INTRODUCTION

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ACTIVATION OF the -adrenoceptor-linked stimulatory guanosine nucleotide-binding protein (G_s) (7) is a key event in the modulation of cardiac membrane currents by sympathetic stimulation (cf. Ref. 9). Ionic current regulation is generally ascribed to enhanced adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation of channel proteins by protein kinase A (PKA; e.g., Ref. 5) after -adrenoceptor activation of G_s and G_s activation of the adenylyl cyclase (AC)-cAMP-PKA cascade (cf. Ref. 7). This slow cytoplasmic cascade of reactions has been invoked to explain the stimulation of the L-type Ca²⁺ current (I_Ca; 20), pacemaker current (4), and delayed rectifier K⁺ current (32) by -adrenoceptor agonists as well as their inhibitory effects on the Na⁺ current (I_Na; 23). Additionally, -adrenoceptor-activated G_s also seems to have a fast, membrane-delimited, cAMP-independent action on I_Ca (20), pacemaker current (31), and I_Na (25).

In guinea pig ventricular and other mammalian cardiac tissues, -adrenoceptor agonists also activate a time- and voltage-independent Cl current (I_Cl; e.g., Refs. 2, 10, 12, 19), which seems to play an important role in the regulation of action potential duration and resting membrane potential in these cells (cf. Ref. 11). Current activation seems to require PKA-mediated phosphorylation as well as nucleotide binding and nucleotide hydrolysis (see Ref. 6) and is curtailed by dephosphorylation by okadaic acid-sensitive and okadaic acid-insensitive phosphatases (14). The contribution of membrane-delimited, cAMP-independent G_s pathways to the regulation of I_Cl is not entirely clear. Although Hwang et al. (15) failed to find evidence for direct G_s modulation of I_Cl, Kozlowski et al. (18) reported that nonhydrolyzable GTP analogs accelerated I_Cl activation in response to photolysis of caged cAMP.

The objective of this study was to further characterize the role of G_s in I_Cl regulation. To this end, we compared I_Cl magnitude as well as I_Cl activation and deactivation time courses after -adrenoceptor activation by isoproterenol (Iso) and AC stimulation by forskolin (Fsk) in guinea pig ventricular cardiomyocytes. Iso or Fsk was step applied by a rapid perfusion technique (cf. Ref. 24). We found that Fsk increased steady-state I_Cl and accelerated I_Cl activation in a concentration-dependent manner. The decay of I_Cl after agonist removal was little affected by variations in the Fsk concentration; however, with supramaximal concentrations (>1 µM Fsk), the delay between agonist washout and the onset of I_Cl decay was significantly prolonged. Stimulation of G_s with 1 µM Iso elicited an I_Cl that activated faster and was larger in size than the maximal I_Cl induced by direct stimulation of AC with Fsk. However, Iso failed to augment I_Cl activated by 1 µM Fsk, and current activation by both Iso and Fsk was abolished when cAMP-dependent phosphorylation was inhibited. Threshold stimulation of -adrenoceptor-coupled G_s protein by 1 nM Iso, which was not sufficient to induce a detectable I_Cl, accelerated the Fsk-induced activation of I_Cl but did not alter I_Cl magnitude and I_Cl deactivation time course. The effect of low Iso was abolished by cell dialysis with guanosine 5'-O-(2-thiodiphosphate) (GDPS). We conclude that cAMP-independent G_s protein pathways do not activate I_Cl but enhance cAMP-mediated current activation in guinea pig ventricular heart cells. Attempts to duplicate the above observations in cardiomyocytes from guinea pig atrium, where the presence of a small cAMP-activated I_Cl had been reported (17), failed because cAMP-activated I_Cl could not be detected in this preparation. cAMP-activated I_Cl was also found to be absent in cardiomyocytes from rat and frog ventricles.

