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OtherANALGESIA AND DRUGS OF ABUSE

Cocaine Blockade of the Acetylcholine-Activated Muscarinic K+ Channel in Ferret Cardiac Myocytes

Yong-Fu Xiao and James P. Morgan
Journal of Pharmacology and Experimental Therapeutics January 1998, 284 (1) 10-18;

Abstract

The effects of cocaine on the acetylcholine(ACh)-activated muscarinic K+ current (IK(ACh)) were assessed with the whole-cell patch-clamp technique in single atrial and left ventricular myocytes enzymatically isolated from adult ferret hearts. The density of IK(ACh) is almost 5 times greater in atrial cells than in left ventricular myocytes. Cocaine reversibly blocked IK(ACh) in a dose-dependent manner. Methylecgonidine (MEG), the major product of pyrolysis of cocaine base, also produced similar effects onIK(ACh). The concentration to produce 50% inhibition of IK(ACh) was 25 μM and 12 μM for cocaine and MEG, respectively. Cocaine at micromolar concentrations also significantly inhibited the adenosine-activated purinergic K+ current (IK(Ado)), which has the same electrophysiological properties asIK(ACh). Furthermore, cocaine inhibitedIK(ACh) activated by GTPγS, which evokesIK(ACh) by bypassing the muscarinic receptor and directly activating the G-protein, GK. These results suggest that cocaine-induced suppression ofIK(ACh) is caused by its interactions beyond the binding site of muscarinic receptors. The antimuscarinic effect of cocaine may play an important role in cocaine cardiotoxicity by reducing the membrane electrical stability and acting synergistically with other actions of cocaine to facilitate the occurrence of lethal cardiac arrhythmias.

The early work of Trautwein and Dudel (1958) revealed that atrial, but not ventricular, myocytes were sensitive to the neurotransmitter acetylcholine. ACh decreases heart rate, slows atrial-ventricular conduction, reduces atrial contractility and attenuates catecholamine-stimulated effects (Loffelholz and Pappano, 1985). All these effects predominately arise from membrane hyperpolarizationvia an activation of IK(ACh). Patch-clamp studies have demonstrated the direct activation ofIK(ACh) by application of ACh in mammalian heart cells (Sakmann et al., 1983). Boyett and his colleagues (Boyett et al., 1988; McMorn et al., 1993) reported that ACh has a direct chronotropic and negative inotropic effect on ferret and rat ventricular myocytes. They also found that IK(ACh) is present in both atrial and ventricular myocytes. Further, Koumi and Wasserstrom (1994)identified and characterized IK(ACh) in isolated cat, guinea pig and human ventricular myocytes. These data indicate that IK(ACh) is present in ventricular myocytes and may play important roles in cardiac function.

Cocaine abuse has presented a major health problem in many countries for more than a century. In North America, the incidence of cocaine use has climbed rapidly in recent years, especially in young adults. Serious medical consequences of cocaine cardiotoxicity, including myocardial ischemia and infarction, ventricular fibrillation and sudden cardiac death, are serious health issues. Existing data show that reduced cardiac vagal tone increases susceptibility to ventricular arrhythmias (Corr and Gillis, 1974; Vanoli et al., 1991). Early studies demonstrated (Wilkerson, 1989) that atropine significantly enhanced cocaine-induced cardiovascular toxicity. In humans cocaine also suppressed cardiac vagal tone in Newlin’s recent study (1994). Furthermore, cocaine inhibited ion flux controlled by the ACh receptor in membrane vesicles of Torpedo californica, Electrophorus electricus and in PC-12 cells, a sympathetic neuronal cell line (Karpen et al., 1982; Karpen and Hess, 1986). Binding studies indicate that cocaine acts as an antimuscarinic agent, particularly at higher doses, in heart and brain (Sharkeyet al., 1988). A recent report shows that cocaine interacts with primary and allosteric recognition sites on muscarinic receptors in membrane homogenates from postmortem human brainstem (Flynn et al., 1992). The hypothesis of cocaine as an antagonist of cardiac muscarinic receptors is further confirmed by other studies (Ritz and George, 1993; Tan and Costa, 1994).

Cardiac intoxication with cocaine has been linked to the inhibition of cardiac neuronal uptake of norepinephrine and its local anesthetic effects on Na+ channels. The effects of cocaine on the cholinergic system of the heart have not been well investigated, however, especially at the cellular and molecular levels. Because muscarinic receptors play a crucial role in modulation of heart rate and stabilization of membrane electrical excitability (Billman and Hoskins, 1989), it is important to evaluate the effects of cocaine on cardiac IK(ACh). Here, we report that cocaine strongly suppresses the K+ current activated by carbachol, adenosine and GTPγS in adult ferret atrial and left ventricular myocytes. The results suggest that cocaine-induced inhibition of IK(ACh) involves block of the channel pore or the receptor binding site, as well as an inhibition of the G-protein (GK). Some of the data in this manuscript have been presented in brief abstract form (Xiao and Morgan, 1996).

