol/L) was added at the peak of the response to (-)-isoproterenol.

View this table: [in this window] [in a new window]   Table 9. Characteristics of Human Atrial Myocytes From Non-ßB and ßB Patients

Enhanced Myocyte Inotropic Responsiveness to Serotonin Induced by Chronic ß-Blockade
Myocytes obtained from ßB patients were sensitized to the effects of serotonin (Figs 9 and 10, Table 9). Serotonin was four times more potent as an inotropic stimulant of myocytes from ßB patients than of those from non-ßB patients. One patient, treated with celiprolol, was excluded from the ßB group because celiprolol has ß-adrenergic stimulant effects, and desensitization of ß-adrenergic responses has been reported for celiprolol.^{29 30 31} There was no significant difference in the characteristics of basal contraction or serotonin-stimulated contraction between the two groups of myocytes (Table 9). The majority of patients taking ß-blockers were also on L-type calcium channel blockers, as were some of those not taking ß-blockers. In Fig 10, patients from either group who were receiving calcium channel blocker therapy are indicated by solid symbols. Comparison of the distribution of the -log EC₅₀ values for serotonin between calcium blocker–treated and –untreated patients in either group shows that it is unlikely that the calcium blocker therapy was the cause of the inotropic difference between non-ßB and ßB patients.

View larger version (15K): [in this window] [in a new window]   Figure 9. Graph showing effect of chronic ß-blocker treatment on the inotropic responsiveness to serotonin of human atrial myocytes. Concentration–effect curves for serotonin on atrial myocytes from non–ßB (, n=12) and ßB (, n=12) patients. The -log EC50 values of the curves are significantly different (P<.01).

View larger version (16K): [in this window] [in a new window]   Figure 10. pD2 (-log EC50) values for serotonin concentration–effect curves on human atrial myocytes from non–ßB (, ) and ßB (, ) patients. Open symbols indicate patients not receiving calcium channel blocker therapy; filled symbols show patients receiving calcium channel blocker therapy. The mean±SEM values for all non-ßB or all ßB patients are also shown, with the exception of the patient receiving celiprolol. *P<.01 ßB vs non-ßB patients.

Effects of the Partial Agonist Renzapride
The serotonin-evoked increases in myocyte contractility we observed have been reported to be mediated by 5-HT₄ receptors.⁹ The gastrokinetic drug renzapride has been reported to be a partial agonist at 5-HT₄ receptors of human atrium.^{4 8} Renzapride enhanced contractile amplitude in atrial myocytes obtained from both non-ßB and ßB patients with partial agonist activity relative to serotonin (Fig 11) that tended to be greater in cells obtained from the latter patients (Fig 11). The difference was significant (P<.05) at 300 nmol/L of the compound. The stimulant effects of renzapride were completely reversed by the 5-HT₄ receptor antagonist^{8 9 27} SB 203186, 300 nmol/L (not shown), consistent with an interaction of renzapride with 5-HT₄ receptors.

View larger version (13K): [in this window] [in a new window]   Figure 11. Graph showing effect of chronic ß-blocker treatment on myocyte responses to renzapride. Cumulative renzapride concentration–effect curves for myocytes from 5 non–ßB () and 7 ßB () patients. Results are expressed as a percentage of the maximum (max) effect of serotonin in the same myocyte. *P<.05 ßB vs non-ßB patients.

Effects of Forskolin
The potency and efficacy of forskolin as an inotropic agent were unchanged in myocytes from atria of ßB patients compared with non-ßB patients (Fig 12, Table 9). The supersensitivity of ß₂AR and 5-HT₄ receptors induced by ß-blockers thus appears to be confined to receptor-mediated effects, since responses to forskolin were not significantly affected.

View larger version (14K): [in this window] [in a new window]   Figure 12. Graph showing lack of effect of chronic ß-blocker treatment on myocyte responses to forskolin. Cumulative concentration–effect curves for forskolin on human atrial myocytes from 6 non–ßB () and 5 ßB () patients. The two curves are not significantly different.

