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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2003 Feb 5;138(3):521–529. doi: 10.1038/sj.bjp.0705065

The preferential β3-adrenoceptor agonist BRL 37344 increases force via β1-/β2-adrenoceptors and induces endothelial nitric oxide synthase via β3-adrenoceptors in human atrial myocardium

C Pott 1, K Brixius 1, A Bundkirchen 1, B Bölck 1, W Bloch 2, D Steinritz 2, U Mehlhorn 3, R H G Schwinger 1,*
PMCID: PMC1573689  PMID: 12569077

Abstract

  1. The present study investigated the effects of the preferential β3-AR agonist BRL 37344 (BRL) on force of contraction (FOC), Ca2+-transient and eNOS-activity in human right atrial myocardium.

  2. BRL concentration-dependently caused an increase in FOC that was paralleled by an increase in Ca2+-transient and a shortening of time to half peak relaxation (T0.5T). These effects were abolished in the presence of propranolol (0.3 μM).

  3. BRL acted as a competitive antagonist towards isoprenaline and in binding experiments it was shown to have a distinct affinity towards β1/2-AR.

  4. In immunohistochemical experiments BRL (10 μM) increased detection of activated eNOS. This effect remained constant in the presence of propranolol (0.3 μM).

  5. BRL increased directly detected NO in DAF-staining experiments. This increase was significantly smaller in the presence of the NO-inhibitor L-NAME.

  6. The inotropic effects of BRL were not changed in the presence of L-NMA.

  7. These results suggest that the inotropic effects of BRL in human atrium are mediated via β1/2-AR, whereas the increase of atrial eNOS-activity is due to β3- adrenergic stimulation. This increase in eNOS-activity did not influence atrial myocardial contractility. In conclusion, this study shows that β3-adrenergic stimulation is present in human atrium, but may not be functionally as significant as in the left ventricle.

Keywords: BRL 37344, β3-adrenoceptor, β3-adrenoceptor agonists, β3-selectivity, DAF, human atrial myocardium, eNOS, NO, L-NMA, L-NAME

Introduction

BRL 37344 (BRL, Figure 1), a preferential β3-AR agonist (Arch et al., 1984; Arch & Wilson, 1996; Balligand et al., 2000), has widely been used to characterize the human and animal β3-adrenergic system. BRL was first referred to in 1984 when it was described to mediate thermogenesis in brown adipocytes via the β3-AR (Arch et al., 1984). Since then it has been described to mediate lipolysis in human and animal adipose tissue (Zaagsma, 1990; Langin et al., 1991; Lowell & Flier, 1995; Tavernier et al., 1996), and it has furthermore been shown to cause relaxation of vasculature (Berlan et al., 1994; Shen et al., 1994) and intestinal smooth muscle (Bond & Clarke, 1988; Manara & Bianchetti, 1990; Koike et al., 1994; Pietri-Rouxel & Strosberg, 1995) via the β3-AR.

Figure 1.

Figure 1

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Chemical structure of the preferential β3-agonist BRL 37344 ((RR+SS)- (±)-4-[2-(2-(3-chlorophenyl)-2-hydroxyethyl)amino)propyl]phenoxyacetate).

Only recently the protein expression of β3-AR was shown in human cardiac tissue (Moniotte et al., 2000). In human left ventricular myocardium BRL and other β3-AR agonists have been described to decrease FOC (force of contraction) via the β3-AR (Gauthier et al., 1996; 1999; Moniotte et al., 2000). The observation, that these effects were blunted in the presence of NO-antagonists, led to the suggestion of an NO dependent pathway mediating β3-adrenergic negative inotropic effects in human myocardium (Gauthier et al., 1998).

Both functional and binding studies have shown that the affinity of standard of β1- and β2-AR antagonists such as propranolol towards the β3-AR is 100–1000 fold lower than towards β1- and β2-AR (Arch & Kaumann, 1993). Accordingly in human left ventricular myocardium (Gauthier et al., 1996) as well as in vascular tissue (Trochu et al., 1999) and intestinal smooth muscle (Roberts et al., 1997; Sennit et al., 1998) β3-adrenergic stimulation has been described to remain unchanged in the presence of β1- and β2-AR antagonists. Arch & Kaumann (1993) even established this aspect as an obligatory criterion to define β3-adrenergic stimulation.

