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Am J Physiol Heart Circ Physiol 279: H1329-H1337, 2000;
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Vol. 279, Issue 3, H1329-H1337, September 2000

Conditioning of beta 1-adrenoceptor effect via beta 2-subtype on L-type Ca2+ current in canine ventricular myocytes

Zsolt Nagykaldi1, David Kem1,3, Ralph Lazzara2,3, and Bela Szabo2,3

1 Sections of Endocrinology and 2 Cardiology, Department of Internal Medicine, and 3 Cardiac Arrhythmia Research Institute, University of Oklahoma Health Sciences Center and Veterans Affairs Medical Center, Oklahoma City, Oklahoma 73104


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the roles of beta 1- and beta 2-receptors (beta -AR) in adrenergic enhancement of L-type Ca2+ current (ICaL) in canine ventricular myocytes. Isoproterenol and l-norepinephrine produced a monophasic and a biphasic concentration-ICaL relationship (CR), respectively. alpha 1-AR inhibition with prazosin and beta 2-AR stimulation with zinterol or l-epinephrine shifted the CR of l-norepinephrine leftward. Zinterol (50 nM) and l-epinephrine (10 nM), but not prazosin, altered the biphasic CR of l-norepinephrine to a monophasic CR. Zinterol and l-epinephrine applied after l-norepinephrine had no effect on ICaL. beta 2-AR inhibition with ICI-118551 reduced the Emax of isoproterenol and l-norepinephrine by 60% and abolished the augmentation of l-norepinephrine by zinterol and l-epinephrine. Carbachol (100 nM) modestly reduced the ICaL response to beta 1-AR stimulation but abolished the enhancement via beta 2-AR. Zinterol augmented the enhancement of ICaL by forskolin, IBMX, and theophylline, but not in the presence of CGP-20712A. We conclude that selective beta 2-AR stimulation does not increase ICaL but enhances adenylyl cyclase activity when stimulated via beta 1-AR and with forskolin. beta 2-AR activity preconditions adenylyl cyclase for beta 1-AR stimulation.

norepinephrine; carbachol; 3-isobutyl-1-methylxanthine; adenylyl cyclase; forskolin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PHYSIOLOGICAL CATECHOLAMINES, l-norepinephrine (NE) and l-epinephrine (Epi), increase cAMP accumulation and cAMP-dependent phosphorylation of the L-type Ca2+ channel and the current through it (ICaL) by activating beta -adrenoceptors (beta -AR) (18). Earlier studies demonstrated expression of several beta -AR subtypes in the heart (9, 11, 33). However, the relative role of beta 1- and beta 2-AR in the cellular response to adrenergic stimuli and whether they have an identical or a different role in increasing ICaL have not been fully clarified in mammalian ventricular myocytes.

Several differences have been observed between beta 1- and beta 2-AR. Their relative abundance is 70-95 and 5-30%, respectively, in the sarcolemma of adult mammalian ventricular myocytes (11, 33). They bind agonists and antagonists with different affinities. NE, the dominant adrenergic neurotransmitter, binds with a greater affinity to beta 1-AR than to beta 2-AR (13, 29, 45). In contrast, Epi (13, 29) and isoproterenol (Iso) bind with similar affinities to both receptor subtypes, whereas zinterol (Zin) binds with a greater affinity to beta 2-AR than to beta 1-AR (24, 25). NE, Epi, and Iso, but not Zin, are full agonists for increasing cAMP and ICaL in mammalian cardiomyocytes (13, 44).

Stimulation of beta 1-AR increases ICaL consistently (21, 26, 34). Variable reports of the effect of beta 2-AR stimulation on ICaL (21, 26, 34, 42) may result from differences among animal species (38, 39), myocytes at various anatomic locations in the heart (1, 7), developmental stages (25), and other less-defined experimental conditions. beta 2-AR is more dominant than beta 1-AR in the frog (38). The coupling and function of beta 2-AR are prominent during neonatal stages in mammalian ventricular myocytes (25), but beta 1-AR is dominant in the adult (2, 9, 21, 25, 26, 34).

