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Cardiovascular Research 2002 56(3):393-403; doi:10.1016/S0008-6363(02)00601-6
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Copyright © 2002, European Society of Cardiology

β3-Adrenergic regulation of an ion channel in the heart—inhibition of the slow delayed rectifier potassium current IKs in guinea pig ventricular myocytes

Ralph F. Boscha,*,1, Alexander C. Schnecka,1, Johann Kiehnc, Wei Zhangc, Annette Hambrockb, Bernd W. Eigenbergera, Norman Rüba, Jeannette Gogela, Christian Mewisa, Ludger Seipela and Volker Kühlkampa

aDepartment of Cardiology, University of Tuebingen, Otfried-Mueller-Strasse 10, D-72076 Tuebingen, Germany
bDepartment of Pharmacology, University of Tuebingen, Tuebingen, Germany
cDepartment of Cardiology, University of Heidelberg, Heidelberg, Germany

* Corresponding author. Tel.: +49-7071-298-2712; fax: +49-7071-295-285

Received 22 January 2002; accepted 12 July 2002


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 Acknowledgments
 References
 
Objectives: IKs, the slow component of the delayed rectifier potassium current, underlies a strong β-adrenergic regulation in the heart. Catecholamines, like isoproterenol, induce a strong increase in IKs. Recent work has pointed to an opposing biological effect of β1- and β3-adrenoceptors in the heart. However the role of these subtypes in the regulation of cardiac ion channel function is unknown. Methods: We investigated the effects of β1- and β3-adrenoceptor modulation on IKs in guinea-pig ventricular myocytes, using patch-clamp techniques. Results: Superfusion with 100 nmol/l isoproterenol increased the step current amplitude by 81.3±8.0%. In contrast, after block of β1- (1 µmol/l atenolol) and β2-receptors (1 µmol/l ICI118,551), isoproterenol induced a reduction of the step current amplitude by 34.3±3.5%. The β3-selective agonist BRL37344 significantly reduced the IKs step current at +70 mV in a concentration-dependent manner (IC50: 5.01 nmol/l). In the presence of bupranolol (β1-, β2- and β3-adrenoceptor antagonist), the effect of BRL37344 was markedly attenuated, from 27.3±5.6% (100 nmol/l BRL37344 alone) to 4.0±1.3% (100 nmol/l BRL37344+1 µmol/l bupranolol). BRL37344 (100 µmol/) did not alter current amplitudes of KvLQT1/minK expressed in CHO cells or in Xenopus oocytes, excluding a direct effect of BRL37344 on the channel. 1 µmol/l BRL37344 mildly prolonged action potentials in guinea pig ventricle (APD90:+7.8%) Conclusions: We have demonstrated a functional coupling between the β3-adrenoceptor and ion channel function in the mammalian heart. Our findings point to a potential role for β3-adrenoceptors in cardiac electrophysiology and pathophysiology.

KEYWORDS Adrenergic (ant)agonists; Ion channels; K-channel; Membrane currents; Myocytes; Receptors

This article is referred to in the Editorial by C.E. Conrath and T. Opthof (pages 353–356) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 Acknowledgments
 References
 
β-Adrenergic receptors (β-ARs) mediate the effects of epinephrine and norepinephrine. Hence, regulation of β-ARs plays an important role in cardiac physiology and pathophysiology via stimulation by the sympathetic transmitters. In the human heart, three subtypes of β-ARs, β1, β2 and β3, have been identified and modulate cardiac function. A fourth adrenergic receptor has been described. However, there is now evidence, that the ‘β4-AR’ is an atypical state of the β1-AR [1,2]. The presence of the β3-AR has been confirmed in a variety of tissues [3–5], including the heart [6–8].

The role of the cardiac β3-AR is not sufficiently understood and the available data are partially conflicting. Some reports have pointed to opposing effects of β1- and β3-AR on cardiac hemodynamics. In human and canine heart preparations, selective β3-agonists produced a profound dose-dependent decrease in cardiac contractility and shortened the action potential. This effect could also be elucidated by intrinsic catecholamines in the presence of β1- and β2-AR antagonists [7]. More recently, the depression of ventricular force of contraction by β3-AR stimulation was confirmed by Kitamura et al. in guinea pig hearts [9]. Other groups however, did not observe inotropic effects of β3-AR stimulation in human ventricle or mouse atrium [10–13]. It has also been reported that the L-type calcium current (ICa,L) [14] and the cystic fibrosis transmembrane conductance regulator (CFTR) [15] are modulated by β3-AR activation.

IKs, the slow component of the delayed rectifier K+ current, is a major outward current in determining the AP-plateau in the myocardium [16–18]. It is well established that the IKs channel is strongly modified by β-adrenergic activation: stimulation of the predominant β-adrenergic subtype (β1) in the heart with catecholamines (noradrenaline, isoproterenol) is associated with a marked increase in IKs current amplitudes [19–21]. In contrast, the role of the β3-AR in the modulation of cardiac potassium channel function has not been investigated in cardiac myocytes.