Although Chamberlain et al. (1999) were able to detect β3-AR in human right atrium, functional results in human atrium remain quite unclear. In contrast to left ventricular myocardium, specific β3-AR agonists, among them BRL, produce positive inotropic effects in isolated right atrium (Arch & Kaumann, 1993; Sennit et al., 1998), as well as an increase of sinual heart rate during in vivo experiments (Wheeldon et al., 1994). Consistently a baroreflex mechanism was suggested to be responsible for the in vivo increase of heart rate, due to β3-adrenergic mediated vasorelaxation (Tavernier et al., 1992), but this, however, does not explain the increase of inotropy during in vitro experiments with isolated atrial myocardial tissue.

In addition, the increase of FOC in human atrial myocardium is prevented in the presence of β1- and β2-antagonists, such as propranolol (Kaumann & Sanders, quoted in Arch & Kaumann, 1993), however it is unknown by which receptor system these effects are mediated. Furthermore it is unclear whether BRL causes β3-adrenergic ‘hidden' effects on the enzymatic level that do not directly influence FOC and are therefore not detectable by functional methods.

It is therefore unclear whether β3-adrenergic stimulation is existent in human atrial myocardium, by which intracellular messenger it is mediated and which influence it may have on atrial myocardial function.

The present study therefore investigates the inotropic effects of BRL in human right atrium by simultaneous measurements of intracellular Ca2+-transient and FOC. Furthermore, it characterizes the affinity of BRL towards classical AR by binding experiments in human right atrium and investigates BRL induced activation of eNOS.

Methods

Tissue Collection

Tissue from patients was taken during sternotomy from 45 individuals (33 male, 12 female; age 68.9±1.2 years) with either coronary heart disease (n=32) or valvular disease (n=13). Medical treatment consisted of diuretics, nitrates, ACE inhibitors, cardiac glycosides and β-adrenoceptor antagonists. Drugs used for general anaesthesia were flunitrazepam, fentanyl and pancuronium bromide with propofol. Immediately after excision the tissue was placed in ice-cold cardioplegic solution containing (in mmol l−1) NaCl 15, KCl 10, MgCl2 4, histidine HCl 180, tryptophane 2, mannitol 30 and potassium dihydrogen oxoglutarate 1, and was delivered to the laboratory within 15 min. The study was approved by the local ethics committee.

Electrically stimulated human right atrial trabeculae

During preparation trabeculae of less than 0.5 mm thickness and 3–6 mm in length, with the muscle fibres running approximately parallel to the length of the trabeculae, were isolated from right atrial tissue. Force of contraction (FOC) and time to half peak relaxation (T0.5T) were measured as described previously (Schwinger et al., 1990b; 1992).

Concentration-response curves for BRL (0.01–0.1 mmol) or isoprenaline (1.0–0.01 mmol) were determined by adding the drugs cumulatively to the organ bath after apparent equilibration of the effects of the previous concentration. When dose response curves with BRL were performed in presence of NO-inhibition, the NO-antagonist L-NMA was added to the organ bath 90 min before experiments were conducted.

To investigate the effects of BRL in presence of propranolol, propranolol was applied to the organ bath at least 45 min before a single dose of BRL (10 μM) was added.

Ca2+- and force measurements

Intracellular Ca2+-transient was measured via fura-2 loading in isolated, electrically driven right atrial trabeculae. Experiments included parallel measurements of FOC and were performed as described previously (Brixius et al., 1997).

Membrane preparation and binding experiments

Preparation of right atrial membranes and detection of radioactivity were performed as described before (Schwinger et al., 1991; Brixius et al., 2001).

To investigate the affinity of BRL towards atrial β-adrenoceptors, the assays were incubated with BRL in rising concentrations (10 fM–1 mM). 3H-CGP 12177 (0.6 nm; specific activity 50 Ci mmol−1), a β1/2/3-AR radiolabelling ligand was then given to the membrane preparations for 90 min at 37°C. These conditions allowed complete equilibration of the receptors with the radioligand. The reaction was terminated by rapid vacuum filtration, and then radioactivity was determined. Thus, the displacement of 3H-CGP 12177 from β1/2/3-AR by rising concentrations of BRL could be documented. Nonspecific binding of 3H-CGP 12177 was measured in the presence of propranolol (10 μmol l−1). All experiments were performed in triplicate.