A difference in the coupling of beta 1- and beta 2-AR to signal transduction mechanisms has been proposed (4, 10, 15, 40, 41, 43). Pretreatment with pertussis toxin (PTX) enhanced the effect of beta 2-AR stimulation (42), and it has been proposed that this receptor subtype may be coupled to stimulatory as well as inhibitory mechanisms capable of activating or inhibiting cellular effectors (40). Several studies have demonstrated that beta 2-AR stimulation increases ICaL and/or kinetics of contraction in a cAMP-dependent manner (6, 24, 30). One study has observed increased ICaL without increased cAMP accumulation during beta 2-AR stimulation (2). However, others have demonstrated that selective beta 2-AR stimulation does not produce a generalized cAMP accumulation in cardiomyocytes but, rather, is confined in regions localized to subsarcolemmal compartments (44, 46).

Recent studies have demonstrated that a fraction of beta -AR exists in an active state in the absence of cognate agonists (27). Spontaneously active beta 1-AR increases cAMP accumulation and enhance ICaL under baseline conditions (14, 27, 31, 34). Therefore, it is likely that spontaneously active beta 1-AR increases ICaL in the presence of beta 2-AR agonists (34). To investigate this possibility, in this study we used an inverse agonist or "negative antagonist" to inhibit spontaneous and agonist-stimulated activity of beta 1-AR (5, 14, 32) during beta 2-AR stimulation with a "pure agonist."

Several studies have demonstrated that beta 1- and beta 2-AR are coupled to adenylate cyclase (AC) via the alpha -subunit of GTP-binding stimulatory protein (Gsalpha ) and that muscarinic M2 receptors (MR) are coupled via the inhibitory protein alpha -subunit (Gialpha ) (22). It has also been demonstrated that the inhibitory effect of MR activity is accentuated in the presence of beta -AR stimulation. However, it has not been elucidated whether beta 1- and beta 2-AR are acting redundantly as two parallel systems or are cooperatively integrated into a common mechanism of signal transmission between beta -AR subtypes and the L-type Ca2+ channel. Furthermore, it has not been clarified whether the accentuated antagonism exists between MR and beta 1- or beta 2-AR when these beta -AR subtypes are stimulated selectively or jointly in adult mammalian ventricular myocytes.

In this study we investigated the interaction between the effect of selective stimulation of beta 1- and beta 2-AR and stimulation of MR as well as direct stimulation of AC with forskolin (19, 37) on ICaL. We also investigated the effect of beta -AR agonists when the breakdown of cAMP is inhibited by phosphodiesterase (PDE) inhibitors. The results suggest that stimulation of beta 1- and beta 2-AR increases ICaL in a cooperative manner, and MR activity antagonizes predominantly beta 2-AR and, less effectively, beta 1-AR. Selective stimulation of beta 2-AR does not increase ICaL, except when PDE inhibitors are also present. However, stimulation of beta 2-AR enhances the effect of beta 1-AR stimulation. The described effects of beta 1-AR, beta 2-AR, and MR activity are likely converging on the AC.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The animal protocol of this study was approved by the Animal Studies Subcommittee of the Department of Veterans Affairs (Oklahoma City, OK) and conformed with the Guide for the Care and Use of Laboratory Animals (7th ed., Washington, DC, National Academy Press, 1996), approved by the Council of the American Physiological Society (1996).

Cell isolation procedure. Myocytes were isolated as previously reported (34). Briefly, the heart of adult mongrel dogs anesthetized with pentobarbital sodium (30 mg/kg iv) was quickly removed and washed with a cold (10°C) modified buffer solution (BS) in which KCl was increased to 8.0 mM. The left anterior descending branch of the coronary artery was cannulated and perfused sequentially with the following solutions at 5-min intervals: modified BS at 10°C, BS at 37°C, and Ca2+-free BS at 37°C. The heart was then perfused with low-Ca2+ (60 µM) BS containing collagenase (1 mg/ml, type II; Worthington Biochemical, Freehold, NJ) at 37°C for 30 min and finally with BS at 37°C for 5 min. The tissue was then minced into a suspension, filtered with a nylon mesh (200-µm pore size), washed, and resuspended five times in BS. Isolated myocytes were stored in MEM with Hanks' salt at 17°C.

