INTRODUCTION

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PHARMACOLOGICALLY DISTINCT ₁-adrenergic receptor (AR) subtypes have been described (6, 13, 18, 24, 28), and molecular cloning and expression of the cDNA for three ₁-AR subtypes has been reported in rat myocytes and in the human heart (13, 28). Most studies that have examined the functional role of ₁-AR subtypes have relied upon nonselective ₁-AR stimulation in the presence of receptor subtype antagonists such as chloroethylclonidine (CEC; see Ref. 19) and 2-(2,6-dimethoxyphenoxyethly)aminomethyl-1,4-benzodioxane hydrochloride (WB-4101; see Ref. 19). Several studies have described the effect of phenylephrine plus CEC and phenylephrine plus WB-4101 on cardiac cell electrophysiological properties (1, 2, 7, 15, 20, 30) and growth (25). In the continued presence of phenylephrine, WB-4101 decreased (1, 2, 20) and CEC enhanced (1) phenylephrine-mediated arrhythmias. In a different study, CEC increased spontaneously beating rate of canine Purkinje fibers, whereas the opposite occurred upon WB-4101 addition (7).

Despite these reports, the functional role of ₁-AR subtypes sensitive either to CEC or to WB-4101 is still incompletely characterized in the heart. Specifically, the effects of these receptor subtypes on contraction, cell Ca²⁺ homeostasis, cytosolic pH (pH_i), and myofilament responsiveness to Ca²⁺ of the myocardium are still unknown. Furthermore, the interaction of these receptor subtypes during nonselective ₁-AR stimulation, as it normally occurs in vivo, still remains to be determined. These questions were addressed in the present study utilizing indo 1- or SNARF-1-loaded adult rat ventricular myocytes.

	METHODS

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Myocyte isolation procedure. Ventricular myocytes were enzymatically dissociated with minor modifications of a technique previously described (27). Briefly, 2- to 4-mo-old male Wistar rats were anesthetized with an intraperitoneal injection of pentobarbital sodium. The heart was quickly excised and retrogradely perfused with 25 ml of a nominally Ca²⁺-free bicarbonate buffer at 36 ± 1°C, continuously gassed with 95% O₂ and 5% CO₂ to keep the pH at ~7.4. The perfusate was then switched to a similar solution to which collagenase (1 mg/ml), protease (0.04 mg/ml), and bovine serum albumin (1 mg/ml) had been added. After ~20 min of perfusion, the left ventricle was isolated, and cardiac myocytes were mechanically disaggregated and resuspended in a bicarbonate buffer with 1.0 mM bathing Ca²⁺ concentration ([Ca²⁺]).

Simultaneous measurement of length and indo 1 fluorescence. Cell length and indo 1 fluorescence were measured simultaneously, as previously described (27). Briefly, single myocytes bathed in bicarbonate-buffered medium were loaded with the ester derivative [acetoxymethyl (AM) form] of the fluorescent Ca²⁺ probe indo 1. After loading, cells were transferred to a Lucite chamber with a glass coverslip on the stage of an inverted microscope and were continuously superfused with buffer composed of (in mM) 116.4 NaCl, 5.4 KCl, 1.6 MgSO₄, 26.2 NaHCO₃, 1.2 NaH₂PO₄, 5.6 D-glucose, and 0.5 CaCl₂. The buffer was gassed with 95% O₂-5% CO₂ (pH 7.36). Two platinum electrodes placed in the bathing fluid and connected to a stimulator (SD9; Grass Instrument, Quincy, MA) were used to field stimulate the myocyte to twitch with pulses of 2-4 ms in duration at a rate of 0.5 Hz. Indo 1 fluorescence was excited by epi-illumination with 10-ms flashes of 350 ± 5 nm light. Paired photomultipliers collected indo 1 fluorescence emission by simultaneously measuring spectral windows of 391 ± 434 and 457 ± 507 nm selected by bandpass interference filters. The ratio of indo 1 emission at the two wavelengths was calculated using a pair of fast integrator sample and hold circuits under the control of a VAX 11/730 computer, and it was taken as an index of cytosolic Ca²⁺ concentration ([Ca²⁺]_i). When isolated cardiac myocytes are loaded with indo 1-AM, there is variable compartmentalization of the indicator into the mitochondria (27) that prevents the use of a standard calibration curve. Thus the present results in indo 1-AM-loaded myocytes are expressed as fluorescence ratio rather than as absolute [Ca²⁺] values. Cell length was monitored simultaneously with indo 1 fluorescence ratio using red light (650-750 nm) to form a bright-field image of the cell, which was projected onto a photodiode array. Myofilament responsiveness to Ca²⁺ was assessed by the cell length-[Ca²⁺]_i relation in the diastolic interval during 50 ms before the delivery of the electrical stimulus (26). Myofilament Ca²⁺ binding and [Ca²⁺]_i achieve a quasi-equilibrium during the relaxation phase of a twitch, and the phase-plane diagram of the cell length-[Ca²⁺]_i relation, at the time of cell relengthening during a twitch, does not change under a variety of conditions that alter the amplitude and time course of the contraction and of the [Ca²⁺]_i transient without affecting myofilament Ca²⁺ sensitivity. Instead, the cell length-[Ca²⁺]_i relation is shifted in opposite directions by interventions that either increase or decrease myofilament responsiveness to Ca²⁺ (26).