	MATERIALS AND METHODS

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Cell preparation. Single ventricular myocytes from adult guinea pig, rat (Sprague-Dawley), and frog (Rana pipiens) hearts were enzymatically dissociated by a previously described method (21) with Yakult collagenase (0.1 mg/ml for guinea pig and rat, 0.2 mg/ml for frog) and stored in a high-K⁺-low-Na⁺ solution (16). Atrial myocytes from guinea pigs were isolated as described by Wu et al. (33). After digestion of the heart, the atria were removed, minced, and further digested in a shaker bath at 37°C with an enzyme solution containing 0.1 mg/ml of dispase, 0.12 mg/ml of trypsin, 0.32 mg/ml of collagenase (type II), and 2.12 mg/ml of albumin. The cells were harvested from the supernatant at 15-min intervals, resuspended in a solution containing (in mM) 1.1 CaCl₂, 127 NaCl, 4.6 KCl, 1.1 MgSO₄, 2 Na-pyruvate, 10 glucose, 10 creatine, 20 taurine, 5 ribose, 0.01 adenine, 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 0.1 allopurinol, pH 7.4, and maintained at room temperature.

Solutions and chemicals. The cells were superfused with an extracellular solution containing (in mM) 140 or 105 NaCl (frog), 5.4 CsCl, 1.8 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose, pH 7.4 with NaOH, and dialyzed via a patch pipette with a solution containing (in mM) 50 CsCl, 80 or 60 Cs-aspartate (frog), 1 MgCl₂, 5 Mg-ATP, 1 Na₂-GTP, 10 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 10 HEPES, pH 7.4 with CsOH. For measurements of Cl selectivity, Cl in the dialysate was partially substituted for by aspartate. In experiments designed to inhibit G protein activation (3), the pipette solution was supplemented with 0.1 mM GDPS (Na₂-GTP was omitted). To inhibit cAMP-dependent phosphorylation, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; 0.1 mM; 26) was added to the pipette solution (Mg-ATP was omitted). Iso (1 mM stock in ascorbic acid-containing H₂O solution) and Fsk (10 mM stock in dimethyl sulfoxide) were added in appropriate amounts singly or in combination to the extracellular solution. Dimethyl sulfoxide (up to 0.1%) had no effect on I_Cl and I_Ca.

Electrophysiology and concentration-clamp method. Isolated cardiomyocytes were transferred to the recording chamber and continuously superfused with the extracellular solution at room temperature (22 ± 1°C). Low-resistance patch pipettes (1-3 M) were prepared in the usual two-step process (8). After gigaseal formation and patch breakthrough, voltage clamp was established by an EPC 9 amplifier controlled by an Atari Mega 4 computer with the E9SCREEN acquisition software (Heka, Lamprecht/Pfalz). Current was measured 1) at a holding potential of 40 mV; 2) during voltage ramps (ramp speed 1 V/s) consisting of three phases: a depolarizing phase from the holding potential of 40 to +80 mV, a hyperpolarizing phase to 80 mV, and a return to the holding potential; and 3) during 200-ms voltage pulses applied from 40 mV to various test potentials at 0.1-0.33 Hz. Cell capacitance was determined with software routines incorporated in E9SCREEN as the time integral of the capacitive current surge measured in response to 5-mV hyperpolarizing steps from a holding potential of 40 mV after correction for series resistance. Currents and voltages were recorded on Atari Megafile 44 disks and analyzed with the Atari data-analysis software 3.11 (Instrutech, Elmont, NY). I_Cl was determined as the Iso- or Fsk-induced change in current under conditions expected to minimize contaminating currents (K⁺ currents were inhibited by replacement of external and internal K⁺ by Cs⁺, I_Na and T-type I_Ca were inactivated by the 40-mV holding potential, and L-type I_Ca was blocked by 1 mM CdCl₂ added to the superfusate). Current density (in pA/pF) is expressed as the ratio of agonist-induced change in current and cell capacitance (90-170 pF). Rapid applications of Iso and Fsk were produced by an electronically operated multibarrel microperfusion system (24) that allows the complete exchange of extracellular solution around heart cells within <30 ms.

Data are expressed as means ± SD. Statistical significance was assessed with the unpaired, two-tailed t-test.

