Introduction

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Beta-1 and beta-2 adrenergic receptors coexist in the heart of various animal species, including man. Both receptors are positively coupled to the adenylyl cyclase system and participate in the mediation of the positive chronotropic and inotropic effects of catecholamines (for reviews, see Stiles et al., 1991). However, the relative amount of each receptor subtype as well as the postreceptor cellular signaling pathways may differ significantly depending on the cardiac tissue, the animal species, the pathophysiological state, the age or the developmental stage (for reviews see Stiles et al., 1984; Brodde, 1991; 1993; Hieble and Ruffolo, 1991 and refs therein). Competitive radioligand binding studies performed in membranes from homogenized hearts have shown that only 20 to 30% of the total beta adrenergic receptors are of the beta-2 subtype in adult mammalian ventricular tissue (Stiles et al., 1984; Brodde, 1991; Hieble and Ruffolo, 1991). This number is even further reduced when purified cardiac myocytes rather than homogenized tissues are used (Freissmuth et al., 1986; Lau et al., 1980; Buxton and Brunton, 1985; Kuznetsov et al., 1995; Cerbai et al., 1995). Yet, selective activation of beta-2 adrenergic receptors produces a large increase in the amplitude of contraction in intact mammalian cardiac muscle (Cerbai et al., 1990; Lemoine and Kaumann, 1991; Brodde, 1991) as well as in isolated ventricular myocytes (del Monte et al., 1993; Xiao and Lakatta, 1993; Xiao et al., 1994; 1995; Altschuld et al., 1995; Kuznetsov et al., 1995). When compared to the effect produced by nonselective beta adrenergic receptor agonists such as isoprenaline, the beta 2-response may represent 25 to 100% of the isoprenaline response (Xiao and Lakatta, 1993; Altschuld et al., 1995). This suggests that the two receptors may differ in their signaling cascade or in the post-receptor amplification mechanisms. In that regard, beta-2 adrenergic receptors were shown to be more tightly coupled to the adenylyl cyclase system than beta-1 receptors (Waelbroeck et al., 1983; Bristow et al., 1989; Green et al., 1992; Levy et al., 1993). Surprisingly, however, the positive inotropic effect mediated by a beta-2 adrenergic receptor agonists is not always correlated with changes in cAMP concentration (Xiao et al., 1994; Altschuld et al., 1995; Kuznetsov et al., 1995), nor is it always accompanied by a positive lusitropic effect that should result from a cAMP-dependent phosphorylation of phospholamban and/or troponin I (Lemoine and Kaumann, 1991; Borea et al., 1992; Xiao et al., 1994). These discrepancies have led the authors to postulate that beta-2 adrenergic receptors, unlike beta-1 receptors, may be coupled to other mechanisms in addition to the adenylyl cyclase system (Lemoine and Kaumann, 1991; Borea et al., 1992; Xiao and Lakatta, 1993; Xiao et al., 1994; 1995; Kuznetsov et al., 1995). In that regard, beta-2 adrenergic receptors have been shown recently to be functionally coupled to pertussis toxin-sensitive G proteins in rat ventricular myocytes (Xiao et al., 1995).

Because the positive inotropic effect of beta adrenergic agonists is generally associated with a stimulation of the I_Ca (Hartzell, 1988; McDonald et al., 1994), it was of interest to examine the respective contribution of beta-1 and beta-2 adrenergic receptors in this effect and to compare the cellular mechanisms involved. Selective beta-2 adrenergic receptor activation was found to produce a stimulation of I_Ca in guinea pig atrial myocytes (Iijima and Taira, 1989), and in rat (Xiao and Lakatta, 1993; Xiao et al., 1994; 1995; Cerbai et al., 1995), guinea pig (Wang and Pelzer, 1995; but see Iijima and Taira, 1989), dog (Altschuld et al., 1995) and frog ventricular myocytes (Skeberdis et al., 1997). The signaling cascade involved in this stimulation has been studied in detail only in rat (Xiao and Lakatta, 1993; Xiao et al., 1994; 1995) and dog ventricular myocytes (Altschuld et al., 1995) using zinterol as a selective beta-2 adrenergic agonist (Minneman et al., 1979). It was concluded that stimulation of I_Ca by zinterol was not mediated by cAMP-dependent mechanisms. This conclusion was based on phenomenological differences between the effects of zinterol and beta-1 adrenergic agonists on I_Ca, cytoplasmic Ca⁺⁺ concentration transients, and cell shortening, and their respective correlation and lack of correlation with changes in the concentration of cAMP (Xiao and Lakatta, 1993; Xiao et al., 1994; 1995; Altschuld et al., 1995).