The present study characterizes the effect of β1-and β3-AR activation on IKs current in isolated ventricular myocytes.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusions
 Acknowledgments
 References
 
2.1 Cell isolation and solutions
Experiments in cardiac myocytes were performed in adult male Hartley albino guinea pigs (400–500 g). All procedures followed were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and with the institutional guidelines of the University of Tübingen. Animals were killed by cervical dislocation, left ventricular myocytes were isolated by enzymatic dissociation as described previously [22]. The cells were superfused at 6 ml/min with a solution containing (mmol/l) NaCl 136, KCl 5.4, CaCl2 2.0, MgCl 1.0; NaH2PO4 0.33, HEPES 5 and glucose 10 (pH adjusted to 7.35 with NaOH). Bath temperature was 36±1 °C. ICa,L and IKr were blocked with 1 µmol/l nisoldipine (Sigma) and 1 µmol/l dofetilide (Pfizer), respectively, unless stated otherwise. Atenolol (Sigma), ICI118,551 (Tocris), noradrenaline (Sigma), isoproterenol (ICN Biomedicals), BRL37344 (Tocris) and bupranolol (Schwarz Pharma, Monheim, Germany) were dissolved in distilled water. The pipette solution contained (mmol/l) KCl 20, K-aspartate 110, MgCl 1.0, HEPES 10, EGTA 5, Mg2ATP 5, GTP 0.1, phosphocreatine 5 (pH adjusted to 7.2 with KOH), unless stated otherwise. The liquid junction potential for our bath and pipette solutions was –11.5 mV.

A CHO cell line that stable expresses KvLQT1/minK channels was a gift from Klaus Steinmeyer (Aventis Pharma, Frankfurt, Germany). The bath and pipette solutions were identical to those used for the myocyte experiments; the bath temperature was 36 °C.

2.2 Voltage-clamp technique
IKs currents were recorded using the whole-cell configuration of the voltage clamp technique. Pipettes with resistances from 2 to 5 M{Omega} when filled with pipette solution were connected to a patch clamp amplifier (Axopatch 200B, Axon Instruments). The sampling frequency was 0.4 kHz. Recordings were low-pass filtered at 1 kHz. Membrane capacitance averaged 112.9±9.7 pF (n = 20). Before compensation, Rs averaged 7.2±0.2 M{Omega}. Corresponding values for Rs after compensation were 3.5±0.2 M{Omega}.

2.3 Xenopus oocytes
The cDNA clone encoding human KvLQT1 was a gift from M.T. Keating (Harvard Medical School, Children's Hospital, Boston, MA, USA). The human minK clone was a gift from A.M. Brown (Case Western Reserve University, Cleveland, OH, USA) and the human β3-AR clone was a gift from R.J. Lefkowitz (Duke University, Durham, NC, USA). Complementary cRNAs were prepared and injected (46 nl in each oocyte) as described previously [23]. The concentrations of the injected cRNAs were: 500 ng/µl KvLQT1, 200 ng/µl minK and 100 ng/µl β3-AR. Two-microelectrode voltage clamp measurements of Xenopus oocytes were performed in a low K+ solution containing (in mmol/l) 5 KCl, 100 NaCl, 1.5 CaCl2, 2 MgCl2 and 10 HEPES (pH 7.3) at room temperature (20 °C).

2.4 Data analysis
Group data are expressed as the mean±S.E.M. Student's paired t-test was used to compare means before and after treatment. A two-tailed P value<0.05 was considered statistically significant. Nonlinear least-square curve-fitting was performed in PCLAMP 6.0 or with SIGMA PLOT software.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusions
 Acknowledgments
 References
 
3.1 β-Adrenergic modulation of IKs by isoproterenol and noradrenaline
Baseline measurements of IKs were performed at least 5 min after cell membrane rupture and a repeating pulse to the most positive potential (+70 mV) was applied to each cell in 30-s intervals to detect rundown of IKs. Initial measurements were followed by a 10-min drug wash in period, after which equilibration of effect was seen in all experiments. Representative recordings are shown in Fig. 1.


Figure 1
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  		Fig. 1 Effects of isoproterenol, an unselective β-adrenoceptor agonist on the slow component of the delayed rectifier K+ current, in guinea pig ventricular myocytes. Currents were elicited with 3-s pulses (0.1 Hz) to voltages between +10 and +70 mV in 10-mV increments from a holding potential of –50 mV; tail currents were recorded on 2-s repolarization to –40 mV. Step currents were measured as the difference between current immediately after the decay of the capacitive surge and the steady-state current at the end of the 3-s pulse. Bath temperature was 36 °C, 1 µmol/l nisoldipine and 1 µmol/l dofetilide were added to block ICa,L and IKr, respectively. Recordings are presented without blockade of β1- and β2-AR (A and B) and after addition of 1 µmol/l atenolol and 1 µmol/l ICI118,551 to block β1- and β2-AR, respectively (C–E). Augmented tail currents of the respective recording are shown below each panel.