Coexistence of Functional 5-HT₄ Receptors, Muscarinic Receptors, and ß-Adrenergic Receptors in the Same Myocyte
To examine the question of the functional coexistence of 5-HT₄ receptors, muscarinic receptors, and ß-adrenergic receptors in the same cell, we challenged different myocytes with different sequential additions of serotonin and/or (-)-isoproterenol and carbachol.

Fig 7 illustrates an experiment in which the atrial myocyte was challenged with (-)-isoproterenol after the washout of inotropically effective serotonin. Carbachol (1 µmol/L) was then added at the height of the inotropic response to (-)-isoproterenol. Similar experiments for myocytes from five non-ßB patients and one ßB patient are summarized in Fig 8. In each case, the presence of carbachol decreased the contraction amplitude to levels seen in the absence of (-)-isoproterenol. Similarly, carbachol added in the presence of serotonin completely abolished its positive inotropic effect (Fig 13). This effect was consistently found in all myocytes studied (Fig 14) and was not different for the maximally activating concentration of carbachol used in non-ßB (n=3) compared with ßB (n=3) patients. Interestingly, when carbachol was washed out and the myocytes were superfused with serotonin once more, the maximum positive inotropic effect of serotonin showed a tendency to be greater than that achieved before the addition of carbachol (Figs 13 and 14). It is possible that this indicates that serotonin induces some rapid desensitization that is reversed by exposure to negative inotropic agents such as carbachol. It was indeed occasionally noted that the positive inotropic effect of serotonin reached a peak for a certain concentration and then fell. This was particularly noticeable for non–maximally effective concentrations of serotonin (Fig 6).

View larger version (16K): [in this window] [in a new window]   Figure 13. Representative tracing of the effect of carbachol in reducing the response to serotonin in a human atrial myocyte (obtained from patient 16, Table 4).

View larger version (32K): [in this window] [in a new window]   Figure 14. Bar graph showing contraction amplitude (percentage shortening of the cell with each beat) of six human atrial myocytes, each from a different patient (3 ßB), with a maximally inotropic concentration of serotonin (0.1 to 1 µmol/L), with carbachol added at the peak of the maximum response to serotonin and after return to serotonin alone, after washout.

These experiments, taken together, clearly demonstrate that the corresponding receptors for serotonin, carbachol, and (-)-isoproterenol coexist and function in the same human atrial cell. In fact, we have not yet encountered an atrial myocyte that lacks functional responses mediated through any one of these receptors.

Serotonin-Induced Arrhythmic Contractions
Serotonin, administered at relatively high concentrations, can elicit arrhythmias in isolated human atria.^{8 27} High concentrations of serotonin (100 to 1000 nmol/L) caused arrhythmic contractions in three myocytes that quickly disappeared after removal of serotonin (Fig 15). The arrhythmic contractions elicited by serotonin had characteristics similar to those caused by (-)-isoproterenol (compare Figs 7 and 15).