The frog heart is a rather unique preparation in which the beta adrenergic receptor population is composed of a majority (80%) of beta-2 subtype (Hancock et al., 1979; Hieble and Ruffolo, 1991). Moreover, a recent competition curve analysis of the effects of various beta-1 and beta-2 agonists and antagonists on I_Ca led to the findings that only beta-2 adrenergic receptors are coupled to I_Ca in this preparation (Skeberdis et al., 1997). Thus, we anticipated that this preparation might be valuable in getting some additional insights on the coupling mechanisms between these receptors and the L-type Ca⁺⁺ channels. For this reason, we investigated the effects of zinterol on I_Ca in whole-cell patch-clamped single frog ventricular myocytes. For comparison, we also examined the effect of zinterol on I_Ca in rat ventricular and human atrial myocytes and tested the hypothesis that a cAMP-independent mechanism may be involved in these effects by directly dialyzing the myocytes with a peptide inhibitor of cAMP-dependent protein kinase. Preliminary results have appeared in abstract form (Skeberdis et al., 1996).

	Methods

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The investigation conforms with the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J no. L358, December 18, 1986) and the French decree no. 87/748 of October 19, 1987 (J Off République Française, October 20, 1987, pp. 12245-12248). Authorizations to perform animal experiments according to this decree were obtained from the French Ministère de l'Agriculture et de la Forêt (no. 04226, April 12, 1991). All protocols for obtaining human cardiac tissue were approved by the ethics committee of our institution (GREBB, Hôpital de Bicêatre, Université de Paris-Sud). [h]Experimental Solutions and Drugs

For the preparation of frog ventricular cells, the ionic composition of Ca⁺⁺-free Ringer solution was (mM): NaCl 88.4; KCl 2.5;NaHCO₃ 23.8; NaH₂PO₄ 0.6; MgCl₂ 1.8; creatine 5; D-glucose 10; 1 mg.ml¹ fatty acid-free bovine serum albumin; 50 I.U.ml¹ penicillin; 50 µg.ml¹ streptomycin; pH 7.4 maintained with 95% O₂, 5% CO₂. Storage Ringer solution was Ca⁺⁺-free Ringer solution to which was added 0.9 mM CaCl₂ and 10 µl ml¹ nonessential and essential amino acid and vitamin solution (minimal essential medium 100x). Dissociation medium was composed of Ca⁺⁺-free Ringer solution to which was added 0.2 mg.ml¹ trypsin, 0.14 mg.ml¹ collagenase (Yakult, Tokyo, Japan), and 10 µl.ml¹ M199 medium. For the preparation of rat and human cardiomyocytes, the ionic composition of the Ca⁺⁺-free Tyrode solution was (mM): NaCl 117; KCl 5.7; NaHCO₃ 4.4; KH₂PO₄ 1.5; MgCl₂ 1.7; HEPES 21.1; creatine 10; D-glucose 11.7; taurine 20; pH adjusted to 7.1 with NaOH. For electrophysiology, the control external solution contained (in mM): NaCl 107; HEPES 10; CsCl 20 (for frog and rat) or 40 (for human); NaHCO₃ 4; NaH₂PO₄ 0.8; MgCl₂ 1.8; CaCl₂ 1.8; D-glucose 5; sodium pyruvate 5; tetrodotoxin 3 × 10⁴ (for frog) or 6 × 10³ (for rat and human); pH 7.4 adjusted with NaOH. Patch electrodes (0.6-2.0 Mohms) were filled with control internal solution which contained (mM): CsCl 119.8; EGTA (acid form) 5; MgCl₂ 4; creatine phosphate disodium salt 5; Na₂ATP 3.1; Na₂GTP 0.42; CaCl₂ 0.062 (pCa 8.5); HEPES 10; pH 7.1 (frog) or 7.3 (rat and human) adjusted with CsOH. Collagenase type IV and protease type XXIV used for human atrial cells dissociation were purchased from Sigma (L'Isle d'Abeau Chesnes, France). Collagenase type A for rat cardiac myocyte dissociation and fetal calf serum were from Boehringer Mannheim (Germany). Collagenase for frog ventricular myocyte dissociation was from Yakult. Delbecco's minimal essential medium was obtained from Gibco-BRL. Tetrodotoxin was from Latoxan (Rosans, France). Zinterol was a generous gift of Bristol Myers Squibb (Evansville, IN). CGP 20712A was a generous gift from Novartis Pharma AG (Basel, Switzerland). ICI 118551 was from Tocris Cookson (Bristol, UK). All other drugs were from Sigma Chemical Co. (St. Louis, MO). All drugs tested in patch-clamp experiments were solubilized in experimental solutions just before application onto the cell studied, i.e., only fresh solutions were tested.