Both loading of the Ca²⁺ probe and experiments were performed at 25°C to minimize loss of the Ca²⁺ indicator from the cells (27). Additionally, some experiments were performed with cells that had not been loaded with indo 1, and only cell length was measured.

pH_i measurements. After enzymatic isolation, myocytes were bathed in /CO₂ buffer, unless indicated otherwise. Alternatively, cells were superfused with a /CO₂-free buffer of the following composition (in mM): 137.0 NaCl, 5.0 KCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 16 D-glucose, and 0.5 CaCl₂ (pH 7.35). Experiments were performed at 25°C. Cells were loaded with the AM form of the fluorescent H⁺-sensitive indicator SNARF-1. pH_i and cell length were monitored on the stage of a modified inverted microscope, as previously described (3). After excitation at 530 ± 5 nm, the ratio of SNARF-1 emission at 590 ± 5 nm to that at 640 ± 5 nm was used as a measure of pH_i according to an in vivo calibration.

	RESULTS

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Effect of phenylephrine plus WB-4101 or CEC on contraction. As in previous studies (4, 10, 29), in the present experimental conditions, superfusion of single ventricular myocytes with phenylephrine increased contraction amplitude, and the effect was maximal and achieved steady state in ~15 min (not shown). The concentration dependence of the effect of WB-4101 and CEC on the positive inotropic action of nonselective ₁-adrenergic stimulation is depicted in Fig. 1. On average, nonselective ₁-adrenergic stimulation caused a twofold increase in twitch amplitude. After addition of WB-4101 in the continued presence of phenylephrine, there was a concentration-dependent decrease in twitch amplitude (Fig. 1A). The peak response was achieved at 2 µM WB-4101, and, at this concentration, twitch amplitude was ~50% lower than in control. In contrast, CEC added in the continued presence of phenylephrine caused a further increase in twitch amplitude (Fig. 1B). This effect saturated at 2 µM, and, at this concentration, twitch amplitude was enhanced ~30% compared with phenylephrine alone. Neither substance alone had any appreciable effect on cell function, and, for all subsequent experiments reported in the present study, either 2 µM WB-4101 or 2 µM CEC were used.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effect of phenylephrine alone and phenylephrine plus different 1-adrenergic receptor (AR) antagonists on contraction of myocytes not loaded with indo 1. A: WB-4101 () had a concentration-dependent effect to oppose the positive inotropic action of 10 µM phenylephrine (). Statistically significant effect was achieved for concentrations of WB-4101 above 0.5 µM; WB-4101 decreased twitch amplitude to control at 1 µM and below control at 2 µM. B: chloroethylclonidine (CEC; ) had a concentration-dependent effect to further increase the positive inotropic action of 10 µM phenylephrine (), and its effect saturated at 2 µM. Concentrations of 2 µM and above reached statistical significance. Both WB-4101 and CEC were added at the concentrations indicated after achieving a maximal and steady response to phenylephrine. Data are plotted as %control (C; ; twitch amplitude in control was 4.1 ± 0.8% of diastolic cell length, n = 9). Each point represents average data obtained from at least 4 cells, and each cell was exposed to only one concentration of phenylephrine and WB-4101 or phenylephrine and CEC.

