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(Circulation Research. 1995;77:565-574.)
© 1995 American Heart Association, Inc.

Articles

ß-Adrenergic Stimulation Induces Acetylcholine to Activate ATP-Sensitive K⁺ Current in Cat Atrial Myocytes

Presented in part in abstract form (Biophys J. 1995;68:12).

Yong Gao Wang, Stephen L. Lipsius

From the Department of Physiology, Loyola University of Chicago, Stritch School of Medicine, Maywood, Ill.

Correspondence to Stephen L. Lipsius, PhD, Department of Physiology, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153.

	Abstract

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Abstract Our previous work on atrial myocytes suggested that the effect of acetylcholine (ACh) to increase K⁺ conductance can be potentiated by prior loading of the sarcoplasmic reticulum (SR) with Ca²⁺. The present study, therefore, sought to determine whether prior exposure to isoproterenol (ISO) potentiates ACh-induced increases in K⁺ conductance and the underlying mechanisms. A nystatin–perforated patch whole-cell configuration was used to record from cat atrial myocytes. Voltage-clamp ramps (40 mV/s) were used to assess total membrane conductance. The experimental protocol consisted of two consecutive 30-second ACh exposures (ACh₁ and ACh₂) separated by a 6-minute recovery period in ACh-free solution. In general, experimental interventions, such as exposure to ISO, were imposed during the period between ACh₁ and ACh₂ to determine their effects on the response to ACh₂ in relation to ACh₁. Under control conditions, K⁺ conductances induced by ACh₁ and ACh₂ were not different from one another with or without activation of L-type Ca²⁺ current (I_Ca,L) during the recovery period. When 1 µmol/L ISO plus I_Ca,L activation was imposed during the recovery period, ACh₂ induced a significantly larger increase in K⁺ conductance than ACh₁. The ACh₂-induced K⁺ current potentiated by ISO was time independent and selectively blocked by 10 µmol/L glibenclamide and therefore identified as ATP-sensitive K⁺ current (I_K,ATP). The effect of ISO to induce ACh₂-activated I_K,ATP was mimicked by 1 µmol/L forskolin or 200 µmol/L 8-(4-chlorophenylthio)-cAMP, but not by 0.5 µmol/L BAY K 8644, and was selectively abolished by (1) 5 µmol/L thapsigargin or 1 µmol/L ryanodine, agents that prevent accumulation of SR Ca²⁺, (2) inhibition of L-type Ca²⁺ current (I_Ca,L) by 1 µmol/L nisoldipine or zero external Ca²⁺, (3) 50 µmol/L Rp-cAMPs, an inhibitor of cAMP-dependent protein kinase A, or (4) 2 µmol/L propranolol. Atropine (1 µmol/L) abolished all ACh-induced currents. Moreover, ACh₂-activated I_K,ATP was selectively blocked by 0.2 µmol/L pirenzepine, an M₁ muscarinic receptor antagonist, or 0.1 µmol/L calphostin C, a selective inhibitor of protein kinase C. AFDX116 (100 µmol/L), an M₂ muscarinic receptor antagonist, blocked the conventional ACh-activated K⁺ current and revealed ACh₂-activated I_K,ATP. These results indicate that prior exposure to ISO potentiates ACh-induced increases in K⁺ current via ACh-activated I_K,ATP. ISO acts via ß-adrenergic receptors and cAMP to enhance both Ca²⁺ influx via I_Ca,L and SR Ca²⁺ uptake. By loading SR Ca²⁺, ISO facilitates ACh-induced stimulation of the M₁ receptor/phosphoinositol signaling pathway to activate I_K,ATP. These findings suggest that prior ß-adrenergic stimulation accentuates subsequent cholinergic inhibition of atrial function via ACh-induced activation of ATP-sensitive K⁺ channels.

Key Words: perforated patch • muscarinic receptors • cAMP • forskolin • phosphoinositol • calcium

	Introduction

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ACh affects cardiac function through a variety of signal transduction pathways. Thus, ACh acts via muscarinic receptors coupled to PTX-sensitive G proteins to inhibit adenylate cyclase activity¹ and thereby inhibit cardiac responses mediated by cAMP. This mechanism contributes to the accentuated inhibitory effects of ACh observed in the presence of ß-adrenergic receptor stimulation, ie, accentuated antagonism.^{2 3} ACh can also act via muscarinic receptors and pertussis toxin–sensitive G proteins to directly activate K⁺ channel currents (I_K,ACh).^{4 5} This mechanism is membrane-delimited and therefore not mediated via second messengers. Recent reports,^{6 7 8} including our own,^{9 10} indicate that ACh also can activate cardiac I_K,ATP. However, the mechanisms underlying cholinergic regulation of I_K,ATP are not well understood. Work on ventricular cell membrane patches suggests that ACh may activate I_K,ATP via G proteins that lower the sensitivity of ATP-sensitive K⁺ channels to inhibition by ATP.⁸ Our previous studies in atrial myocytes indicate that ACh activates I_K,ATP via second messengers generated by the PI signaling pathway.¹⁰ Moreover, these studies suggest that the ability of ACh to activate I_K,ATP depended on prior loading of SR Ca²⁺ induced by an initial ACh exposure. This latter finding has led to the present hypothesis that other agents that raise SR Ca²⁺ can stimulate ACh-induced activation of I_K,ATP. In the present study, therefore, we sought to determine whether prior ß-adrenergic stimulation by ISO can induce ACh to activate I_K,ATP.

The response of atrial myocytes to ISO can be altered by the ruptured-patch recording method, presumably because of cell dialysis.¹¹ In the present study, therefore, we used a nystatin–perforated patch¹² whole-cell recording method to minimize alterations in the intracellular milieu and thereby preserve intracellular signaling processes. The present experiments indicate that prior exposure to ISO induces ACh to activate a GLIB-sensitive K⁺ current, presumably I_K,ATP. The effect of ISO to potentiate ACh-induced increases in K⁺ conductance is a novel mechanism that may contribute to accentuated cholinergic antagonism of atrial function.