 
Fig. 1A and B shows the effect of β-adrenergic stimulation using isoproterenol. Superfusion with isoproterenol (100 nmol/l) led to a strong increase in IKs step and tail currents. At a test potential of +70 mV, the increase of IKs tail current amplitude averaged 108.5±12.7%. The IKs step current amplitude was similarly increased by 81.3±8.0% over control (n = 5, P<0.05). However, after blocking the β1- and β2-ARs with 1 µmol/l atenolol and 1 µmol/l ICI118,551, respectively, 1 µmol/l isoproterenol decreased the IKs step and tail current amplitudes significantly (step current amplitude: –41.9±3.0%, P<0.05), (Fig. 1C–E, n = 6). The inhibitory effect of isoproterenol, when stimulating β3-ARs was almost completely reversible upon washout (87.7±4.3% of control) (Fig. 1E).

The isoproterenol and noradrenaline dose–response effect in the presence of atenolol and ICI118,551 is illustrated in Fig. 2. Isoproterenol produced a maximum inhibition of IKs of 42.1±3.0% (P<0.05, n = 6) at a concentration of 1 µmol/l (Fig. 2A). Higher concentrations (>1 µmol/l) reversed the block and even increased the current again, i.e. 10 µmol/l increased currents by 60.0±9.6% (n = 4). The physiological β-agonist noradrenaline caused effects that were comparable to those observed with isoproterenol. Inhibition of IKs was also concentration-dependent as is illustrated in Fig. 2B. The amount of inhibition was comparable to that observed with isoproterenol (31.6±2.0% IKs inhibition at a concentration of 1 µmol/l noradrenaline). Fig. 2C shows an example of the time course of changes in IKs step current amplitude after application of 10 nmol/l isoproterenol. In our preparations, the maximum effect was observed after 2 min (65.1% in this cell). However, a decay of isoproterenol induced inhibition of IKs was seen in all cells until a steady state was reached after about 10 min of drug superfusion. All results given refer to steady state inhibition which was 31.8% in the preparation shown in Fig. 2.


Figure 2
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  		Fig. 2 Properties of IKs block by isoproterenol and noradrenaline. Concentration dependence of isoproterenol (A) and noradrenaline (B) induced inhibition of step currents. The numbers in brackets indicate the number of cells studied at each concentration. (C) Time course of changes in IKs step current at a test potential of +70 mV after application of 10 nmol/l isoproterenol. Horizontal bars indicate the application of atenolol, ICI118,551 and isoproterenol. After 10 min of drug perfusion, isoproterenol was washed out for 5 min. *, P<0.05 vs. control.

 
3.2 Selective β3-adrenergic stimulation inhibits IKs current
To confirm the indirect observation that β3-AR stimulation decreases IKs, we studied the effect of the highly selective β3-receptor agonist BRL37344. Fig. 3A–C shows the effects of 1 µmol/l BRL37344 on IKs, in the presence of 1 µmol/l atenolol and ICI118,551. The drug inhibited IKs step and tail current amplitudes (step: –39.2±6.3%, tail: –39.6±7.1%), (P<0.05 vs. control, n = 4). Like in the isoproterenol experiments, effects on step and tail currents were comparable. The effect of BRL37344 was partially reversible after 10 min perfusion with BRL37344–free external solution and reached an average of 77.7±5.5% of the control value. Fig. 3D illustrates the time course of IKs step current reduction after superfusion with 1 nmol/l BRL37344. Similar to the findings with isoproterenol, there was a fast onset of inhibition after 2 min (48.6%), followed by a slow decline of the inhibitory effect. Steady state was reached after 10 min (33.3%). This behavior of inhibition was noted in all cells studied (n = 31).


Figure 3
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  		Fig. 3 Effect of 1 µmol/l BRL37344 on IKs. (A–C) Representative measurement of activating currents and subsequent deactivating tails (pulse protocol and experimental conditions identical to Fig. 1). Augmented tail currents of the respective recording are shown below each panel. (D) Time course of onset and resolution of 1 nmol/l BRL37344 block of IKs step current. BRL37344 was washed out for 10 min.

 
Concentration dependence of inhibition and the effects on voltage-dependent activation of IKs are shown in Fig. 4. BRL37344 decreased IKs step current amplitude at +70 mV in a concentration-dependent manner with a 50%-effective inhibitory concentration (EC50) of 5.01 nmol/l (Fig. 4A). The maximum inhibition (41.0±8.1%, P<0.05) was reached with a concentration of 10 µmol/l BRL37344, higher concentrations did not further inhibit the currents. The voltage-dependent activation of IKs in the presence of 1 µmol/l BRL37344 was studied by normalizing IKtail at a given test potential to IKtail at +70 mV. The data were fitted with a Boltzmann distribution (Fig. 4B).