Composition of the solutions. The standard BS contained (in mM) 140.0 NaCl, 4.0 KCl, 1.8 CaCl2, 1.0 MgCl2, 1.0 Na2HPO4, 15.0 HEPES, and 11.0 glucose; pH was adjusted with NaOH to 7.36 at 37°C, and the solution was saturated with 100% O2. BS with Cs+ consisted of (in mM) 100.0 NaCl, 20.0 CsCl, 1.0 CaCl2, 1.0 MgCl2, 15.0 HEPES, 11.0 glucose, and 30.0 tetraethylammonium chloride; pH was adjusted to 7.36 with NaOH at 37°C, and the solution was saturated with 100% O2. Pipette filling solution in voltage-clamp experiments was composed of (in mM) 140.0 CsCl, 4.0 Mg-ATP, 10.0 EGTA, 15.0 HEPES, 0.1 Na2HPO4, and 5.0 glucose; pH was adjusted to 7.36 with CsOH at 37°C. MEM with Hanks' salts (catalog no. 41600-073, GIBCO) was enriched with (in mM) 24.0 NaHCO3, 11.0 glucose, 10.0 taurine, 2.0 pyruvic acid, 5.0 ribose, and 0.1 allopurinol; the solution was equilibrated with 5% CO2-95% O2, and pH was 7.4 at 17°C.

Iso, NE, Epi, Zin, prazosin, carbachol, CGP-20712A (CGP), and ICI-118551 (ICI) were diluted from freshly prepared stock solutions in distilled water also containing 30 µM ascorbic acid and kept at 5°C in the dark. The salts, Iso, NE, Epi, prazosin, and IBMX were purchased from Sigma Chemical (St. Louis, MO); CGP, ICI, and carbachol from Research Biochemicals (Natick, MA); and forskolin from Calbiochem (La Jolla, CA). Zin HCl was kindly provided by Bristol-Myers Squibb Pharmaceutical Research Institute (Princeton, NJ).

Voltage-clamp experiments. A sample of the cell suspension was transferred to a water-jacketed and temperature-controlled (37°C) perfusion chamber (0.5 ml) and superfused with the appropriate buffer solution at 3 ml/min. The perfusion chamber was fixed to the stage of an inverted microscope (Nikon, Melville, NY). After sedimentation, a myocyte having sharp and regular striations, clear contours, and a transparent cytoplasm without granulation and blebs was selected for electrophysiological study. The myocytes were used 3-36 h after isolation. Patch-type glass microelectrodes were pulled from 1.0-mm-OD borosilicate blanks (catalog no. 1B100F-4, World Precision Instruments, Sarasota, FL) by means of a programmable puller (model P-87, Sutter Instruments), having a resistance of 1.5-3.5 MOmega . Whole cell voltage-clamp experiments were performed with an Axopatch-1C unit (Axon Instruments, Foster City, CA) interfaced with a TL-1 analog-to-digital/digital-to-analog converter (Lab Master DMA, Scientific Solutions, OH) to a Pentium personal computer (Gateway 2000) with use of pCLAMP 6.03 software (Axon Instruments). The junction potential between the pipette tip and bath solution was compensated before the start of seal formation. ICaL was activated by step depolarization from a holding level of -40 mV to between -40 and +40 mV or between -20 and +5 mV in 5-mV increments for 300 ms, repeated at 5-s intervals. Peak ICaL was determined as the difference between the peak inward current and the current at the end of each 300-ms step depolarization. The greatest peak ICaL obtained (usually at a step depolarization to 0 ± 5 mV in controls) is referred to as ICaL (see Fig. 2, B and C). K+ currents were inhibited by using Cs+ and tetraethylammonium chloride. The concentration of Cl- in the pipette approximated that in the extracellular solution; therefore, Cl- current was assumed to be negligible at membrane potentials at which maximal ICaL was determined (Emax). The inward current observed under these conditions was fully inhibited by >= 0.3 mM CdCl3 and by 3.0 µM D-600, and it is considered ICaL.

