Introduction

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Parasympathetic regulation of the heart rate is mediated by acetylcholine (ACh) (Loewi and Navratil, 1926; Löffelholz and Pappano, 1985). ACh activates M2 muscarinic receptors and the heterotrimeric G_i-2, G_i-3 and/or G_o proteins in cardiac myocytes (Luetje et al., 1988). The  subunits of the G_i proteins inhibit the adenylyl cyclase (Sunahara et al., 1996), whereas the subunits of the G_i proteins directly activate the inwardly rectifying muscarinic K⁺ (K_ACh) channel in sinoatrial (SA), atrial, and atrioventricular (AV) nodes, and Purkinje myocytes (Logothetis et al., 1987; Sowell et al., 1997; Yamada et al., 1998).

K_ACh channels cause the negative chronotropic effect by hyperpolarizing SA node cells (del Castillo and Katz, 1955; Hutter and Trautwein, 1955; Noma and Trautwein, 1978; Sakmann et al., 1983). On the other hand, the suppression of adenylyl cyclase decreases the heart rate by inhibiting such inward currents in SA node cells as the hyperpolarization-activated (I_f) current, the L-type calcium current, and the sustained inward current, all of which are activated by cAMP or cAMP-dependent protein kinase, especially in the presence of -adrenergic stimulation (Irisawa et al., 1993). It is unknown to what extent each of the signal transduction pathways is responsible for the negative chronotropic effect of ACh. This is partly because there have been no selective inhibitors of every signal transduction pathway evoked by muscarinic stimulation.

A peptidyl honeybee toxin tertiapin was recently found to selectively inhibit K_ACh channels in cardiac myocytes (Jin and Lu, 1998; Drici et al., 2000; Kitamura et al., 2000). Tertiapin fully inhibits ACh-induced whole-cell K_ACh channel currents in rabbit atrial myocytes in a concentration-dependent manner in a range of concentrations between 10 pM and 10 µM through ~1:1 stoichiometry (Kitamura et al., 2000). The half-maximum IC₅₀ of tertiapin is ~8 nM independent of the membrane potential. Tertiapin also inhibits the K_ACh channel current activated by a hydrolysis-resistant GTP analog applied to the cytosol, indicating that the effect of tertiapin is not mediated by a muscarinic receptor. At the single-channel level, tertiapin inhibits the K_ACh channel from the outside of the membrane by reducing the NP_o (N is the number of functional channels, and the P_o is the open probability of each channel) without affecting the single-channel conductance or fast open-close kinetics. Thus, tertiapin is a slow blocker of the K_ACh channel.

In this study, I examined the effect of tertiapin on the sinus bradycardia caused by carbachol (CCh) in the presence of a -adrenergic blocker in the Langendorff preparation of rabbit hearts with ECG. I divided the effect of CCh into the tertiapin-sensitive (i.e., K_ACh current-dependent) and -insensitive (i.e., K_ACh current-independent) components. The tertiapin-sensitive component was ~10 times less sensitive to CCh but ~3 times more effective than the tertiapin-insensitive component in decreasing the sinus rate. The tertiapin-insensitive component seemed to be mediated by inhibition of I_f currents probably through suppression of the basal cAMP level.

	Materials and Methods

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Langendorff Preparation of Rabbit Hearts. Male Japanese-White rabbits weighing 1.5 to 1.7 kg were injected with heparin sodium (200 U/kg b.wt.) through an ear vein. Approximately 20 min later, the rabbits were anesthetized with pentobarbital sodium or thiopental sodium (30 mg/kg b.wt.) injected through a vein in another ear. As soon as the rabbits lost a nociceptive response, the heart was quickly removed and immediately reperfused with oxygenated modified Tyrode's solution (for composition, see below) at 37°C in the Langendorff apparatus in a retrograde manner.

Recording Electrocardiogram from Isolated Rabbit Hearts. The isolated heart was hung over a glass funnel ~5 cm in diameter in such a way that its apex lightly touched the inner surface of the lowest and narrowest part of the funnel. A few sheets of thin paper (Kimwipes; Kimberly-Clark Co., Tokyo, Japan) filled the space between the ventricular wall and the inner surface of the funnel. After the paper was wetted with the cardiac effluent (i.e., the modified Tyrode's solution), the standard bipolar lead ECG was recorded from the electrodes connected to the paper with a conventional ECG recorder (Cardiofax; Nihon Kohden Co. Ltd., Tokyo, Japan).