Frog Ventricular Myocytes

Ventricular cells were enzymatically dispersed from frog (Rana esculenta) heart, by a combination of collagenase (Yakult) and trypsin (type III or XIII, Sigma) as described (Fischmeister and Hartzell, 1986). Frogs were decapitated and double pithed. The isolated cells were stored in storage Ringer solution, and kept at 4°C until use (2-48 hr after dissociation). In some isolations, amino acids were omitted from the dissociation and storage solutions, with no change in the results.

Human Atrial Myocytes

Surgery. Specimens of right atrial appendages were obtained from two patients (one male aged 44, one female aged 73) undergoing heart surgery for coronary artery disease at the Hôpital Marie-Lannelongue, Le Plessis-Robinson, France. Both patients received a pharmacological pretreatment composed of a Ca-channel blocker (diltiazem), a -adrenergic antagonist (atenolol) and a NO-donor (molsidomine). In addition to these medications, both patients received sedatives, anesthesia, and antibiotics. Dissociation of the cells was realized immediately after surgery.

Cell dissociation. Myocytes were isolated as described previously (Kirstein et al., 1995; Rücker-Martin et al., 1993). The cell suspension was filtered, centrifuged and the pellet resuspended in DMEM supplemented with 10% fetal calf serum, nonessential amino acids, 1 nM insulin and antibiotics (penicillin, 100 IU/ml and streptomycin, 0.1 µg/ml). For patch-clamp experiments 100 to 200 µl of this cell suspension were put in a Petri dish containing control external solution.

Rat Ventricular Myocytes

Rat cardiomyocytes were obtained by retrograde perfusion from hearts of male Wistar rats (180-220 g) as previously described (Pucéat et al., 1990) with slight modifications. Briefly, the rats were subjected to anesthesia by intra-peritoneal injection of urethane and the hearts were rapidly excised. The hearts were perfused retrogradely at a constant flow and at 37°C by an oxygenated Ca-free Tyrode solution during 5 min followed by 1 hr perfusion with the same solution containing 1 mg/ml collagenase A (Boehringer-Mannheim, Indianapolis, IN) and 300 µM EGTA (free Ca⁺⁺ concentration adjusted to 20 µM). The ventricles and atria were then separated. Ventricles were chopped finely and agitated gently to dissociate individual cells. The resulting cell suspension was filtered and the cells settled down. The supernatant was discarded and cells resuspended a further four times in Tyrode solution containing a progressively increasing calcium concentration. The myocytes were maintained at 37°C until use.

Electrophysiological Experiments

The whole-cell configuration of the patch-clamp technique was used to record the high-threshold calcium current (I_Ca) on Ca⁺⁺-tolerant frog ventricular, human atrial and rat ventricular myocytes. In the routine protocols the cells were depolarized every 8 sec from a holding potential of -80 to 0 mV for 200 or 400 msec. In human and rat cardiomyocytes, the test pulse to 0 mV was preceded by a short pre-pulse (50 msec) to -50 mV. The pre-pulse and/or the application of tetrodotoxin (0.3 µM for frog, 6 µM for human and rat) was used to eliminate fast sodium currents. K⁺ currents were blocked by replacing all K⁺ ions with intracellular and extracellular Cs⁺. For the determination of current-voltage relationships for I_Ca (see fig. 2A) and I_Ca inactivation curve (see fig. 2B) in frog ventricular myocytes, a double pulse voltage-clamp protocol was used (Argibay et al., 1988). Briefly, every 4 sec, the membrane potential of the cell, which was normally maintained at its holding value of -80 mV, experienced the following sequence of events: different potentials values ranging from -100 to +100 mV for 200 msec, -80 mV for 3 msec and 0 mV for 200 msec (see inset in fig. 2B). In few experiments in rat, the holding potential was maintained at -60 mV with no difference in results. Voltage-clamp protocols were generated by a challenger/09-VM programmable function generator (Kinetic Software, Atlanta, GA). The cells were voltage-clamped using a patch-clamp amplifier (model RK-400; Bio-Logic, Claix, France). Currents were sampled at a frequency of 10 kHz using a 16-bit analogue-to-digital converter (PCL816, Advantech France, Levallois Perret, France) connected to a PC compatible micro computer.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Voltage-dependence of the stimulatory effect of zinterol on ICa. Current-voltage (I-V) curves (A) and inactivation curves (B) of ICa in the presence () or absence () of 100 nM zinterol. A, The current-voltage relationships of ICa were measured during 200 msec depolarizations to various potentials (see "Methods"). B, Inactivation curves of ICa were obtained using a double-step protocol consisting of a 200-msec duration conditioning pulse (CP) to various potentials followed by a 200-msec test potential (TP) to 0 mV as indicated in the inset of B. ICa measured during a 200-msec test pulse at 0 mV is plotted as a function of the conditioning pulse potential and expressed as a percentage of ICa at 0 mV in the absence of conditioning pulse.