Immunohistochemistry

Tissue pretreatment

Investigation of β3-AR mediated changes in eNOS-activity by use of an eNOS-antibody, which only detected the activated eNOS (Bloch et al., 2001), made it necessary to conduct preincubation procedures with freshly obtained tissue. Therefore, two pieces of myocardial tissue, obtained from one patient were suspended in separated organ baths for at least 45 min. Then, one of the pieces was taken from the bath and was immediately placed in 4% paraformaldehyde, whereas the remaining piece of myocardium was incubated with BRL (10 μM) within the organ bath for another 5–10 min and then also fixed in 4% paraformaldehyde. When experiments were performed in the presence of β1/2-AR inhibition, propranolol (0.3 μM) was added to the organ bath at least 45 min before experiments were conducted.

Fixation procedures

The pretreated tissue was kept in 4% paraformaldehyde for 4 h and then rinsed in 0.1 M phosphate-buffered saline (PBS) for 24 h. Tissues were then stored for 12 h in PBS solution with 18% sucrose for cryoprotection and frozen at −80°C.

Immunocytochemistry

Prior to immunohistochemical examination 20 μM slices from pretreated human tissue were placed in a bathing solution of 3% H2O2 and 60% methanol PBS for 30 min, then permeabilized with 0.2% TritonX-100 in 0.1 M PBS. Thereafter, specimens were treated with 5% normal goat serum (NGS) and 5% bovine serum (BSA) solution in PBS. Prior to each step the sections were rinsed in PBS buffer three times. Incubation with primary antibody was performed in a PBS-based solution of 0.8% BSA and 20 mM NaN3 for 12 h at 4°C. The polyclonal rabbit anti-eNOS antibody (Biomol) was applied at a dilution of 1 : 1500. After rinsing with PBS the sections were incubated with the corresponding secondary biotinylated goat anti-rabbit antibody for 1 h at room temperature. A streptavidin-horseradish peroxidase complex was then applied as a detection system (1 : 100 dilution) for 1 h. Finally, staining was developed for 3–5 min with 3,3-diaminobenzidine tetrahydrochloride (DAB) in 0.05 M TRIS-HCl buffer and 0.1% H2O2. Negative control sections were incubated without the primary antibody.

Semi-quantitative analysis

For semi-quantitative analysis of the human cardiac tissue, all slices were incubated and stored under identical conditions. An individual, semi-quantitative score was used to differentiate between negative (−), slightly positive (+), and clearly positive (++). All specimens were judged by two independent investigators in a double blinded fashion. Data were only accepted as altered if both investigators agreed upon the score.

TV-densitometry

For intensity analysis of immunostaining in cardiomyocytes we measured the grey values of 30 cardiomyocytes from three randomly selected areas of each slice. The intensity of immunostaining was reported as the mean of measured cardiomyocyte grey value minus background grey value. The background grey value was measured at a cell free area of the slice. For staining intensity detection a Zeiss Axiophot microscope coupled to a 3-chip CCD-camera was used and the analysis was performed using the Optimas 6.01 image analysis program.

DAF-Fluorometry

Diaminofluorescein (DAF-FM) is converted via an NO-specific mechanism to an intensely fluorescent triazole derivative (Kojima et al., 1998; Itoh et al., 2000). We used DAF-FM DA to detect changes of the NO level induced by BRL. Therefore right atrial myocardial tissue was collected as described above. It was then shock frozen and stored at−80°C. For experiments the tissue was equilibrated at −20°C for at least 1 h and sliced to 25 μM thickness. Slices were fixed to a plastic scale which had been coated with 15% gelatine. Incubation medium was added immediately containing CaCl2, MgCl2, KCl, NaCl, NaH2PO4, Glucose, NaHCO3 and L-Arginine 1 mM.

For measurements DAF (10 μM), BRL (10 μM), NONOate (10 μM) and L-NAME (100 μM) were added for the relevant experiment. In the following intensity of DAF-FM fluorescence was measured each 10 s for 10 min. The intensity of DAF-FM in absence of any of the agents listed above was set 100% as reference value for each timepoint. The effects of BRL and of BRL+L-NAME on the NO-level were then investigated in relation to the DAF-FM fluorescence intensity. For negative and positive controls we repeated experiments with L-NAME or the NO donor NONOate instead of BRL.