1

Isolation of Atrial Myocytes and Patch-Clamp Study. The methods of isolation of atrial myocytes and patch-clamp experiments were precisely described in a previous paper (Kitamura et al., 2000). Briefly, the heart in the Langendorff apparatus was perfused with 150 ml of the nominally calcium-free Tyrode's solution (for composition, see below) and then that with 0.04% (w/v) collagenase (Yakult Pharmaceutical, Tokyo, Japan) for 10 to 15 min. The digested heart was stored in the KB solution (for composition, see below) at 4°C. Atrial myocytes were isolated from the digested atrial tissue in the modified Tyrode's solution in a recording chamber set on an inverted microscope (Axiovert 135; Carl Zeiss, Jana, Germany).

Solutions and Chemicals. The modified Tyrode's solution contained 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.53 mM MgCl₂, 5.5 mM glucose, and 5.5 mM HEPES (pH adjusted to 7.4 with NaOH). For the nominally calcium-free Tyrode's solution, CaCl₂ was simply omitted from the modified Tyrode's solution. The KB solution contained 10 mM taurine, 10 mM oxalic acid, 70 mM glutamic acid, 25 mM KCl, 10 mM KH₂PO₄, 11 mM glucose, 0.5 mM EGTA, and 10 mM HEPES (pH adjusted to 7.3 with KOH). The pipette solution contained 140 mM KCl, 5 mM MgCl₂, 5 mM EGTA, 3 mM ATP, 0.1 mM GTP, and 5 mM HEPES (pH adjusted to 7.4 with KOH). The calculated free Mg²⁺ concentration in this solution was 1.6 mM. In the ECG experiments, 5 mM ascorbic acid was added to the modified Tyrode's solution (pH readjusted to 7.4 with NaOH) to prevent oxygenation of tertiapin. Ascorbic acid (5 mM) alone did not affect the ECG. Tertiapin was purchased from Peptide Institute Inc. (Osaka, Japan). Tertiapin was dissolved in distilled water at 100 µM immediately before use, further diluted to desired concentrations with the oxygenated modified Tyrode's solution with ascorbic acid, and immediately applied to the hearts. CCh and propranolol were purchased from Sigma Chemical Co. (St. Louis, MO). Atropine was from Wako Pure Chemical (Osaka, Japan).

Statistical Analysis. All statistical values are indicated as mean ± S.E. The statistical difference was evaluated by Student's t test. For multiple comparison between pairs, difference was assessed with analysis of variance and the Bonferroni method. A value of p < 0.05 was considered statistically significant.

	Results

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Concentration-Dependent Effect of Carbachol on the Sinus Rate of a Rabbit Heart. I first examined the concentration-dependent effect of CCh on the sinus rate in the Langendorff preparation of a rabbit heart by monitoring the ECG (Fig. 1). At the beginning of each experiment, a -adrenergic blocker propranolol (100 nM) was applied to eliminate the effect of residual catecholamine in the heart (Fig. 1A). Propranolol (100 nM) decreased the sinus rate to 85.7 ± 1.9% of the control on average and increased the PQ interval significantly (Table 1). After the sinus rate became stable (from recordings 1 and 2), CCh was applied in a cumulative manner. CCh decreased the sinus rate in a concentration-dependent manner in the range of concentration between 10 nM and 1 µM (recordings 3-6). CCh caused sinus arrest in eight of nine hearts at 1 µM and in all four hearts at 10 µM. The PQ interval slightly but significantly increased from 51.12 ± 2.78 ms in controls to 59.22 ± 1.98 ms in the presence of 300 nM CCh (n = 9). However, CCh did not cause the second or third degree of AV conduction block before inducing the sinus arrest in 39 of 40 hearts examined. Thus, I did not further analyze the effect of CCh and/or tertiapin on the AV conduction.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Carbachol-induced sinus bradycardia and effect of tertiapin in an isolated rabbit heart. A, in the Langendorff preparation of a rabbit heart, drugs indicated above the graph were applied through coronary arteries in a retrograde manner. Throughout the experiment, ECG was monitored. The measured sinus rate (ordinate) is plotted against the experimental time in minutes (abscissa). The numbers in parentheses correspond to those in B. B, representative ECG recordings obtained at the timings indicated by numbers in A. Note a single ventricular escaped beat in (6).