Control or drug-containing solutions were applied to the exterior of the cell by placing the cell at the opening of 300-µm inner diameter capillary tubings flowing at a rate of 50 µl/min (Fischmeister and Hartzell, 1986). Changes in extracellular solutions were automatically achieved using a rapid solution changer (RSC100, Bio-Logic, Claix, France). Drug-containing solutions were applied to the interior of the cell by a system that permitted perfusion of the patch-electrode with different solutions (Fischmeister and Hartzell, 1987). The dead volume of the intracellular perfusion system was such that 30 to 50 sec were needed for an air bubble to travel from one end to the other end of the system. Perfusion time depended on patch-electrode resistance, access to the cell and the molecular weight of the molecule tested. Typically, with the cAMP-dependent protein kinase inhibitor peptide PKI(15-22) (MW = 2222.4), the beginning of I_Ca inhibition occurred 3 to 5 min after the beginning of intracellular perfusion with this compound (see e.g., fig. 7). All experiments were done at room temperature (19-25°C), and the temperature did not change by more than 1°C in a given experiment.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effect of intracellular perfusion with PKI(15-22) on the stimulatory effect of zinterol on ICa in rat ventricular myocytes. In A and B, a rat ventricular myocyte was initially superfused with control external solution and internally dialyzed with control intracellular solution. Each symbol represents the maximal peak amplitude of ICa obtained by depolarizing the cell every 8 sec to 0 mV over a period of 400 msec from a holding potential of -80 mV. During the periods indicated, the cells were superfused with zinterol (10 µM). In A, the intracellular solution was changed at the arrow to one containing 20 µM PKI(15-22) which perfused the cell throughout the rest of the experiment as indicated by the upper horizontal line. In B, the control intracellular solution dialyzed the cell throughout the entire experiment. The current traces shown on top of the graphs in A and B were recorded in the different experimental conditions, at the times indicated by the corresponding letters on the main graphs. The dotted lines on the main graphs indicate the exponential rundown of ICa.

Data Analysis

The maximal amplitude of whole-cell I_Ca was measured as previously described (Fischmeister and Hartzell, 1986; Argibay et al., 1988). Currents were not compensated for capacitive and leak currents. On-line analysis of the recordings was made possible by programming a PC-compatible 486/66 microcomputer in Assembling language (Borland, USA) to determine, for each membrane depolarization, peak and steady-state current values (Fischmeister and Hartzell, 1986). The results are expressed as mean ± S.E.M. Differences between means were tested for statistical significance by Student's t test. In the text, the "basal" condition refers to the absence of beta adrenergic agonist. In the case of single applications, the effect of a compound is referred to as the percent variation over the basal amplitude of I_Ca.