Methods

Isolation of single myocytes.

Single atrial and left ventricular myocytes were enzymatically isolated from adult ferret (male, 6–10 weeks of age, Marshall Farm, North Rose, NY) hearts by a method similar to that described previously (Xiao and McArdle, 1994). Animal care and experimental procedures were performed according to the guidelines of Institutional Animal Care and Use Committee in compliance with the US Public Health Service Policy as stated in The Guide for the Care and Use of Laboratory Animals (HHS, NIH Publication No. 85–23, 1985). After a ferret was deeply anesthetized with chloroform, the heart was rapidly removed and washed in ice-cold, oxygenated Tyrode’s solution containing (in mM): NaCl,137; CaCl2, 2; KCl, 5; MgCl2, 1; CaCl2, 1.8; HEPES, 10; glucose, 10, pH 7.4. The aorta was quickly connected to a modified Langendorff system. This perfusion system had a hydrostatic pressure of 80 cm and a flow rate of 8 to 10 ml/min. The heart was initially perfused with the oxygenated 37°C Ca++-free Tyrode’s solution for 6 min. The heart was then perfused and recirculated for 38 to 45 min with 50 ml Ca++-free Tyrode’s solution containing 45 to 50 mg collagenase (CLS 2, Worthington Biomedical Corporation, Freehold, NJ), 1 to 2 mg protease Type XIV and 0.1% bovine serum albumin (Sigma Chemical Company, St. Louis, MO). After the enzyme treatment, the heart was washed sequentially with 50 ml 0.2 mM Ca++and 50 ml 0.4 mM Ca++ Tyrode’s solution plus 1 mg/ml bovine serum albumin. When the enzymatic solution was completely washed out, several pieces of atrial and left ventricular tissue were cut off and placed separately into two Petri dishes (60 × 15 mm) containing 0.4 mM Ca++ Tyrode’s solution plus 1 mg/ml bovine serum albumin. The cardiac tissue was further sliced into finer pieces and gently agitated for 1 to 2 min. The dispersed cells and tissues were filtered through a 250-μm polypropylene mesh. The effluent containing dissociated myocytes was kept in 0.4 mM Ca++ Tyrode’s solution plus 1 mg/ml bovine serum albumin at 22–23°C room temperature for 1 to 2 hr before beginning patch-clamp experiments.

Recording of ACh-activated whole-cell currents.

A small volume (25 μl) of the effluent solution containing cardiac myocytes was pipetted into a chamber (0.5 ml) with a coverglass bottom mounted on an inverted microscope and superfused (1–2 ml/min) with the Tyrode’s solution. Recording pipettes were made from glass tubes (World Precision Instruments, Inc., Sarasota, FL) by a two-stage pull on a David Kopf vertical puller (model 700D, Tujunga, CA). The heat-polished or unpolished electrode with a resistance of 2 to 4 megohm was connected via a Ag-AgCl wire to an Axopatch 1D amplifier (Axon Instruments, Foster City, CA). After forming a conventional “gigaseal” the capacitance of the electrode was compensated. Additional suction was used to form the whole-cell configuration. With the nystatin-perforated patch technique no further suction was applied after forming a conventional high-resistance seal. The whole-cell recording configuration was formed within 15 min (8.7 ± 1.1 min, n = 24) after the gigaseal. A current representing the membrane capacitance was recorded by application of a 5-mV hyperpolarizing pulse from a holding potential of −80 mV (Xiao and McArdle, 1994). Correction of cell capacitance and series resistance was then performed before application of the experimental voltage-clamp protocol. External solutions were exchanged with a fast perfusion system (Xiao et al., 1995).IK(ACh) was evoked by extracellular application of 1 μM carbachol and stored on the hard disk of a personal computer running the Pclamp 5.5.1 data acquisition programs. All experiments were carried out in the 2 mM Ca++Tyrode’s solution at 22–23°C. The pipette solution for the nystatin-perforated patch method (Horn and Marty, 1988) contained (in mM): KCl, 140; EGTA, 0.5; HEPES, 5, pH 7.3; and 100 μg/ml nystatin which was freshly added before each experiment. The intracellular solution for the classic whole-cell recording method (Hamill et al., 1981) contained (in mM): KCl, 80; KOH, 60; MgCl2, 1; CaCl2, 1; EGTA, 10; HEPES, 10; MgATP, 5; and pH 7.3.

Recording of the action potential.

Electrically stimulated action potentials of ferret atrial cells were recorded with the current-clamp method described elsewhere (Xiao and McArdle, 1995). The resting membrane potential was approximately −80 mV after obtaining the classical whole-cell recording configuration. Action potentials were elicited by intracellular injection of 30 to 50 pA depolarizing current for 10 ms.

Materials.

