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1 University of Otago, Dunedin, New Zealand
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(Received 13 June 2007;
accepted after revision 20 August 2007; first published online 24 August 2007)
Corresponding author C. P. Bolter: Department of Physiology, School of Medical Sciences, University of Otago, PO Box 913, Dunedin 9054, New Zealand. Email: chris.bolter{at}stonebow.otago.ac.nz
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Introduction |
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It remains unclear how the various candidate currents participate in vagal slowing of the in situ mammalian pacemaker. A more-or-less general view holds that activation of IK,ACh is a primary mechanism and that its effects on the slow diastolic depolarization (SDD; pacemaker potential) are supported by a shift of the reactivation potential for If to more negative values. However, the participation of IK,ACh and If in vagal slowing has been questioned by Hirst and colleagues. In a series of articles, they presented evidence that ACh released from the parasympathetic varicosities occupies a set of muscarinic receptors (junctional receptors) that is physically distinct from a set of receptors (extrajunctional receptors) occupied by ACh that has been applied to a preparation through the bathing medium or by ionophoresis (Klemm et al. 1992; Demir et al. 1999). In particular, they conclude that these two separate sets of muscarinic receptors are coupled to quite different types of membrane channels/currents. Several separate pieces of evidence support these ideas. Micrographic evidence shows that parasympathetic varicosities in the SAN form well-organized synapses with pacemaker cells (Klemm et al. 1992; Choate et al. 1993; Hirst et al. 1996). Experiments on vagally innervated beating and arrested amphibian (cane toad; Bywater et al. 1989, 1990; Edwards et al. 1993; Bramich et al. 1994) and mammalian (guinea-pig; Campbell et al. 1989) pacemaker preparations showed that changes in membrane potential in response to neurally released ACh were caused, at least in part, by different membrane currents to changes in membrane potential produced by applied ACh. When Ba2+ was applied to block IK,ACh, the responses to exogenous ACh were abolished (amphibian) or partly blocked (guinea-pig), while responses to vagal stimulation were little affected. More recently, it has been shown that responses to vagal stimulation persist in the beating and arrested guinea-pig SAN preparation after both IK,ACh and If have been blocked (Bolter et al. 2001). From these experiments, Hirst and colleagues proposed that junctional receptors were coupled to neither IK,ACh nor If. In addition, in the amphibian, vagal stimulation caused an increase in membrane resistance of arrested pacemaker cells while bath-applied ACh caused a reduction in resistance, suggesting that the vagus exerted its influence through a reduction in inward current (Bywater et al. 1990). One extensive analysis (Demir et al. 1999) showed that these ideas are consistent with the pacemaker responses to vagal stimulation observed in a wide range of studies. In contrast, in an analysis of the effects of ACh on the rabbit SAN, Zhang et al. (2002) concluded that its primary effect was through activation of IK,ACh and that effects on other currents were permissive.
Several problems are encountered when attempting to identify the roles played by candidate currents for the pacemaker response to vagal stimulation. A primary difficulty is the choice of appropriate channel blockers and antagonists of signalling pathways. Barium is frequently applied to obtain block of IK,ACh. However, Ba2+ can produce a general block of inwardly rectified K+ channels present in heart tissues. This may alter the membrane potential and firing rate of the pacemaker cells and make subsequent experimental observations more difficult to interpret. A further problem with Ba2+ as a block for IK,ACh stems from the conditions under which it blocks this channel. From experiments on guinea-pig atrial cells, Zang et al. (1995) concluded that block of IK,ACh by Ba2+ was both state and voltage dependent. In the absence of ACh, the open probability of IK,ACh is low, and the indirect depolarization of pacemaker cells by Ba2+ may reduce further the chance of Ba2+ blocking IK,ACh.
In the study reported here we have re-examined the role of IK,ACh in the vagal slowing of the in situ pacemaker. To block IK,ACh we have use tertiapin-Q, a stable analogue of the bee venom toxin, tertiapin. When applied to the heart, tertiapin-Q can produce a relatively specific and complete block of IK,ACh (Kitamura et al. 1996; Yamada, 2002; Benavides-Haro et al. 2003). It therefore possesses considerable advantages over Ba2+ for investigating the role of IK,ACh in vagal slowing; complete block of IK,ACh can be obtained without altering baseline values of membrane potential or beating frequency.