	Results

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Zinterol stimulates frog I_Ca. A typical experiment using zinterol as a selective beta-2 adrenergic agonist in a frog ventricular myocyte is shown in figure 1A. I_Ca was measured every 8 sec by depolarizing the cell over a period of 200 msec to 0 mV from a holding potential of -80 mV. Zinterol produced a clear increase in I_Ca at concentrations > 1 nM. At 10 nM, the current increased more than 2-fold, and a maximal stimulation of 300% was reached between 100 nM and 1 µM zinterol. Upon washout of the drug, I_Ca returned progressively to its basal amplitude. Figure 1B shows the results of several similar experiments as the one shown in figure 1A. The data are presented as a dose-response curve for the effect of zinterol on I_Ca. The dose-response curve was fitted using a nonlinear least-mean-squares regression of the means to the Michaelis equation. The concentration of zinterol (EC₅₀) required for half-maximal stimulation of I_Ca was derived from this analysis: EC₅₀ = 2.2 nM. Thus, zinterol was highly potent in stimulating I_Ca in frog ventricular myocytes.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of zinterol on ICa in frog ventricular cells. A, A frog ventricular myocyte was initially superfused with control external solution and internally dialyzed with control intracellular solution. Each symbol represents the maximal peak amplitude of ICa obtained by depolarizing the cell every 8 sec to 0 mV over a period of 200 msec from a holding potential of -80 mV. During the periods indicated by the horizontal lines, the cell was successively exposed to five increasing concentrations of zinterol (0.001, 0.01, 0.1, 1 and 10 µM). The current traces shown on top of the graph were recorded in the different experimental conditions, at the times indicated by the corresponding letters on the graph. B, Concentration-response curve for the stimulatory effect of zinterol on ICa. The effect of increasing concentrations of zinterol on ICa were obtained in several experiments performed as in A. The points show the mean ± S.E.M. of the number of cells indicated near the symbols. The continuous line was derived from a nonlinear least-mean-squares regression of the means to the Michaelis equation. The concentration of zinterol (EC50) required for half-maximal stimulation of ICa was derived from this analysis: EC50 = 2.2 nM.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Antagonist effects of ICI 118551 on the stimulatory effect of zinterol on ICa in frog ventricular myocytes. A frog ventricular myocyte was initially superfused with control external solution and internally dialyzed with control intracellular solution. Each symbol represents the maximal peak amplitude of ICa obtained by depolarizing the cell every 8 sec to 0 mV over a period of 200 msec from a holding potential of -80 mV. During the periods indicated by the horizontal lines, the cell was successively exposed to four decreasing concentrations of ICI 118551 (1, 0.1, 0.01 and 0.001 µM) and/or to zinterol (0.1 µM). The current traces shown on top of the graph were recorded in the different experimental conditions, at the times indicated by the corresponding letters on the graph.

Zinterol produces a maximal stimulation of frog I_Ca. We then examined whether zinterol was capable to produce a maximal stimulation of I_Ca in frog ventricular myocytes, e.g., comparable to the maximal effect of isoprenaline or forskolin, a direct adenylyl cyclase activator. To answer this question, we performed five experiments like the one illustrated in figure 4. After stimulation of I_Ca with a saturating concentration (1 µM) of zinterol, 3-isobutyl-1-methyxanthine (IBMX, 100 µM), a nonselective phosphodiesterase inhibitor, or forskolin (10 µM) was added to the solution containing zinterol to maximally increase the concentration of cAMP within the cell, either by blocking its degradation (IBMX) or by maximally stimulating its synthesis (forskolin). We found that neither IBMX nor forskolin were able to increase I_Ca above the level reached in the presence of zinterol alone. In five other experiments, the effect of isoprenaline (1 µM) was tested after a stimulation of I_Ca with 1 µM zinterol. In these experiments, zinterol alone increased I_Ca by 418 ± 54% above basal level, and the current was not further increased by addition of isoprenaline (442 ± 51%). Thus, activation of the 2-adrenergic receptors with zinterol is sufficient to maximally stimulate I_Ca in frog ventricular myocytes.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of IBMX and forskolin on ICa in the presence of a saturating concentration of zinterol. A frog ventricular myocyte was initially superfused with control external solution and internally dialyzed with control intracellular solution. Each symbol represents the maximal peak amplitude of ICa obtained by depolarizing the cell every 8 sec to 0 mV over a period of 200 msec from a holding potential of -80 mV. During the periods indicated by the horizontal lines, the cell was exposed to zinterol (1 µM) alone or to zinterol in the presence of IBMX (100 µM) or forskolin (10 µM). The current traces shown on top of the graph were recorded in the different experimental conditions, at the times indicated by the corresponding letters on the graph.