Materials

The β3-AR agonist BRL 37344 ((RR+SS)-(±)-4-[2-(2-(3-chlorophenyl) -2 -hydroxyethyl)amino)propyl]phenoxyacetate) was obtained by Tocris (Bristol, U.K.). Further substances used were isoprenaline and propranolol (Sigma, St. Louis, U.S.A.), the radioligand 3H-CGP 12177 (Amersham, Braunschweig, Germany), forskolin (Sigma-Aldrich, St. Louis, U.S.A.), the NO-donor NONOate (Alexis), DAF (Alexis) and the NO inhibitor N-Nitro-L-Arginine (L-NMA) (Buchs, Switzerland). Rabbit anti-eNOS antibody against the bovine eNOS peptide (599-613) plus additional C-terminal Cys conjugated to KLH (PYNSSPREQHKSYKC) was obtained from Biomol (Hamburg, Germany), the secondary biotinylated goat anti-rabbit antibody was ordered from Vector Laboratories (Burlingame, CA, U.S.A.).

BSA and normal goat serum as well as chemicals required for staining with the avidin-biotin-peroxidase complex were purchased from Sigma (Deisenhofen, Germany). All other chemicals were of analytical grade or the best grade commercially available. For studies with isolated myocardium and trabeculae, stock solutions were prepared and added to the organ bath. All compounds were dissolved in twice distilled water and did not change the pH of the medium.

Statistical analysis

All data are presented as mean±s.e.mean. Data analysis was performed using Student's t-test for paired and unpaired data, where appropriate. Significance was considered at a P value <0.05. Data obtained via DAF-fluorometry was analysed using Mann–Whitney-test with statistical significance considered at P<0.05.

Results

Effects of BRL on force of contraction and on intracellular calcium-transient

The present study investigates the inotropic effects of the preferential β3-AR agonist BRL 37344 (BRL) in human atrial myocardium (Figure 2). Figure 2a shows original tracings of force of contraction under cumulative application of BRL (0.01 μM–100 μM). Figure 2b summarizes the results. BRL increased FOC concentration-dependently (0 μM BRL: 13.6±2.28 mN/mm2; +BRL (100 μM): 20.8±3.24 mN/mm2; +54.6±16.1%; P<0.01; n=19) compared to control (n=6). This positive inotropic effect was associated with an abbreviation of time to half peak relaxation (0 μM BRL: 193.7±5.24 ms; +BRL (100 μM): 173.4±5.66 ms; −10.22±2.33%; P<0.01; n=19; Figure 3) compared to control (n=6).

Figure 2.

Figure 2

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Effects of BRL 37344 (BRL) on the twitch tension of human right atrial trabeculae. Dose-response curve and controls for the effects of BRL on peak tension. (a) Original tracings of the effects of BRL in human right atrial trabeculae. Figure 2a is representative for experiments shown in Figure 2b. (b) Values are the means±s.e.mean of 19 experiments for BRL and six for controls. The response is expressed as absolute values of peak tension in mN per mm2 of trabecula diameter. *=Significant statistical difference (P<0.01) from basal peak tension.

Figure 3.

Figure 3

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Effects of BRL on time to half peak relaxation (T0.5T) in human right atrial trabeculae. Values are the means±s.e.mean of 19 experiments for BRL and six for controls. The response is expressed as absolute values of T0.5T in ms. *: P<0.01 vs basal T0.5T.

Figure 4 gives representative original tracings of force of contraction and Ca2+-transient under basal conditions and after 5 min of incubation with BRL. Growth of contractile force induced by BRL (0 μM BRL: 11.25±3.75 mN/mm2; +BRL (10 μM): 13.85±3.65 mN/mm2; +26.3±9.7%) was accompanied by an increase of intracellular Ca2+-transient (+86.0±32.2%) in right atrial trabeculae.

Figure 4.

Figure 4

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Ca2+ -transient and force of contraction. Ca2+-transient was measured by fura-2 fluorescence. Original tracings of basal contraction and Ca2+-transient (above) and after treatment with BRL (below).

