We investigated the roles of β1- and β2-receptors (β-AR) in adrenergic enhancement of L-type Ca2+ current (I CaL) in canine ventricular myocytes. Isoproterenol and l-norepinephrine produced a monophasic and a biphasic concentration-I CaL relationship (CR), respectively. α1-AR inhibition with prazosin and β2-AR stimulation with zinterol orl-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 afterl-norepinephrine had no effect onI CaL. β2-AR inhibition with ICI-118551 reduced the E max of isoproterenol andl-norepinephrine by 60% and abolished the augmentation ofl-norepinephrine by zinterol and l-epinephrine. Carbachol (100 nM) modestly reduced the I CaLresponse to β1-AR stimulation but abolished the enhancement via β2-AR. Zinterol augmented the enhancement of I CaL by forskolin, IBMX, and theophylline, but not in the presence of CGP-20712A. We conclude that selective β2-AR stimulation does not increaseI CaL but enhances adenylyl cyclase activity when stimulated via β1-AR and with forskolin. β2-AR activity preconditions adenylyl cyclase for β1-AR stimulation.
- adenylyl cyclase
physiological catecholamines, l-norepinephrine (NE) andl-epinephrine (Epi), increase cAMP accumulation and cAMP-dependent phosphorylation of the L-type Ca2+ channel and the current through it (I CaL) by activating β-adrenoceptors (β-AR) (18). Earlier studies demonstrated expression of several β-AR subtypes in the heart (9, 11, 33). However, the relative role of β1- and β2-AR in the cellular response to adrenergic stimuli and whether they have an identical or a different role in increasing I CaLhave not been fully clarified in mammalian ventricular myocytes.
Several differences have been observed between β1- and β2-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 β1-AR than to β2-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 β2-AR than to β1-AR (24, 25). NE, Epi, and Iso, but not Zin, are full agonists for increasing cAMP and I CaL in mammalian cardiomyocytes (13, 44).
Stimulation of β1-AR increasesI CaL consistently (21,26, 34). Variable reports of the effect of β2-AR stimulation on I CaL(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. β2-AR is more dominant than β1-AR in the frog (38). The coupling and function of β2-AR are prominent during neonatal stages in mammalian ventricular myocytes (25), but β1-AR is dominant in the adult (2, 9, 21,25, 26, 34).
A difference in the coupling of β1- and β2-AR to signal transduction mechanisms has been proposed (4, 10, 15, 40,41, 43). Pretreatment with pertussis toxin (PTX) enhanced the effect of β2-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 β2-AR stimulation increases I CaL and/or kinetics of contraction in a cAMP-dependent manner (6, 24,30). One study has observed increasedI CaL without increased cAMP accumulation during β2-AR stimulation (2). However, others have demonstrated that selective β2-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 β-AR exists in an active state in the absence of cognate agonists (27). Spontaneously active β1-AR increases cAMP accumulation and enhance I CaL under baseline conditions (14, 27, 31, 34). Therefore, it is likely that spontaneously active β1-AR increases I CaL in the presence of β2-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 β1-AR (5, 14, 32) during β2-AR stimulation with a “pure agonist.”
Several studies have demonstrated that β1- and β2-AR are coupled to adenylate cyclase (AC) via the α-subunit of GTP-binding stimulatory protein (Gsα) and that muscarinic M2 receptors (MR) are coupled via the inhibitory protein α-subunit (Giα) (22). It has also been demonstrated that the inhibitory effect of MR activity is accentuated in the presence of β-AR stimulation. However, it has not been elucidated whether β1- and β2-AR are acting redundantly as two parallel systems or are cooperatively integrated into a common mechanism of signal transmission between β-AR subtypes and the L-type Ca2+ channel. Furthermore, it has not been clarified whether the accentuated antagonism exists between MR and β1- or β2-AR when these β-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 β1- and β2-AR and stimulation of MR as well as direct stimulation of AC with forskolin (19, 37) on I CaL. We also investigated the effect of β-AR agonists when the breakdown of cAMP is inhibited by phosphodiesterase (PDE) inhibitors. The results suggest that stimulation of β1- and β2-AR increases I CaL in a cooperative manner, and MR activity antagonizes predominantly β2-AR and, less effectively, β1-AR. Selective stimulation of β2-AR does not increase I CaL, except when PDE inhibitors are also present. However, stimulation of β2-AR enhances the effect of β1-AR stimulation. The described effects of β1-AR, β2-AR, and MR activity are likely converging on the AC.