Long-lasting, adenylyl-cyclase-mediated effects of zinterol on frog I_Ca. Although the effects of zinterol on I_Ca resemble in their amplitude those of other beta adrenergic agonists such as isoprenaline or salbutamol, they differed markedly in their kinetics of action and washout. For example, with isoprenaline, the time for half maximal stimulation of I_Ca (t_on) was <20 sec and was essentially independent of the concentration used (Méry et al., 1993). This shows that the rate-limiting step for the effect of isoprenaline is beyond agonist binding to the receptor (Frace et al., 1993). However, with zinterol, t_on was at least an order of magnitude larger and was strongly dependent on the drug concentration. For instance, t_on was 153 ± 22.5 sec (n = 7) with 10 nM zinterol and 71.3 ± 10.5 sec with 100 nM zinterol (n = 5). Thus, unlike with isoprenaline, binding of the agonist to its receptor was rate-limiting in the case of zinterol action. Moreover, during washout of the drug, I_Ca recovered much faster after stimulation with isoprenaline or salbutamol than after stimulation with zinterol. Indeed, the time for 50% recovery from the stimulation of I_Ca (t_off) was < 80 sec with isoprenaline (Méry et al., 1993) as well as with salbutamol (Skeberdis et al., 1997) although t_off was an order of magnitude larger with zinterol (16.5 ± 2.1 min, n = 5). Actually, figure 1A shows that complete washout of zinterol effect required a period of almost an hour.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Long-lasting effect of zinterol on ICa in frog ventricular myocytes. A frog ventricular myocyte was initially superfused with control external solution and internally dialyzed with control intracellular solution. Each symbol represents the maximal peak amplitude of ICa obtained by depolarizing the cell every 8 sec to 0 mV over a period of 200 msec from a holding potential of -80 mV. During the periods indicated by the horizontal lines, the cell was exposed to zinterol (1 µM) or to acetylcholine (ACh, 1 µM). The first application of ACh was obtained right after washout of zinterol. The dotted line is a mono-exponential fit of the time course of recovery of ICa from zinterol stimulation using a 18-min time constant. The current traces shown on top of the graph were recorded in the different experimental conditions, at the times indicated by the corresponding letters on the graph.

Role of cAMP-dependent phosphorylation in the stimulatory effect of zinterol on I_Ca in frog, rat and human cardiomyocytes. Because the stimulatory effect of zinterol on I_Ca in frog ventricular myocytes 1) is maximal, 2) is not additive with the stimulatory effects of isoprenaline, forskolin or IBMX and 3) is inhibited by ACh, the most likely hypothesis is that this effect is mediated by activation of adenylyl cyclase and subsequent cAMP-dependent phosphorylation of L-type Ca⁺⁺ channels. However, recent studies indicate that cAMP-independent mechanisms may also participate in the stimulatory effect of zinterol on I_Ca in mammalian cardiac preparations (Xiao and Lakatta, 1993; Xiao et al., 1994; 1995; Altschuld et al., 1995). Indeed, in rat (Xiao et al., 1994) and dog ventricular myocytes (Altschuld et al., 1995), the stimulation of I_Ca by zinterol as well as the positive inotropic effect of the drug were shown to be independent of cAMP concentration. If there were a cAMP-independent mechanism involved in the effect of zinterol on I_Ca, one would predict that an inhibitor of cAMP-dependent protein kinase would not completely block the stimulatory effect of zinterol. However, none of these studies examined the effect of zinterol in the presence of such inhibitors. For this reason, we reexamined the effect of zinterol on I_Ca in the presence of an intracellular application of a highly selective cAMP-dependent protein kinase peptide inhibitor, PKI(15-22) (Walsh et al., 1990). These experiments were performed in frog ventricular myocytes as well as in two different mammalian species, rat and human, using an intracellular perfusion system (Fischmeister and Hartzell, 1987). Figure 6A shows a typical experiment performed in frog. After I_Ca had been enhanced by extracellular application of 0.1 µM zinterol, 20 µM PKI(15-22) were added to the intracellular solution which started to dialyze the cell. Few min after addition of PKI(15-22), I_Ca decreased dramatically, although zinterol was still present in the extracellular solution. As seen in figure 6A, the current returned to basal level in the presence of zinterol and PKI(15-22). On average, in four cells in which 0.1 µM zinterol increased I_Ca by 993 ± 18% over basal level, intracellular application of 20 µM PKI(15-22) reduced the stimulatory effect of zinterol by 99.1 ± 1.1% after 15 to 20 min. The control experiment illustrated in figure 6B shows that the strong reduction in I_Ca observed in the presence of PKI(15-22) was not a consequence of rundown or desensitization of the zinterol effect on I_Ca because I_Ca decreased by less than 25% from its maximal amplitude after a 20 min continuing exposure to 0.1 µM zinterol in the absence of PKI(15-22) (on average-23.5 ± 2.9% decrease, n = 4).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effect of intracellular perfusion with PKI(15-22) on the stimulatory effect of zinterol on ICa in frog ventricular myocytes. In A and B, a frog ventricular myocyte was initially superfused with control external solution and internally dialyzed with control intracellular solution. Each symbol represents the maximal peak amplitude of ICa obtained by depolarizing the cell every 8 sec to 0 mV over a period of 200 msec from a holding potential of -80 mV. During the periods indicated, the cells were superfused with zinterol (0.1 µM). In A, the intracellular solution was changed at the arrow to one containing 20 µM PKI(15-22) which perfused the cell throughout the rest of the experiment as indicated by the upper horizontal line. In B, the control intracellular solution dialyzed the cell throughout the entire experiment. The current traces shown on top of the graphs in A and B were recorded in the different experimental conditions, at the times indicated by the corresponding letters on the main graphs.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effect of intracellular perfusion with PKI(15-22) on the stimulatory effect of zinterol on ICa in human atrial myocytes. In A and B, a human atrial myocyte was initially superfused with control external solution and internally dialyzed with control intracellular solution. Each symbol represents the maximal peak amplitude of ICa obtained by depolarizing the cell every 8 sec to 0 mV over a period of 400 msec from a holding potential of -80 mV. During the periods indicated, the cells were superfused with zinterol (1 µM). In A, the intracellular solution was changed at the arrow to one containing 20 µM PKI(15-22) which perfused the cell throughout the rest of the experiment as indicated by the upper horizontal line. In B, the control intracellular solution dialyzed the cell throughout the entire experiment. The current traces shown on top of the graphs in A and B were recorded in the different experimental conditions, at the times indicated by the corresponding letters on the main graphs.