Effect of Tertiapin on the Sinus Bradycardia Induced by Lower Concentrations of Carbachol. I further examined the effect of tertiapin in the presence of lower concentrations of CCh (Fig. 2). Tertiapin (100 nM) reversed the negative chronotropic effect of 300 nM CCh by ~41% (Fig. 2A). The same concentration of tertiapin, however, antagonized the effect of 100 nM CCh only by ~16% (Fig. 2B) and barely antagonized that of 30 nM CCh (Fig. 2C). In the absence of CCh, 100 or 300 nM tertiapin did not significantly alter the sinus rate, although it reduced the effect of subsequently applied 1 µM CCh by 73% (Fig. 2D and data not shown). Therefore, the effect of tertiapin was less as the concentration of CCh decreased.

View larger version (56K): [in this window] [in a new window]   Fig. 2.   Effect of tertiapin in the presence and absence of 300, 100, and 30 nM carbachol. Tertiapin (100 nM) was applied in the presence of 300 nM (A), 100 nM (B), and 30 nM (C) of CCh or in the absence of CCh (D).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Carbachol-induced negative chronotropic effect in the presence and absence of tertiapin, and carbachol-induced muscarinic K+ channel currents in rabbit atrial myocytes. A, relationship between the sinus rate and concentrations of CCh in the absence (circles) and the presence of 100 nM (triangles) or 300 nM (squares) of tertiapin. The sinus rate is indicated as a percentage of the control. , p < 0.05 versus control; , p < 0.025 versus control; and , p < 0.025 versus 100 nM tertiapin. Symbols and bars indicate the mean and S.E., respectively. The number of observations at each point was 4 to 10. Inset, the negative chronotropic effect of CCh in the absence of tertiapin (open circles), and its tertiapin-sensitive (diamonds) and -insensitive (squares) components are depicted as a percentage of the maximum effect of CCh. The tertiapin-sensitive component was calculated by subtracting the percentage of the sinus rate in the presence of tertiapin from that in the absence of tertiapin. The tertiapin-insensitive component was calculated by subtracting the percentage of the sinus rate in the presence of tertiapin from 100%. The data in the presence of 1 µM CCh and 100 nM tertiapin were omitted from the calculation. The broken line is the curve shown in panel B, b (see below). Panel B: a, in the whole-cell configuration of the patch-clamp method, CCh-induced KACh channel currents (bottom panel) were recorded from a rabbit atrial myocyte. From the holding potential of 40 mV, a set of three 300 ms voltage pulses to 100, 40, and +10 mV were applied every 3 s (top panel). Arrowheads and dotted lines indicate the zero potential (top panel) and the zero current (bottom panel) levels. b, relationship between concentrations of CCh and the steady-state CCh-induced KACh channel currents at 40 mV expressed as a percentage of the maximum (I). Symbols and bars indicate the mean and S.E., respectively. The solid line is the fit of the data with the following Hill equation: I = 100/(1 + (4.35 × 107/[CCh])1.52).

Concentration-Dependent Effect of Carbachol on the K_ACh Channel. I next examined the effect of CCh on K_ACh channel currents in atrial myocytes (Fig. 3B). In the whole-cell configuration of the patch-clamp method, CCh (>10 nM) induced K_ACh channel currents with characteristic slow relaxation (Fig. 3B, a). The current flowed inward at 100 mV and outward at 40 and +10 mV. CCh induced K_ACh channel currents in a concentration-dependent manner in the range of concentrations between 1 nM and 10 µM (Fig. 3B, b). The line indicates the fit of the results with the Hill equation (see figure legend), which provided an estimate of the half-maximum effective concentration of 435 nM and the Hill coefficient of 1.52. This curve is replotted in the inset of Fig. 3A. This curve was very similar to that of the tertiapin-sensitive component in a range of CCh concentrations of 1 µM. However, in the presence of >1 µM CCh, the K_ACh current increased in a concentration-dependent manner, whereas that for the tertiapin-sensitive component reached a plateau because >1 µM CCh caused sinus arrest.