Our experiments were performed on Langendorff-perfused hearts of young adult guinea-pigs, rather than on isolated right atrial preparations. The choice of this preparation was dictated by the necessity of ensuring that tertiapin-Q (
2500 Da) was accessible to cells. This should be readily achieved in a vascularly perfused preparation, but not necessarily in one that is superperfused.
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Experimental preparations
Experiments were performed on guinea-pig hearts perfused by the Langendorff technique. Animals of either sex, 160–250 g (3–4 weeks old), were injected with heparin (500 units kg–1, I.P.) and anaesthetized with pentobarbitone sodium (60 mg kg–1, I.P.). The trachea was cannulated and mechanical ventilation applied. Both right and left vagus nerves were cut high in the neck and separated from the common carotid artery and cervical sympathetic trunk. The chest was opened in the mid-line, and the ascending aorta was separated from the pulmonary trunk. The heart was cooled rapidly and respiration terminated. A metal cannula was inserted retrograde into the ascending aorta, and coronary flow re-established with Krebs–Henseleit solution, filtered (0.45 µM), and saturated with carbogen (95% CO2–5% O2) at 37°C. A bubble trap was located directly ahead of the aortic cannula, which the perfusate entered at 36°C. Perfusion pressure was held at 50 mmHg.
Once sepearted from other tissues, the experimental preparation consisting of the heart and vagus nerves was transferred to a 20 ml organ bath and immersed in Krebs–Henseleit solution maintained at 35°C. The right vagus nerve was drawn through a pair of platinum electrodes for electrical stimulation. An ECG was recorded from a pair of electrodes inserted into the perimeter of the bath; the metal aortic cannula acted as the earth electrode.
Solutions and drugs
The composition of the Krebs–Henseleit solution was (in mmol l–1): Na+, 143.3; K+, 5.3; Mg2+, 1.2; Ca2+, 1.5; Cl–, 127.8; HCO3–, 25.1; SO42–, 1.2; H2PO4, 0.9; D-glucose, 10.0; and the β-adrenergic antagonist propranolol, 1 µM. Other drugs used in these experiments were tertiapin-Q, acetylcholine iodide, bethanecol and atropine. All drugs were obtained from Sigma (St Louis, MO, USA). Tertiapin-Q, Cs+ and atropine were added to the bulk perfusate. Bethanecol and ACh were introduced to the perfusate as a bolus injection into the bubble trap located immediately upstream of the aortic cannula. The step function of a calibrated infusion pump (ktS model 100, IITC Life Science, Woodland Hills, CA, USA) provided a reproducible bolus injection.
Measurements, data acquisition and protocols
Coronary flow was measured from timed volume collections of the overflow of the organ bath. The ECG signals were amplified, filtered, and the R wave was detected using a custom-designed window discriminator. The output of the window discriminator triggered an instantaneous rate-meter (MacLab/8s, ADInstruments, Castle Hill, NSW, Australia) to generate the heart rate. Heart rate and a signal indicating the application of individual electrical stimuli were recorded at 100 s–1 (Chart v4.2.3, ADInstruments). In addition, the ECG signal was recorded continuously to tape for off-line analysis. To ensure that data were interpreted correctly, the ECG records from all experiments were re-recorded through the MacLab system, sampling at 1000 s–1, and atrial rate was calculated from the P–P interval.
After a 30 min stabilization period, one of a number of experiments was performed. Each experiment consisted of measurements made first in control conditions, then after 24 min in tertiapin-Q, and finally after a 1 h period in the original control solution (washout period). Preliminary experiments showed that it required 20 min for the full effects of tertiapin-Q to become established. Each set of measurements included the responses to 10 s trains of supramaximal electrical stimulation of the vagus nerve (vagal stimulation) at 2, 5 and 10 Hz (2 ms rectangular pulses), with 1 min recovery between trains. Next we recorded the responses to bolus injection of 3 nmol of ACh (30 µl of a 10–4
M solution over 1.29 s), or 30, 50 and 70 nmol bethanecol (30, 50 and 70 µl of a 10–3
M solution), delivered in succession at 1 min intervals.