	Materials and Methods

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Details of the isolation and recording methods have been published previously.¹³ Briefly, adult cats of either sex were anesthetized with sodium pentobarbital (70 mg/kg IP). Hearts were mounted and perfused via a Langendorff apparatus. The initial perfusion was a bicarbonate-buffered Tyrode's solution for 5 minutes followed by a 5-minute perfusion with a nominally Ca²⁺-free Tyrode's solution and then a 30- to 40-minute perfusion with low (36 µmol/L) Ca²⁺ Tyrode's solution containing 0.06% collagenase (Worthington Biochemical, type II). After collagenase perfusion, both atria were cut into small pieces and agitated in fresh collagenase and 0.01% protease. Experiments were performed on either right or left atrial cells, with no discernible differences.

Cells used for study were transferred to a small (0.3-mL) tissue bath on the stage of an inverted microscope (Nikon Diaphot) and superfused with a modified Tyrode's solution containing (in mmol/L) NaCl 137, KCl 5.4, MgCl₂ 1.0, CaCl₂ 2.5, HEPES 5, and glucose 11 and titrated with NaOH to a pH of 7.4. Solutions were perfused by gravity at 5 mL/min, and experiments were performed at 35±1°C. Cells studied were isolated on the same morning as each experiment, and those selected for study were elongated and quiescent. Voltage and ionic currents were recorded by using a nystatin–perforated patch¹² whole-cell recording method.¹⁴ This method minimizes dialysis of intracellular constituents with the internal pipette solution. Nystatin was dissolved in dimethyl sulfoxide at a concentration of 50 mg/mL and then added to the internal pipette solution to yield a final nystatin concentration of 150 µg/mL. The internal pipette solution contained (in mmol/L) potassium glutamate 100, KCl 40, MgCl₂ 1.0, Na₂-ATP 4, EGTA 0.5, and HEPES 5 and was titrated with KOH to a pH of 7.2. Pipettes had inner diameters of 1 to 1.5 µm and, when filled with internal solution, had resistances of 2 to 3 M. Once a gigaseal was formed, access resistance decreased to as low as 15 M after 5 to 10 minutes.

Our standard experimental protocol involved an initial 30-second ACh exposure (ACh₁) followed by a 6-minute recovery period in ACh-free solution and then a second 30-second ACh exposure (ACh₂) (see Fig 1). In most experiments, I_Ca,L was activated by depolarizing voltage-clamp pulses imposed during the recovery period between ACh₁ and ACh₂. In general, experimental interventions such as exposure to ISO, forskolin, and BAY K 8644 were also imposed during the 6-minute recovery period, after ACh₁ and before ACh₂, unless stated otherwise. In this way, we determined the effect of each experimental intervention on ACh-induced K⁺ conductances by comparing the response to ACh₂ in relation to ACh₁. Voltage-clamp ramps (40 mV/s) between -130 and +30 mV were imposed during and after each ACh exposure to assess changes in total membrane conductance. Voltage ramps were delivered during the administering of ACh at the peak increase in ACh-induced K⁺ conductance, ie, within 1 second. In some experiments, steady state I-V relations were generated by clamping from a holding potential of -40 mV to more positive and negative voltages for 2 seconds in 10-mV increments. Steady state currents were measured at the end of each clamp step. In these experiments, external solutions contained 1 µmol/L verapamil to block I_Ca,L and 1 mmol/L 4-aminopyridine to block transient outward current, respectively. Activation of I_Ca,L was elicited by voltage-clamp steps from a holding potential of -40 to 0 mV for 200 milliseconds at 0.5 Hz. Current densities (pA/pF) were determined by normalizing currents to total cell capacitance.¹⁵

View larger version (14K): [in this window] [in a new window]   Figure 1. I-V relations of original K+ currents elicited by two consecutive ACh exposures separated by a 6-minute recovery period. A, ICa,L was activated by depolarizing pulses during the recovery period between ACh1 and ACh2. ACh1 and ACh2 elicited similar increases in K+ current. B, ISO (1 µmol/L) plus ICa,L activation were imposed during the recovery period. ACh2 elicited a larger increase in K+ current than did ACh1 at voltages positive and negative to the reversal potential. Inset, ISO increased basal ICa,L amplitude (328%) and slowed inactivation. c,w indicates background currents recorded during the control period (c) shortly before ACh1 and during the recovery period (w) shortly before ACh2.

A single suction pipette recorded voltage (bridge mode) or ionic currents (discontinuous voltage-clamp mode) by use of an Axoclamp 2A amplifier (Axon Instruments, Inc). In the voltage-clamp mode, the sample rate was 10 to 12 kHz. A second scope was used to monitor the duty cycle to ensure complete settling of the voltage transient between samples. Computer software (PCLAMP, Axon Instruments, Inc) was used to deliver voltage protocols and to acquire and analyze data. All signals were digitally recorded on VCR tape.

Drugs included ACh (Sigma Chemical Co), ISO (Sigma), forskolin (Sigma), Rp-cAMPs (LC Laboratories), ryanodine (Progressive Agri-Systems, Inc), 8-CPT-cAMP (Sigma), BAY K 8644 (a generous gift from Miles, Inc), thapsigargin (Sigma), atropine (Sigma), AFDX116 (a generous gift from Boehringer Ingelheim), calphostin C (Kamiya Biomedical Co), pirenzepine (Sigma), propranolol (Sigma), and GLIB (Sigma). Statistical significance of paired and unpaired data was determined by Student's t test and at P<.05. Data are expressed as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of ISO on I_K,ACh
Fig 1A shows original currents elicited by voltage-clamp ramps during the two consecutive ACh exposures (ACh₁ and ACh₂) that were separated by a 6-minute recovery period. I_Ca,L was activated by depolarizing pulses every 2 seconds during the recovery period between ACh₁ and ACh₂ (not shown). The I-V relation shows that ACh₁ elicited an increase in membrane conductance typical of I_K,ACh, ie, a reversal potential close to the K⁺ equilibrium potential, and inward rectification at positive voltages. ACh₂ elicited essentially the same increase in K⁺ current as did ACh₁; ie, ACh₁- and ACh₂-induced currents were superimposed. In a total of four cells, there were no significant differences between currents induced by ACh₁ and ACh₂. Similar results were obtained when the same protocol was performed without activation of I_Ca,L during the recovery period; ie, ACh₁- and ACh₂-induced currents were superimposed (not shown) (n=4). These findings indicate that an initial 30-second exposure to ACh has no influence on the effect of a second 30-second ACh exposure to increase K⁺ conductance. Furthermore, the additional Ca²⁺ influx resulting from activation of basal I_Ca,L has no influence on the ability of ACh₂ to increase K⁺ conductance. In Fig 1B, the same protocol was repeated, including activation of I_Ca,L, with the addition of 1 µmol/L ISO during the recovery period between ACh₁ and ACh₂. ISO had no effect on background current (w in Fig 1B). As expected, ISO markedly increased peak I_Ca,L amplitude and slowed inactivation (inset). ISO increased mean peak I_Ca,L by 220% (n=6). After exposure to ISO and I_Ca,L activation for 6 minutes, ACh₂ induced a markedly larger increase in K⁺ conductance than ACh₁ at voltages both positive and negative to the reversal potential. In a total of 15 cells, after subtraction of control background currents, ACh₁- and ACh₂-induced K⁺ currents elicited at -130 mV were 10.8±1.0 and 14.7±1.2 pA/pF, respectively (P<.005), and those elicited at +30 mV were 11.7±1.0 and 15.2±1.5 pA/pF, respectively (P<.005). The percent changes between ACh₁- and ACh₂-induced K⁺ currents for this series of experiments and the subsequent experiments described below are summarized in the Table. The present findings indicate that ISO potentiates ACh₂-induced increases in K⁺ conductance. The purpose of the present study was to determine the nature of the ACh-induced K⁺ current potentiated by ISO and the underlying mechanisms responsible for its activation.