A rundown in ICaL has been observed during the initial 5 min of whole cell voltage clamp. In our experiments under control conditions the current was relatively stable during the 5-30 min after rupture of the cell membrane in the patch under the microelectrode. In untreated myocytes the difference was <= 5% in ICaL during the 5-30 min after the microelectrode penetration. Therefore, all the tests were performed during this window of time. The drug effects were tested at steady state, generally at the 5th min of treatment if not otherwise stated. The concentration-ICaL relationship was determined in a noncumulative manner, each data point was obtained in a different myocyte, and data were accepted only if the effect was fully reversible during the washout of drugs.

Statistical analysis. For statistical analysis we used Jandel Scientific Software Sigmastat 5.1. The data were analyzed by one-way ANOVA. Values are means ± SE. Differences are accepted to be significant when P < 0.05. We used Prism 3.0 (GraphPad Software) for the concentration-ICaL relationship nonlinear regression analysis. The best fit was achieved by monophasic and biphasic logistic equations. The program compared the fits of the two equations by performing an F test and by calculating the P value (significance of better fit). We set the level of significance at P = 0.05. The best fit was usually achieved within 25 iterations.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Concentration-dependent effect of adrenergic agonists on ICaL in the presence and absence of ICI. To determine the relative role of beta 1- and beta 2-AR in the effect of adrenergic agonists on ICaL, we established the concentration-ICaL relationship in a noncumulative manner of agonists that are nonselective (Iso), predominantly selective for beta 1-AR (NE), and predominantly selective for beta 2-AR (Zin). The nonlinear regression analysis of the data produced a monophasic sigmoidal function for Iso and a biphasic function for NE (Fig. 1). The maximal effect (Emax) of Iso and NE was similar (Emax = 335 ± 15 and 318 ± 14% of baseline, respectively, n = 6). The concentration-ICaL relationship of Zin was profoundly different from that of Iso or NE, exhibiting a relatively small increase and only at relatively high concentrations. At lower concentrations acting selectively on beta 2-AR, Zin did not increase ICaL. In the presence of 50 nM Zin, ICaL was 105 ± 2% of the baseline in 40 myocytes; the difference was statistically not significant.


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  		Fig. 1.   Noncumulative concentration-L-type Ca2+ current (ICaL) relationship for isoproterenol (Iso), l-norepinephrine (NE), NE + ICI-118551 (ICI), and zinterol (Zin). Values are means ± SE in 3-6 myocytes. Parameters of the concentration-ICaL relationship for Iso were as follows: EC50 = 3.0 nM and maximum effect (Emax) = 335% of control. For NE there was a biphasic relationship: EC(1)50 and EC(2)50 = 11 and 380 nM, respectively, and E(1)max and E(2)max = 170 and 311% of control, respectively. Parameters for Zin were as follows: EC50 = 506 nM and Emax = 149 ± 11% of control. Parameters of "goodness of fit" and "comparison of fits" for the concentration-ICaL relationship of NE were as follows: R2 = 0.9976, absolute sum of squares = 133.9, F = 10.47, and P = 0.0318.

When NE was applied in the presence of ICI and in other myocytes in the presence of ICI and Zin, the second phase of the concentration-ICaL relationship of NE was virtually abolished, and Emax was reduced by ~60%. The Emax of Iso, observed at 20 nM, was reduced by ICI in a similar manner. In contrast, beta 1-AR blockade by CGP (300 nM) fully abolished the effect of 20 nM Iso (Table 1).

                              
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  		Table 1.   Effects of adrenergic agonists on ICaL

Concentration-ICaL relationship of NE in the presence of prazosin and Zin. The concentration-ICaL relationship of NE was shifted to the left by prazosin (1.0 µM), but there was no change in Emax or in the biphasic character of the curve (Fig. 2). When NE and prazosin were applied in the presence of Zin (50 nM), the concentration-ICaL relationship was shifted further leftward with no increase in Emax. However, the curve was altered from biphasic to monophasic, resembling that of Iso. Prazosin or prazosin + Zin increased ICaL most conspicuously at 50-100 nM NE (Table 1). In five myocytes, Zin (50 nM) was applied after the effect of NE and prazosin had developed, and under these conditions, Zin had no effect on ICaL. Representative traces of (maximal) ICaL recorded under control conditions, in the presence of NE + prazosin in Zin-pretreated and nonpretreated myocytes, are shown in Fig. 2B. The voltage-current relationship of peak ICaL under the same conditions is shown in Fig. 2C.