A single preparation received all the treatments and tests described for one of the following three protocols. These protocols examined: (i) the effect of either 300 nM or 1 µM tertiapin-Q on the responses to vagal stimulation and application of ACh; (ii) the effect of 300 nM tertiapin-Q on the responses to vagal stimulation and application of ACh in preparations exposed to 2 mM Cs+; and (iii) the effect of 300 nM tertiapin-Q on the responses to vagal stimulation and application of bethanecol in preparations exposed to 2 mM Cs+. The effect of the vehicle was tested in three preparations. The maximum change in atrial rate in response to 30, 50 and 70 µl injectates of water were –1.2 ± 0.6, –0.1 ± 0.9 and –1.2 ± 0.7%, respectively (P> 0.5 for all volumes).
Statistical analysis
Data are presented as group means ± 1 S.D. For most experiments, statistical analysis was carried out using ANOVA for repeated measures with Fisher's PLSD applied post hoc. For simple comparison between group means, Student's t test was applied.
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In the rat, the blood supply to the SAN originates from the internal thoracic artery, rather than from the coronary arteries (Halpern, 1957). We were unable to find a definitive study on the origin of the vascular supply to the guinea-pig sino-atrial node. Several studies have described branches of the coronary arteries that passed into the atria (e.g. Flores et al. 1984). In four preliminary experiments, we added cardio-green dye to the perfusate as it passed through the bubble trap, then arrested the heart 10 s later by adding K+ to the perfusate. The heart chambers were rinsed with saline, opened, and inspected under a microscope. In all four preparations, the right atrium, including the posterior wall between the crista terminalis and the septum, was fully stained by the dye. We concluded that perfusate entering the aortic cannula had reached all parts of the right atrium, including the sino-atrial node, through vessels originating from the coronary arteries.
Electrocardiogram and atrial rate during vagal stimulation and the application of ACh
Electrocardiogram recordings from Langendorff preparations showed normal waveforms, with clear P waves and QRS complexes (Fig. 1A). At higher frequencies of vagal stimulation, and briefly after the application of acetylcholine, periods of third degree heart block were often observed, during which the R–R interval no longer reflected the sino-atrial pacemaker frequency (Fig. 1B). In our final analysis, atrial rate was calculated from the P–P interval. Atrial rate was reduced by both acetylcholine and vagal stimulation (Fig. 1C and D). To quantify the response to vagal stimulation we compared the atrial rate during the 10 s immediately before stimulation with the rate recorded during the final 4 s of stimulation. To quantify the response to ACh we compared the atrial rate during the 10 s immediately before application of the drug with the lowest rate achieved during the next 30 s. The record presented in Fig. 1B shows a typical response to ACh. Initially, atrial rate fell, and this was accompanied by third degree atrioventricular (AV) block.
Effects of tertiapin-Q on responses to vagal stimulation and acetylcholine
In control conditions, vagal stimulation reduced atrial rate in a frequency-dependent manner (Figs 2A; P < 0.0001). In these preparations, subsequent exposure to tertiapin-Q (300 nM and 1 µM) had little or no significant effect on baseline values of either atrial rate or coronary flow (179 ± 14, 179 ± 13 and 172 ± 13 beats min–1*, and 2.4 ± 0.9, 2.5 ± 0.7 and 2.3 ± 0.7 ml min–1 in control conditions, tertiapin-Q and after washout, respectively; n = 10; * significantly lower than in control and tertiapin-Q, P < 0.005). The responses to both vagal stimulation and ACh were attenuated by tertiapin-Q. Exposure to 300 nM (n = 4) and 1 µM tertiapin-Q (n = 6) caused similar attenuation of the responses to vagal stimulation (P =0.7) and ACh (P =0.94). Consequently, data from preparations treated with either 300 nM or 1 µM tertiapin-Q were pooled (Fig. 2). We also calculated the ratio of the responses obtained in the presence and absence of tertiapin-Q. There was no significant difference in this ratio at the three different frequencies of vagal stimulation (mean ratio = 0.42 ± 0.12). Tertiapin-Q attenuated the peak response to ACh more than it attenuated the responses to vagal stimulation (mean ratio = 0.24 ± 0.24; P = 0.006).