View this table: [in this window] [in a new window]   Table 1. Percent Changes in K+ Currents Induced by ACh2 in Relation to ACh1

Effect of GLIB
Our previous study¹⁰ indicates that under appropriate conditions ACh can activate two separate K⁺ currents: I_K,ACh and a K⁺ current that is inhibited by GLIB, a specific blocker of I_K,ATP in cardiac muscle.^{16 17} In Fig 2A, we determined whether the ACh₂-induced K⁺ current potentiated by ISO was selectively blocked by 10 µmol/L GLIB. GLIB was administered throughout the experiment, and 1 µmol/L ISO and I_Ca,L activation were imposed during the recovery period. Although GLIB had no effect on background K⁺ currents, it selectively blocked the ACh₂-induced K⁺ current potentiated by ISO. In the four cells studied, the ACh₁- and ACh₂-induced K⁺ currents at -130 mV were 11.3±1.7 and 11.6±1.8 pA/pF, respectively, and those at +30 mV were 19.5±3.9 and 19.8±3.9 pA/pF, respectively. These results suggest that the ACh₂-induced K⁺ current potentiated by ISO is I_K,ATP.

View larger version (12K): [in this window] [in a new window]   Figure 2. Effect of GLIB on ACh2-induced K+ currents potentiated by ISO. A, I-V relation of original K+ currents elicited by ACh1 and ACh2 separated by a 6-minute recovery period. ICa,L activation and 1 µmol/L ISO were imposed during the recovery period. GLIB (10 µmol/L) was administered throughout the experiment. GLIB selectively abolished the ACh2-induced K+ current potentiated by ISO. B, Mean I-V relations of ACh2-induced K+ current potentiated by ISO () and GLIB-sensitive K+ current () (n=4). c,w indicates background currents recorded during the control period (c) shortly before ACh1 and during the recovery period (w) shortly before ACh2.

In Fig 2B, we compared the mean I-V relations for the ACh₂-induced K⁺ current potentiated by ISO () and the GLIB-sensitive K⁺ current (). The I-V relation of the potentiated ACh₂-induced K⁺ current was determined from the mean data presented in Fig 1B. Background currents were subtracted from the respective ACh-induced K⁺ currents, and then the currents induced by ACh₁ were subtracted from those induced by ACh₂. The GLIB-sensitive K⁺ current was determined by using the mean data from two different groups of cells. The GLIB-insensitive K⁺ current, as shown in Fig 2A, was subtracted from the total K⁺ current induced by ACh₂ under control conditions, as shown in Fig 1B. It is apparent that both I-V relations exhibit similar features. Both currents exhibited a linear conductance at voltages negative to -50 mV and inwardly rectified at more positive voltages. Moreover, the reversal potential and slope conductance of the potentiated ACh₂-induced K⁺ current (-83±2 mV, 133±17 pS/pF) were not statistically different from those of the GLIB-sensitive K⁺ current (-79±1 mV, 110±27 pS/pF) (n=4).

In Fig 3, we further characterized the ACh-activated GLIB-sensitive K⁺ current by determining its steady state I-V relation by use of voltage-clamp steps. In these experiments, cells were exposed to 1 µmol/L ISO plus I_Ca,L activation for 6 minutes, followed by exposure to 10 µmol/L ACh. Clamp steps between -120 and +20 mV were imposed during continuous exposure to ACh in the absence and then presence of 10 µmol/L GLIB. Fig 3A shows original ACh-activated GLIB-sensitive K⁺ currents determined by subtracting currents obtained in the presence from those in the absence of GLIB. As shown, the currents are time independent and inwardly rectifying and are relatively noisy at positive voltages. These features are consistent with those of I_K,ATP.¹⁸ In Fig 3B, the steady state I-V relation shows a reversal potential of -82 mV and a slope conductance of 151 pS/pF. In a total of three cells, the mean slope conductance was 147±6 pS/pF, and the reversal potential was 80±1 mV. These values are similar to those of the ACh-activated GLIB-sensitive K⁺ currents obtained with voltage ramps (Fig 2B).

View larger version (18K): [in this window] [in a new window]   Figure 3. Steady state ACh-activated GLIB-sensitive K+ currents. A, An atrial cell was exposed to 1 µmol/L ISO for 6 minutes, followed by exposure to 10 µmol/L ACh. During ACh, voltage-clamp steps were imposed from a holding potential of -40 mV to voltages between +20 and -120 mV for 2 seconds in the absence and presence of 10 µmol/L GLIB. External solutions contained 1 µmol/L verapamil and 1 mmol/L 4-aminopyridine. B, Steady-state I-V relation of ACh-activated GLIB-sensitive K+ currents.

In four additional cells, we used DNP, a metabolic inhibitor that lowers intracellular ATP levels and thereby activates I_K,ATP in cardiac muscle.¹⁹ Voltage-clamp ramps were used to assess K⁺ conductances. DNP (100 µmol/L) induced an increase in current at voltages both negative and positive to the reversal potential that was blocked by 10 µmol/L GLIB (not shown). The mean slope conductance (148±15 pS/pF) and reversal potential (82±1 mV) of the DNP-induced K⁺ currents were not statistically different from those of the ACh₂-induced K⁺ currents potentiated by ISO (see Fig 2B).