Subsequent experiments were aimed at determining whether, under our experimental conditions, CEC and WB-4101 were selective antagonists of ₁-AR receptor subtypes. Myocytes were incubated with 10 µM CEC for 30 min at 37°C. Such an exposure to CEC is expected to fully and irreversibly inactivate CEC-sensitive ₁-AR subtypes (19). Subsequently, CEC was extensively washed, and myocytes were studied with the protocol depicted in Fig. 1. These cells responded to phenylephrine with an increase in twitch amplitude, and the subsequent addition of 2 µM CEC had no effect on contraction (Fig. 2). However, the response to WB-4101 was preserved, and it reversed the effect of phenylephrine (Fig. 2). This result indicates that, under our experimental conditions, 2 µM CEC had no functional effect on the WB-4101-sensitive receptor subtype. Crossover binding of WB-4101 to the CEC-sensitive ₁-AR receptor subtype also appears unlikely because this should enhance rather than decrease contraction amplitude.

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Representative example of the effect of phenylephrine and of the subsequent addition of CEC or WB-4101. Before exposure to phenylephrine, the cell was incubated for 30 min at 37°C with 10 µM CEC and extensively washed thereafter. Under these conditions, phenylephrine had a positive inotropic action; the addition of CEC (2 µM) did not exert any effect, whereas WB-4101 (2 µM) decreased twitch amplitude. Four myocytes were studied with this experimental protocol.

The results in Figs. 1 and 2 indicate that phenylephrine plus CEC and phenylephrine plus WB-4101 have opposite effects on myocardial contraction. The possible mechanisms for these responses were further examined in indo 1- and SNARF-1-loaded myocytes.

Effect of phenylephrine plus WB-4101 or CEC on contraction and indo 1 fluorescence. Figure 3 shows the effect of phenylephrine alone and of phenylephrine plus WB-4101 on the simultaneously recorded contraction and indo 1 fluorescence transient and on myofilament Ca²⁺ response of a representative myocyte. Nonselective ₁-AR stimulation with phenylephrine was associated with an increase in twitch amplitude and [Ca²⁺]_i transient amplitudes (Fig. 3A, compare tracings a and b). In addition, the inotropic effect of phenylephrine was associated with a shift of the cell length-[Ca²⁺]_i relation leftward of control (Fig. 3B) and with a reduction in diastolic cell length, which occurred without a change in diastolic indo 1 fluorescence ratio (Fig. 3A, tracings a and b), another effect that suggests an enhanced myofilament response to Ca²⁺. These results are in agreement with prior studies that have shown that the positive inotropic action of ₁-AR stimulation is due both to an increase in the amplitude of the [Ca²⁺]_i transient (4, 8, 10) and to an increased myofilament response to Ca²⁺ (8, 10, 22, 29). Figure 3A (tracing c) shows that WB-4101 in the continued presence of phenylephrine decreased both contraction and [Ca²⁺]_i transient amplitudes to values lower than control. In contrast, WB-4101 did not reverse the effect of phenylephrine on the myofilament response to Ca²⁺ (Fig. 3B) and on diastolic cell length. Figure 4 shows a representative example of the effect of phenylephrine and phenylephrine plus CEC on contraction and indo 1 fluorescence and shows the myofilament Ca²⁺ response of a single myocyte. Similar to the result in Fig. 3, the positive inotropic effect of phenylephrine alone was associated with an increase in [Ca²⁺]_i transient amplitude, with diastolic cell shortening without a rise in diastolic indo 1 fluorescence (Fig. 4A, compare traces a and b) and with a leftward shift of the cell length-[Ca²⁺]_i relation (Fig. 4B). CEC superfusion in the continuing presence of phenylephrine caused a further increase in twitch amplitude and a further decrease in diastolic length without changing either systolic or diastolic indo 1 fluorescence (Fig. 4A, compare traces b and c); in addition, it also caused a further shift of the cell length-[Ca²⁺]_i relation (Fig. 4B).