Figure 4
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  		Fig. 4 BRL37344 concentration-and voltage-dependent effects on IKs. (A) Inhibition of step current, plotted as a function of BRL37344 concentration. Experimental data were fit with a Hill equation of the following form: F = Bmax/{1+(EC50/[BRL37344]h}, where Bmax indicates the maximal inhibition, and h, the Hill coefficient. Numbers in parentheses indicate the number of cells studied at each concentration. The calculated EC50 was 5.01 nmol/l. (B) Effects of 1 µmo/l BRL37344 on IKs voltage dependence of activation. The activation curves were obtained by normalizing tail currents at a given potential to those at the most positive test potential (+70 mV). Data were fit with a Boltzmann function of the following form: A = 1/{1+exp [Vh–Vm]/k}, where A is the activation variable (tail current at test potential Vm divided by tail current at +70 mV), Vm is the test potential, Vh and k are the half-activation voltage and the slope factor, respectively.

 
The β3-AR mediated decrease of IKs current was accompanied by a significant shift of the half activation voltage (Vh) to more positive potentials (from +31.6±1.3 mV to +41.5±1.0 mV, n = 6, P<0.05). The slope factor k of the activation curve remained unchanged (kcontrol=+13.8±1.4 mV versus kBRL37344=+11.9±1.1 m V; P = ns).

Under control conditions, IKs current showed a typical activation and deactivation kinetic, with a fast activation time constant (tfast) of 230.4±10.3 ms and a slow time constant (tslow) of 2004.9±278.8 ms at +70 mV. The fast deactivation time constant (tfast) at –40 mV averaged 107.4±13.0 ms and tslow 482.9±90.7 ms, values similar to those previously published [22]. BRL37344 markedly altered the activation kinetics of IKs with time constants averaging 32.4±0.3 ms (tfast) and 254.3±14.5 ms (tslow) (n = 7, P<0.05 vs. control), whereas the deactivation time constant remained unchanged (tfast=98.6±36.4 ms and tslow=533.6±130.3 ms; P = ns). The same effect on activation kinetics of IKs was observed when isoproterenol was added after blockade of β1- and β2-ARs (see Fig. 1D). The effects of β3-adrenergic stimulation on fully activated IKs were evaluated with a 5-s depolarizing pulse to +50 mV, followed by a 2-s repolarizing step to potentials between –110 and –40 mV to record tail currents. The reversal potential Vrev,IKs was determined by measuring tail current amplitudes of fully activated currents. Amplitudes were plotted as a function of the repolarization potential. Superfusion with 1 µmol/l BRL 37344 did not alter Vrev,IKs, which was –81.0±3.3 mV in control and –80.9±3.4 mV after BRL 37344 (corrected for the liquid junction potential, n = 7, P = ns).

To exclude an effect of atenolol and ICI118,551 on our results, the experiments with BRL37344 were also performed without blocking β1- and β2-AR. BRL37344 inhibited IKs to a comparable extent under these experimental conditions (data not presented).

In the presence of 1 µmol/l bupranolol, a β3-antagonist [24], the inhibition of IKs current amplitudes at a concentration of 100 nmol/l BRL37344 was markedly reduced from 27.3±6.6% to 4.0±1.3% (n = 4, P<0.05), further strengthening the evidence that the inhibition of the current was mediated by activation of β3-adrenergic receptors.

3.3 BRL37344 effects on KvLQT1/minK channels expressed in a CHO cell line and in Xenopus oocytes
To exclude a direct inhibitory effect of BRL37344 on IKs channels, we investigated the drug's effect on human KvLQT1/minK channels, stably coexpressed in a CHO cell line. In cells with a stable current, measurements were performed before and after a 5–10 min wash in period of 100 µmol/l BRL37344. As shown in Fig. 5A and B, BRL37344 did not change the KvLQT1/minK current amplitude. Similar results were obtained in a total of six cells, step current amplitudes at +70 mV averaged 2220±400 pA for control and 2160±400 pA after superfusion with BRL37344, tail currents amplitudes were 411±78 pA and 398±77 pA before and after BRL37344 superfusion, respectively (P = ns for both).


Figure 5
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  		Fig. 5 Measurement of KvLQT1/minK current in stable transfected CHO cells before (A) and after (B) superfusion with 100 µmol/l BRL37344 at 36 °C. The pulse protocol was identical to the one used for IKs recordings in cardiac myocytes (Fig. 1). (C and D) Measurement of the effects of BRL37344 (100 µmol/l) on KvLQT1/minK channels in Xenopus oocytes at room temperature (20 °C): test pulses from a holding potential of –60 to 100 mV in 20-mV increments (2 s), return pulses back to –40 mV (2 s) to measure tail current.