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  		Fig. 2.   A: effect of Zin (50 nM) on the noncumulative concentration-ICaL relationship of NE in the presence of prazosin (Praz, 1.0 µM). For comparison, the concentration-ICaL relationship of NE is shown in the absence of other drugs. Treatment with Zin was started 2 min before NE + Praz. Values are means ± SE in 3-6 myocytes. Parameters of the concentration-ICaL relationship of NE + Praz were as follows: EC(1)50 and EC(2)50 = 12 and 323 nM, respectively, and E(1)max and E(2)max = 235 and 325% of control, respectively. Parameters of goodness of fit and comparison of fits for the same concentration-ICaL relationship were as follows: R2 = 0.9946, absolute sum of squares = 274.3, and P < 0.05. Parameters of the concentration-ICaL relationship of NE + Praz in the presence of Zin were as follows: EC50 = 15 nM and Emax = 310% of control. Parameters of goodness of fit and comparison of fits for the same concentration-ICaL relationship were as follows: R2 = 0.9900, absolute sum of squares = 457.3, and P < 0.05. B: ICaL in the 5th min of NE + Praz treatment. Representative superimposed traces of ICaL are as follows: control ICaL (a), ICaL in the 5th min of NE (100 nM) treatment in the presence of Praz (b), and ICaL in the 5th min of NE (100 nM) + Praz applied in the 2nd min of Zin (50 nM) treatment (c). C: current-voltage relationship of ICaL in control (a), in the 5th min of NE (100 nM) + Praz (1 µM) treatment (b), and in the 5th min of NE (100 nM) + Praz applied in the 2nd min of Zin (50 nM) treatment (n = 6, P < 0.0005, with vs. without Zin). Em, membrane potential.

Enhancement of the effect of NE by Epi and antagonism by carbachol. Epi (10 nM) that was subthreshold for increasing ICaL increased the effect of a superimposed treatment with NE (100 nM) ~1.6-fold (Fig. 3). When Epi was applied to five myocytes after the effect of NE had developed, it did not increase ICaL further (data not shown).


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  		Fig. 3.   Effect of NE (100 nM) on ICaL in the presence and absence of l-epinephrine (Epi, 10 nM) and/or carbachol (CC, 100 nM). Data were obtained in 8 myocytes for NE and Epi, in 5 myocytes for NE + Epi, and in 4 myocytes for NE + CC and NE + Epi + CC.

The effect of NE (100 nM) was antagonized by carbachol (100 nM). However, the decrease in ICaL was relatively small (<10%). In contrast, carbachol abolished the enhancing effect of Epi, as shown by the difference between ICaL in the presence of Epi and NE with or without carbachol (Fig. 3). The effects of Epi and carbachol on NE stimulation of ICaL are summarized in Table 2.

                              
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  		Table 2.   Effects of Epi and carbachol on NE stimulation of ICaL

Enhancement of the effect of forskolin and IBMX by Zin. Forskolin (100 nM) increased ICaL ~1.8-fold more in the presence than in the absence of Zin (Fig. 4). Similarly, IBMX (10 µM) increased ICaL ~2.2-fold more in the presence than in the absence of Zin (Fig. 5). In contrast to the enhancement of the NE effect by Zin, which was observed when Zin was applied before but not after NE, the effect of IBMX was enhanced by Zin regardless of the sequence of drug application. The increase by Zin, which had no effect alone, was 2.15-fold greater in the presence of IBMX compared with a 1.21-fold increase caused by IBMX alone.


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  		Fig. 4.   Effect of forskolin (100 nM) in the presence and absence of Zin (50 nM) on ICaL. Data were obtained in 4 myocytes in each condition.