View larger version (15K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   Fig. 3.   Representative example of the effect of phenylephrine and of the addition of WB-4101 (WB) in the continued presence of phenylephrine on an indo 1-loaded myocyte. A: continuous cell length tracing (top) and tracings of simultaneously recorded indo 1 fluorescence and cell length (bottom). The latter are on an expanded time scale and were obtained at the times indicated by the letters below the upper continuous length record: a, control; b, effect of phenylephrine was maximal; and c, after addition of WB-4101. Phenylephrine increased both twitch and [Ca2+]i transient amplitudes. Additionally, phenylephrine decreased diastolic cell length without raising diastolic indo 1 fluorescence (compare traces a and b). Addition of WB-4101 decreased systolic [Ca2+]i and twitch amplitudes to values lower than in control (compare traces a and c). Reduction in diastolic length without an increased indo 1 fluorescence ratio persisted during WB-4101 exposure (trace c). Four myocytes were studied with this experimental protocol. B: phase-plane diagram of the length-[Ca2+]i relation for the same cell shown in A. Note the effect of phenylephrine to shift the relaxation phase of this relation above and leftward of control, which is indicative of an enhanced myofilament response to Ca2+. Addition of WB-4101 did not reverse the effect of phenylephrine alone. , 1-AR stimulation.

View larger version (16K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   Fig. 4.   Representative example of the effect of phenylephrine and of the addition of CEC in the continued presence of phenylephrine on contraction and indo 1 fluorescence. A: display of the results is similar to that in Fig. 3. Phenylephrine alone increased twitch and [Ca2+]i transient amplitudes, decreased diastolic length, and did not raise diastolic indo 1 fluorescence. CEC addition caused a further increase in twitch amplitude and a decrease in diastolic cell length, which were not associated with significant changes in systolic [Ca2+]i and diastolic indo 1 fluorescence (compare traces b and c). Five myocytes were studied with this experimental protocol. B: phase-plane diagram of the length-[Ca2+]i relation. Relaxation phase of this relationship was shifted leftward and above control by nonselective 1-AR stimulation (). This shift was enhanced after addition of CEC.

In other experiments, in contrast to those depicted in Figs. 3 and 4 in which phenylephrine was given before subtype antagonists, myocytes were first exposed either to WB-4101 or CEC alone, and phenylephrine was added subsequently. Under these conditions, there was no effect of either WB-4101 or CEC alone on contraction or indo 1 fluorescence. However, phenylephrine plus WB-4101 decreased twitch and [Ca²⁺]_i transient amplitudes below control without affecting the myofilament Ca²⁺ response (n = 11, not shown). Phenylephrine in the continued presence of CEC had a positive inotropic action associated with an increase in [Ca²⁺]_i transient amplitude, and this was also accompanied by an enhanced myofilament response to Ca²⁺ (n = 4, not shown).

The data presented so far show that nonselective ₁-AR stimulation increases contraction and [Ca²⁺]_i transient amplitudes and are in agreement with prior reports that have demonstrated an enhanced myofilament response to Ca²⁺ (8, 10, 22, 29). These effects are further enhanced by the addition of CEC in the continued presence of phenylephrine. In contrast, phenylephrine plus WB-4101 has a negative inotropic action that is due to a decreased [Ca²⁺]_i transient amplitude.

Effect of phenylephrine plus WB-4101 and CEC on contraction and indo 1 fluorescence. Because multiple ₁-AR subtypes have been identified in the rat heart (13, 28), it is necessary to establish whether the effects of nonselective ₁-AR stimulation with phenylephrine on contraction and [Ca²⁺]_i could be prevented by the simultaneous presence of both WB-4101 and CEC. If WB-4101 and CEC, at the concentrations used in this study, were truly selective blockers of only two ₁-AR subtypes and if other ₁-AR subtypes do not modulate contraction, then the combined use of WB-4101 and CEC would be expected to prevent all inotropic effects of nonselective ₁-AR stimulation. This issue was examined with the experimental protocol depicted in Fig. 5. The simultaneous addition of WB-4101 and CEC had no effect on contraction and [Ca²⁺]_i, and, in the continued presence of both substances, there was no effect of phenylephrine on twitch amplitude, diastolic length, and indo 1 fluorescence. This result is representative of the average effect in eight myocytes (see Table 1) and suggests that only WB-4101- and CEC-sensitive ₁-AR subtypes modulate myocardial contraction and [Ca²⁺]_i.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Representative example of the effect of combined WB-4101 and CEC exposure and of subsequent phenylephrine addition. Display is similar to that of Fig. 3. There was no effect of WB-4101 and CEC, and no response occurred upon the addition of phenylephrine. Eight myocytes were studied with this experimental protocol.