Cocaine and MEG were obtained from Sigma (St. Louis, MO) and dissolved weekly in deionized water at a concentration of 10 mM and stored at −20°C before use. The experimental concentrations of cocaine and MEG were obtained by dilution of the stocks. Carbachol, nystatin, adenosine, ATP and GTPγS were obtained from Sigma (St. Louis, MO).

Data analysis and statistics.

IK(ACh) was recorded at different holding potentials and measured as the amplitude of the carbachol-activated current minus the steady-state holding current. All myocytes were held at −80 mV for 1 to 2 min before changing holding potential and reapplication of carbachol. Recordings ofIK(ACh) were made from the same myocytes before, during and after drug treatment. A washout was conducted to determine whether IK(ACh) returned toward the pretreatment value and to ensure that changes ofIK(ACh) were not caused by functional damage. The current density was calculated as the current amplitude divided by the cell membrane capacitance to exclude the effect of different cell size. Data are presented as the mean ± S.E. The Student’s t test was used to evaluate the difference between two values. One-way analysis of variance and Dunnett’s test for critical difference were used for data derived from more than two groups. A difference of P < .05 was considered statistically significant.

Results

IK(ACh) of ferret cardiomyocytes.

Both atrial and left ventricular myocytes isolated from ferret hearts have clear striations, but atrial cells appear spindle-shaped and narrower in width than left ventricular myocytes, which are rod-shaped. Almost all cells examined in this study were quiescent before and after forming a whole-cell configuration when the holding potential (Vh) was set at −80 mV. The resting membrane potential measured by the zero-current clamp method was −80 ± 1 and −81 ± 3 mV for the atrial (n = 13) and left ventricular (n = 6) myocytes, respectively. IK(ACh) was evoked by rapid application of carbachol and recorded via a glass electrode with the nystatin perforated-patch technique. With this technique IK(ACh) was observed in each patch after application of 1 μM carbachol to either atrial or left ventricular myocytes.

Figure 1 shows that extracellular application of 1 μM carbachol evokedIK(ACh) in ferret atrial and left ventricular myocytes. In atrial myocytes, especially in those with a larger current (fig. 1A), muscarinic receptors were desensitized to the stimulation of carbachol during a sustained superfusion. This desensitization had a rapid initial phase, 17 ± 3 s (n = 5), followed by a slower one in atrial cells (fig.1A). In contrast, the rapid initial phase of the desensitization was not obvious in ventricular myocytes (fig. 1B). Although the membrane capacitance was about 2.5-fold greater in the ventricular myocytes (P < .0001, 200 ± 16 pF, n = 12) than in the atrial cells (78 ± 6 pF, n = 21), the averageIK(ACh) for the left ventricular myocytes recorded at the holding potential of 0 mV was 116 ± 18 pA, which was only half of the current for the atrial cells, 231 ± 39 pA (P < .01). Compared with atrial cells (2.9 ± 0.3 pA/pF), however, the current density of IK(ACh) was almost 5 times lower (P < .0001, 0.6 ± 0.1 pA/pF) in single left ventricular myocytes (fig. 1B). The current density varied markedly among individual cells, from 1.0 to 5.0 pA/pF for the atrial myocytes (n = 21) and 0.3 to 0.9 pA/pF for the left ventricular cells (n = 12).

Figure 1
Figure 1

Comparison of IK(ACh)between ferret atrial and left ventricular myocytes. Original current traces recorded from an atrial (A) and a left ventricular (B) myocyte.IK(ACh) was activated by extracellular application of 1 μM carbachol and recorded at a holding potential of 0 mV with the nystatin-perforated patch-clamp method. (C) Comparison of the current density between atrial and ventricular myocytes. The current density is calculated by the amplitude ofIK(ACh) divided by the cell membrane capacitance. ****P < .0001; between the atrial (open bar, A. cell, n = 21) and left ventricular (hatched bar, V. cell, n = 12) myocytes.

The current evoked by carbachol was completely blocked by extracellular application of 1 μM atropine in both atrial and left ventricular myocytes (data not shown). With 5 mM extracellular and 140 mM intracellular K+ solutions this current reversed polarity at about −80 mV (n = 19), which is close to the calculated K+ equilibrium potential of −84 mV. The inward part of the carbachol-activated current was abolished and became an outward current after removing extracellular K+. Increasing extracellular K+ from 5 to 20 mM shifted the reversal potential (ERev) from −80 mV to −46 mV. Extracellular perfusion of 1 mM K+ solution altered ERev to −126 mV, which is very close to the theoretical ERev of −125 mV (data not shown). These results demonstrate that the current studied in the present experiments is carried by K+ and activated by muscarinic receptors.

Fade of IK(ACh).