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).
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 MΩ. 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.I CaL 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. PeakI CaL 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 I CaL obtained (usually at a step depolarization to 0 ± 5 mV in controls) is referred to as I CaL (see Fig. 2, Band 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 maximalI CaL was determined (E max). 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 I CaL.
A rundown in I CaL 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% inI CaL 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-I CaL 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.
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-I CaL 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 theP value (significance of better fit). We set the level of significance at P = 0.05. The best fit was usually achieved within 25 iterations.
Concentration-dependent effect of adrenergic agonists on ICaL in the presence and absence of ICI.
To determine the relative role of β1- and β2-AR in the effect of adrenergic agonists onI CaL, we established the concentration-I CaL relationship in a noncumulative manner of agonists that are nonselective (Iso), predominantly selective for β1-AR (NE), and predominantly selective for β2-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 (E max) of Iso and NE was similar (E max = 335 ± 15 and 318 ± 14% of baseline, respectively, n = 6). The concentration-I CaL 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 β2-AR, Zin did not increase I CaL. In the presence of 50 nM Zin,I CaL was 105 ± 2% of the baseline in 40 myocytes; the difference was statistically not significant.
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-I CaL relationship of NE was virtually abolished, and E max was reduced by ∼60%. The E max of Iso, observed at 20 nM, was reduced by ICI in a similar manner. In contrast, β1-AR blockade by CGP (300 nM) fully abolished the effect of 20 nM Iso (Table1).
Concentration-ICaL relationship of NE in the presence of prazosin and Zin.
The concentration-I CaL relationship of NE was shifted to the left by prazosin (1.0 μM), but there was no change inE max 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-I CaL relationship was shifted further leftward with no increase in E max. However, the curve was altered from biphasic to monophasic, resembling that of Iso. Prazosin or prazosin + Zin increasedI CaL 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 I CaL. Representative traces of (maximal) I CaL recorded under control conditions, in the presence of NE + prazosin in Zin-pretreated and nonpretreated myocytes, are shown in Fig. 2 B. The voltage-current relationship of peak I CaL under the same conditions is shown in Fig. 2 C.
Enhancement of the effect of NE by Epi and antagonism by carbachol.
Epi (10 nM) that was subthreshold for increasingI CaL 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 increaseI CaL further (data not shown).
The effect of NE (100 nM) was antagonized by carbachol (100 nM). However, the decrease in I CaL was relatively small (<10%). In contrast, carbachol abolished the enhancing effect of Epi, as shown by the difference between I CaLin the presence of Epi and NE with or without carbachol (Fig. 3). The effects of Epi and carbachol on NE stimulation ofI CaL are summarized in Table2.
Enhancement of the effect of forskolin and IBMX by Zin.
Forskolin (100 nM) increased I CaL ∼1.8-fold more in the presence than in the absence of Zin (Fig.4). Similarly, IBMX (10 μM) increasedI CaL ∼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.
The effect of Zin on I CaL developed in the presence of IBMX at a slow rate. To determine whether Zin increasesI CaL independently of β1-AR activity or enhances the baseline activity of nonstimulated β1-AR, we investigated the effect of IBMX and Zin in the presence of CGP, an inverse agonist on β1-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.