Effects of BRL in the presence of propranolol

To investigate whether the positive inotropic effect of BRL is due to β1/2-adrenergic stimulation, experiments were performed in the presence of propranolol. After application of propranolol, FOC declined in right atrial trabeculae (0 μM propranolol: 17.6±3.59 mN/mm2; +propranolol (0.3 μM): 13.71±2.98 mN/mm2; −22.3±4.37%; P<0.05; n=7). Incubation with propranolol continued (at least 45 min) until a steady state in force of contraction was reached. The following application of BRL (10 μM) did not change isometric force of contraction (0 μM BRL: 13.71±2.98 mN/mm2; +BRL (10 μM): 13.77±2.86 mN/mm2; +1.02±1.28%; P>0.05; n=7). Trabeculae obtained from the same patients showed a distinct increase in FOC, when not pretreated with propranolol (0 μM BRL: 9.07±3.39 mN/mm2; +BRL (10 μM): 17.47±6.56 mN/mm2; +96.7±14.42%; P<0.05; n=6). Figure 5 shows original tracings of BRL induced effects on FOC in the absence and presence of propranolol. These results indicate that the positive inotropic effect of BRL measured in right auricular trabeculae may be mediated via β1- and β2-AR.

Figure 5.

Figure 5

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Effects of BRL on the twitch tension in presence of propranolol. Original tracings on the effects of BRL (10 μM) on right atrial trabeculum in the absence (above) and in the presence of propranolol (0.3 μM; below). This figure is representative for six experiments in the absence and seven in the presence of propranolol.

Influence of BRL on isoprenaline induced inotropic effects

To investigate whether BRL acts as a competitive antagonist towards isoprenaline, concentration response curves of isoprenaline (isoprenaline: 10−10 –10−5 M) were measured in isolated, electrically stimulated right atrial trabeculae in the absence (n=11) and in the presence (n=11) of BRL (10 μM). In presence of BRL the maximum isoprenaline-induced increase in force of contraction was slightly decreased (isoprenaline: (10−5 M): +11.16±2.35 mN/mm2; isoprenaline (10−5M)+BRL (10−5M): +8.58±1.78 mN/mm2; P=0.132; n=11; Figure 6). Yet, in the presence of BRL, the concentration of isoprenaline, needed to achieve a 50% increase of the maximum isoprenaline-induced positive inotropic effect (EC50 isoprenaline) was significantly shifted to the right (EC50 isoprenaline: control : 28.4±8.2 nM, +BRL (10 μM): 144.7±53.6 nM; P<0.05; n=11).

Figure 6.

Figure 6

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Dose-response-curves for the positive inotropic effects of isoprenaline in non-pretreated trabeculae (11 experiments; control) and trabeculae pretreated with BRL (10 μM; 11 experiments). Values are the means±s.e.mean and are expressed in percentage of basal peak tension.

Affinity of BRL towards β1- and β2-AR

To study possible affinities of the preferential β3-adrenergic agonist BRL towards classical AR, competition experiments to the β1/2/3-AR ligand 3H-CGP 12.177 were performed in human right atrial membrane preparations (three experiments; triple measurements each). Membranes were incubated with cumulative concentrations of BRL (10 fM–1 mM) and 3H-CGP 12.177 (0.6 nM) was added. Figure 7 shows the competition curve obtained with BRL in percentage of basal specific 3H-CGP 12.177 binding±s.e.mean. In concentrations from 1 μM–1 mM BRL was able to displace 3H-CGP 12.177 from 96.3% of all labelled β-AR (i.e. β1/2- and β3-AR). Since only a small fraction of all labelled β-AR can account for β3-AR we have to assume that BRL largely displaced the radioligand from β1/2-AR indicating a distinct affinity towards β1- and β2-adrenoceptors.

Figure 7.

Figure 7

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Binding: Displacement of 3H-CGP 12.177 by BRL 37344. Values are the means±s.e.mean of three experiments (triple measurements each). Atrial membrane preparations were saturated with increasing concentrations of BRL and incubated with the β1-, β2- and β3-radioligand 3H-CGP 12·177. Note that displacement of 3HCGP 12.177 takes place within the same range of concentrations in which significant change in force of contraction is caused (Figure 2).