 
The effects of BRL37344 were also tested on KvLQT1/minK channels expressed in Xenopus oocytes as illustrated in Fig. 5C and D. Addition of 100 µmol/l BRL37344 did not change the KvLQT1/minK current amplitude. Step current amplitudes at +100 mV were 98.6±7.3% and those of tails 101.5±5.5% of controls (n = 6, P = ns).

3.4 Effects of β3-adrenergic stimulation on action potentials in isolated guinea pig ventricular myocytes
The effects of β3-adrenergic stimulation on action potential properties were tested in isolated guinea pig ventricular myocytes in the current clamp mode of the patch clamp technique at a stimulation frequency of 1 Hz. After a control period of 5 min with stable action potentials, 1 µmol/l BRL 37344 was superfused for 10 min before recordings were repeated. A typical recording is shown in Fig. 6. Only cells with a stable resting membrane potential (Vm) below –82 mV (corrected for the liquid junction potential) were included. Vm averaged –85.3±0.6 mV under control conditions and was not altered by superfusion with BRL 37344 (–86.1±0.8 mV (n = 9, P = ns vs. control, Table 1). Action potential amplitudes were also not affected by BRL 37344. Action potential duration (APD) was assessed as duration to 20, 50 and 90% repolarization (APD20, APD50 and APD90, respectively). β3-AR activation was associated with a mild prolongation of the APD in all phases of repolarization, the increase averaged 10.4, 8.8 and 7.8% for APD20, APD50 and APD90 vs. control (P<0.05 for all, Table 1).


Figure 6
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  		Fig. 6 Representative recordings of action potentials in a guinea pig ventricular myocyte before (control) and after superfusion with 1 µmol/l BRL37344. Recordings were done in the current clamp mode of the patch clamp technique at 36 °C in normal Tyrode's solution. Membrane potentials are displayed without correction for the liquid junction potential. See text and Table 1 for details.

 

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  		Table 1 Effects of 1 µmol/l BRL37344 on action potential characteristics in guinea pig ventricular myocytes

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusions
 Acknowledgments
 References
 
In the present study, we have demonstrated that β3-AR activation is associated with a strong inhibition of the slow delayed rectifier K+ current, IKs, in guinea pig ventricular myocytes. In contrast, predominant β1-AR stimulation increased the IKs current. The reduction of IKs was induced by isoproterenol and by noradrenaline when β1- and β2-AR were blocked as well as by addition of a highly selective β3-AR agonist (BRL37344). A direct block of IKs could be excluded by heterologous expression of the channel in a CHO cell line and in Xenopus oocytes. β3-AR activation was associated with a mild prolongation of the action potential duration in guinea pig ventricular myocytes.

4.1 β-Adrenergic regulation of IKs channels
Stimulation of β-AR by the unselective β-agonist isoproterenol led to a marked increase in IKs current amplitudes (Fig. 1). This finding is consistent with the results of earlier studies which demonstrated an increase in IKs in different species [19–21]. After expression of the IsK channel in Xenopus oocytes, the current is also increased after administration of second messengers like c-AMP or PKA [25].

When isoproterenol or noradrenaline was added after block of β1- and β2-AR by atenolol and ICI118,551, we observed a reversible decrease of IKs. This can be explained by activation of the β3-AR. In concordance with this explanation is the inhibition of IKs by the specific β3-agonist BRL37344 over a wide range of concentrations (Fig. 4). This effect was almost completely abolished by the β3-antagonist bupranolol. Inverted catecholamine effects in the heart, mediated by β3-AR activation, have been reported by Gauthier et al. and Kitamura et al. [7,9]. They found a negative inotropic effect of isoproterenol after block of β1- and β2-AR in human and guinea pig hearts, as well in nanomolar potencies. However, this work is in strong contrast to reports that were not able to observe a significant contractile response following stimulation of β3-AR [10–13]. Isoproterenol, in concentrations up to 1 µmol/l inhibited IKs when β1- and β2-AR were blocked by competitive antagonists (atenolol and ICI118,551). When higher concentrations of isoproterenol were administered, this effect could be overcome and IKs current amplitudes were even increased. Probably, this reflects a functional antagonism of β1/β2- vs. β3-AR activation on IKs. As atenolol and isoproterenol are competitors at the β1-AR and ICI118,551 and isoproterenol at the β2-AR, very high concentrations of isoproterenol may lead to partial activation of β1- or β2-AR which could counterbalance and even reverse the inhibitory effect of β3-AR activation.