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  		Fig. 5.   Effect of Zin (50 nM) on ICaL in the presence and absence of IBMX (10 µM). Each curve is representative of experiments in 4 myocytes with similar results.

The effect of Zin on ICaL developed in the presence of IBMX at a slow rate. To determine whether Zin increases ICaL independently of beta 1-AR activity or enhances the baseline activity of nonstimulated beta 1-AR, we investigated the effect of IBMX and Zin in the presence of CGP, an inverse agonist on beta 1-AR. In the presence of CGP (300 nM), the effect of Zin + IBMX was inhibited (Fig. 6). The effects of Zin, IBMX, and CGP are summarized in Table 3.


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  		Fig. 6.   Effect of CGP-20712A (CGP, 300 nM) on ICaL in the presence of Zin (50 nM) and IBMX (10 µM). Each curve is representative of experiments in 4 myocytes with similar results. WO, washout of the drug.


                              
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  		Table 3.   Effects of Zin, IBMX, and CGP on ICaL


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

beta 1- and beta 2-AR coexist in the mammalian ventricular myocardium (9, 11, 33). This study demonstrates that these beta -AR subtypes are functionally integrated into a common signaling system acting cooperatively in enhancing ICaL when both are stimulated by the physiological catecholamines Epi and NE in ventricular myocytes of the dog. The observation that selective stimulation of beta 2-AR when spontaneous and agonist-stimulated beta 1-AR activity is inhibited does not increase ICaL in mammalian ventricular myocytes confirms earlier reports (21, 26, 34, 40). However, a new finding of this study is that preceding and coinciding beta 2-AR stimulation enhances the effect of spontaneous and pharmacologically stimulated beta 1-AR activity on ICaL. The enhancement is not due to a summation of two effects via beta 1- and beta 2-AR but, rather, to beta 2-AR conditioning of the signal transduction mechanism from beta 1-AR to ICaL. This conditioning by beta 2-AR is shown by the absence of an increase in ICaL when beta 2-AR is stimulated alone or when beta 2-AR is stimulated after beta 1-AR stimulation has developed. The dependence of beta 2-AR stimulation on beta 1-AR activation indicates a cooperative relationship between beta 1- and beta 2-AR and supports their different roles in adrenergic enhancement of ICaL rather than their function as parallel redundant systems.

This and other studies (2, 21, 26, 34, 40, 43) have demonstrated that selective pharmacological stimulation of beta 1-AR produces a different response from that of beta 2-AR. Selective stimulation of beta 1-AR consistently increases AC, ICaL, and contractility in cardiac myocytes (9, 21, 26, 34, 40). The effects of beta 2-AR stimulation are different in different animal species (39), age-related developmental stages (25), anatomic locations in the heart (1, 28), and other less-defined conditions.

Spontaneously active beta 1-AR contributes to baseline activity of AC and ICaL, which is inhibited by CGP (34), a highly selective inverse agonist on beta 1-AR, or atenolol, but not ICI (31). In this study we investigated the interaction between beta 2-AR stimulation and spontaneously active beta 1-AR in the presence of PDE inhibitors. Under these conditions, cAMP accumulation and ICaL are increased (35, 36), and in the present experiments, beta 2-AR stimulation had an additional increasing effect on ICaL. The increasing effect of PDE inhibition with IBMX and beta 2-AR stimulation on ICaL was inhibited by CGP, demonstrating the role of beta 1-AR; therefore, we conclude that beta 2-AR is not acting independently of beta 1-AR.

In the absence of PDE inhibitors, the uninhibited cAMP degradation could offset the beta 2-AR-mediated augmentation of the ICaL response to spontaneously active beta 1-AR. PDE inhibition rescued the effect beta 2-AR stimulation on ICaL. The slowly increasing ICaL in the presence of beta 2-AR stimulation may reflect a slow accumulation of cAMP when no beta 1-AR agonists are present. These observations are consistent with an earlier demonstration of beta 2-AR acting on ICaL via the cAMP-dependent signaling pathway (40).