Effects of ISO Are Mediated via cAMP
The next several experiments were designed to determine the possible mechanisms responsible for the effects of ISO to elicit ACh-activated I_K,ATP. If the effects of ISO are mediated, as expected, via stimulation of cAMP, then forskolin should mimic the effects of ISO. In Fig 4A, the experimental protocol was repeated with 1 µmol/L forskolin plus I_Ca,L activation imposed during the recovery period. Forskolin increased mean basal I_Ca,L amplitude by 260% (n=3), which was comparable to that induced by ISO (not shown). Like ISO, forskolin had no effect on background current. After the 6-minute exposure to forskolin, ACh₂ induced a larger increase in K⁺ conductance than ACh₁ at voltages both positive and negative to the reversal potential. In the five cells studied, after subtraction of background currents, ACh₁- and ACh₂-induced K⁺ currents elicited at -130 mV were 18.7±4.0 and 23.3±3.4 pA/pF, respectively (P<.02), and those at +30 were 20.4±2.5 and 25.4±2.4 pA/pF, respectively (P<.03). These values are comparable to those elicited by ISO (Fig 1B and Table) and strongly suggest that the effects of ISO are mediated via cAMP.

View larger version (13K): [in this window] [in a new window]   Figure 4. Effects of forskolin and BAY K 8644. A, Forskolin (1 µmol/L) plus ICa,L activation were imposed during the recovery period. Forskolin mimicked the effects of ISO. B, BAY K 8644 (0.5 µmol/L) plus ICa,L activation were imposed during the recovery period. ACh2 elicited only a modestly larger increase in K+ current than did ACh1. c,w indicates background currents recorded during the control period (c) shortly before ACh1 and during the recovery period (w) shortly before ACh2.

If cAMP mediates the effects of ISO, then directly increasing intracellular cAMP should also mimic the effects of ISO. Therefore, we determined whether 8-CPT-cAMP, a membrane-permeable analogue of cAMP that directly activates PKA, could potentiate ACh₂-induced increases in K⁺ current. I_Ca,L activation and exposure to 200 µmol/L 8-CPT-cAMP were imposed during the period between ACh₁ and ACh₂. 8-CPT-cAMP increased mean basal I_Ca,L by 203% (n=5), which was comparable to the effects of ISO or forskolin. Under these conditions, ACh₂ elicited a significantly larger increase in K⁺ current than ACh₁ at voltages both positive and negative to the reversal potential. In a total of five cells, after subtraction of background currents, ACh₁- and ACh₂-induced K⁺ currents elicited at -130 mV were 13.0±1.5 and 17.2±2.2 pA/pF, respectively (P<.01), and those elicited at +30 were 13.4±1.1 and 17.2±1.6 pA/pF, respectively (P<.02).

One possible explanation of these findings is that ISO, forskolin, and 8-CPT-cAMP each stimulate Ca²⁺ influx via I_Ca,L, and the additional intracellular Ca²⁺ somehow stimulates ACh₂ to activate I_K,ATP. If this hypothesis is correct, then stimulation of I_Ca,L by BAY K 8644 should also mimic the effects of each of these agents. BAY K 8644 is a direct L-type Ca²⁺ channel agonist²⁰ that does not act via cAMP. In Fig 4B, 0.5 µmol/L BAY K 8644 and I_Ca,L activation were imposed during the recovery period. Although BAY K 8644 increased mean basal I_Ca,L by +197% (not shown), ACh₂-induced activation of K⁺ current was only modestly potentiated compared with ACh₁. In the three cells studied, K⁺ currents induced by ACh₁ and ACh₂ at -130 mV were 12.1±1.4 and 14.0±1.8 pA/pF, respectively, and those at +30 mV were 13.2±2.0 and 15.0±2.5 pA/pF, respectively. These findings suggest that enhanced Ca²⁺ influx via I_Ca,L alone cannot account for the effects of ISO to induce ACh-activated I_K,ATP.

Contribution of I_Ca,L
In the experiments shown in Fig 5A, we assessed the contribution made by I_Ca,L to the effects of ISO. ACh was administered three consecutive times for 30 seconds each. ISO was administered during both 6-minute recovery periods between each ACh exposure. I_Ca,L activation, however, was imposed only during the second recovery period between ACh₂ and ACh₃. After the initial exposure to ISO alone, ACh₂ elicited an increase in K⁺ conductance that was modestly larger than ACh₁ at voltages negative and positive to the reversal potential. In a total of eight cells, ISO alone potentiated ACh₂-induced compared with ACh₁-induced K⁺ current by +12.5±5.5% (-130 mV) and +13.0±2.1% (+30 mV). When I_Ca,L was activated in conjunction with exposure to ISO during the second recovery period, the ACh₃-induced increase in K⁺ conductance was markedly potentiated. Compared with the response to ACh₂, ACh₃ elicited a further increase in K⁺ current of +55.7±8.3% (-130 mV) and +31.5±11.5% (+30 mV) (n=5). When compared with ACh₁, ACh₃ increased K⁺ current by +76.5±9.3% (-130 mV) and +49.1±15.7% (+30 mV) (n=5). Clearly, the ability of ISO to potentiate ACh-induced increases in K⁺ conductance is significantly enhanced by I_Ca,L activation.

View larger version (13K): [in this window] [in a new window]   Figure 5. Contribution of ICa,L. A, Effect of ISO alone and ISO plus ICa,L activation on ACh-induced K+ currents. I-V relation of original K+ currents showing the response to three consecutive exposures to 1 µmol/L ACh separated by two 6-minute recovery periods. ISO (1 µmol/L) was administered during both recovery periods, and ICa,L activation was imposed only during the second recovery period. B, Effect of 1 µmol/L nisoldipine on ACh2-induced K+ currents potentiated by ISO. I-V relation of original K+ currents elicited by ACh1 and ACh2 separated by a 6-minute recovery period is shown. ICa,L activation and 1 µmol/L ISO were imposed during the recovery period. Nisoldipine was administered throughout the experiment. Nisoldipine selectively abolished the ACh2-induced K+ current potentiated by ISO. c,w indicates background currents recorded during the control period (c) shortly before ACh1 and during the recovery period (w) shortly before ACh2 (and ACh3 in panel A).