The β3-induced inhibition of IKs (Figs. 2B and 3DGo) displayed a fast onset within 2 min, implicating a high affinity coupling of the β3-AR to the IKs channel and a rapid triggering of the involved signaling pathway. This was followed by a decay of inhibition until a steady state was reached after 10 min, a possible mechanism being a desensitization of the signal transduction pathway. Nantel et al. have also reported a modest decrease in the potency of isoproterenol to stimulate the β3-AR after short-term isoproterenol pretreatment [26]. Reduced expression of the receptor and of G-proteins seem to be involved in this process [27]. As an agonist-induced downregulation of receptors has been observed for β1- and β2-, but not for β3-AR [28,29] a possible mechanism for the weakening of the effect over time includes an agonist-induced selective decrease in G-proteins [28] or a cellular redistribution of G-proteins from membranes to the cytosol [27]. An alternative explanation for our observation includes overlap of several signaling pathways distal to the receptor. Most currently available data on β3-AR-mediated intracellular signaling are generated in adipocytes and in the gastrointestinal tract. In these tissues, an involvement of both Gs and Gi/o protein have been described [4,30]. In human ventricular tissues, Gauthier et al. demonstrated that Gi/o{alpha} subunits and a NO-pathway are involved in β3-AR mediated cardiodepression and Gs-proteins have also been reported as coupling proteins [7,23]. Several signaling pathways could therefore participate in the functional coupling of β3-ARs to IKs channels. Promiscuous coupling of β-ARs to several types of G proteins has also been demonstrated with other G protein-coupled receptors including the β2-adrenoceptor [31,32]. Further studies will be necessary to characterize the intracellular β3-AR downstream signaling, leading to a regulation of cardiac ion channel function.

Our results regarding the β3-AR-mediated inhibition of IKs current differ from a recent report by Kathoefer et al. [23]. After stimulation with isoproterenol, they observed an increase of human KvLQT1/minK current coexpressed with β3-ARs in Xenopus oocytes mediated via Gs proteins. The reasons for these discrepant results remain speculative. First, there were deviations in temperature (we used 36 °C for the cardiomyocytes, in contrast to room temperature for the oocytes). It has been demonstrated, that the kinetics of IKs are highly temperature dependent over the range of 20–37 °C [33,34]. Second, important species differences in the expression and the structure of the β3-AR have been reported. The guinea pig β3-AR is structurally different from AR in humans in transmembrane regions that are considered important for ligand binding and G-protein interaction [35]. However, similar effects on contractility have been observed in human and guinea pig preparations after β3-AR stimulation, indicating similar functional properties of the receptor [7,9]. Finally, the signal cascade by which β3-AR effects are mediated might be different between guinea pigs and Xenopus oocytes. These results point to important differences in the coupling of β3-AR to IKs channels in cardiac myocytes and Xenopus oocytes. These differences should be considered when studying β3-AR channel interaction in different experimental conditions.

β3-AR was associated with a mild prolongation of the action potential duration in isolated guinea pig ventricular myocytes, consistent with a block of a potassium current like IKs. The extent of prolongation by high dosages of BRL 37344 was 8–10% in all phases of repolarization. This is the range that would have been expected as maximum inhibition of IKs achieved by β3-AR activation was 40%. Complete block of the current by the chromanol 293B [36] or chromanol 1556 [37] increased APD by 35–40%. The results are in contrast to a shortening of APD as demonstrated by Gauthier et al. and Leblais et al. in human ventricle [7,15]. First, this might represent a difference in the contribution of IKs to repolarization in different species. IKs is the predominant repolarizing current in guinea pig ventricle. In human ventricle, existence of the current was confirmed [38], but current densities were much smaller, the relative contribution as well as the expression along the different ventricular layers are currently not known. Secondly, other ionic currents are modulated by β3-AR stimulation. These include the L-type Ca2+ current (ICa,L) [14] as well as CFTR [15]. Thus, differences in the balance between inward and outward currents by β3-AR activation can have distinct effects on action potential duration in different species.


   5 Conclusions
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
Acknowledgments
 References
 
We have demonstrated that β3-adrenoceptors are functionally coupled to a potassium channel in the heart. A stimulation of the β3-AR markedly reduced IKs currents which is a counterregulatory effect to the formerly investigated β1-AR mediated increase of the current. Our findings, together with the results of β3-AR influence on cardiac contractility and hemodynamics, point to a potential role of β3-AR in cardiac physiology and pathophysiology.

Time for primary review 30 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 Acknowledgments
References
 
The work was supported by grants of the Deutsche Forschungsgemeinschaft [DFG-Bo1396/3 (RFB); DFG-Ki663/1-1 (JK)], the Bundesministerium für Bildung und Forschung (BMBF)/University of Tübingen (IZKF) (Fö. 01KS9602) (RFB), the fortuene program of the University of Tuebingen (640-0-0 RFB), the Pinguin Stiftung (Henkel AG), Duesseldorf, Germany (RFB, ACS) and the Franz-Loogen-Stiftung, Duesseldorf, Germany (LS, VK).