Several studies have demonstrated that beta 2-AR is coupled to ICaL and other effectors via a cAMP/protein kinase A-dependent pathway (40). In this study, beta 2-AR stimulation enhanced the ICaL response to direct stimulation of AC by forskolin, similar to the response to beta 1-AR stimulation. It demonstrates that beta 2-AR stimulation increases the response of AC to other stimuli. The enhancement of the AC response by beta 2-AR stimulation may explain the findings by Bristow et al. (6), who found that when beta 1- and beta 2-AR are stimulated with Iso in human ventricular myocardium, the majority of the AC response is related to beta 2-AR. It is contrary to the smaller abundance of beta 2-AR relative to beta 1-AR (beta 2-AR/beta 1-AR = 19/81%) (5, 6, 9, 33) and the finding that stimulation of beta 2-AR alone does not increase cAMP accumulation (2, 26, 40). When beta 1- and beta 2-AR were maximally stimulated with NE in our experiments, 60% of the maximal ICaL response was related to beta 2-AR activity. This indicates the similarity between the AC and ICaL responses to beta 2-AR stimulation. In earlier studies, the concentration-response relationship for stimulation of AC and cAMP accumulation in human atrium and ventricle was biphasic for NE and monophasic for Epi (13). In our study the concentration-ICaL relationship of NE was strikingly similar to that of NE for AC stimulation described by others (13). This similarity between the effects of NE on AC and on ICaL suggests that the change in ICaL reflects the change in cAMP accumulation in a cellular compartment associated with the L-type Ca2+ channel.

The biphasic character of the concentration-ICaL curve of NE is consistent with NE acting on two types of receptors with different affinities. The affinity of NE is greater for beta 1-AR than for beta 2-AR (29). At <= 100 nM the effect of NE was not inhibited by beta 2-AR inhibition with ICI, indicating no role for beta 2-AR in this effect. However, at maximally effective concentrations of NE, 60% of the ICaL response was inhibited by ICI, indicating the magnitude of beta 2-AR enhancement of the ICaL response to beta 1-AR stimulation. Inhibition of beta 1-AR with CGP abolished the ICaL response at all concentrations of NE, indicating that beta 2-AR stimulation had no effect on ICaL independent of beta 1-AR stimulation.

Similar to our data in the dog, Xiao et al. (40) observed no increase in cAMP and ICaL and other effector responses during beta 2-AR stimulation in the presence of CGP in wild-type and transgenic (TG4) mice overexpressing human beta 2-AR. However, they observed that beta 2-AR stimulation increases [gamma -32P]GTP incorporation in stimulatory Gsalpha and inhibitory Gialpha 2-3 proteins. Inhibition of Gi proteins with PTX rescued the stimulatory effects of beta 2-AR stimulation on cAMP, ICaL, and other effectors. They postulated that beta 2-AR is coupled to Gs and Gi proteins and that the concurrently activated stimulatory and inhibitory mechanisms produce a zero net effect in the mouse or limit the positive effect of beta 2-AR stimulation to a subsarcolemmal compartment in the rat (42, 46).

There are no data demonstrating that beta 2-AR stimulation is coupled to Gi in canine ventricular myocytes. In the sinoatrial node, atrial tissues and specialized conducting system beta 2-AR agonists produce stimulatory effects in the absence of PTX in different mammalian species (1, 7, 24). Similarly, in the present study, beta 2-AR stimulation enhanced the ICaL response to beta 1-AR stimulation in the absence of PTX. However, in the absence of beta 1-AR stimulation, beta 2-AR stimulation did not increase ICaL in our experiments and did not increase cAMP accumulation in the experiments by Altschuld et al. (2) in the dog, and there was only a small increase in adult rat ventricular myocytes (26). Therefore, the absence of an ICaL response to beta 2-AR stimulation can be explained by the absence of an AC response.

The absence of a direct effect of beta 2-AR stimulation on AC and ICaL and the enhancement of the ICaL response to beta 1-AR by beta 2-AR stimulation may be explained by several mechanisms. beta 1- and beta 2-AR could interact at the receptor level. Receptor dimerization has been shown to increase the efficacy of beta -AR coupling to Gs proteins and to increase the AC response (20). However, beta 2-AR stimulation augmented the effect of forskolin that directly activates AC (12, 17). Therefore, beta 1- and beta 2-AR are more likely to converge on AC.