Effect of CsCl on the Sinus Rate and Carbachol-Induced Bradycardia. The tertiapin-insensitive component may be mediated by suppression of the basal cAMP level. It is reported that ACh inhibits I_f currents at lower concentrations than activating K_ACh channels by reducing the basal cAMP level (DiFrancesco and Tromba, 1988; DiFrancesco et al., 1989). Thus, I examined the effect of CsCl on the heart rate because CsCl is reported to selectively inhibit I_f currents at 1 to 2 mM in rabbit SA node cells (Denyer and Brown, 1990). As shown in Fig. 4A, 2 mM CsCl reduced the sinus rate to ~66% of the control (from recordings 1 and 2), indicating that I_f currents contribute to but are not essential for sinus automaticity (Irisawa et al., 1993). CsCl (2 mM) did not significantly change the QRS or QTc intervals (Table 1), indicating that it did not block the voltage-dependent or inwardly rectifying K⁺ currents in ventricular and Purkinje myocytes at least effectively in the physiological range of membrane potentials. CCh (1 µM) applied after washout of CsCl caused sinus arrest (recording 3), and 300 nM tertiapin applied under this condition increased the sinus rate to the level similar to that in the presence of 2 mM CsCl alone (recordings 2-4). On average, the sinus rate was 78.5 ± 4.2% of controls in the presence of 2 mM CsCl alone (n = 12) and 72.1 ± 4.3% in the presence of 1 µM CCh and 300 nM tertiapin (n = 5) (Fig. 3A). These values were not significantly different (p = 0.382). In Fig. 4B, 1 µM CCh was added in the presence of 2 mM CsCl. CCh decreased the sinus rate more slowly than in the absence of CsCl probably because K_ACh channels were blocked by CsCl more potently as the membrane potential was hyperpolarized (Argibay et al., 1983). In the presence of 2 mM CsCl, 300 nM tertiapin completely antagonized the effect of 1 µM CCh (recordings 2-4). Thus, it is likely that the tertiapin-insensitive component of the CCh-induced sinus bradycardia results from inhibition of the I_f current.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effect of tertiapin on carbachol-induced sinus bradycardia in the presence and absence of CsCl. A, CsCl (2 mM) was first applied alone. After washout of CsCl, CCh and tertiapin were applied. B, CCh and tertiapin were applied in the presence of 2 mM CsCl.

	Discussion

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By using tertiapin as a selective K_ACh channel blocker in the heart (Kitamura et al., 2000), I divided the negative chronotropic effect of CCh into the tertiapin-sensitive (i.e., K_ACh current-dependent) and -insensitive (i.e., K_ACh current-independent) components. The K_ACh current-dependent component exhibited a steep concentration-response curve in the range of CCh concentrations between 100 nM and 1 µM, and accounted for ~75% of the maximum effect of CCh. On the other hand, the K_ACh current-independent component was induced by 1 to 300 nM CCh and accounted for ~25% of the maximum effect of CCh. Thus, the K_ACh current-dependent component was ~10 times less sensitive to CCh but ~3 times more effective than the K_ACh current-independent component in decreasing the sinus rate.

The concentration-response curve for the CCh-induced K_ACh current closely resembled that for the tertiapin-sensitive component in a range of CCh concentrations of 1 µM with a similar steepness (Figs. 3A, inset; the Hill coefficient 1). This positive cooperativity may arise from the interaction between the G protein subunits and K_ACh channels (Hosoya et al., 1996). In the presence of >1 µM CCh, the CCh-induced K_ACh current increased in a concentration-dependent manner, whereas that for the tertiapin-sensitive component reached a plateau. This is because >1 µM CCh caused sinus arrest.

Recently, Drici et al. (2000) identified the role of K_ACh channels in the parasympathetic regulation of AV conduction by showing that tertiapin attenuated the ACh-induced advanced AV block in guinea pig hearts. They also briefly mentioned the effect of tertiapin on the negative chronotropic effect of ACh. In guinea pig hearts, they found that tertiapin almost completely reversed the effect of 0.5 µM ACh. In rabbit hearts, however, they found that 5 µM ACh reduced the sinus rate by only 20%, and that this response was insensitive to tertiapin. This is very different from my observation. I found in preliminary experiments that >1 µM ACh usually caused sinus arrest as is the case for CCh. At lower concentrations, however, ACh caused more variable effect than CCh (data not shown), indicating that cholinesterase significantly affects the effect of ACh in whole heart preparation (Löffelholz and Pappano, 1985). Thus, it is possible that the maximum effective concentration of ACh in their rabbit heart preparation might have been much higher than 5 µM.