Activation of the endothelial NO-synthase (eNOS)

There is evidence that cardiac effects of the β3-adrenoceptor are mediated by activation of the endothelial NO-Synthase (Gauthier et al., 1998; Moniotte et al., 2000). To investigate whether the preferential β3-adrenoceptor agonist BRL induces an activation of the eNOS, that may be functionally overruled by its agonism at the β1-/β2-adrenoceptors in human right atrium, immunohistochemical stainings of activated eNOS were performed in isolated right atrial trabeculae in the absence and presence of BRL (10 μM), using an antibody which only detect eNOS after activation of the enzyme (Bloch et al., 2001). Figure 8 presents pictures taken from original immunostainings. We observed a distinct cytosolic increase in eNOS immunoreaction in myocardial tissue preincubated with BRL (Figure 8b) compared to non pretreated tissue (Figure 8a). Besides atrial cardiomyocytes, atrial vascular tissue also showed an increase in eNOS, when treated with BRL (data not shown). This is consistent with reports on β3-adrenergic vasorelaxation and increase of cGMP (Trochu et al., 1999) and can in this context be evaluated as a further control for a factual stimulation via β3-AR.

Figure 8.

Figure 8

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Influence of BRL on intensity of immunostaining for eNOS proteins in human right atrial myocardium. Cytosolic staining is observed in myocytes under basal conditions (a) and is increased after 5 min of incubation with BRL (b). Experiments were repeated in the presence of propranolol (0.3 μM). Staining again increased from basal (c) to pretreatment with BRL (10 μM) (d). Incubation with forskolin (0.3 μM; 5 min; (f)) did not increase detection of activated eNOS compared to basal level (e). Pictures are representative for 11 experiments with BRL (five in absence and six in presence of propranolol) and three experiments with forskolin.

To investigate whether these effects were indeed mediated by β3-AR and not by β1/2-AR, we repeated experiments in presence of propranolol (0.3 μM). Increase of eNOS staining detection was unchanged in presence of propranolol (see Figure 8c (basal) and d (+BRL)).

Positive inotropic substances have also been observed to stimulate eNOS (Tsukahara et al., 1994). To exclude that the observed activation of eNOS is due to Ca2+-increase or myocardial share-stress induced by the β1/2-adrenergic component of BRL, we performed stainings of activated eNOS in atrial trabeculae in the absence (see Figure 8e) and presence (see Figure 8f) of forskolin (0.3 μM), a potent positive inotrope. In contrast to BRL forskolin did not cause an increase of eNOS.

Microscopic slides were evaluated via TV densitometry and increase of staining intensity was documented in per cent of basal densitometric units for non-pretreated tissue (+BRL (10 μM); +53.52±15.21%; P<0.05; n=5) and tissue preincubated with propranolol (+BRL (10 μM); +75.08±17.6%; P<0.05; n=6).

The evaluation of atrial tissue in the presence and absence of forskolin was performed equally (+forskolin (0.33 μM); −20.2±12.3%; P>0.05; n=3).

Effects of BRL on NO-release

We measured NO-detection in right atrial myocardium via DAF-staining method. Figure 9 shows fluorescence images taken after reaction time of 150 μM) that are representative for these experiments.

Figure 9.

Figure 9

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Bioimaging of BRL induced changes in NO release in right atrial myocardium via DAF fluorometry. (a–b): Controls: NO-detection via fluorescence intensity basal (a) and after 150 s of reaction time (b) in absence of BRL. (c–d): NO-Detection in presence of BRL (10 μM) basal (c) and after 150 s (d). (e–f): NO-Detection in presence of BRL (10 μM) and L-NMA basal (e) and after 150 s (f).

Change in NO release in control time courses in absence of BRL was taken as reference value (Figure 9a and b; reaction time 300 s: 100%±0.00; n=8). Compared to controls NO-detection significantly increased in presence of 10 μM BRL (Figure 9c and d; reaction time 300 s: 216±36%; n=7). This BRL induced increase in NO-release was significantly diminished in the presence of the NO-antagonist L-NAME (100 μM; Figure 9e and f; reaction time 300 s: 121±20%; n=7).

To demonstrate the reliability of these findings we performed DAF control curves without BRL in the presence of L-NAME (100 μM) and in the presence of the NO-donor NONOate (10 μM). As expected NO-release was decreased in the presence of L-NAME (89±8%; n=4) and distinctly increased by NONOate (218±59%; n=3).