View larger version (18K): [in this window] [in a new window]   Figure 15. Tracing showing that arrhythmic contractions (arrows) induced by serotonin in a human atrial myocyte (obtained from patient 1, Table 4) are reversible upon washout.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have demonstrated that isolated atrial tissues and myocytes obtained from patients chronically treated with ß-blockers, usually selective for ß₁AR, exhibit 5-HT₄ receptor hyperresponsiveness compared with those of patients not treated with ß-blockers. The inotropic hyperresponsiveness to serotonin of atrial tissues from ßB patients appeared to be unrelated to the age or degree of heart failure (mild to moderate) of the patients, to the nature of the cardiac disease suffered by the patients, or to chronic treatment with an L-type calcium channel blocker, which was also probably not a factor for the hypersensitivity to serotonin of atrial myocytes obtained from ßB patients. We found that an equilibrium inotropic response to serotonin is accompanied by an elevated level of cAMP in atrial tissues obtained from non-ßB patients as well as from ßB patients and that the serotonin-evoked increase in cAMP levels tends to be greater in tissues from ßB patients, being significant at 10 µmol/L serotonin. In contrast to the 5-HT₄ receptor hyperresponsiveness, the effects of calcium in atrial tissues and of forskolin in atrial myocytes (this report) are unchanged by chronic treatment with ß-blockers, the latter observation being in line with unchanged effects of dibutyryl cAMP found previously in atrial tissues.¹³ These findings make it unlikely that chronic ß₁-selective blocker treatment induces perturbed responses of the contractile proteins to calcium or changes in the biochemical cascade downstream of the catalytic unit of the adenylyl cyclase, at least in the case of ß₂AR and 5-HT₄ receptors. Our findings that serotonin, (-)-isoproterenol, and carbachol can affect the function of the same myocyte allow for the possibility of cross talk between 5-HT₄ receptors and ß₁AR, as well as muscarinic receptors, within the same cell. We suggest that the 5-HT₄ receptor hyperresponsiveness is the result of a modification of cross talk between ß₁AR and 5-HT₄ receptors caused by chronic ß₁AR blockade.

Although the ß-blocker–induced 5-HT₄ receptor hyperresponsiveness we observed was quite marked, multivariate regression analysis revealed that the degree of hyperresponsiveness to serotonin in tissues from ßB (compared with non-ßB) patients was more pronounced at weak basal forces and correlated with the magnitude of (-)-isoproterenol–induced force (Tables 7 and 8). We interpret this pattern by assuming that when basal force is high, presumably due to high intramyocyte Ca²⁺ levels, further increases in Ca²⁺ levels caused by receptor stimulation saturate the cascade leading to increased contractile force. The resultant ceiling in contractile force reduces visualization of serotonin hyperresponsiveness.

View this table: [in this window] [in a new window]   Table 7. Predicted Serotonin Force Levels (From the Optimum Model of the Equation) for a Range of Basal Contractile Forces and Forces Achieved in the Presence of 200 µmol/L (-)-Isoproterenol in Tissues From non-ßB and ßB Patients

View this table: [in this window] [in a new window]   Table 8. Geometric Means of Experimental Serotonin Force Levels Calculated for a Range of Basal Contractile Forces and Forces Achieved in the Presence of 200 µmol/L (-)-Isoproterenol in Tissues From Non-ßB and ßB Patients

To account for the hyperresponsiveness to serotonin of atrial tissues obtained from ßB patients, we considered the possibility that serotonin may exert its effects indirectly through release of norepinephrine, thereby activating atrial ßAR. However, the ßAR antagonists (-)-pindolol and (±)-propranolol, at concentrations that block both ß₁AR and ß₂AR on human atrium,^{32 33} did not prevent the increase in inotropic efficacy of serotonin caused by chronic ß-blocker treatment. In addition, because the hyperresponsiveness to serotonin was also observed in myocytes studied in the absence of nerves, we rule out an indirect effect of this nature.

Another explanation for the hyperresponsiveness of tissues to serotonin that we considered and rejected is the possibility that neuronal uptake of serotonin²⁸ is more pronounced in atria from non-ßB patients than in atria from ßB patients. If this were the case, a greater potentiation of the responses to serotonin by cocaine would be expected in non-ßB tissues than in tissues from ßB patients, but in fact the opposite was observed (Fig 2). Even in the presence of cocaine, however, the inotropic efficacy of serotonin was greater in tissues from ßB patients than in tissues from non-ßB patients (Fig 3). Furthermore, our observation that 5-HT₄ receptor hyperresponsiveness also occurs in isolated myocytes precludes the involvement of the neuronal uptake of serotonin and/or the release of neuronal or extraneuronal transmitters in this phenomenon.