β1- and β2-AR coexist in the mammalian ventricular myocardium (9, 11,33). This study demonstrates that these β-AR subtypes are functionally integrated into a common signaling system acting cooperatively in enhancing I CaL when both are stimulated by the physiological catecholamines Epi and NE in ventricular myocytes of the dog. The observation that selective stimulation of β2-AR when spontaneous and agonist-stimulated β1-AR activity is inhibited does not increase I CaL in mammalian ventricular myocytes confirms earlier reports (21, 26,34, 40). However, a new finding of this study is that preceding and coinciding β2-AR stimulation enhances the effect of spontaneous and pharmacologically stimulated β1-AR activity on I CaL. The enhancement is not due to a summation of two effects via β1- and β2-AR but, rather, to β2-AR conditioning of the signal transduction mechanism from β1-AR to I CaL. This conditioning by β2-AR is shown by the absence of an increase in I CaL when β2-AR is stimulated alone or when β2-AR is stimulated after β1-AR stimulation has developed. The dependence of β2-AR stimulation on β1-AR activation indicates a cooperative relationship between β1- and β2-AR and supports their different roles in adrenergic enhancement of I CaL 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 β1-AR produces a different response from that of β2-AR. Selective stimulation of β1-AR consistently increases AC, I CaL, and contractility in cardiac myocytes (9, 21,26, 34, 40). The effects of β2-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 β1-AR contributes to baseline activity of AC and I CaL, which is inhibited by CGP (34), a highly selective inverse agonist on β1-AR, or atenolol, but not ICI (31). In this study we investigated the interaction between β2-AR stimulation and spontaneously active β1-AR in the presence of PDE inhibitors. Under these conditions, cAMP accumulation and I CaL are increased (35,36), and in the present experiments, β2-AR stimulation had an additional increasing effect onI CaL. The increasing effect of PDE inhibition with IBMX and β2-AR stimulation onI CaL was inhibited by CGP, demonstrating the role of β1-AR; therefore, we conclude that β2-AR is not acting independently of β1-AR.
In the absence of PDE inhibitors, the uninhibited cAMP degradation could offset the β2-AR-mediated augmentation of theI CaL response to spontaneously active β1-AR. PDE inhibition rescued the effect β2-AR stimulation on I CaL. The slowly increasing I CaL in the presence of β2-AR stimulation may reflect a slow accumulation of cAMP when no β1-AR agonists are present. These observations are consistent with an earlier demonstration of β2-AR acting on I CaL via the cAMP-dependent signaling pathway (40).
Several studies have demonstrated that β2-AR is coupled to I CaL and other effectors via a cAMP/protein kinase A-dependent pathway (40). In this study, β2-AR stimulation enhanced theI CaL response to direct stimulation of AC by forskolin, similar to the response to β1-AR stimulation. It demonstrates that β2-AR stimulation increases the response of AC to other stimuli. The enhancement of the AC response by β2-AR stimulation may explain the findings by Bristow et al. (6), who found that when β1- and β2-AR are stimulated with Iso in human ventricular myocardium, the majority of the AC response is related to β2-AR. It is contrary to the smaller abundance of β2-AR relative to β1-AR (β2-AR/β1-AR = 19/81%) (5, 6, 9, 33) and the finding that stimulation of β2-AR alone does not increase cAMP accumulation (2, 26,40). When β1- and β2-AR were maximally stimulated with NE in our experiments, 60% of the maximalI CaL response was related to β2-AR activity. This indicates the similarity between the AC andI CaL responses to β2-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-I CaL 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 I CaL suggests that the change inI CaL reflects the change in cAMP accumulation in a cellular compartment associated with the L-type Ca2+channel.
The biphasic character of the concentration-I CaLcurve of NE is consistent with NE acting on two types of receptors with different affinities. The affinity of NE is greater for β1-AR than for β2-AR (29). At ≤100 nM the effect of NE was not inhibited by β2-AR inhibition with ICI, indicating no role for β2-AR in this effect. However, at maximally effective concentrations of NE, 60% of the I CaL response was inhibited by ICI, indicating the magnitude of β2-AR enhancement of theI CaL response to β1-AR stimulation. Inhibition of β1-AR with CGP abolished theI CaL response at all concentrations of NE, indicating that β2-AR stimulation had no effect onI CaL independent of β1-AR stimulation.