The classic whole-cell recording technique can cause “wash-out” of the muscarinic response to ACh (Horn and Marty, 1988). Therefore, the effects of the nystatin perforated-patch method and the classic whole-cell recording technique, with or without intracellular dialysis 5 mM ATP, were compared. Figure 2 (open circles) shows that IK(ACh) evoked by repeated application of carbachol did not significantly fade in the nystatin perforated-patch myocytes (n = 8) during 60 min. By use of the classic whole-cell recording method, the amplitude of IK(ACh) decreased by 23 ± 5% (n = 9) of the control within 30 min when the intracellular solution contained 5 mM ATP (open triangles). In contrast, IK(ACh) was significantly reduced (P < .01, n = 5) within 5 min and only left 6 ± 4% of the control current within 30 min in the myocytes intracellularly dialyzed with ATP-free solution (closed triangles).

Figure 2
Figure 2

Fade of IK(ACh) in ferret atrial cells. Currents were recorded at 0 mV holding potential and normalized by the value of the first exposure to 1 μM carbachol.IK(ACh) was elicited by repeated application of 1 μM carbachol and recorded by use of the nystatin-perforated patch technique (○) and the classic whole-cell recording method with (▵) and without (▴) intracellular dialysis of 5 mM MgATP.

Cocaine blockade of IK(ACh).

Binding data have shown that cocaine blocked muscarinic cholinergic receptors in heart and brain (Sharkey et al., 1988). Furthermore, atropine enhances the cardiovascular effects of cocaine (Wilkerson, 1989). It is known that cocaine exacerbates catecholamine-induced ventricular fibrillation (Inoue and Zipes, 1988), but the mechanism has not been delineated. Therefore, we examined the effects of cocaine on IK(ACh) in both atrial and left ventricular myocytes. Figure3 demonstrates that extracellular application of 50 μM cocaine significantly decreasedIK(ACh) of atrial myocytes. Cocaine suppressed both inward and outward portions ofIK(ACh), but altered neither the current-voltage relation nor the ERev(n = 10). In atrial cells, cocaine at a concentration as low as 1 μM produced 25 ± 8% (fig.4, P < .05, n = 6) inhibition of IK(ACh); 100 μM cocaine completely blocked IK(ACh). The concentration of cocaine necessary to produce 50% suppression ofIK(ACh) (IC50) is 25 μM in atrial cells (fig. 4).

Figure 3
Figure 3

Suppressant effect of cocaine onIK(ACh) in ferret atrial myocytes with the nystatin-perforated patch-clamp method. Extracellular application of 50 μM cocaine strongly suppressed IK(ACh)activated by 1 μM carbachol at all holding potentials (○, control; •, cocaine) and altered neither the current-voltage relation nor the reversal potential in atrial cells (n = 10). *P < .05; **P < .01; between carbachol (○) and carbachol plus cocaine (•).

Figure 4
Figure 4

Dose-response curve for cocaine-induced suppression of IK(ACh) in ferret atrial cells with the nystatin-perforated patch-clamp method. The current was activated at 0 mV by extracellular application of 1 μM carbachol (as the control) or 1 μM carbachol plus various concentrations of cocaine. Mean values ± S.E. from five to eight individual cells are plotted. *P < .05; **P < .01; significantly different between the values of the control and cocaine treatment.

Ventricular fibrillation is a common cause of cocaine-induced sudden cardiac death (Isner et al., 1986; Lathers et al., 1988). We further assessed the effects of cocaine onIK(ACh) in single left ventricular myocytes and compared the data with those obtained from atrial cells. Figure5 shows that cocaine significantly suppressed IK(ACh) in both atrial and left ventricular myocytes. Twenty and 50 μM cocaine produced 45 ± 4% and 65 ± 7% inhibition ofIK(ACh) in the atrial myocytes and 40 ± 1% and 53 ± 1% inhibition ofIK(ACh) in the left ventricular myocytes, respectively. Suppression of IK(ACh) was significantly different (P < .05) between the control and treatment groups of both atrial and left ventricular myocytes, but the degree of cocaine-induced inhibition was not significantly different between these two cell types (fig. 5C).

Figure 5
Figure 5

Comparison of cocaine-induced suppression ofIK(ACh) in atrial and left ventricular myocytes with the nystatin-perforated patch-clamp method.IK(ACh) was activated by 1 μM carbachol and suppressed by 50 μM cocaine in an atrial (A) and a left ventricular (B) myocyte. Note the different current scale in A and B. The oscillation of the base line in A was caused by spontaneous contraction of the cell when it was held at 0 mV. Bars represent perfusion period of carbachol (open) and plus cocaine (solid). (C) Comparison of inhibition of IK(ACh) at 0 mV in atrial (open bars, n = 7) and left ventricular (hatched bars, n = 7) myocytes exposed to 20 and 50 μM cocaine. The normalized suppression is almost the same between two groups of myocytes.

MEG suppression of IK(ACh).