Because inotropic responses to serotonin are enhanced in tissues and myocytes obtained from ßB patients compared with those from non-ßB patients, it seemed plausible that serotonin would induce larger increases in cAMP levels in tissues from ßB patients than from non-ßB patients, which was indeed seen with both 1 and 10 µmol/L serotonin, becoming significant at the latter concentration. The enhanced serotonin-induced cAMP levels we have found in tissues from ßB patients could arise from an increased density of 5-HT₄ receptors and/or enhanced coupling of 5-HT₄ receptors to G_s protein and adenylyl cyclase and perhaps ion channels.

Although most of our patients were treated with a ß₁-selective blocker, a few were treated with a ß-blocker not selective for ß₁AR, ie, one that blocks ß₂AR as well as ß₁AR. There appeared to be no difference in the inotropic responsiveness to serotonin of tissues obtained from patients treated with ß-blockers not selective for ß₁AR compared with the responsiveness of tissues obtained from patients treated with ß₁-selective blockers. Similarly, there was no difference in the potency of serotonin between these two groups of tissues. We interpret these results as being a manifestation of the same phenomenon, ß₁AR blockade. The ß₁AR-blocking component of the ß-blockers not selective for ß₁AR would cause the same attenuation of tonic activation of the cardiac ß₁AR by neuronally released norepinephrine as the ß₁-selective blockers, and there would be no additional manifestation of ß₂AR blockade. This is perhaps not surprising, since it is unlikely that there is usually tonic in vivo activation of the ß₂AR.

We recently described serotonin-evoked inotropic responses, mediated through 5-HT₄ receptors, in left atrial tissue obtained from non-ßB patients with terminal heart failure.⁵ Interestingly, when expressed as a percentage of the response to 200 µmol/L (-)-isoproterenol, the maximum response to serotonin of this left atrial tissue was similar to that reported here for right atrial tissue obtained from non-ßB patients without terminal heart failure (left atrium, 24±5%, n=5⁵ ; right atrium, 19±6%, n=13, this study). Thus, it is plausible that the 5-HT₄ receptor hyperresponsiveness we observed in tissues derived from the right atrial appendages of patients receiving chronic ß-blocker treatment is generally applicable across the whole of both atria. It is also possible that in vivo left and right atrial 5-HT₄ receptors could be activated by platelet-derived serotonin, which could conceivably give rise to serotonin-induced arrhythmias,^{8 27} with a higher incidence in patients chronically treated with ß-blockers. This suggestion is supported by the ability of serotonin to elicit atrial arrhythmic contractions in vitro and the finding that the incidence of the arrhythmias is higher in atria obtained from ßB patients than in atria obtained from non-ßB patients.^{8 27} In the present study, we provide evidence that serotonin can elicit arrhythmic contractions in a single human atrial myocyte (Fig 15).

We conclude that chronic treatment of patients with a ß-blocker, selective or not selective for ß₁AR, causes 5-HT₄ receptor inotropic hyperresponsiveness in isolated human right atrium and myocytes associated with an enhanced ability of serotonin to increase atrial cAMP levels. 5-HT₄ receptors, ßARs, and muscarinic receptors cofunction in the same atrial myocyte, raising the possibility of chronic cross talk between different receptor populations within a single cell. Chronic blockade of ß₁AR could therefore enhance 5-HT₄ receptor responsiveness by modifying the cross talk between the receptor populations, perhaps by improving the coupling of the 5-HT₄ receptors to adenylyl cyclase and/or ionic channels.

	Selected Abbreviations and Acronyms

 

ß1AR = ß1-adrenergic receptors ßB = patients chronically treated with a ß-blocker ß2AR = ß2-adrenergic receptor ßAR = ß-adrenergic receptor 5-HT = 5-hydroxytryptamine (serotonin) non-ßB = patients not treated with a ß-blocker NYHA = New York Heart Association

	Acknowledgments

 
We wish to thank the surgeons of Papworth and the Royal Brompton Hospitals for the supply of atrial tissues. We also thank Dr J.A. Hall for determining the NYHA degree of heart failure status for some patients. We thank Crispin Davies and Kerry Davia for preparing cells.

Received May 24, 1994; revision received April 20, 1995; accepted June 12, 1995.

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