Similar to our data in the dog, Xiao et al. (40) observed no increase in cAMP and I CaL and other effector responses during β2-AR stimulation in the presence of CGP in wild-type and transgenic (TG4) mice overexpressing human β2-AR. However, they observed that β2-AR stimulation increases [γ-32P]GTP incorporation in stimulatory Gsα and inhibitory Giα2–3 proteins. Inhibition of Gi proteins with PTX rescued the stimulatory effects of β2-AR stimulation on cAMP, I CaL, and other effectors. They postulated that β2-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 β2-AR stimulation to a subsarcolemmal compartment in the rat (42, 46).
There are no data demonstrating that β2-AR stimulation is coupled to Gi in canine ventricular myocytes. In the sinoatrial node, atrial tissues and specialized conducting system β2-AR agonists produce stimulatory effects in the absence of PTX in different mammalian species (1, 7,24). Similarly, in the present study, β2-AR stimulation enhanced the I CaL response to β1-AR stimulation in the absence of PTX. However, in the absence of β1-AR stimulation, β2-AR stimulation did not increase I CaL 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 I CaL response to β2-AR stimulation can be explained by the absence of an AC response.
The absence of a direct effect of β2-AR stimulation on AC and I CaL and the enhancement of theI CaL response to β1-AR by β2-AR stimulation may be explained by several mechanisms. β1- and β2-AR could interact at the receptor level. Receptor dimerization has been shown to increase the efficacy of β-AR coupling to Gs proteins and to increase the AC response (20). However, β2-AR stimulation augmented the effect of forskolin that directly activates AC (12, 17). Therefore, β1- and β2-AR are more likely to converge on AC.
It has been demonstrated that agonist-activated MR inhibit β-AR stimulation of cAMP accumulation and I CaL by activating Giα, which inhibits β-AR activation of AC (18, 19, 22). We have found that the antagonism between β-AR and MR stimulation with carbachol occurred predominantly between β2-AR and MR, theI CaL response to β1-AR stimulation being only marginally reduced. Other studies have demonstrated that the signals of β-AR and MR merge on AC via Gsα and Giα, respectively (17, 18,22). Two kinetically different Gsα- 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 β1- and β2-AR exhibit a different susceptibility to muscarinic antagonism in neonatal rat ventricular myocytes (3). However, it has not been clarified whether β1- and β2-AR-activated Gsα- subunits bind to different sites and produce different effects on AC. Our observation that the effect of β2-AR stimulation can be completely abolished by stimulating MR, whereas the effect of β1-AR without stimulation of β2-AR is only minimally reduced, raises the possibility that the muscarinic activity is in a more specific antagonism with the β2-AR than the β1-AR effect on AC in canine ventricular myocytes. It is possible that β2-AR and MR modulate the sensitivity of AC to β1-AR activity, although in an opposite direction (22).
Recently, it has been demonstrated that β2-AR, but not β1-AR, stimulation causes Gβγtranslocation from cytosolic to sarcolemmal compartments in the rat heart (23). These data demonstrate a different binding between β-AR subtypes and G proteins. In mouse ventricular myocytes, β2-AR, but not β1-AR, was shown to activate Giα2–3 (40). This also demonstrates a different coupling between β-AR subtypes and G proteins. In our experiments, β2-AR stimulation enhanced the I CaL response to β1-AR when β2-AR stimulation preceded and/or coincided with β1-AR stimulation but had no effect when applied after the effect of β1-AR stimulation had developed or when applied in the absence of β1-AR activity. From this we may conclude that β2-AR stimulation does not produce an active conformation of AC, but it preconditions AC for β1-AR-mediated signals and direct stimulation with forskolin.
The molecular mechanism of β2-AR-mediated enhancement of the I CaL response, and possibly the AC response, to β1-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 I CaL response to stimulation of β-AR subtypes could be affected by buffering intracellular Ca2+with EGTA in our experiments. However, it is unlikely that the functional interaction between β-AR subtypes observed in this study is related to particular conditions of our experiments. The observed interaction between β1- and β2-AR-linked responses may have a great physiological relevance, because the stimulation of β2-AR by a low concentration of Epi enhances the magnitude of the effect of NE onI CaL by more than twofold and the interaction between β1- and β2-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-I CaL response curves.
The authors appreciate the excellent technical assistance of Eva Szabo.
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:).
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.
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