Taken together, the data presented so far show that phenylephrine plus WB-4101 and phenylephrine plus CEC have different effects on myocardial cell contraction on systolic [Ca²⁺]_i and on the myofilament response to Ca²⁺. Because pH_i is a major determinant of myofilament sensitivity to Ca²⁺ and because the nonselective ₁-AR stimulation increases pH_i (10, 29), additional experiments examined the effect of phenylephrine plus CEC or WB-4101 on pH_i and contraction.

Effect of phenylephrine plus CEC or WB-4101 on contraction and pH_i. Neither CEC nor WB-4101 alone had an effect on pH_i. The addition of phenylephrine in the continuing presence of CEC increased twitch amplitude and caused cytosolic alkalinization (Fig. 6A). The increase in twitch amplitude was correlated to that in pH_i (Fig. 6B). In contrast, phenylephrine plus WB-4101 decreased pH_i and contraction below control values (Fig. 6C). The degree of cytosolic acidification exhibited cell-to-cell variability and was related to the magnitude of the decrease in twitch amplitude (Fig. 6D). Previous studies had shown that nonselective ₁-AR stimulation increases pH_i in myocardial cells via activation of sarcolemmal Na⁺/H⁺ exchange (10, 29). Therefore, the results of the present study suggest that the CEC-sensitive receptor may be responsible for the effect of nonselective ₁-AR stimulation to enhance pH_i. In contrast, that a different ₁-AR subtype could decrease pH_i was unexpected.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effects of phenylephrine plus either CEC or WB-4101 on intracellular pH (pHi) and contraction of isolated myocardial cells. A: phenylephrine plus CEC enhanced both pHi and contraction amplitude (n = 7). There was no effect of CEC alone. Average increase in pHi was 0.06 ± 0.01 pH units (P < 0.005; pHi in control 7.33 ± 0.06). Average increase in twitch amplitude was 269.3 ± 59.9% of control (P < 0.01; twitch amplitude in control was 5.9 ± 1.9% of diastolic cell length). B: cytosolic alkalinization was correlated to the increase in twitch amplitude in response to phenylephrine plus CEC (r = 0.72, P < 0.05). C: phenylephrine plus WB-4101 decreased both pHi and contraction amplitude (n = 9). There was no effect of WB-4101 alone. Average decrease in pHi was 0.04 ± 0.01 pH units (P < 0.05; pHi in control 7.36 ± 0.02), and, in the same myocytes, twitch amplitude was 75.5 ± 6.3% of control (P < 0.05; twitch amplitude in control was 6.5 ± 1.2% of diastolic cell length). D: cytosolic acidification was correlated with the decrease in contraction amplitude in response to WB-4101 and phenylephrine (r = 0.79, P < 0.05).  TA, difference in twitch amplitude, expressed as % of twitch amplitude under control conditions.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Effects of WB-4101 plus phenylephrine in a /CO2-free buffer. A: representative example of the effect of WB-4101 plus phenylephrine to decrease pHi and contraction in /CO2-free buffer. Similar result was obtained in 6 myocytes; average pHi was 0.03 ± 0.01 pH units (P < 0.05; pHi in control 7.39 ± 0.05), and twitch amplitude was 78.5 ± 7.5% of control (P < 0.05; twitch amplitude in control was 6.2 ± 1.5% of diastolic cell length). Both responses were comparable to those in /CO2-buffered solution. B: representative example of a myocyte preincubated for 3 h with 5 nM staurosporine. This cell did not respond to phenylephrine plus WB-4101 with a decrease in pHi or contraction amplitude, and a similar result was obtained with 3 other myocytes. C: representative example of prolonged (12-24 h) incubation with 0.2 µM PMA. This myocyte did not respond to phenylephrine plus WB-4101 with a decrease in pHi and contraction amplitude. Four myocytes were studied with this experimental protocol.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Representative tracings of the effect of phenylephrine plus WB-4101 on pHi recovery from an acid load induced by exposure and washout of a solution containing 20 mM NH4Cl. All experiments were implemented in /CO2-free buffer. Arrows indicate exposure to either phenylephrine plus WB-4101 (B and D) or EIPA (C); thereafter, 10 min were allowed before the next NH4Cl pulse. A: under control conditions, a second exposure to NH4Cl yielded effects on pHi similar to the initial exposure (n = 6). B: left-hand tracing shows the effect of NH4Cl exposure and washout in control. In the same myocyte, phenylephrine plus WB-4101 enhanced and delayed the peak decrease in pHi upon NH4Cl washout. In addition, there was a sustained decrease in pHi at a time when full recovery had already occurred in control (n = 5). C: Na+/H+ exchange inhibition with 10 µM EIPA abolished pHi recovery and enhanced cytosolic acidification after NH4Cl washout (n = 4); these effects were qualitatively similar to those of phenylephrine plus WB-4101. D: effects of phenylephrine plus WB-4101 on pHi were abolished by preincubation with 5 nM staurosporine for 3 h (n = 5).