Tertiapin-Q altered the form as well as the magnitude of the responses to both vagal stimulation and ACh. Much of the rapid response seen on application and termination of vagal stimulation, and the oscillatory response often seen during vagal stimulation at 2 Hz, were eliminated by tertiapin-Q (Fig. 3A). In the presence of tertiapin-Q, it took longer for heart rate to recover to baseline when vagal stimulation was terminated. Although the application of ACh did not always produce a rapid initial response in atrial rate, addition of tertiapin-Q usually removed most of any initial abrupt response (Fig. 3B).
In the preparation shown in Fig. 3, the responses recovered fully after a 60 min period of washout. Complete restoration of control responses was not always observed, particularly after 1 µM tertiapin-Q. For the pooled data, the response obtained after washout expressed as a fraction of the control response was between 76 and 82% for vagal stimulation (P <0.001), with no significant influence of stimulation frequency on the extent of recovery, and 75% for ACh (P <0.0001; Fig. 3A and B). Under control conditions, ACh induced a brief period of third degree AV block. This response was abolished by 300 nM and 1 µM tertiapin-Q, and returned after washout.
We examined the time course of responses to 10 s trains of vagal stimulation at 2 and 5 Hz. Figure 4 shows the effects of tertiapin-Q on the responses recorded at 1, 2 and 10 s of a 10 s train of stimulation. The decrease in atrial rate produced by vagal stimulation has been expressed in two ways: (i) as a fraction of baseline atrial rate (Fig. 4A); and (ii) as a fraction of the change in rate established by the end of the stimulus train (Fig. 4B). Figure 4B depicts clearly the effect of tertiapin-Q on the speed of the response to vagal stimulation. In addition to reducing substantially the size of the steady-state response to vagal stimulation, block of IK,ACh by tertiapin-Q also resulted in a more slowly evolving response (P =0.03 and 0.0004, for 2 and 5 Hz, respectively; n = 8).
In three preparations, following the tests performed after washout of tertiapin-Q, we examined the effect of atropine on the heart rate responses to vagal stimulation and ACh. Atropine (10–6 M) virtually abolished the responses to 5 Hz vagal stimulation and 7 nmol ACh (reduced to 2.0 ± 0.7 and 0.8 ± 0.7%, respectively, of the values recorded immediately before exposure to atropine; P <0.0001 for each test).
Effects of tertiapin-Q on responses to vagal stimulation, ACh and bethanecol in the presence of 2 mM Cs+
The substantial responses to vagal stimulation and ACh that remained after IK,ACh had been blocked by tertiapin-Q indicated that additional currents were involved in the responses to these stimuli. To examine this further, a second series of experiments were performed in Krebs–Henseleit solution containing 2 mM Cs+, which blocks the pacemaker current, If (DiFrancesco, 1985). In 2 mM Cs+, preparations settled to a baseline atrial rate 60% of the value recorded in its absence (111 ± 8 versus 179 ± 14 beats min–1; n
= 9 for both groups; P < 0.0001). In perfusate containing 2 mM Cs+, vagal stimulation produced responses that were frequency dependent and that were attenuated but not abolished by 300 nM tertiapin-Q (P
=0.001; Fig. 5A). The ratio of test and control responses to vagal stimulation was not frequency dependent (mean ratio = 0.47 ± 0.14; n
= 15 observations; P
= 0.59). In 2 mM Cs+, the response to ACh was strongly but not completely blocked by tertiapin-Q (Fig. 5B; P
= 0.001). Washout fully restored responses to both vagal stimulation and ACh. Tertiapin-Q attenuated the peak response to ACh more than it attenuated the responses to vagal stimulation (P
=0.003).
The time course of the responses to vagal stimulation were examined (Fig. 6). Bradycardias took longer to develop fully in tertiapin-Q. During 10 s trains of 2 Hz stimulation, the presence of tertiapin-Q resulted in responses that developed at substantially slower rates after 1 and 2 s of stimulation (P =0.02 and 0.002, respectively). Similar results were obtained with 5 Hz trains (P =0.07 and 0.04, respectively).