BRL in presence of NO inhibition

The dose response curves with BRL were now repeated in the presence of L-NMA, a potent inhibitor of the NO synthesis. Figure 10 shows mean values in percentage of maximum force±s.e.mean. Maximum increase in FOC induced by BRL in presence of L-NMA was not changed (0 μM BRL: 10.30±1.91 mN/mm2; +BRL (100 μM): 14.9±2.58 mN/mm2; +53.43±32.95%; n=4) compared to maximum increase in FOC in absence of L-NMA (0 μM BRL: 13.84±2.40 mN/mm2; +BRL (100 μM): 20.14±2.95 mN/mm2; +55.80±16.99%; n=18).

Figure 10.

Figure 10

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Effects of BRL on trabeculae preincubated with the NO-inhibitor L-NMA (0.1 mM; four experiments). Controls are non-pretreated trabeculae (18 experiments). Dose-response-curves for BRL in the presence and in the absence of L-NMA are shown in percent of basal peak tension.

Discussion and conclusions

The present study investigated the cardiac effects of the preferential β3-adrenoceptor agonist BRL 37344 in human right atrial myocardium. It was shown that BRL induces a positive inotropic effect in human atrial myocardium via stimulation of the β1- and β2-adrenoceptors. Nevertheless, BRL-application was paralleled by an increase in eNOS-activity, that remained unchanged in the presence of propranolol, a β1-/β2-antagonist. Thus, BRL induces an increase in eNOS activity, that is most likely due to β3-adrenergic stimulation. Atrial functional effects due to β3-adrenergic stimulation of eNOS through BRL could not be detected.

β1-/β2-adrenoceptor mediated increase in force of contraction by BRL

Various studies have shown that β3-adrenergic agonistic substances cause positive inotropic and chronotropic effects in human atrium (Arch & Kaumann, 1993; Wheeldon et al., 1994; Sennit et al., 1998). In vivo increase of heart rate induced by BRL has often been attributed to baroreflex mechanisms, due to a β3-adrenergic vasodilatation (Tavernier et al., 1992; Takayama et al., 1993). However this does not explain the positive inotropic effects of BRL detected in isolated atrial myocardium as shown in this study. These findings are supported by previous reports as well (Arch & Kaumann, 1993; Sennit et al., 1998).

In the present study the positive inotropic effect of BRL was abolished after pretreatment with the β1- and β2-AR antagonist propranolol, indicating that the BRL-mediated positive inotropic effect may be due to β1-/β2-adrenergic stimulation. In agreement, the binding experiments of the present study provided evidence that BRL has a distinct affinity towards β1/2-AR and the range of concentration in which BRL acted as an agonist towards classical AR was exactly the same in which it was able to cause significant cardiostimulant effects. Consistently, we could show that in human atrial myocardium BRL acts as a competitive antagonist towards isoprenaline at β1/2-AR. The shortening of time to half peak relaxation (T0.5T) and the increase of intracellular Ca2+-transient induced by BRL are as well consistent with β1/2-adrenergic myocardial stimulation.

From these findings, we conclude that the above described effects were caused by stimulation of atrial β1- and β2-AR. In agreement, β3-adrenergic stimulation has in a variety of tissues been described to be resistant against antagonists possessing only high affinity towards β1- and β2-AR such as propranolol (Kaumann & Molenaar, 1996; Gauthier et al., 1996; Sennit et al., 1998; Trochu et al., 1999).

Yet we observed that the maximum inotropic effect reached by BRL in right atrium is significantly lower than the one reached by isoprenaline (+BRL (100 μM): +57.01±15.77%; n=16; +Isoprenaline (10 μM): +122.09±98.8; n=11 (P<0.05 (1 : 2)). Both isoprenaline and BRL are known to stimulate β3-AR in cardiac tissue, however isoprenaline has a much lower affinity towards β3-AR than BRL (Gauthier et al., 1996). Thus, the minor positive inotropic effect observed after application of BRL in comparison to the application of isoprenaline may be attributed to a BRL-induced stimulation of the β3-AR diminishing its β1- and β2-adrenergic effects. On the other hand this observation may indicate that BRL, although it significantly causes β1- and β2-adrenergic effects in right atrium, still has a lower affinity or a lower intrinsic activity towards β1- and β2-AR than isoprenaline.