   Notes
 
1 Both authors contributed equally. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 Acknowledgments
 References
 

  1. Kaumann A.J., Engelhardt S., Hein L., Molenaar P., Lohse M. Abolition of (–)-CGP 12177-evoked cardiostimulation in double β1/β2-adrenoceptor knockout mice. Obligatory role of β1-adrenoceptors for putative β4-adrenoceptor pharmacology. Naunyn-Schmiedeberg's Arch. Pharmacol. (2001) 363:87–93.[CrossRef][Web of Science][Medline]
  2. Kompa A.R., Summers R.J. Desensitization and resensitization of β1- and putative β4-adrenoceptor mediated responses occur in parallel in a rat model of cardiac failure. Br. J. Pharmacol. (1999) 128:1399–1406.[CrossRef][Web of Science][Medline]
  3. Atgie C., Tavernier G., D'Allaire F., et al. β3-Adrenoceptor in guinea pig brown and white adipocytes: low expression and lack of function. Am. J. Physiol. (1996) 271:R1729–1738.[Web of Science][Medline]
  4. Emorine L.J., Marullo S., Briend Sutren M.M., et al. Molecular characterization of the human β3-adrenergic receptor. Science (1989) 245:1118–1121.[Abstract/Free Full Text]
  5. Granneman J.G., Lahners K.N., Chaudhry A. Molecular cloning and expression of the rat β 3-adrenergic receptor. Mol. Pharmacol. (1991) 40:895–899.[Abstract]
  6. Chamberlain P.D., Jennings K.H., Paul F., et al. The tissue distribution of the human β3-adrenoceptor studied using a monoclonal antibody: direct evidence of the β3-adrenoceptor in human adipose tissue, atrium and skeletal muscle. Int. J. Obes. Relat. Metab. Disord. (1999) 23:1057–1065.[CrossRef][Web of Science][Medline]
  7. Gauthier C., Tavernier G., Charpentier F., Langin D., Le Marec H. Functional β3-adrenoceptor in the human heart. J. Clin. Invest. (1996) 98:556–562.[Web of Science][Medline]
  8. Moniotte S., Kobzik L., Feron O., et al. Upregulation of β3-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium. Circulation (2001) 103:1649–1655.[Abstract/Free Full Text]
  9. Kitamura T., Onishi K., Dohi K., et al. The negative inotropic effect of β3-adrenoceptor stimulation in the beating guinea pig heart. J. Cardiovasc. Pharmacol. (2000) 35:786–790.[CrossRef][Web of Science][Medline]
  10. Kaumann A.J., Molenaar P. Modulation of human cardiac function through 4 β-adrenoceptor populations. Naunyn Schmiedebergs Arch. Pharmacol. (1997) 355:667–681.[CrossRef][Web of Science][Medline]
  11. Oostendorp J., Kaumann A.J. Pertussis toxin suppresses carbachol-evoked cardiodepression but does not modify cardiostimulation mediated through β1- and putative β4-adrenoceptors in mouse left atria: no evidence for β2- and β3-adrenoreceptor function. Naunyn Schmiedebergs Arch. Pharmacol. (2000) 361:134–145.[CrossRef][Web of Science][Medline]
  12. Harding S.E. Lack of evidence for β3-adrenoceptor modulation of contractile function in human ventricular myocytes. Circulation (1997) 96:I53.
  13. Molenaar P., Sarsero D., Kaumann A.J. Proposal for the interaction of nonconventional partial agonists and catecholamines with the ‘putative β 4-adrenoceptor’ in mammalian heart. Clin. Exp. Pharmacol. Physiol. (1997) 24:656.
  14. Cheng H.J., Zhang Z.S., Onishi K., et al. Upregulation of functional β3-adrenergic receptor in the failing canine myocardium. Circ. Res. (2001) 89:599–606.[Abstract/Free Full Text]
  15. Leblais V., Demolombe S., Vallette G., et al. β3-adrenoceptor control the cystic fibrosis transmembrane conductance regulator through a cAMP/protein kinase A-independent pathway. J. Biol. Chem. (1999) 274:6107–6113.[Abstract/Free Full Text]
  16. Li G.R., Feng J., Yue L., Carrier M., Nattel S. Evidence for two components of delayed rectifier K+ current in human ventricular myocytes. Circ. Res. (1996) 78:689–696.[Abstract/Free Full Text]
  17. Sanguinetti M.C., Jurkiewicz N.K. Delayed rectifier outward K+ current is composed of two currents in guinea pig atrial cells. Am. J. Physiol. (1991) 260:H393–399.[Web of Science][Medline]
  18. Wang Z., Fermini B., Nattel S. Rapid and slow components of delayed rectifier current in human atrial myocytes. Cardiovasc. Res. (1994) 28:1540–1546.[Abstract/Free Full Text]