Another important way of assessing the role of I_Ca,L in the effects of ISO is to repeat the standard protocol in the presence of nisoldipine, a selective blocker of I_Ca,L. Nisoldipine (1 µmol/L) blocked all inward current via I_Ca,L stimulated during the recovery period (not shown). The original records in Fig 5B show that in the presence of nisoldipine, ACh₁ and ACh₂ induced a similar increase in K⁺ current; ie, ACh₂-activated I_K,ATP was selectively abolished by blocking I_Ca,L. In a total of four cells, there were no differences between currents induced by ACh₁ and ACh₂ in the presence of nisoldipine. In another series of experiments, we determined the effect of omitting Ca²⁺ from the external solution throughout the experiment (data not shown). Removing external Ca²⁺ also abolished inward current via I_Ca,L. Without external Ca²⁺, ACh₁ and ACh₂ elicited increases in K⁺ current that were not significantly different from one another at voltages positive and negative to the reversal potential (n=5). These results indicate that blocking Ca²⁺ influx via I_Ca,L selectively inhibits ISO-induced ACh₂-activated I_K,ATP. Moreover, these findings suggest that although Ca²⁺ influx via I_Ca,L alone is not sufficient, it is essential for ISO to induce ACh-activated I_K,ATP.

Accumulation of SR Ca²⁺
Clearly, the present experiments indicate that the effects of ISO are mediated by cAMP. In addition to stimulating I_Ca,L amplitude, cAMP enhances SR Ca²⁺ uptake. Therefore, in two series of experiments we tested either thapsigargin, an inhibitor of SR Ca²⁺ uptake,²¹ or ryanodine, an alkaloid that opens SR Ca²⁺ release channels²² and prevents accumulation of SR Ca²⁺. Each drug was administered from the beginning of its respective experiment, ie, throughout both ACh₁ and ACh₂. As usual, ISO and I_Ca,L activation were imposed during the period between ACh₁ and ACh₂. In the presence of 5 µmol/L thapsigargin, K⁺ currents induced by ACh₁ and ACh₂ at -130 mV were 14.2±1.4 and 15.2±1.3 pA/pF, respectively, and those at +30 mV were 15.8±1.8 and 15.1±1.5 pA/pF, respectively (n=5) (see Table). In the presence of 1 µmol/L ryanodine, K⁺ currents induced by ACh₁ and ACh₂ at -130 mV were 13.6±1.5 and 14.1±1.5 pA/pF, respectively, and those at +30 mV were 16.4±3.5 and 15.2±2.4 pA/pF, respectively (n=4) (see Table). These results indicate that by inhibiting the accumulation of SR Ca²⁺, these agents selectively abolished the effect of ISO to potentiate ACh₂-induced K⁺ current. It is important to note that in the presence of either thapsigargin or ryanodine, ISO elicited a typical increase in I_Ca,L amplitude (not shown). In another series of experiments, we tested the effects of Rp-cAMPs, a specific inhibitor of cAMP-dependent PKA.²³ Rp-cAMPs was administered throughout both ACh₁ and ACh₂. In the presence of 50 µmol/L Rp-cAMPs, ACh₁- and ACh₂-induced K⁺ currents at -130 mV were 12.3±1.6 and 12.9±1.6 pA/pF, respectively, and those at +30 mV were 16.2±2.2 and 15.7±2.4 pA/pF, respectively (n=5). These results indicate that inhibition of cAMP-dependent PKA selectively abolished the ACh₂-induced increase in K⁺ current potentiated by ISO. Although Rp-cAMPs also prevented ISO-induced stimulation of I_Ca,L (not shown), these experiments collectively suggest that the conditioning effect of ISO requires cAMP-mediated stimulation of SR Ca²⁺ uptake and accumulation.

ß-Adrenergic and Muscarinic Receptor Subtypes
In the next few experiments, we examined the surface receptors mediating the effects of ISO and ACh. As shown in Fig 6A, 1 µmol/L ACh was administered three consecutive times separated by two 6-minute recovery periods. ISO (1 µmol/L) and I_Ca,L activation were imposed during the recovery periods between each ACh exposure. Propranolol (2 µmol/L) was administered after ACh₂, throughout the second recovery period, and during ACh₃. As shown, ACh₁ elicited a typical increase in K⁺ conductance. After exposure to ISO plus I_Ca,L activation, ACh₂ also elicited a typical potentiated increase in K⁺ conductance compared with ACh₁. In the presence of propranolol, the response to ACh₃ was similar to that of ACh₁; ie, propranolol selectively blocked ACh₃-activated I_K,ATP. In the four cells studied, ACh₁ and ACh₂ elicited an increase in K⁺ current at -130 mV of 6.3±1.8 and 8.7±2.3 pA/pF, respectively (P<.5), and at +30 mV the currents were 9.1±1.8 and 12.0±2.1 pA/pF, respectively (P<.01). There were no significant differences, however, between ACh₁- and ACh₃-induced K⁺ currents. These results indicate that the effect of ISO to elicit ACh-activated I_K,ATP is mediated via ß-adrenergic receptors.

View larger version (20K): [in this window] [in a new window]   Figure 6. The role of ß-adrenergic and muscarinic receptors. A, Original current records showing the effect of ß-adrenergic receptor block by propranolol. Propranolol (2 µmol/L) was administered during the second recovery period and during ACh3. ACh2 induced a typical potentiated increase in K+ current compared with ACh1. Propranolol selectively abolished the potentiated K+ current during exposure to ACh3. The responses to ACh3 and ACh1 were not different. B, Original current records showing the effect of 0.2 µmol/L pirenzepine. Pirenzepine was administered throughout the experiment. Pirenzepine selectively abolished the ACh2-induced K+ current potentiated by ISO. C, Original current records showing the effect of AFDX116. AFDX116 (100 µmol/L) was administered after ACh1 and during ACh2. ACh2 induced a smaller, rather than larger, increase in K+ current compared with ACh1. In each series of experiments, ISO (1 µmol/L) and ICa,L activation were imposed during the recovery period. c,w indicates background currents recorded during the control period (c) shortly before ACh1 and during the recovery period (w) shortly before ACh2 (and ACh3 in panel A).