We also examined the effect of blocking If with 2 mM Cs+ on the time course of responses to vagal stimulation at 2 and 5 Hz before (Fig. 7A) and during (Fig. 7B) block of IK,ACh with tertiapin-Q. Removing If increased the speed of the response in both conditions. In the absence of tertiapin-Q, preparations exposed to 2 mM Cs+ showed a faster response to vagal stimulation at both 2 and 5 Hz (P <0.0001 at both frequencies). The difference in speed of response was also evident at 2 and 5 Hz stimulation during block of IK,ACh (P <0.0001 at both stimulation frequencies).
Responses to vagal stimulation and ACh were larger when 2 mM Cs+ was present in the perfusate (Figs 2 and 5), and the response to vagal stimulation at 10 Hz was substantially different (82 ± 15 versus 56 ± 14% in 2 mM Cs+ and standard solutions, respectively; n = 8 and 9; P = 0.002).
Applied acetylcholine may produce its effects on heart rate by binding both to muscarinic receptors on sino-atrial cells and to nicotinic receptors on postganglionic parasympathetic neurones. To see whether the reduction of responses to ACh produced by tertiapin-Q was attributable primarily to a direct effect on muscarinic receptors in the sino-atrial node, we performed a set of experiments (n = 4) in which we recorded the heart rate responses to bolus application of bethanecol, a selective muscarinic agonist (Benavides-Haro et al. 2003). The perfusate contained 2 mM Cs+. Tertiapin-Q (300 nM) antagonized the graded responses to nerve stimulation (P =0.073) and bethanecol (P <0.0001; Fig. 8B and C) to a similar degree to that seen previously. Antagonism of the response did not depend on the concentration of bethanecol (mean ratio = 0.36 ± 0.21; n = 16 observations; P = 0.92). Responses to bethanecol were prolonged, and the effects of sequential bolus administrations were additive. Figure 8A illustrates the responses in one preparation. In the other preparations, responses to bethanecol recovered incompletely after washout, although the responses to vagal stimulation recovered fully (Fig. 8B and C).
Effect of tertiapin-Q on the response of atrial rate to stimulation of the right cardiac sympathetic nerves
We considered the possibility that tertiapin-Q may antagonize the heart rate response to vagal stimulation partly through an effect somewhere along the parasympathetic neuronal pathway. To investigate this further, in three preparations we examined the heart rate responses to electrical stimulation of the right thoracic sympathetic chain. Heart rate responses to 10 s trains of sympathetic nerve stimulation at 5 Hz were not altered by the presence of 300 nM tertiapin-Q (P <0.001).
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Originally, tertiapin was considered to be a ‘specific’ blocker of IK,ACh, and current research articles often cite the use of tertiapin because it possesses this specific action. Tertiapin, however, can block additional potassium channels, including a myocardial hyperpolarization-activated time-dependent potassium channel (Ehrlich et al. 2004), a human Ca2+-activated large-conductance K+ channel, and mouse inwardly rectifying Kir3.1 and Kir3.2 heteromultimeric K+ channels (Kanjhan et al. 2005). We consider it unlikely that these other potassium channels play a significant role in sinoatrial pacemaking in the guinea-pig (there was no change in atrial rate after application of tertiapin-Q), or in the muscarinic modulation of pacemaking.
The participation of IK,ACh in vagal slowing
The influence of tertiapin-Q on the responses to vagal stimulation did not depend on stimulation frequency. In isolated guinea-pig atria, we have recorded a similar effect of tertiapin-Q on vagal slowing produced by stimulus trains at 1 Hz (C. P. Bolter and M.J. Turner, unpublished observations). Our results are not predicted by a recent model of parasympathetic control of SAN pacemaker (Demir et al. 1999) that incorporated observations by Hirst and colleagues (Campbell et al. 1989; Bywater et al. 1989, 1990; Edwards et al. 1993; Bramich et al. 1994; reviewed by Hirst et al. 1996). This model predicts that at lower frequencies of vagal stimulation, a bradycardia is produced by a reduction of inward current through channels coupled to junctional muscarinic receptors, and that at lower frequencies, ACh is restricted to acting at the junction (i.e. adjacent to a varicosity) because of its infrequent release and a high rate of hydrolysis. In this model, higher frequencies of vagal stimulation must occur for ACh to escape the junction and act on extrajunctional receptors, including those coupled to IK,ACh (Hirst et al. 1996; Demir et al. 1999). Our results suggest that either a significant number of junctional receptors are coupled to IK,ACh or that, even with 2 Hz stimulation, a significant amount of ACh escapes hydrolysis and leaves the junction to bind with extrajunctional receptors.