MEG is the major product of pyrolysis of cocaine base during smoking (Coneet al., 1994). Extracellular application of 20 μM MEG produced 53 ± 5% inhibition ofIK(ACh) (n = 9, P < .01, fig. 6) in ferret atrial myocytes. Figure 6A shows the original current trace ofIK(ACh) recorded from an atrial cell. The current was markedly reduced in the presence of 20 μM MEG. The inhibition of IK(ACh) is concentration-dependent (fig. 6B). The IC50 is 12 μM for MEG (solid line) and 25 μM for cocaine (dotted line), respectively. Thus, compared with cocaine, MEG had a greater effect onIK(ACh).

Figure 6
Figure 6

Effects of MEG onIK(ACh) with the nystatin-perforated patch-clamp method. (A) The original traces of Ik(ACh) were recorded from an atrial myocyte at a holding potential of 0 mV. Bars represent a perfusion period for carbachol alone (1 μM, open bar) and carbachol (1 μM) plus MEG (20 μM, solid bar). (B) The dose-response curve for MEG-induced inhibition of IK(ACh)in ferret atrial cells. The current was activated at 0 mV by extracellular application of 1 μM carbachol (as the control) or 1 μM carbachol plus various concentrations of MEG. Mean ± S.E. from four to nine individual cells are plotted. *P < .05; **P < .01. The dotted curve in panel B represents the dose-response of cocaine-induced suppression ofIK(ACh) in ferret atrial myocytes (see fig.4).

Effects of cocaine on adenosine-activated K+ currents.

Stimulation of A1-purinergic receptors with adenosine can elicit the same K+ current as activation of muscarinic receptors in atrial cells (Kurachi et al., 1986; Kurachi, 1994; Ito et al., 1995). Figure7 summarizes the effects of cocaine on this adenosine-activated K+ current (IK(Ado)). Extracellular application of 100 μM adenosine activated IK(Ado), which varied from 71 to 579 pA. The average density ofIK(Ado) at 0 mV holding potential was 2.12 ± 0.46 pA/pF for ferret atrial cells (n = 13). Co-application of 50 μM cocaine suppressed the mean current ofIK(Ado) to 0.90 ± 0.33 pA/pF (P < .001), which is 58 ± 5% inhibition.

Figure 7
Figure 7

Inhibition of adenosine-activated K+currents by cocaine. The current was activated by extracellular application of 100 μM adenosine (open bar) and 100 μM adenosine plus 50 μM cocaine (hatched bar) in ferret single atrial myocytes at 0 mV with the nystatin-perforated patch-clamp method. Values represent mean ± S.E. of 13 individual experiments. ***P < .001;vs. the control.

Effects of cocaine on GTP-activated K+currents.

Intracellular dialysis with GTP or GTPγS activates a K+ current which is the same K+ current activated by ACh (Yatani et al., 1987; Koumi and Wasserstrom, 1994). Figure8 depicts an example ofIK(ACh) activated by intracellular dialysis with 100 μM GTPγS. The superimposed current traces in figure 8A (a) are the inwardly rectifier K+ current (IK1) recorded immediately after forming the whole-cell configuration and without stimulation of muscarinic receptors. Cocaine had no significant effect onIK1 (data not shown), which is consistent with an previous study (Kimura et al., 1992). After the initial three applications (each for 5 s) of 1 μM carbachol and 10 min after dialysis with 100 μM GTPγS,IK(ACh) was enhanced and maintained at a high level without carbachol stimulation (fig. 8A, b). The net currents activated by GTPγS are shown in fig. 8A (d) which is the result of b minus a. Extracellular application of 50 μM cocaine markedly reducedIK(ACh) activated by GTPγS (fig. 8A, c). After subtraction of c from b, the net suppression ofIK(ACh) by cocaine was shown in e. AlthoughIK(ACh) was significantly suppressed by cocaine, the current-voltage relationship was not altered (fig. 8B). The average inhibition of GTPγS-activatedIK(ACh) by 50 μM cocaine was 42 ± 4% (P < .001, n = 8). These results indicate that because cocaine inhibits GTPγS-activatedIK(ACh), which bypasses the muscarinic receptor, the cocaine-induced suppression ofIK(ACh) may be mostly by block of the channel pore or inhibition of GK.

Figure 8
Figure 8

Cocaine blockade of the K+ current in a GTPγS-dialyzed atrial myocyte with the classical whole-cell recording technique. (A) Superimposed original current traces ofIK1 (a) were recorded at 0.5 min after rupture of the patched membrane. After three repeated exposures (each for 5 s duration) to 1 μM carbacholIK(ACh) was recorded at 10 min after 100 μM GTPγS dialysis in the absence (b) and presence of 50 μM cocaine (c). The net IK(ACh) activated by GTPγS was shown in d (b − a) and inhibited by cocaine in e (b − c). The currents were activated by 250-ms pulses from a holding potential of −40 mV down to −130 mV up to 20 mV with 10 mV increments every 5 s. Note GTPγS-enhanced L-type Ca++ currents in b and d, and cocaine inhibited the current in c and e. (B) Current-voltage relationships of the K+currents measured at the 200-ms time point for the superimposed traces in a (○), b (•) and c (▴).