It is widely accepted that the negative chronotropic effect of vagal nerve activity or parasympathomimetics results from hyperpolarization of sinus node cells due to activation of K_ACh channels (Gaskell, 1887; Burgen and Terroux, 1953; del Castillo and Katz, 1955; Hutter and Trautwein, 1955 and 1956; Harris and Hutter, 1956; Hutter, 1957; Trautwein an Dudel, 1958; Noma and Trautwein, 1978; Sakmann et al., 1983). On the other hand, weak vagal stimulation or low concentrations of ACh is known to cause the negative chronotropic effect by decreasing the slope of diastolic depolarization without causing hyperpolarization of the maximum diastolic potential (Hutter and Trautwein, 1955 and 1956; West, 1955; Bouman et al., 1963; Shibata et al., 1985; Campbell et al., 1989; DiFrancesco, 1993). This phenomenon may be at least in part explainable in terms of the slow relaxation of K_ACh channels (Yamada et al., 1998). ACh-induced inhibition of the L-type calcium channel current and/or the sustained inward current may also be responsible for this phenomenon (Irisawa et al., 1993). On the other hand, DiFrancesco et al. ascribed the phenomenon to inhibition of the I_f current resulting from the ACh-induced suppression of the basal cAMP level (DiFrancesco and Tromba, 1988; DiFrancesco et al., 1989; DiFrancesco, 1993). They showed that ACh inhibited I_f currents and slowed the spontaneous firing rate of isolated rabbit SA node cells at ~20 times lower concentrations than activating K_ACh channels. Although there is some controversy on their hypothesis and contribution of I_f currents to the basal sinus automaticity (Irisawa et al., 1993), 2 mM CsCl indeed reduced the sinus rate by ~20% (Fig. 4) without causing a significant change in QRS or QTc intervals (Table 1). Thus, the I_f current seems to contribute to the basal cardiac pacemaking probably by counteracting the hyperpolarizing influence of the atrial myocytes connected to SA node cells (DiFrancesco, 1993; Irisawa et al., 1993). Furthermore, the sinus rate was similar in the presence of 2 mM CsCl alone and in the presence of 1 µM CCh plus 300 nM tertiapin (Fig. 4A), and there was no tertiapin-insensitive component in the presence of 2 mM CsCl (Fig. 4B). Therefore, the tertiapin-insensitive component seems to result from suppression of I_f currents.

It was recently demonstrated that GIRK4 knock-out mice deficient of K_ACh channels exhibited a normal mean resting heart rate with impaired baroreflex (Wickman et al., 1998). In view of the present study, the mean resting heart rate may be regulated by relatively low concentrations of ACh through the suppression of I_f currents (Fig. 3A). The study also indicates that the vagal activity under baroreflex provides sufficiently high concentrations of ACh to activate K_ACh channels in the SA node cells (Fig. 3A). In the baroreflex, the efferent cardiac vagal activity occurs more or less fixed to the cardiac cycle (Jewett, 1964; Katona et al., 1970), and the effectiveness of vagal activity on the sinus automaticity is strongly dependent on the relationship between the pacemaker cycle and the duration, amplitude, and timing of the hyperpolarizing effect of vagal impulses (Jalife and Moe, 1979). K_ACh channels possess a sufficiently fast response time to ACh to mediate such a phasic effect of vagal activity (Breitwieser and Szabo, 1988; Inomata et al., 1989), at least in part due to the direct coupling of the channels with G proteins and the regulator of G protein signaling proteins (Doupnik et al., 1997; Yamada et al., 1998). The positive cooperativity found in activation of K_ACh channels (Fig. 4B) will also help the channels to promptly open when ACh concentration rises and suddenly close when it decreases even slightly. It is, therefore, plausible that K_ACh channels play an essential role in the baroreflex.

To summarize, I quantitatively assessed the contribution of K_ACh channels to the negative chronotropic effect of CCh in rabbit hearts by using tertiapin. I found that K_ACh currents mediate the negative chronotropic effect of 300 nM CCh and account for up to ~75% of the maximum effect of CCh. However, suppression of I_f currents might play a major role for the effect of 1 to 100 nM CCh and account for ~25% of the maximum effect of CCh. The K_ACh current-dependent mechanism may play an important role in the beat-to-beat regulation of the sinus rate under baroreflex, whereas suppression of I_f currents may participate in regulation of the mean resting heart rate. Tertiapin is a useful pharmacological tool to identify the physiological and pathophysiological roles of K_ACh channels in parasympathetic regulation of the heart rate of various animals under ex vivo and in vivo conditions.