	Discussion

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In our study, we examined the effect of the beta-2 adrenergic receptor agonist zinterol on the L-type Ca⁺⁺ current (I_Ca) in frog ventricular myocytes and, to a lesser extent, in rat ventricular and human atrial myocytes. In all three preparations, zinterol produced a large increase in I_Ca and the maximal stimulation was comparable to that produced by a saturating concentration of isoprenaline, a nonselective beta adrenergic agonist. A precise characterization of the effect of zinterol on I_Ca in frog ventricular myocytes demonstrated that this effect was 1) concentration-dependent (EC₅₀ = 2.2 nM); 2) independent of the membrane potential; 3) long lasting; 4) antagonized by ICI 118551, a beta-2 adrenergic receptor antagonist; 5) not additive with the effects of IBMX or forskolin and 6) reversed by ACh. Moreover, in all three preparations tested, the stimulatory effect of zinterol on I_Ca was fully antagonized by intracellular perfusion with PKI(15-22), a highly selective peptide inhibitor of the cAMP-dependent protein kinase. We conclude that, in frog, rat and human cardiac myocytes, beta-2 adrenergic receptor activation induces an increase in I_Ca which is mediated by activation of adenylyl cyclase, and subsequent activation of cAMP-dependent protein kinase and phosphorylation of L-type Ca⁺⁺ channels.

Although initial competitive binding studies concluded to the absence of beta-2 adrenergic receptors in purified ventricular myocytes from mammalian hearts (Freissmuth et al., 1986; Lau et al., 1980; Buxton and Brunton, 1985), more recent studies have clearly established the presence of these receptors in ventricular myocytes from several mammals, such as rats (Kuznetsov et al., 1995; Cerbai et al., 1995), dogs (Murphree and Saffitz, 1988), baboons (Cui et al., 1996) and humans (del Monte et al., 1993). However, the 1/2 ratio may vary somewhat from one study to the other in a given animal species (e.g., 80/20 to 92/8 in rat myocytes: Cerbai et al., 1995; Kuznetsov et al., 1995; Cui et al., 1996) and from one species to the other (e.g., 85/15 in dog: Murphree and Saffitz, 1988; 59/41 in baboon: Cui et al., 1996; 20/80 in frog: Hancock et al., 1979). The 1/2 ratio may also vary depending on the pathophysiological state (Brodde, 1993; Ihl-Vahl et al., 1996), the age (White et al., 1994; Cerbai et al., 1995) or the developmental stage (Kuznetsov et al., 1995; for reviews, see Stiles et al., 1984; Brodde, 1991, 1993; Hieble and Ruffolo, 1991 and references therein). Finally, in mammals, the proportion of beta-2 adrenergic receptors was shown to be somewhat larger in atrial compared to ventricular tissues (Carlsson et al., 1977; Hedberg et al., 1980; Molenaar and Summers, 1987), and more so in human where beta-2 adrenergic receptors may account for 35 to 50% of the total number of beta adrenergic receptors (Robberecht et al., 1983; Brodde, 1991; Hieble and Ruffolo, 1991). The latter finding may explain why beta-2 adrenergic agonists exert preferentially positive chronotropic rather than inotropic effects in humans (Brodde, 1991).