Although IK(ACh) was always elicited by re-stimulation of muscarinic receptors in ATP-loaded cells, in the myocytes intracellularly dialyzed with ATP plus GTPγS the response to carbachol stimulation decreased gradually. Figure9 illustrates this effect. In figure 9A an atrial cell loaded with 5 mM ATP responded well to stimulation of 1 μM carbachol every time. The holding current at 0 mV holding potential did not change during the observation period. Furthermore, cocaine suppressed IK(ACh) every time when 50 μM cocaine plus 1 μM carbachol was applied. In contrast, in another atrial cell loaded with 5 mM ATP plus 100 μM GTPγS,IK(ACh) was gradually reduced when 1 μM carbachol was reapplied extracellularly (fig. 9B). The holding current increased continuously and reached an elevated level within 10 to 15 min after forming the classic whole-cell recording configuration. In eight atrial myocytes IK(ACh) was 334 ± 36 pA after 10 to 15 min intracellular dialysis with 100 μM GTPγS. Compared with 509 ± 57 pA ofIK(ACh) elicited during the first exposure to carbachol, the current activated by GTPγS was reduced 33 ± 3% (P < .01, n = 8). Extracellular application of 50 μM cocaine suppressed IK(ACh) by 57 ± 3% (activated by carbachol) and by 42 ± 4% (activated by GTPγS), respectively. Cocaine had less effect, 15 ± 2%, on IK(ACh) activated by GTPγS than by carbachol. The difference of the values suppressed by cocaine between carbachol- and GTPγS-evoked K+ currents is statistically significant (n = 8, P < .05). These results suggest that cocaine-induced inhibition ofIK(ACh) is mostly by block of the channel pore or inhibition of GK, and partially caused by block of the ACh binding site of muscarinic receptors.

Figure 9
Figure 9

Desensitization ofIK(ACh) in GTPγS-dialyzed atrial cells with the classical whole-cell recording technique.IK(ACh) was activated by extracellular perfusion of 1 μM carbachol (open bars) and 1 μM carbachol plus 50 μM cocaine (solid bars). The recordings were made with 10-s pulses at 0 mV holding potential from one atrial cell dialyzed with 5 mM ATP (A) and another atrial cell dialyzed with 5 mM ATP plus 100 μM GTPγS (B). Currents of a, b, c and d were recorded at 0.5, 3, 5 and 10 min after forming the whole-cell recording configuration, respectively. Note the increase of the outward steady current after the time of intracellular dialysis with GTPγS (B).  

Effects of ACh and cocaine on the action potential.

Boyett and co-workers (1988) reported that ACh significantly reduced the action potential duration because of activation ofIK(ACh). Because cocaine blockedIK(ACh) in the present experiments, we further examined the effects of cocaine on carbachol-induced shortening of the action potential duration. Figure10 illustrates that extracellular application of 1 μM carbachol profoundly shortened action potential duration (from 406 ms of the control to 138 ms, measured at 75% repolarization), increased the threshold for initiation of an action potential (from 16 pA of the control to 24 pA), and prolonged the cycle length of excitability (from 520 ms of the control to 920 ms) in a ferret atrial cell. Co-application of 40 μM cocaine partially reversed the carbachol-induced effects on evoked action potentials. The duration, threshold and cycle length of excitability of action potentials were 186 ms, 20 pA and 420 ms, respectively, when both carbachol and cocaine were perfused. After washout of carbachol and cocaine all the parameters of the action potential recovered completely. Similar results were observed from a total of five atrial and three left ventricular myocytes. These results further suggest that cocaine antagonizes the effects produced by stimulation of cardiac muscarinic receptors.

Figure 10
Figure 10

Effects of carbachol and cocaine on the duration, threshold and cycle length of the action potential recorded with the classical whole-cell patch-clamp method. (A) The atrial cell was held at approximately −80 mV by injection of a negative constant current (6 pA) and was stimulated with single 20-ms depolarization currents (28 pA) every 10 s. (B) The atrial cell was held at approximately −80 mV by injection of a negative constant current (6 pA) and was stimulated with a series of depolarization currents (50 ms) in 4-pA increments at 10-s intervals. (C) The cell was held at −80 mV by constant injection of −6 pA current and given a pair of superthreshold electrical stimuli (30 ms and 22 pA for each) at 0.1 Hz with various intervals in 100-ms increments. All the recordings were made from the same myocyte. The duration, threshold and the cycle length of recovery of the action potential is shown in the absence (Control) and presence of 1 μM carbachol (Carb), as well as 1 μM carbachol plus 40 μM cocaine (Carb + cocaine) and washout (Washout).

Discussion

IK(ACh) of ferret cardiomyocytes.