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Representative example of the effects of CEC added in the continued presence of phenylephrine on contraction and pHi. Phenylephrine enhanced contraction amplitude and pHi. Upon addition of 2 µM CEC, both pHi and contraction amplitude increased further. Four myocytes were studied with this experimental protocol. Display of results is similar to that in Fig. 3.

	DISCUSSION

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Although recent evidence indicates that ₁-AR subtypes are present on adult rat myocytes, the pharmacological properties of these receptors have not been completely elucidated, and no specific agonists are available. As in prior studies, the effects of ₁-AR subtypes were dissected in the present studies using nonselective ₁-AR stimulation with phenylephrine in conjunction with ₁-AR subtype blockers WB-4101 and CEC, at concentrations that provided maximal and opposite physiological effects. Because confusion still exists between the pharmacological properties and the molecular classification of ₁-AR subtypes, in the present study, ₁-AR subtypes have been referred to as WB-4101- and CEC-sensitive, and attribution of the physiological effects to a specific receptor subtype has been avoided.

The results of the present study provide the first evidence of the functional role of WB-4101- and CEC-sensitive ₁-AR subtypes on myocardial contraction, cell Ca²⁺ homeostasis, myofilament responsiveness to Ca²⁺, and pH_i. The specific following conclusions can be drawn from the results: 1) the positive inotropic action of nonselective ₁-AR stimulation is associated with both an increased [Ca²⁺]_i transient amplitude, pH_i, and an enhanced myofilament response to Ca²⁺; 2) a similar response is obtained with phenylephrine plus CEC; 3) phenylephrine plus WB-4101 causes a negative inotropic action that is associated with a decrease in [Ca²⁺]_i transient amplitude and pH_i; 4) the decrease in pH_i caused by phenylephrine plus WB-4101 appears to be due to inhibition of Na⁺/H⁺ exchange and is abolished by interventions that either inactivate or downregulate PKC; 5) when ₁-AR subtypes are stimulated simultaneously, they effect opposing actions on cell Ca²⁺ homeostasis and pH_i, and the CEC-sensitive receptor attenuates the positive inotropic action of the WB-4101-sensitive receptor subtype; and 6) because all effects of nonselective ₁-AR stimulation with phenylephrine are abolished by simultaneous addition of WB-4101 plus CEC, it is likely that only two ₁-AR subtypes modulate myocardial inotropy in rat ventricular myocytes.