	RESULTS

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cAMP-dependent I_Cl in guinea pig ventricular cardiomyocytes. Figure 1 demonstrates the effect of step applications of Iso and Fsk on the background current in guinea pig ventricular heart cells. At a holding potential of 40 mV, a saturating concentration of Iso (1 µM) consistently induced a substantial inward holding current (Fig. 1Aa). Current activation by Iso was abolished when cAMP-dependent phosphorylation was inhibited by cell dialysis with 0.1 mM Rp-cAMPS (Fig. 1Ab). A threshold concentration of Iso (1 nM), which has been reported to exert Rp-cAMPS-resistant, GDPS-sensitive effects on I_Ca in guinea pig ventricular cardiomyocytes (cf. Ref. 24), had no detectable effect on the holding current (see Fig. 1Ac). Fsk (1 µM) mimicked the effect of Iso in generating an inward holding current (Fig. 1Ad); however, in response to stimulation of AC with Fsk, the current activated slower and reached a smaller steady-state amplitude than that activated by -adrenoceptor stimulation with Iso. At a holding potential of 40 mV, current activation with 1 µM Iso was preceded by a latency of 3 ± 2 s and reached 50 and 95% completion in 16 ± 3 and 44 ± 9 s, respectively (Fig. 1B). After exposure of the myocytes to 1 µM Fsk, current activation was detectable after a latency of 28 ± 8 s and reached 50 and 95% completion only after 62 ± 13 and 112 ± 8 s, respectively (Fig. 1B). The average steady-state current activated by 1 µM Fsk (0.57 ± 0.30 pA/pF, Fig. 1B, inset) was significantly smaller than the current activated by 1 µM Iso (0.89 ± 0.32 pA/pF; P = 0.005). However, 1 µM Iso failed to further augment the current stimulated by 1 µM Fsk (Fig. 1C).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effect of isoproterenol (Iso) and forskolin (Fsk) on holding current in guinea pig ventricular myocytes. A: chart recordings of holding current at 40 mV. Arrows, rapid application of 1 µM Iso (a), 1 µM Iso after block of protein kinase A by cell dialysis with 0.1 mM Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; b), 1 nM Iso (c), and 1 µM Fsk (d). Dashed line, 0-current level. B: normalized average time course of changes in holding current I/Imax at 40 mV in response to rapid application at 0 time of 1 µM Iso (n = 9 myocytes) or 1 µM Fsk (n = 10 myocytes). Horizontal and vertical lines, SDs of time and current density, respectively, at 50% activation. Current activation with Iso is significantly faster (P < 0.0001). Inset, average steady-state density of holding current activated by 1 µM Iso (n = 14 myocytes) and 1 µM Fsk (n = 21 myocytes). Iso activates significantly more current than Fsk (P = 0.005). All data in Figs. 1-8 are from a 1st exposure of cells to agonist. C: current density recorded at 40 and +40 mV.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Cl-sensitive nature of Fsk-induced current. A: current-voltage (I-V) relationships recorded from cells superfused with extracellular solution containing 150 mM Cl and dialyzed with solution containing 120 (a) or 22 mM Cl (b) before (control), during, and after (washout) exposure to 1 µM Fsk. Erev, reversal potential of Fsk-induced current; [Cl]e, extracellular Cl concentration; [Cl]i, intracellular Cl concentration. B: dependence of Erev on [Cl]i in 150 mM extracellular Cl solution. Solid line, fit of data points (from Figs. 2A and 3A) by the Goldman equation with an aspartate-to-Cl-permeability ratio of 0.17; dotted line, relationship between Erev and [Cl]i as calculated with the Goldman equation for a pure Cl conductance.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Dependence of steady-state ICl density and ICl activation time course on concentration of Fsk. A: averaged I-V relationships of ICl recorded from cells under standard conditions in response to stimulation with 0.03 µM Fsk (n = 3 myocytes), 0.3 µM Fsk (n = 4 myocytes), 1 µM Fsk (n = 4 myocytes), 3 µM Fsk (n = 3 myocytes), and 10 µM Fsk (n = 5 myocytes). B: dose-response relationship for Fsk and ICl density at 40 () and +40 mV (). Each point represents average ICl density measured in 3-10 cells. Smooth curve, least-squares fit of data at 40 mV with the Hill equation with a half-maximal concentration of Fsk of 0.39 ± 0.03 mM and a Hill coefficient of 2.43 ± 0.55. C: correlation between time of half-maximal activation of ICl (1/2) and concentration of Fsk determined at 40 mV.