Some early functional studies demonstrated that both atrial and ventricular myocytes receive cholinergic innervation (Kent et al., 1974; Goldman et al., 1983;Takahashi et al., 1985). Recently, cholinergic innervation in the cardiac ventricles was confirmed in rats with the viral tracing method (Standish et al., 1994). The neurons that innervate the ventricles are numerous, and their distribution within the nucleus ambiguus and dorsal motor nucleus of the vagus is similar to that of neurons innervating other cardiac targets, such as the sinoatrial node. Our present study demonstrates that both atrial and ventricular myocytes of ferrets have muscarinic receptors. These results support the previous findings (Boyett et al., 1988) that ACh has a negative inotropic effect on ferret ventricular myocytes and add to the mounting body of evidence that mammalian ventricular myocytes from different species, including humans, contain muscarinic receptors (Loffelholz and Pappano, 1985; Litovsky and Antzelevitch,1990; McMornet al., 1993; Koumi and Wasserstrom, 1994; Ito et al., 1995). The density of IK(ACh)varied greatly between individual cells isolated from the same atria or from the same ventricle. This variability is probably caused by the heterogeneity of cholinergic innervation within the myocardium (Loffelholz and Pappano, 1985; Litovsky and Antzelevitch, 1990). The density of IK(ACh) in ferret ventricular myocytes was 5 times lower than that in atrial cells, which is consistent with the finding of a lower density ofIK(ACh) in human ventricular myocytes (Koumi and Wasserstrom, 1994).

In the heart, activation of K+ channels affects the resting membrane potential and action potential duration. Boyettet al. (1988) reported that ACh significantly hyperpolarized the membrane potential and decreased action potential duration. Our observation indicates that extracellular application of carbachol resulted in not only a decrease in the action potential duration, but also an increase in the threshold and the effective refractory period of action potentials. These findings agree with studies in vivo that application of cholinergic agonists or vagal stimulation prolongs the ventricular effective refractory period in animal models (Inoue and Zipes, 1988). All these actions of ACh are believed to be related to the activation of IK(ACh).

Previous experiments have shown that activation of theIK(ACh) channel did not depend on cytoplasmic factors, but instead was via the muscarinic receptor-coupled G-proteins in mammalian atrial myocytes (Soejima and Noma, 1984; Pfaffinger et al., 1985; Kurachi et al., 1986). Evidence for direct coupling of G-proteins to the K+-ACh channel was established in experiments applying exogenous G-protein, GK, to cell-free membrane patches (Codina et al., 1987; Yatani et al., 1987). In the present experimentsIK(ACh) did not fade in the myocytes with the nystatin-perforated patch method. This result suggests that the regulation of IK(ACh) involves some important intracellular factors. Furthermore,IK(ACh) ran down to a very low level within 30 min in the myocytes by use of the classic whole-cell recording method without intracellular dialysis with ATP. In contrast, with use of the same recording method IK(ACh)remained at 80% of the control if ATP was included in the internal solution. These data indicate that intracellular ATP is critical for maintaining the response of muscarinic receptors to ACh, which is consistent with the results found in guinea pig atrial cells (Zanget al., 1993).

Cocaine blockade of IK(ACh).

We are the first to report that cocaine strongly suppressed the ACh-activated muscarinic K+ current. A concentration as low as 1 μM cocaine significantly reducedIK(ACh) in ferret single heart cells. This concentration is much lower than the concentration, 10 μM, of cocaine required to produce significant blockade of the Na+ channel (Crumb and Clarkson, 1990; Xiao and Morgan, unpublished data). The concentration of cocaine to produce 50% inhibition of IK(ACh) activated by carbachol is 25 μM in ferret atrial cells, which is only half of the concentration (50 μM) of the drug to produce 50% inhibition ofINa in rat ventricular myocytes (Renardet al., 1994; Xiao and Morgan, unpublished data). Therefore, cocaine blockade of cardiac IK(ACh) can be an important mechanism for its cardiotoxicity.