eNOS activation by BRL in human myocardium

This study is the first to show a direct stimulation of eNOS by the β3-AR agonist BRL followed by an increase of NO in human atrial myocardium. The existence of eNOS in human cardiovascular tissue and its involvement in β3-adrenergic mediation have been evaluated in earlier studies (Balligand et al., 1995; Gauthier et al., 1998; Trochu et al., 1999), however evidence for a direct influence of β3-AR agonists on the activity level of the eNOS enzyme has still been lacking. We detected a distinct increase in eNOS immunoreaction and in directly detected NO in presence of BRL, and we related this effect to β3-adrenergic stimulation. This assumption was secured by the observation that forskolin as a positive inotrope could not induce similar effects and that BRL induced effects on eNOS remained unchanged in the presence of propranolol. Therefore we conclude that the effects of BRL on eNOS are, in contrast to its inotropic effects, mediated via atrial β3-AR.

Functional implication of atrial β3-adrenergic stimulation

This study shows that in human atrium BRL increases eNOS-activity and in the following NO via the β3-AR, but fails to directly induce negative inotropic effects as it does in the left ventricle (Gauthier et al., 1996; Moniotte et al., 2000). Even in the presence of propranolol, a potent β1/2-antagonist, a negative inotropic effect of BRL could not be detected. Consistently, after inhibition of the β3-adrenergic second messenger NO by the NO-inhibitor L-NMA the dose response curve of BRL remained unchanged, indicating that β3-adrenergic stimulation may not directly influence contraction in human atrial myocardium. It is therefore possible that α3- adrenergic stimulation in human atrium is not of the same functional significance as in left ventriculum. These regional differences may be attributed by the different kind of β-adrenoceptor coupling in right atrial and left ventricular myocardium (Schwinger et al., 1991). Another possible explanation is that the atrial β3-AR might be expressed in much lower numbers compared to the left ventricle. In addition, an overexpression of the eNOS has been described for atrial myocardium compared to the left ventricle (Bloch et al., 1999). Thus, β3-adrenergic stimulation may not be sufficient to elicit the already high basal atrial eNOS activity significantly over its basal level, thereby failing to increase the cGMP-level and to elicit measurable negative inotropic effects.

Thus, the role of eNOS as a second messenger for β3-adrenergic stimulation may be blunted in human atrium, compared to left ventriculum. However, from our findings we cannot exclude that atrial β3-adrenergic stimulation may oppose β1/2-adrenergic chronotropic and dromotropic effects via an increase of NO and by this way possibly decreases sinual frequency. Thus, the atrial β3-adrenergic system might provide a protective negative feed back mechanism against atrial arrhythmia and sinual tachycardia, resulting from excessive β1/2-adrenergic stimulation. Furthermore atrial β3-adrenergic stimulation might become functionally significant in the setting of heart failure, where increased expression of β3-AR has recently been reported, at least for the ventricle (Moniotte et al., 2000). β3-adrenergic stimulation may as well influence cell growth and differentiation.

In conclusion, this work shows that NO-mediated β3-adrenergic stimulation is present in human atrium, but, at least under normal conditions, does not directly influence atrial contractility. In addition we could provide further information about the β3-adrenergic intracellular mediation pathway by showing that β3-adrenergic stimulation directly and time dependently activates eNOS which again induces a release of NO.

Limitation of the study

The present study was performed with right atrial tissue from patients with either valvular or coronary disease. Furthermore the patients received medical and anaesthetical treatment. It therefore cannot be excluded that the observed effects may have been influenced by disease state or medication.

Acknowledgments

We are indebted to all colleagues of the Department of the Cardiothoracic Surgery of the University of Cologne for providing us with human myocardial samples. We are grateful to S. Danneschewski, K. Rösler and M. Ghilav for technical assistence. The work was gratefully supported by the Köln-Fortune programme of the University of Cologne. This work contains data from the doctoral thesis of C. Pott.

Abbreviations

AR

adrenoceptor

BRL

BRL 37344

cGMP

cyclic guanosine monophosphate

DAF

diaminofluorescein

eNOS

endothelial nitric oxide synthase

FOC

force of contraction

L-NMA

N-nitro-L-arginine

L-NAME

N-Nitro-L-arginine methylester hydrochloride

NO

nitric oxide

T0.5T

time to half peak relaxation

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