Dependence of I_Cl on the concentration of Fsk. To examine why the stimulation of AC with 1 µM Fsk was less efficient in activating I_Cl than -adrenoceptor stimulation with 1 µM Iso (see Fig. 1B), we next determined the dependence of I_Cl density, I_Cl activation time course, and I_Cl deactivation on the concentration of Fsk. Figure 3A shows averaged current-voltage (I-V) relationships of I_Cl induced by different concentrations of Fsk. Over a potential range from 80 to +80 mV, 0.03 µM Fsk mostly failed to activate I_Cl. Significant current activation occurred with 0.3 µM Fsk. With 1 µM Fsk, I_Cl density was maximal and indistinguishable from that obtained with 3 and 10 µM Fsk. The relationship between Fsk concentration and current density at 40 mV was identical to that at +40 mV (Fig. 3B) and well fitted by a Hill equation with a coefficient of ~2 and a half-maximal concentration of 0.39 µM.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Dependence of ICl deactivation on concentration of Fsk. A: time course of ICl deactivation in 3 myocytes after withdrawal of 0.6, 1, and 10 µM Fsk at 0 time. ICl was measured at +40 mV and normalized to ICl density at time of Fsk withdrawal. B: average delay between Fsk withdrawal and onset of ICl decay (left) and average time of half-maximal ICl decay (1/2 measured from onset of current decay; right) with 0.6 (n = 3 myocytes), 1 (n = 5 myocytes), and 10 µM Fsk (n = 6 myocytes).

Effect of -adrenoceptor-coupled G_s on Fsk activation of I_Cl. Low concentrations of Iso (~1 nM) that have been reported to exert Rp-cAMPS-resistant, GDPS-sensitive effects on I_Ca (cf. Ref. 24) did not induce detectable activation of I_Cl (see Fig. 1Ac) nor did the application of 1 µM Iso when cAMP-dependent phosphorylation was inhibited (see Fig. 1Ab). On the other hand, cAMP-mediated activation of both I_Ca and I_Cl was found to be accelerated by nonhydrolyzable GTP analogs (18). To further examine the role of -adrenoceptor-coupled G_s in the regulation of I_Cl, we studied the effect of 1 nM Iso on Fsk-induced I_Cl. The recordings in Fig. 5A show that I_Cl on step application of 1 µM Fsk (top) activated slower than if applied together with 1 nM Iso (bottom). In all myocytes examined, the coapplication of low Iso had no significant effect on the size of I_Cl (Fig. 5B) but accelerated the 1 µM Fsk-induced activation of I_Cl by ~35% (Fig. 5C; times to current onset and to 50 and 95% activation being 19 ± 6, 42 ± 11, and 74 ± 10 s, respectively), an effect that was not observed when 1 nM Iso was coapplied with 10 µM Fsk (see Fig. 5C, inset; n = 4 myocytes).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effect of low concentrations of Iso on activation of ICl by Fsk in guinea pig ventricular myocytes. A: chart recordings of ICl (at 40 mV) activated by 1 µM Fsk alone (top) and by simultaneous application of 1 µM Fsk together with 1 nM Iso (bottom). Arrows, time of application. Dashed line, 0-current level. B: average steady-state density of current activated by 1 µM Fsk (n = 21 myocytes) and 1 µM Fsk + 1 nM Iso (n = 18 myocytes). Difference in current density is statistically not significant (P = 0.24). C: normalized average activation time course of ICl at 40 mV in response to rapid application at 0 time of 1 µM Fsk (n = 10 myocytes) or simultaneous application of 1 µM Fsk + 1 nM Iso (n = 13 myocytes). Horizontal and vertical lines, SDs in time and current density, respectively, at 50% ICl activation. Difference in times of 50% activation is statistically significant (P = 0.0028). Inset, 1/2 with 1 and 10 µM Fsk in absence or presence of 1 nM Iso.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Low Iso-induced acceleration of ICl activation by Fsk in guinea pig ventricular myocytes is abolished by guanosine 5'-O-(2-thiodiphosphate) (GDPS). A: chart recordings of ICl (at 40 mV) activated by 1 µM Fsk alone (top) and by simultaneous application of 1 µM Fsk together with 1 nM Iso (bottom) after cell dialysis with GTP-free solution containing 0.1 mM GDPS. Arrows, time of application. Dashed line, 0-current level. B: normalized average activation time course of ICl at 40 mV in response to rapid application at 0 time of 1 µM Fsk (n = 6 myocytes) or 1 µM Fsk + 1 nM Iso (n = 8 myocytes) after cell dialysis with GTP-free solution containing 0.1 mM GDPS. Means ± SD at 50% deactivation are omitted for clarity because they were virtually identical. Inset, average steady-state magnitude of current activated by 1 µM Fsk alone or 1 µM Fsk + 1nM Iso after GDPS dialysis (P = 0.018).