To determine the receptors mediating the effects of ACh, we repeated the standard protocol of ISO and I_Ca,L activation in the presence of 1 µmol/L atropine. Atropine blocked all ACh-induced changes in current (not shown) (n=2). Although these results indicate that ACh is acting via muscarinic receptors, they do not distinguish among multiple subtypes of muscarinic receptors.²⁴ Our previous work indicated that ACh can act via M₂ muscarinic receptors to activate I_K,ACh and via M₁ muscarinic receptors to activate I_K,ATP.¹⁰ Therefore, as shown in Fig 6B, we used pirenzepine, an M₁ receptor antagonist, to determine whether ACh was acting via M₁ receptors to elicit the potentiated K⁺ current. Pirenzepine (0.2 µmol/L) was administered throughout both ACh₁ and ACh₂. ACh₁ elicited a typical increase in K⁺ current. After exposure to ISO plus I_Ca,L activation, ACh₂ failed to elicit the potentiated increase in K⁺ current. In the five cells studied, the ACh₁- and ACh₂-induced K⁺ currents at -130 mV were 13.5±3.5 and 11.5±3.4 pA/pF, respectively, and those at +30 mV were 19.6±2.3 and 15.5±0.8 pA/pF, respectively. Values at each voltage were not significantly different from one another. These results suggest that after exposure to ISO, ACh₂ is acting via M₁ receptors to activate I_K,ATP.

If ACh₂ is acting via M₂ and M₁ receptors to activate I_K,ACh and I_K,ATP, respectively, then blocking M₂ receptors should allow selective ACh₂-induced activation of I_K,ATP. As shown in Fig 6C, the standard protocol was performed with ISO plus I_Ca,L activation imposed during the recovery period. AFDX116 (100 µmol/L), a selective M₂ antagonist,²⁵ was administered during the recovery period and during ACh₂. ACh₁ elicited a typical increase in K⁺ current. In the presence of AFDX116, however, ACh₂ elicited a smaller, rather than larger, increase in K⁺ conductance than ACh₁ at voltages both positive and negative to the reversal potential. In addition, the smaller current induced by ACh₂ reversed at -80 mV and inwardly rectified at positive voltages. In the five cells studied, ACh₁- and ACh₂-induced K⁺ currents at -130 mV were 8.3±1.5 and 2.1±0.5 pA/pF, respectively (P<.005), and those at +30 mV were 10.0±1.8 and 2.8±0.3 pA/pF, respectively (P<.01). The AFDX116-insensitive current induced by ACh₂ was 75% smaller than the ACh₁-induced K⁺ current (see Table). In four additional cells, we repeated the same protocol with 100 µmol/L AFDX116 administered throughout both ACh₁ and ACh₂. Under these conditions, ACh₁ failed to elicit any change in current, and ACh₂ elicited a small current with features similar to those described in the preceding experiments.

To determine whether the AFDX116-insensitive current was inhibited by GLIB, we performed our standard protocol of exposure to ISO and I_Ca,L activation during the recovery period and then added 100 µmol/L AFDX116 plus 10 µmol/L GLIB to the external solution immediately after the administration of ACh₁ and during the recovery period and ACh₂ administration (not shown). Under these conditions, ACh₂ failed to activate any significant increase in current. At -130 mV, ACh₁ and ACh₂ elicited an increase in K⁺ current of 11.5±0.9 and 0.5±0.2 pA/pF, respectively, and at +30 mV, the currents were 16.2±1.8 and 1.0±0.4 pA/pF, respectively (see Table). Taken together, these experiments indicate that after exposure to ISO, ACh is acting via M₁ muscarinic receptors to activate I_K,ATP.

Role of PKC
M₁ muscarinic receptors are coupled to PI hydrolysis,²⁶ the production of DAG, and stimulation of PKC. Therefore, we determined whether calphostin C, a selective PKC antagonist,²⁷ could inhibit ACh₂-induced activation of I_K,ATP. ISO (1 µmol/L) plus I_Ca,L activation were imposed during the recovery period, and calphostin C (0.1 µmol/L) was administered after ACh₁, during the recovery period, and during ACh₂. In the three cells studied, ACh₁- and ACh₂-induced increases in K⁺ current at -130 mV were 11.9±1.6 and 12.0±1.5 pA/pF, respectively, and at +30 mV, they were 15.8±3.2 and 15.9±2.9 pA/pF, respectively (see Table). Values measured at either voltage were not statistically different. These results indicate that inhibition of PKC selectively abolished ACh₂-activated I_K,ATP and support the idea that ACh is acting via the PI signaling pathway.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The main finding of the present work is that by elevating SR Ca²⁺, ISO conditions atrial cells so that a subsequent exposure to ACh elicits a potentiated increase in K⁺ conductance due to activation of I_K,ATP. The hypothesis underlying the present experiments was based on our previous studies showing that an initial prolonged exposure to ACh conditioned atrial cells by raising SR Ca²⁺ and thereby potentiated the response to a subsequent ACh exposure.^{9 10} For ACh to condition the cell, the duration of the initial ACh exposure had to be at least 2 minutes.⁹ In the present study, atrial cells were exposed to ACh for only 30 seconds, to avoid the conditioning effects of an initial ACh exposure on subsequent ACh-induced increases in K⁺ conductance. Even when Ca²⁺ influx was increased by activation of I_Ca,L during the period between two ACh exposures, the responses to ACh₁ and ACh₂ were not different (Fig 1A). However, the addition of ISO plus I_Ca,L activation during the recovery period potentiated the increase in K⁺ current induced by ACh₂. Several of the present findings identify the ACh₂-induced K⁺ current potentiated by ISO as I_K,ATP. Thus, the current was selectively abolished by GLIB, a selective blocker of I_K,ATP in cardiac muscle.^{16 17} Voltage-clamp steps imposed during exposure to ACh revealed time-independent GLIB-sensitive K⁺ currents that exhibited a steady state I-V relation characteristic of I_K,ATP.¹⁸ Moreover, metabolic inhibition by DNP elicited a GLIB-sensitive K⁺ current that exhibited a reversal potential and slope conductance similar to the ACh₂-induced K⁺ current potentiated by ISO. If one assumes that DNP activated most, if not all, of the available ATP-sensitive K⁺ channels, the fact that the slope conductances of the DNP- and ACh-activated I_K,ATP are similar suggests that ACh also may be activating the majority of available ATP-sensitive K⁺ channels. It is worth noting that the whole-cell slope conductance (normalized for cell capacitance) of DNP-induced I_K,ATP in cat atrial myocytes is 2.5 times smaller than that in cat ventricular myocytes (authors' unpublished data, 1994). Assuming that the open probability of the ATP-sensitive K⁺ channels activated by DNP is similar in both tissues, these findings suggest that the density of ATP-sensitive K⁺ channels in atrial muscle is significantly lower than in ventricular muscle.