Billman and Loppi (1993) found that cocaine decreased the cardiac vagal tone index in conscious dogs. In a recent study cocaine also suppressed cardiac vagal tone in humans (Newlin, 1994). The mechanism of cocaine blockade of the cardiac cholinergic system has not been delineated, but is particularly important, because the cardiac muscarinic receptor plays a crucial role in modulation of heart rate and stabilization of membrane electrical excitability (Billman and Hoskins, 1989). In the present study cocaine suppressed the K+ current activated by stimulation of muscarinic receptors. The cocaine-induced suppression of IK(ACh) is possibly caused by block of the muscarinic receptor binding site and the channel pore, or by a modulation of the coupled G-protein. A binding study (Sharkeyet al., 1988) presented evidence for cocaine as an antimuscarinic agent, particularly at higher doses, in heart and brain. Cocaine inhibited M2 receptor binding measured with [3H]quinuclidinyl benzilate in both tissues with a Ki of 18.8 μM. Cocaine also reversed the methacholine-induced inhibition of guinea pig atrial contractions. Cocaine as a competitive antagonist of muscarinic receptors has been reported in cardiac tissues (Sharkey et al., 1988; Billman and Loppi, 1993). In this study we found that cocaine not only blocked IK(ACh) activated by carbachol, but also blocked the K+ current activated by adenosine or GTPγS. When the K+current is activated by adenosine, by stimulation of purinergic receptors or by GTPγS stimulation of GK(Kurachi et al., 1986), the binding site of muscarinic receptors is bypassed. Although the inhibitory effects of cocaine onIK(Ado) are possibly caused by blocking the A1-receptor binding site, the cocaine-induced suppression of IK(ACh) activated by GTPγS must result from either an inhibition of GKactivity or block of the channel pore. In the present study the same concentration of cocaine produced a greater blocking effect (15% more) on IK(ACh) activated by carbachol than by GTPγS. This suggests that cocaine-induced suppression ofIK(ACh) is partially by blocking the binding site of muscarinic receptors, and mostly by directly blocking the channel pore or by suppressing GK activity. Furthermore, Newman and co-workers (1994) found that anhydroecgonine methyl ester (MEG) and its congeners had negligible affinities to muscarinic receptors expressed in transfected cell lines. This observation, along with our present results, indicates that the pyrolysis product of cocaine is more potent than cocaine not because of its interaction at muscarinic receptors, but because of its interaction at the level of the membrane channel or G-protein. The ability of cocaine, acting as a local anesthetic, to block the channel pore ofIK(ACh) is consistent with its effect on cardiac Na+ channels (Crumb and Clarkson, 1990;Renard et al., 1994).

Significance.

The present data suggest that cocaine blockade of IK(ACh) may have clinical relevance to its cardiotoxicity. Cardiovascular morbidity and mortality from use of cocaine, particularly among young and healthy adults, have increased markedly in the past several years. Although cardiac intoxication with cocaine has been linked to a sympathomimetic effect and its local anesthetic effects of blocking cardiac Na+channels, the precise mechanisms responsible for life-threatening cardiovascular events remain undetermined. ACh released on vagal stimulation produces the negative chronotropic, dromotropic and inotropic effects on the heart by activation ofIK(ACh). Activation of muscarinic K+ channels enhances the membrane stability of cardiac myocytes by hyperpolarization and has a protective effect against some cardiac arrhythmias (Rardon and Bailey, 1983; Rauch and Noroomand, 1991). In this study we found that stimulation of muscarinic receptors resulted in an increase in the stimulatory threshold and the refractory period, which was attenuated by application of cocaine. Wilkerson’s work (1989) found that atropine significantly enhanced the cocaine-induced cardiovascular toxicity. Our present results demonstrate that cocaine has a potent blocking effect on the cardiacIK(ACh), which may enhance cocaine cardiotoxicity.

In humans, cocaine can produce a 30 to 50% increase in heart rate and blood pressure at a low plasma concentration, approximately 1 μM (Fischman et al., 1983). Increasing doses of cocaine further increase heart rate and blood pressure, leading to ventricular fibrillation and cardiac arrest (Gay, 1982). Why cocaine-induced increase in blood pressure is accompanied by tachycardia, rather than being dominated by a vagal reflex slowing the heart rate, as seen with most pressor agents, has always been a dilemma. Our finding that cocaine suppresses IK(ACh) may explain this diminished vagal influence. Autopsy studies (Mittleman and Wetli, 1984;McKelway et al., 1990) have revealed mean plasma concentrations of cocaine of approximately 20 μM. The highest value found in autopsy was 80 μM at whichIK(ACh) was almost completely suppressed according to our present data. It is well established that the parasympathetic innervation of the heart opposes the sympathetic input and plays an important homeostatic role with regard to heart rate by activation of IK(ACh). Our finding of cocaine blockade of the cardiac IK(ACh)indicates that the drug may prevent the slowing effects of ACh on the heart rate, not only under basal conditions, but also during compensatory reflex vagal responses to the sympathomimetic action of cocaine. Because the antimuscarinic effect of cocaine can attenuate the ACh protective action, cocaine blockade of cardiacIK(ACh) may thus play an important role in its cardiotoxicity.

Footnotes

  • Send reprint requests to: Yong-Fu Xiao, M.D., Ph.D., Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215.

  • ↵1 This work was supported by NIH grant HL51307 (to J.P.M).

  • Abbreviations:
    ACh
    acetylcholine
    IK(ACh)
    acetylcholine-activated muscarinic K+ channel
    IK(Ado)
    adenosine-activated purinergic K+ channel
    MEG
    methylecgonidine
    EGTA
    ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
    HEPES
    N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
    • Received March 20, 1997.
    • Accepted September 3, 1997.

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OtherANALGESIA AND DRUGS OF ABUSE

Cocaine Blockade of the Acetylcholine-Activated Muscarinic K+ Channel in Ferret Cardiac Myocytes

Yong-Fu Xiao and James P. Morgan
Journal of Pharmacology and Experimental Therapeutics January 1, 1998, 284 (1) 10-18;
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