Effect of -adrenoceptor-coupled G_s on I_Cl deactivation. To further investigate whether Iso alters the responses of I_Cl to Fsk stimulation via effects on cell cAMP or via cAMP-independent pathways, we tried to assess the cAMP level reached by stimulation with Fsk and/or Iso by measuring I_Cl deactivation. Figure 7A shows typical examples of I_Cl deactivation after stimulation and washout of Iso and Fsk alone or in combination. Compared with 1 µM Fsk (Fig. 7A, ), I_Cl deactivated somewhat faster when 1 µM Iso was used as the agonist (Fig. 7A, ). This was due to a shorter delay to the onset of current decay (56 ± 4 s; Fig. 7B, left) as well as a shorter time to half-maximal current decay (53 ± 9 s; Fig. 7B, right). Current deactivation after exposure to 1 µM Fsk + 1 nM Iso (Fig. 7A, ) was slightly faster than with Fsk alone; however, on average, both the delay to the onset of I_Cl decay (Fig. 7B, left) and the time of half-maximal current decay (Fig. 7B, right) were not significantly different from the values obtained with Fsk alone. After stimulation of AC with 1 µM Iso + 1 µM Fsk (Fig. 7A, ), I_Cl deactivation was the slowest, mostly due to the prolonged delay to the onset of I_Cl decay. Indeed, the delay to the onset of I_Cl decay (321 ± 42 s; Fig. 7B, right) and the time of half-maximal I_Cl decay (106 ± 21 s; Fig. 7B, left) were similar to the values observed with 10 µM Fsk (see Fig. 4B). Compared with the deactivation time course with 1 µM Fsk, the most prominent alteration in the deactivation kinetics of I_Cl was a prolongation of the delay to the onset of I_Cl decay at supramaximal levels of cAMP reached either by increasing the concentration of Fsk to 10 µM (see Fig. 4) or by coapplication of 1 µM Iso. The decay of I_Cl was comparatively little affected by variations in agonist or agonist concentration and seems to be less sensitive to changes in the steady-state level of cAMP.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Time course of ICl deactivation after stimulation with Iso and/or Fsk. A: time course of ICl deactivation after withdrawal of 1 µM Iso, 1 µM Fsk, 1 µM Fsk + 1 nM Iso, and 1 µM Fsk + 1 µM Iso at 0 time. ICl was measured at +40 mV and normalized to ICl density at time of agonist withdrawal. B: average delay between agonist withdrawal and onset of ICl decay (left) and average time of half-maximal ICl decay (1/2 measured from onset of current decay; right) with 1 µM Fsk (n = 5 myocytes), 1 µM Fsk + 1nM Iso (n = 5 myocytes), 1 µM Fsk + 1 µM Iso (n = 7 myocytes), and 1 µM Iso (n = 4 myocytes).

cAMP-activated I_Cl in cardiomyocytes from different species. We tried to determine whether similar differences in I_Cl responses to Fsk and Iso exist in cardiomyocytes from other tissues. Figure 8 demonstrates the effect of Iso (1 µM) on holding current at 40 mV and I_Ca at 0 mV in cardiomyocytes from guinea pig ventricle (Fig. 8A) and atrium (Fig. 8B), rat ventricle (Fig. 8C), and frog ventricle (Fig. 8D). In all cell types, step application of Iso increased I_Ca by more than twofold; however, Iso affected the holding current only in guinea pig ventricular heart cells. These results confirm observations by others (12) that cAMP-dependent I_Cl is not ubiquitously present in mammalian cardiac tissue and show that it is also absent in frog ventricle.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effect of Iso on holding current and Ca2+ current (ICa) in enzymatically isolated cardiomyocytes from different species. A-D, left: chart recordings of ICa and holding current in guinea pig ventricle (A), guinea pig atrium (B), rat ventricle (C), and frog ventricle (D). Membrane was held at 40 mV; ICa was triggered by 40- (C) or 50-ms (A, B, and D) voltage-clamp pulses to 0 mV applied at 0.2 (A and B) or 0.25 Hz (C and D). Arrows, step application of 1 µM Iso. A-D, right: ICa as well as short segments of holding current before and after depolarizing steps at high resolution.