The present findings indicate that the effects of ISO to potentiate ACh-induced increases in K⁺ current are mediated via cAMP. Thus, the effects of ISO were (1) mediated via ß-adrenergic receptors, (2) mimicked by forskolin and 8-CPT-cAMP, and (3) abolished by inhibition of cAMP-dependent PKA with Rp-cAMPs. Moreover, ISO acted to potentiate ACh-induced K⁺ current by stimulating both I_Ca,L and SR Ca²⁺ uptake. Thus, even though BAY K 8644 increased I_Ca,L to about the same extent as ISO, it failed to potentiate ACh-induced K⁺ conductance to the same extent as ISO. This can be explained by the fact that BAY K 8644 is an L-type Ca²⁺ channel agonist that does not stimulate cAMP and therefore, unlike ISO, does not stimulate SR Ca²⁺ uptake. Likewise, thapsigargin or ryanodine selectively abolished the effects of ISO on ACh₂-activated I_K,ATP by depleting SR Ca²⁺ without decreasing ISO-induced stimulation of I_Ca,L. These experiments clearly separate the stimulatory effect of ISO on SR Ca²⁺ uptake from its effect on I_Ca,L. In addition, the potentiating effect of ISO was significantly diminished or abolished without concomitant I_Ca,L activation or when I_Ca,L was blocked by nisoldipine, respectively. In these experiments, the lack of Ca²⁺ influx via I_Ca,L also would be expected to attenuate SR Ca²⁺ uptake. Taken together, these experiments indicate that exposure to ISO conditions the cell through cAMP-induced stimulation of both Ca²⁺ influx via I_Ca,L and SR Ca²⁺ uptake. In addition, the present results support our previous studies,¹⁰ in which we concluded that a prolonged ACh exposure conditions atrial cells by loading SR Ca²⁺.

The present findings also indicate that ACh-induced increases in K⁺ conductance are mediated via both M₂ and M₁ muscarinic receptors; M₂ receptors mediate activation of I_K,ACh, and M₁ receptors mediate activation of I_K,ATP. Moreover, the fact that ACh-induced activation of I_K,ATP was dependent on PKC activity and that M₁ receptors mediate PI hydrolysis²⁶ strongly suggests that after exposure to ISO, ACh is acting via the PI signaling pathway to activate I_K,ATP. PKC is thought to mediate various intracellular processes, including regulation of ion channels, via phosphorylation of protein substrates. It seems unlikely that PKC was activated directly by ISO rather than by ACh because -adrenergic receptors mediate the PI signaling pathway,²⁸ and in the present study the effects of ISO were selectively abolished by ß-adrenergic receptor block. Moreover, these results are consistent with our previous studies showing that ACh acts via M₁ receptors and the PI signaling pathway to activate I_K,ATP and via M₂ receptors to directly activate I_K,ACh.¹⁰

How does ISO induce ACh to activate I_K,ATP? On the basis of the present findings, it seems reasonable to conclude that elevated SR Ca²⁺ induced by ISO- and ACh-induced stimulation of the PI signaling pathway may operate in concert to activate I_K,ATP. One possible response of stimulating the PI signaling pathway is the production of IP₃ and the mobilization of intracellular Ca²⁺ stores.²⁹ It may be that ACh acts via IP₃ to mobilize Ca²⁺ stores filled by prior exposure to ISO, which in turn activates a Ca²⁺-dependent PKC to phosphorylate ATP-sensitive K⁺ channels. Atrial muscle contains a Ca²⁺-dependent PKC- subtype.³⁰ The present findings, however, show that ryanodine selectively abolished ACh-induced activation of I_K,ATP. This indicates that the SR Ca²⁺ stores filled by ISO are mediated via ryanodine receptors and not IP₃ receptors.³¹ In addition, the time course of IP₃-induced mobilization of intracellular Ca²⁺ may be too slow to account for the ACh-induced activation of I_K,ATP. Alternatively, loading of SR Ca²⁺ has been shown to enhance the spontaneous release of SR Ca²⁺ from single Ca²⁺-release channels, ie, Ca²⁺ sparks.³² Moreover, Ca²⁺ sparks³² and the effects of ISO to induce ACh-activated I_K,ATP, as shown in the present study, are both abolished by ryanodine. Spontaneous SR Ca²⁺ release may increase Ca²⁺ concentrations at selected subsarcolemmal sites that may facilitate activation of I_K,ATP. On the basis of work in ventricular membrane patches,³³ it seems unlikely that the effects of elevated Ca²⁺ would be to alter the sensitivity of ATP-sensitive K⁺ channels to inhibition by ATP. However, several steps in the PI signaling pathway, such as stimulation of membrane-bound phospholipase C and the hydrolysis of phosphoinositides, are Ca²⁺-dependent processes.³⁴ Moreover, elevated Ca²⁺ may stimulate Ca²⁺-dependent PKC subtypes found in atrial muscle.³⁰

Therefore, we propose the model shown in Fig 7. A resting atrial myocyte exhibits relatively low SR Ca²⁺ levels and therefore little, if any, spontaneous SR Ca²⁺ release. Under these conditions, an initial brief exposure to ACh primarily acts via M₂ receptors to directly activate I_K,ACh. Subsequent exposure to ISO stimulates cAMP-dependent PKA activity and thereby enhances Ca²⁺ influx via I_Ca,L and SR Ca²⁺ uptake. Both mechanisms contribute to elevating SR Ca²⁺ levels. By raising SR Ca²⁺, ISO enhances spontaneous SR Ca²⁺ release and thereby raises Ca²⁺ concentrations in restricted subsarcolemmal spaces. This local Ca²⁺-enriched environment facilitates the effects of a second ACh exposure to act via M₁ receptors and the PI signaling pathway. As a result, ACh₂-induced activation of PKC, either Ca²⁺ dependent or Ca²⁺ independent, phosphorylates membrane sites to activate I_K,ATP. Phosphorylation has been proposed as an element in the activation of I_K,ATP.^{7 35}

View larger version (16K): [in this window] [in a new window]   Figure 7. Schematic diagram of the proposed mechanisms responsible for ISO to induce ACh-activated IK,ATP. ACh1 indicates initial exposure to 10 µmol/L ACh before ISO; ACh2, second exposure to 10 µmol/L ACh after ISO; ISO, 1 µmol/L ISO administered between ACh1 and ACh2; M2, M2 muscarinic receptor; M1, M1 muscarinic receptor; ß, ß-adrenergic receptor; AC, adenylate cyclase; Gp, GTP-binding protein; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-diphosphate; cAMP/PKA, cAMP-dependent PKA; and SL, sarcolemma.