Release of ACh from an individual varicosity following the passage of an action potential is likely to be intermittent and infrequent (Brock & Cunnane, 1993), and its effect brief (Creed & McDonald, 1975; Tai et al. 2003). The response to a 10 Hz stimulus train would probably result from ACh release at many different varicosities over 10 s, with a low chance that any particular varicosity would be activated repetitively such that ACh could accumulate and escape. The summation of inhibitory junctional potentials (IJPs) that would be seen upon repeated stimulation at the frequencies we applied (2–10 Hz; Bolter et al. 2001) are likely to be a result of the spatial summation of dispersed individual IJPs that depends upon the syncytial properties of the pacemaker (space constant 200–800 µM; Boyett et al. 2000), rather than upon temporal summation of IJPs at individual varicosities.
The time course of the responses to vagal stimulation and the influence of tertiapin-Q provide further evidence against a functionally selective action of vagally released ACh on junctional receptors. Tertiapin-Q suppresses a greater fraction of the response seen early in the stimulus train than at steady state (Fig. 4). Tertiapin-Q abolished about 80% of the response that had evolved after 2 s of stimulation (4 stimuli at 2 Hz or 10 at 5 Hz). This is a similar level of antagonism to that seen against the immediate responses to 3 nmol ACh. Our data show that, for a brief or small stimulus (4 or 10 vagal stimuli over 2 s, or 3 nmol ACh) a greater part of the bradycardia produced was abolished by tertiapin-Q. There was less antagonism of the steady-state responses to vagal stimulation. Under physiological conditions, action potentials arrive at parasympathetic varicosities in groups or bursts, rather than continuously or randomly (Koizumi et al. 1985). The normal pattern of cardiac parasympathetic activity may result in a dynamic control of heart rate by the vagus that depends, predominantly, upon the activation of IK,ACh.
The participation of If in vagal slowing
Tertiapin-Q antagonized the effects of vagal stimulation and ACh to a similar extent, regardless of whether 2 mM Cs+ was present in the perfusate. A simple conclusion can be drawn, that in this preparation If played little or no role in vagal slowing or in the pacemaker response to applied ACh. Whether a reduction in basal If contributes to vagal slowing continues to be debated. Disagreement about the role of If in vagal slowing originates with studies on single pacemaker cells (DiFrancesco, 1985; DiFrancesco & Tromba, 1988; DiFrancesco et al. 1989). In isolated cells, a much lower concentration of ACh is required to reduce If than to activate IK,ACh (DiFrancesco et al. 1989), leading to the conclusion that If should be the major player in vagal slowing of the in situ pacemaker. In our experiments, the time course of responses to vagal stimulation suggests that If may be operating to oppose the responses. Muscarinic stimulation shifts the activation potential for If to a more negative value, and this is thought to occur more slowly than muscarinic activation of IK,ACh (Wallick et al. 1997; Renaudon et al. 1997; Accili et al. 1998). We observed that with 5 Hz stimulation, an initial rapid reduction in atrial rate was sometimes followed by a brief acceleration before the rate declined further (Fig. 3A). A brief secondary acceleration is a well-established phenomenon (Wallick et al. 1997; Demir et al. 1999) and is probably a result of recruitment of If by an initial hyperpolarization (activation of IK,ACh), before muscarinic stimulation shifts the threshold for activation of If to a more negative potential.
Analysis of the development of bradycardia during the 10 s stimulus trains adds support to the idea that shift of the activation potential of If to more negative values is slow. Removal of If was accompanied by a faster response to vagal stimulation (Fig. 7). The smaller and more slowly evolving response that remained after IK,ACh was blocked was still accelerated by the removal of If. This suggests that for brief bursts of vagal activity, If restrains and reduces the efficacy of vagal stimulation on heart rate reduction, rather than being modulated in a way that contributes to slowing.