	DISCUSSION

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Responses of I_Cl to stimulation with Fsk. The above results show that, in guinea pig ventricular myocytes, Fsk affects I_Cl activation time course and steady-state conductance in a dose-dependent manner. The Hill coefficient of ~2 for the steep dose-conductance curve (see Fig. 3B) is similar to that determined with Iso (29) and might result from the stochiometry for PKA activation by cAMP; four cAMP molecules are required to liberate two catalytic subunits (28).

Effects of G_s activation by low Iso on Fsk activation of I_Cl. Activated G proteins modulate the Fsk stimulation of AC in various preparations, usually by altering both the potency and efficacy of Fsk (27); for example, activated G_i protein inhibited the Fsk-induced activation of I_Cl in guinea pig ventricular myocytes (cf. Ref. 13). The inhibition seemed to result from a decrease in the potency of Fsk. An increase in Fsk potency and/or efficacy by activated G_s protein (27) would enhance cAMP production and could explain the acceleration of I_Cl activation by 1 µM Fsk in the presence of 1 nM Iso (see Fig. 5C). If this explanation were correct, the increase in cAMP induced by 1 nM Iso should be similar to that obtained by elevating the Fsk concentration from 1 to 10 µM because coapplication of 1nM Iso accelerated the activation of I_Cl to a similar extent as did the elevation of Fsk from 1 to 10 µM (see Fig. 5C, inset). Direct measurements of cAMP levels in single cardiomyocytes are presently not possible; however, an estimate of relative cAMP levels can be obtained by comparing the time course of I_Cl deactivation under both conditions. After stimulation and washout of 10 µM Fsk, I_Cl deactivated significantly slower than with 1 µM Fsk (see Fig. 7). This would indicate that AC stimulation with 10 µM Fsk elevates the level of cAMP well above the saturating concentration achieved with 1 µM Fsk, which prolongs the delay to the onset of I_Cl decay by the time it takes for the 10 µM Fsk-induced cAMP level to decrease to the level reached with 1 µM Fsk. In contrast, the coapplication of 1 nM Iso did not significantly affect the deactivation of 1 µM Fsk-induced I_Cl (see Fig. 7), which argues against a significant increase in cell cAMP level on G_s activation. This indicates that a G_s-induced increase in Fsk potency and/or efficacy cannot account for the effects of 1 nM Iso on Fsk-induced I_Cl.

Response of I_Cl to stimulation with Iso. Several observations have led to the conclusion that the activation of I_Cl by Iso results exclusively from stimulation of the AC-cAMP-PKA pathway (cf. Ref. 6). Most importantly, 1) I_Cl activation by Iso was abolished when cAMP-dependent phosphorylation was inhibited (see Fig. 1A; cf. Ref. 15) and 2) stimulation with Fsk or other interventions that increase cellular cAMP levels occluded the effect of Iso (see Fig. 1C; cf. Ref. 15). However, when we compared I_Cl densities in response to -adrenoceptor activation with Iso or AC stimulation with Fsk under standardized conditions (high GTP-containing dialysates were used to minimize current rundown and only responses to a first agonist exposure were compared), we found that the maximal steady-state I_Cl in response to Iso (1 µM) was 1.6 times larger than that in response to 1 µM Fsk (see Fig. 1C). We are not aware of another study where I_Cl densities in response to -adrenoceptor stimulation with Iso or AC stimulation with Fsk are compared, paying particular attention to the possible problems arising from repetitive agonist applications, i.e., current rundown or other desensitization processes. Hwang et al. (15), in guinea pig ventricular myocytes at 36°C, also recorded an average I_Cl density in response to 1 µM Iso [1.3 ± 0.5 (SE) µA/µF] that was 1.6 times larger than that in response to 1 µM Fsk (0.8 ± 0.1 µA/µF). Although this difference was not significant compared with the large variations in current density with either agonist, the similarity to our results is obvious.