It is worth noting that the ability of ACh to elicit I_K,ATP specifically required an increase in SR Ca²⁺ rather than simply an increase in cytosolic Ca²⁺. Thus, the ability of ISO to induce ACh-activated I_K,ATP was abolished by agents that specifically deplete SR Ca²⁺, even though the ISO-induced stimulation of Ca²⁺ influx via I_Ca,L was unchanged. Moreover, when Ca²⁺ influx was increased by activation of basal I_Ca,L alone or by BAY K 8644, ACh either failed to activate I_K,ATP or elicited only a modest activation, respectively. These findings are consistent with our previous studies in which depletion of SR Ca²⁺ with either caffeine or ryanodine also prevented ACh-induced activation of I_K,ATP.¹⁰ Therefore, we speculate that the SR may function to distribute Ca²⁺ to specific subsarcolemmal sites at which Ca²⁺ can modulate regulation of ATP-sensitive K⁺ channels via the PI signaling pathway.

In ventricular myocytes, ISO has been shown to augment I_K,ATP but only when I_K,ATP has been previously activated either by pinacidil,³⁶ a K⁺ channel opener, or by lowering intracellular ATP.³⁷ In the former study, it was speculated that the effects of ISO were due to cAMP-dependent phosphorylation of the channel. This mechanism does not appear to contribute to the present results, because the effects of ISO to induce ACh-activated I_K,ATP were selectively blocked by agents such as thapsigargin or ryanodine, which would not be expected to interfere with ISO-induced stimulation of cAMP. In the latter study, ISO enhanced I_K,ATP by metabolically depleting local subsarcolemmal ATP levels via stimulation of adenylate cyclase. This effect of ISO could not be mimicked by direct stimulation of PKA with 8-CPT-cAMP and only occurred when intracellular ATP levels were previously lowered below 1 mmol/L. Several of the present findings make it unlikely that a similar mechanism is responsible for the effects of ISO to induce ACh-activated I_K,ATP. Thus, the use of the perforated-patch method precludes the possibility that ATP levels were below 1 mmol/L before exposure to ISO or that ISO could drop ATP concentrations that low. In addition, the effects of ISO were mimicked by direct stimulation of PKA with 8-CPT-cAMP, which would bypass stimulation of adenylate cyclase. Moreover, the fact that ISO never induced a change in background current suggests that ISO did not decrease ATP, either locally or globally, to levels low enough to directly activate the channel. Furthermore, drugs that specifically abolished the effects of ISO to induce ACh-activated I_K,ATP, such as ryanodine, would not be expected to influence the ability of ISO to stimulate adenylate cyclase or to lower ATP. It is also worth considering that the intracellular ATP concentration that produces half-maximal inhibition of ATP-sensitive K⁺ channels is 25 to 100 µmol/L.³⁸ It could be argued that only a fraction of the channels may need to be activated to elicit the currents reported in the present study and therefore may require only a small decrease in intracellular ATP below normal. This idea has been proposed to explain findings that early ischemia produces significant shortening in action potential duration with little change in intracellular ATP concentrations.³⁸ The present results, however, indicate that ACh activates as many ATP-sensitive K⁺ channels as severe metabolic inhibition induced by DNP, suggesting that ACh is activating a relatively large percentage of the available channels. If this response to ACh is somehow dependent on an ISO-induced depletion of intracellular ATP, it seems likely that it would have to be a relatively large decrease in ATP. This seems unlikely given the considerations discussed above.

In terms of cardiac function, cholinergic inhibition is accentuated by the presence of sympathetic stimulation, ie, accentuated antagonism.^{2 3} At least part of the underlying mechanism is due to cholinergic inhibition of ß-adrenergic–induced stimulation of adenylate cyclase and cAMP.¹ The present results demonstrate a novel mechanism of adrenergic-cholinergic interaction that is unrelated to cholinergic inhibition of cAMP. Thus, ß-adrenergic stimulation of cAMP increases SR Ca²⁺ levels, which in turn enhances ACh-induced increases in K⁺ conductance. This mechanism may be important in the normal reciprocal interplay between sympathetic and parasympathetic regulation of atrial function. Thus, prior ß-adrenergic stimulation may directly accentuate subsequent cholinergically mediated hyperpolarization and shortening of the action potential duration. In this way, atrial activity enhanced by sympathetic stimulation is effectively antagonized by enhanced cholinergic inhibition of atrial activity. This mechanism may contribute to the antiarrhythmic effects of vagal nerve activity.³⁹ On the other hand, this mechanism may underlie the effect of sympathetic stimulation to enhance atrial flutter rate or initiate atrial fibrillation by shortening atrial refractoriness mediated by vagal nerve stimulation. Finally, the present findings, as well as our previous studies,¹⁰ indicate that loading of SR Ca²⁺ can potentiate ACh-induced increases in K⁺ conductance via ACh-activated I_K,ATP. This represents a fundamentally new mechanism of cholinergic regulation that has wide implications with respect to the way in which drugs, neurotransmitters, and physiological or pathophysiological states that modulate SR Ca²⁺ may also modulate ACh-induced activation of ATP-sensitive K⁺ channels.

	Selected Abbreviations and Acronyms

 

ACh = acetylcholine ACh1, ACh2 = two consecutive 30-second ACh exposures 8-CPT-cAMP = 8-(4-chlorophenylthio)-cAMP DAG = diacylglycerol DNP = 2,4-dinitrophenol GLIB = glibenclamide I-V = current-voltage ICa,L = L-type Ca2+ current IK,ACh = ACh-activated K+ current IK,ATP = ATP-sensitive K+ current IP3 = inositol 1,4,5-trisphosphate ISO = isoproterenol PI = phosphoinositol PKA = protein kinase A PKC = protein kinase C SR = sarcoplasmic reticulum

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-27652. We thank C. Rechenmacher for her expert technical assistance.

Received December 27, 1994; accepted May 12, 1995.

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