Other currents
When both IK,ACh and If had been blocked, we still observed substantial steady-state responses to vagal stimulation and muscarinic agonists. Responses to both the onset and the termination of vagal stimulation were much slower when IK,ACh was blocked. The altered kinetics of the off-response in tertiapin-Q suggests that block of IK,ACh may allow the modulation or effect of a current that exerts no influence during normal vagal stimulation. While the rapid activation of IK,ACh at the onset of stimulation could mask the effect of a more slowly responding current, it is difficult to see how the rapid decay of IK,ACh at the termination of stimulation could mask the slower offset of such a current. Our experiments could not identify the residual mechanism(s); ICa,L, Ist, the Na+–K+ pump current (INaK) and the background Na+ current (IB,Na) are all potential candidates for additional currents that may be modulated by vagal stimulation (Demir et al. 1999).
Conclusions and perspective
Our results indicate that IK,ACh is a major and possibly the predominant current responsible for the bradycardia obtained in response to trains of vagal stimulation at 2–10 Hz. This conclusion is supported by evidence from a study on gene knockout mice. Using two simple and indirect estimates of vagal control of heart rate in conscious wild-type mice and in mice with the G-protein - activated, inwardly rectifying K+ channel 4 knocked out, Wickman and co-workers concluded that approximately 50% of vagal control was derived through activation of IK,ACh (Wickman et al. 1998).
While changes in If appear to occur during vagal stimulation, our results do not provide evidence that modulation of If contributes substantially to vagal bradycardia. Rather, when both If and IK,ACh were blocked, we saw evidence of an additional slow component. We suggest that an experimental design which uses vagal stimulation delivered in a more typical physiological pattern may help to further unravel the roles played by the currents in response to neurally released ACh.
Our conclusions are based upon the final output (an action potential conducted through the atria) of a heterogeneous group of cells that comprise the SAN pacemaker complex (Kodama et al. 1996). It is probably inappropriate to analyse the effects of nerve stimulation or muscarinic agonists as if they resulted from the homogeneous population of cells with properties described in reductionist studies. The conclusions of Hirst and co-workers are based on electrophysiological recordings made on individual cells located within the pacemaker complex (primary pacemaker cells were identified from their electrical characteristics in the control conditions; Bolter et al. 2001; Campbell et al. 1989). It is likely that adjacent cells within the complex will demonstrate similar membrane potentials and respond with similar changes in membrane potential to muscarinic stimulation. However, more distant cells may possess quite different properties and may behave very differently. Furthermore, the space constant in the node is small (200–800 µM; Bouman et al. 1989), allowing large variations in membrane potential over short distances. Nodal heterogeneity is a result of the varied spatial distribution of several features (Boyett et al. 2000): the channels involved in pacemaking, the parasympathetic innervation (Roberts et al. 1989), muscarinic receptors (Beau et al. 1995) and the channels that are modified by muscarinic stimulation (Boyett et al. 2000). In addition, the connectivity of cells within the node, which is determined by the distribution of gap junctions (Boyett et al. 2006), also determines the signal that exits the node to depolarize the adjacent atrial myocardium. A recent study that applied optical mapping to identify the origin and route of the cardiac potential within the SAN of the rabbit illustrates how this complexity may give rise to conflicting interpretations on the role of IK,ACh in vagal slowing (Fedorov et al. 2006). In these experiments, large differences in the nature of the membrane potentials from cells within the SAN complex were observed. Brief periods of vagal stimulation (0.2–2.0 s at 200 Hz) produced considerable alterations in these potentials (including substantial hyperpolarization). The origin of the leading pacemaker was shifted, and the sequence of depolarization within the node complex was altered (Fedorov et al. 2006).
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Acknowledgements |
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Author's present address
D. J. English: Medical Officers Unit, Southland District Health Board, PO Box 828, Invercargill, New Zealand.
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), tertiapin-Q (
) and washout conditions (
). Tertiapin-Q produced a substantial but incomplete antagonism of the responses to all frequencies of vagal stimulation and to ACh administration.