It has been demonstrated that agonist-activated MR inhibit beta -AR stimulation of cAMP accumulation and ICaL by activating Gialpha , which inhibits beta -AR activation of AC (18, 19, 22). We have found that the antagonism between beta -AR and MR stimulation with carbachol occurred predominantly between beta 2-AR and MR, the ICaL response to beta 1-AR stimulation being only marginally reduced. Other studies have demonstrated that the signals of beta -AR and MR merge on AC via Gsalpha and Gialpha , respectively (17, 18, 22). Two kinetically different Gsalpha - binding sites have been demonstrated on mammalian cardiac type VI AC, and these sites interact with each other in a cooperative manner (8, 16, 17). It has also been demonstrated that beta 1- and beta 2-AR exhibit a different susceptibility to muscarinic antagonism in neonatal rat ventricular myocytes (3). However, it has not been clarified whether beta 1- and beta 2-AR-activated Gsalpha - subunits bind to different sites and produce different effects on AC. Our observation that the effect of beta 2-AR stimulation can be completely abolished by stimulating MR, whereas the effect of beta 1-AR without stimulation of beta 2-AR is only minimally reduced, raises the possibility that the muscarinic activity is in a more specific antagonism with the beta 2-AR than the beta 1-AR effect on AC in canine ventricular myocytes. It is possible that beta 2-AR and MR modulate the sensitivity of AC to beta 1-AR activity, although in an opposite direction (22).

Recently, it has been demonstrated that beta 2-AR, but not beta 1-AR, stimulation causes Gbeta gamma translocation from cytosolic to sarcolemmal compartments in the rat heart (23). These data demonstrate a different binding between beta -AR subtypes and G proteins. In mouse ventricular myocytes, beta 2-AR, but not beta 1-AR, was shown to activate Gialpha 2-3 (40). This also demonstrates a different coupling between beta -AR subtypes and G proteins. In our experiments, beta 2-AR stimulation enhanced the ICaL response to beta 1-AR when beta 2-AR stimulation preceded and/or coincided with beta 1-AR stimulation but had no effect when applied after the effect of beta 1-AR stimulation had developed or when applied in the absence of beta 1-AR activity. From this we may conclude that beta 2-AR stimulation does not produce an active conformation of AC, but it preconditions AC for beta 1-AR-mediated signals and direct stimulation with forskolin.

The molecular mechanism of beta 2-AR-mediated enhancement of the ICaL response, and possibly the AC response, to beta 1-AR stimulation in the ventricular myocardium requires further studies. Cardiac type V and VI AC isoforms have been shown to be inhibited and PDEs enhanced by Ca2+. Therefore, the ICaL response to stimulation of beta -AR subtypes could be affected by buffering intracellular Ca2+ with EGTA in our experiments. However, it is unlikely that the functional interaction between beta -AR subtypes observed in this study is related to particular conditions of our experiments. The observed interaction between beta 1- and beta 2-AR-linked responses may have a great physiological relevance, because the stimulation of beta 2-AR by a low concentration of Epi enhances the magnitude of the effect of NE on ICaL by more than twofold and the interaction between beta 1- and beta 2-AR effects on AC increases the range of agonist concentration by one or two orders of magnitude in which the system differentially responds, as shown in the agonist concentration-ICaL response curves.


    ACKNOWLEDGEMENTS

The authors appreciate the excellent technical assistance of Eva Szabo.


    FOOTNOTES

This study was supported in part by Merit Review Grants from the Veterans Affairs Medical Center. Other support was provided by Dr. William Talley III, David and Barbara Green, and Paul and Doris Travis.

Address for reprint requests and other correspondence: B. Szabo, Dept. of Veterans Affairs Medical Center (151F), 921 NE 13th St., Oklahoma City, OK 73104 (E-mail: Bela-Szabo{at}ouhsc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 November 1999; accepted in final form 9 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 279(3):H1329-H1337


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