  19. Bennett P.B., Begenisich T.B. Catecholamines modulate the delayed rectifying potassium current (IK) in guinea pig ventricular myocytes. Pflugers Arch. (1987) 410:217–219.[CrossRef][Web of Science][Medline]
  20. Sanguinetti M.C., Jurkiewicz N.K., Scott A., Siegl P.K. Isoproterenol antagonizes prolongation of refractory period by the class III. antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action. Circ. Res. (1991) 68:77–84.[Abstract/Free Full Text]
  21. Yazawa K., Kameyama M. Mechanism of receptor-mediated modulation of the delayed outward potassium current in guinea-pig ventricular myocytes. J. Physiol. Lond. (1990) 421:135–150.[Abstract/Free Full Text]
  22. Bosch R.F., Gaspo R., Busch A.E., et al. Effects of the chromanol 293B, a selective blocker of the slow, component of the delayed rectifier K+ current, on repolarization in human and guinea pig ventricular myocytes. Cardiovasc. Res. (1998) 38:441–450.[Abstract/Free Full Text]
  23. Kathöfer S., Zhang W., Karle C., et al. Functional coupling of human β3-adrenoceptors to the KvLQT1/minK potassium channel. J. Biol. Chem. (2000) 275:26743–26747.[Abstract/Free Full Text]
  24. Arch J.R., Kaumann A.J. β3 and atypical β-adrenoceptors. Med. Res. Rev. (1993) 13:663–729.[CrossRef][Web of Science][Medline]
  25. Suessbrich H., Busch A.E. The IKs channel: coassembly of IsK (minK) and KvLQT1 proteins. Rev. Physiol. Biochem. Pharmacol. (1999) 137:191–226.[Medline]
  26. Nantel F., Bonin H., Emorine L.J., et al. The human β 3-adrenergic receptor is resistant to short term agonist-promoted desensitization. Mol. Pharmacol. (1993) 43:548–555.[Abstract]
  27. Milligan G. Agonist regulation of cellular G protein levels and distribution: mechanisms and functional implications. Trends. Pharmacol. Sci. (1993) 14:413–418.[CrossRef][Medline]
  28. Chambers J., Park J., Cronk D., et al. β3-Adrenoceptor agonist-induced downregulation of Gs {alpha} and functional desensitization in a Chinese hamster ovary cell line expressing a β 3-adrenoceptor refractory to downregulation. Biochem. J. (1994) 303:973–978.[Web of Science][Medline]
  29. Granneman J.G. Effects of agonist exposure on the coupling of β1 and β3 adrenergic receptors to adenylyl cyclase in isolated adipocytes. J. Pharmacol. Exp. Ther. (1992) 261:638–642.[Abstract/Free Full Text]
  30. Chaudhry A., MacKenzie R.G., Georgic L.M., Granneman J.G. Differential interaction of β1- and β3-adrenergic receptors with Gi in rat adipocytes. Cell Signal. (1994) 6:457–465.[CrossRef][Web of Science][Medline]
  31. Gudermann T., Schoneberg T., Schultz G. Functional and structural complexity of signal transduction via G-protein-coupled receptors. Annu. Rev. Neurosci. (1997) 20:1982.
  32. Kilts J.D., Gerhardt M.A., Richardson M.D., et al. β 2-Adrenergic and several other G protein-coupled receptors in human atrial membranes activate both Gs and Gi. Circ. Res. (2000) 87:705–709.[Abstract/Free Full Text]
  33. Busch A.E., Lang F. Effects of [Ca2+]i and temperature on minK channels expressed in Xenopus oocytes. FEBS Lett. (1993) 334:221–224.[CrossRef][Web of Science][Medline]
  34. Walsh K.B., Begenisich T.B., Kass R.S. β-Adrenergic modulation of cardiac ion channels. Differential temperature sensitivity of potassium and calcium currents. J. Gen. Physiol. (1989) 93:841–854.[Abstract/Free Full Text]
  35. Strosberg A.D., Pietri R.F. Function and regulation of the β3-adrenoceptor [see comments]. Trends. Pharmacol. Sci. (1996) 17:373–381.[Medline]
  36. Bosch R.F., Gaspo R., Busch A.E., et al. Effects of the chromanol 293B, a selective blocker of the slow component of the delayed rectifier K+ current, on repolarization in human and guinea pig ventricular myocytes. Cardiovasc. Res. (1998) 38:441–450.[Abstract/Free Full Text]
  37. Gogelein H., Bruggemann A., Gerlach U., Brendel J., Busch A.E. Inhibition of IKs channels by HMR 1556. Naunyn-Schmiedebergs Arch. Pharmacol. (2000) 362:480–488.[CrossRef][Web of Science][Medline]
  38. Li G.R., Feng J., Yue L., Carrier M., Nattel S. Evidence for two components of delayed rectifier K+ current in human ventricular myocytes. Circ. Res. (1996) 78:689–696.[Abstract/Free Full Text]

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