Selective agonists of beta-2 adrenergic receptors were shown earlier to increase I_Ca in guinea pig atrial myocytes (Iijima and Taira, 1989), as well as in rat (Xiao and Lakatta, 1993; Cerbai et al., 1995), dog (Altschuld et al., 1995), guinea pig (Wang and Pelzer, 1995; but see Iijima and Taira, 1989) and frog ventricular myocytes (Skeberdis et al., 1997). The maximal stimulatory effect of these agonists on I_Ca varied from 30% in guinea pig (Wang and Pelzer, 1995) to 100% in rat ventricular myocytes (Xiao and Lakatta, 1993) of the effect of isoprenaline. Here we found that a selective activation of beta-2 adrenergic receptors with zinterol accounts for 100% of the isoprenaline response in frog and rat ventricular and human atrial myocytes. Although both subtypes of beta receptors can increase cardiac I_Ca and force of contraction, some difference may exist in the mechanisms involved. First, Xiao and Lakatta (1993) showed in rat ventricular myocytes a marked prolongation of the I_Ca inactivation phase upon application of zinterol which was not found upon activation of the beta-1 adrenoceptors. This phenomenon, however, was not observed in another study in the same preparation (Cerbai et al., 1995). Although we did not study in details the kinetics of I_Ca in our experiments, we found no drastic change in the inactivation phase of I_Ca in none of the three preparations examined (frog, rat and human, data not shown). Second, unlike isoprenaline or noradrenaline, a more selective beta-1 adrenergic agonist, zinterol did not abbreviate the twitch relaxation or the cytosolic Ca⁺⁺ transient in rat, canine and human isolated cardiomyocytes (Xiao and Lakatta, 1993; Xiao et al., 1994; Altschuld et al., 1995; Kuznetsov et al., 1995). Third, unlike the effects of noradrenaline, the positive inotropic effect of zinterol as well as the increase in Ca⁺⁺ transient were found to be poorly correlated with the increase in cAMP concentration and cAMP-dependent phosphorylation of phospholamban in the same preparations (Lemoine and Kaumann, 1991; Borea et al., 1992; Xiao et al., 1994; Altschuld et al., 1995; Kuznetsov et al., 1995). Finally, unlike the effects of noradrenaline, the stimulatory effects of zinterol on I_Ca, Ca⁺⁺ transient and cell shortening were strongly potentiated by a pertussis toxin pre-treatment in rat ventricular myocytes (Xiao et al., 1995).

Altogether, these studies concluded to the presence of a cAMP-independent mechanism in the stimulatory effects of beta-2, but not beta-1, adrenergic receptor agonists. However, this conclusion was not supported by our current findings. Indeed, we found that when cAMP-dependent phosphorylation is blocked by PKI(15-22), a highly selective peptide inhibitor of cAMP-dependent protein kinase (Walsh et al., 1990), zinterol does not anymore stimulate I_Ca in frog, rat and human cardiomyocytes. On the contrary, we propose that all of the stimulatory effect of beta-2 adrenergic receptor activation on I_Ca is actually mediated by cAMP-dependent phosphorylation. Such a conclusion is consistent with previous studies on I_Ca regulation in frog, rat and rabbit cardiomyocytes in response to the nonselective agonist isoprenaline (Hartzell et al., 1991; Hartzell and Fischmeister, 1992; Tanaka et al., 1996) and with recent contractile and biochemical studies performed in human atrium (Kaumann et al., 1996). This conclusion is not necessarily at variance with the aforementioned lack of correlation between the effect of beta-2 adrenergic receptor activation and measured cAMP levels. Indeed, we have shown recently that the response of I_Ca in frog ventricular myocytes to isoprenaline is mainly due to a local rise in cAMP (Jureviius and Fischmeister, 1996b). Because the beta adrenergic receptor population is composed of 80% of beta-2 subtype in frog cardiomyocytes (Hancock et al., 1979; Hieble and Ruffolo, 1991) and only beta-2 adrenergic seem to be coupled to L-type Ca⁺⁺ channels in this preparation (Skeberdis et al., 1997), the possibility exists that the cAMP generated by beta-2 adrenergic receptor activation may be more localized, and hence less visible in biochemical assays, than the cAMP generated by activation of beta-1 adrenoceptors. Although both pools of cAMP would be efficiently coupled to L-type calcium channels that are sarcolemmal membrane proteins, the pool of cAMP generated by beta-2 adrenoceptor activation may be less efficiently coupled to more distant proteins, such as phospholamban and/or troponin I.