That ₁-AR subtypes have opposing effects on myocardial inotropy, [Ca²⁺]_i, pH_i, and on Na⁺/H⁺ exchange is a novel discovery and may provide an explanation to prior studies which showed that nonselective ₁-AR stimulation can either enhance, have no effect on, or decrease myocardial contraction (4, 9, 23, 31). These different responses, at least in part, may be related to preferential activation of one of the two receptor subtypes under varying experimental conditions or among species under a given experimental condition. Prior studies have shown that nonselective -adrenergic stimulation causes cytosolic alkalinization via PKC-mediated activation of Na⁺/H⁺ exchange (10, 29). Here we report that the WB-4101-sensitive ₁-AR subtype (i.e., phenylephrine plus CEC) mediates such an increase in pH_i. In addition, the effect of the CEC-sensitive subtype (i.e., phenylephrine plus WB-4101) to decrease pH_i seems also to be mediated by PKC modulation of the Na⁺/H⁺ exchange as it is abolished either by staurosporine, prolonged exposure to PMA, or by EIPA. Indeed, in mammalian myocardium, there is evidence that the WB-4101-sensitive subtype is mainly coupled to phosphoinositide hydrolysis and PKC activation (14). This is also confirmed by previous results obtained in similar experimental conditions. In high bathing [Ca²⁺], a condition known to cause a downregulation of PKC, stimulation of the WB-4101-sensitive ₁-AR subtypes had no appreciable effect on twitch amplitude, cytosolic Ca²⁺, and pH_i (11). Interestingly, in the same experimental conditions, the effect of CEC-sensitive subtypes to reduce pH_i was abolished, whereas the residual negative inotropic effect correlated with a reduced cytosolic Ca²⁺ (11). Thus the present results suggest the involvement of PKC in the effect of both CEC- and WB-4101-sensitive ₁-AR receptor subtypes.

It is uncertain how PKC may decrease pH_i via Na⁺/H⁺ exchange. However, recent studies have identified different Na⁺/H⁺ exchanger isoforms (32), and one member of the Na⁺/H⁺ exchanger gene family, NHE-3, has been reported to decrease pH_i upon acute stimulation with PMA (16). Thus it is tempting to suggest that different Na⁺/H⁺ exchanger isoforms may be expressed within the rat myocardium and that CEC- and WB-4101-sensitive ₁-AR subtypes may be coupled via PKC to Na⁺/H⁺ exchangers that possess opposite modulatory effects on pH_i. Alternatively, ₁-AR subtypes may be coupled to different PKC isoforms that have different effects on Na⁺/H⁺ exchange.

Although phenylephrine enhances myofilament responsiveness to Ca²⁺, and this effect can be prevented by preexposure in WB-4101, why this effect was not reversed by WB-4101 remains to be explained. It has been shown that cytosolic alkalinization (10, 29) and possibly a change in the phosphorylation state of myosin light chain 2 (5) are mechanisms for the ₁-AR effect on myofilament responsiveness to Ca²⁺. However, the reversibility of the enhanced myofilament-Ca²⁺ sensitivity due to ₁-AR stimulation has not yet been examined. Thus, once myofilament responsiveness to Ca²⁺ has been increased, it may not be promptly reversed, even if some of the mechanisms for this response are no longer operative.

From our functional studies, phenylephrine plus WB-4101 or CEC appeared to act on specific receptor subtypes without evidence of crossover binding. However, the selectivity of CEC and WB-4101 has been questioned (14, 21). CEC has been reported to bind irreversibly to sites identified as 1B, 1C, and 1D, and WB-4101 has been reported to have a relatively high affinity for both 1A and 1C subtypes (12). Nonetheless, both antagonists have been used extensively, and results similar to ours have been found by several authors employing rat myocardial preparations. In addition, as illustrated in Fig. 2, the adoption of the protocol of incubation and cell pretreatment with CEC as originally devised by Minneman et al. (19) elicited a pattern of responses virtually identical to our conventional preparations.

In summary, the present study indicates that WB-4101- and CEC-sensitive ₁-AR subtypes have opposite modulatory actions on myocardial contraction, cell Ca²⁺ homeostasis, and pH_i. Furthermore, our results complement those of other studies that have shown opposite effects of phenylephrine plus CEC and phenylephrine plus WB-4101 on the electrophysiological properties of different cardiac preparations (1, 2, 7, 20).