Failure of Ba2+ and Cs+ to block the effects of vagal nerve stimulation in sinoatrial node cells of the guinea-pig heart

Autonomic Neuroscience: Basic and Clinical 94 Ž2001. 93–101 www.elsevier.comrlocaterautneu

Failure of Ba2q and Csq to block the effects of vagal nerve stimulation in sinoatrial node cells of the guinea-pig heart C.P. Bolter a , D.J. Wallace b, G.D.S. Hirst b,) a

b

Department of Physiology, UniÕersity of Otago, Dunedin, New Zealand Department of Zoology, UniÕersity of Melbourne, ParkÕille, Melbourne, Victoria 3052, Australia

Received 28 May 2001; received in revised form 20 September 2001; accepted 26 September 2001

Abstract The aim of the study was to evaluate which ionic currents are modified in the sinoatrial node of guinea pigs when the vagus is stimulated. Responses of isolated atrial preparations to bilateral vagus nerve stimulation were examined. In bath-mounted preparations, 10-s trains of vagal stimulation Ž1–50 Hz. slowed the rate at which atrial contractions occurred. After the trains of stimuli, the force generated by each contraction was reduced. Both vago-inhibitory responses persisted in the presence of caesium Ž2 mM. and barium ions Ž1 mM.. Vagal stimulation evoked a similar bradycardia in superperfused preparations in which intracellular recordings were made from pacemaker cells in the sinoatrial node. When pacemaking activity was abolished by adding the organic calcium channel antagonist nifedipine Ž1 mM. to the perfusate, vagal stimulation generated an inhibitory junction potential ŽIJP.. Both the bradycardia and the amplitude of the inhibitory junction potential increased as the frequency of vagal stimulation was increased. The ability of vagal stimulation to produce inhibitory junction potentials was unaffected by the addition of caesium and barium ions to the perfusate. These observations suggest that the negative chronotropic and inotropic responses to vagal stimulation only minimally involve a muscarinically activated potassium current Ž I KACh . or changes in the hyperpolarization-activated pacemaker current I h . q 2001 Elsevier Science B.V. All rights reserved. Keywords: Cardiac pacemaker cells; Parasympathetic nerve; Bradycardia

1. Introduction Cardiac pacemaker cells generate an ongoing discharge of pacemaker action potentials, which involves the interaction of several voltage-dependent and voltage-independent ion channels. In addition to the ion channels involved in pacemaker activity, cardiac pacemaker cells contain a set of barium ŽBa2q .-sensitive inwardly rectifying Kq channels ŽSakmann et al., 1983; Momose et al., 1984.. These are activated by added acetylcholine ŽACh. to produce an outwardly directed potassium current Ž I KA Ch ; Momose et al., 1984.. These channels are usually thought to be activated by neurally released ACh ŽLoffelholz and Pappano, 1985.. However, in both amphibian and mammalian cardiac pacemaker cells, although both vagal stimulation and added ACh activate muscarinic receptors to slow the heart rate, vagal stimulation does not appear to activate I KA Ch . )

Corresponding author. E-mail address: [email protected] ŽG.D.S. Hirst..

In both amphibian and mammalian hearts, when pacemaking activity is abolished by adding an organic Ca2q channel antagonist to the physiological saline, the membrane potential of pacemaker cells stabilises at a potential positive of the peak diastolic potential recorded from the beating heart ŽJalife et al., 1980; Bywater et al., 1990.. In arrested preparations, vagal stimulation and added ACh each produces hyperpolarization and, again, muscarinic receptor antagonists abolish both responses ŽJalife et al., 1980; Bywater et al., 1990; Bramich et al., 1994.. In amphibian pacemaker cells, the inhibitory junction potentials ŽIJPs. produced by vagal stimulation persisted in the presence of sufficient Ba2q to block I KA Ch or sufficient caesium ions ŽCsq. to block I h ŽBywater et al., 1990; Bramich et al., 1994.. In contrast, the hyperpolarization produced by added ACh is completely blocked by Ba2q or Csq ŽBywater et al., 1990; Bramich et al., 1994.. These observations led to the suggestion that vagally released ACh slows pacemaker activity by blocking a background Naq current Ž I bNa ., whereas applied ACh activates a

1566-0702r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 Ž 0 1 . 0 0 3 5 5 - 1

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different set of receptors coupled to I KA Ch ŽBywater et al., 1990; Edwards et al., 1993.. It is not clear how vagally released ACh produces a bradycardia in mammalian pacemaker cells. When trains of high-frequency transmural stimuli were used to initiate IJPs, their amplitudes were reduced by adding sufficient Csq and Ba2q ŽBoyett et al., 1995.. This led to the suggestion that in the mammalian heart, the bradycardia caused by vagal stimulation involves I KA Ch ŽBoyett et al., 1995.. Additional support for the role of I KA Ch in the vagal control of heart rate comes from the finding that reflex vagal control of heart rate control is reduced in GIRK knock-out mice ŽWickman et al., 1998.. More recently, a number of candidate mechanisms for parasympathetic control of mammalian sinoatrial pacemaker activity have been reviewed and incorporated into a model that described pacemaker activity in mammalian hearts ŽDemir et al., 1999.. This analysis again suggested that the bradycardia associated with low-frequency vagal stimulation did not involve either I KA Ch or inhibition of I h , but could be accounted for by changes in I bNa . A similar analysis of pacemaker activity in amphibian heart also concluded that the effects of vagal stimulation on the amphibian cardiac pacemaker could be entirely accounted for by an effect on I bNa ŽEdwards et al., 1993.. The present study has examined the responses to vagal stimulation in the guinea-pig heart, with the aim of testing the hypothesis that vagal inhibition produced by moderate frequencies of stimulation is little changed after I KA Ch and I h are blocked. It was found that the bradycardia produced by vagal stimulation was little affected by the addition of Csq and Ba2q to the physiological saline. A preliminary account of this study has appeared ŽBolter and Hirst, 1998..

2. Materials and methods The procedures described have been approved by the animal experimentation ethics committee at the University of Melbourne. Guinea pigs of either sex, weight 150–200 g, were stunned and exsanguinated, and the heart and both vagus nerves isolated as described by Campbell et al. Ž1989.. Briefly, the cervical and thoracic regions were rapidly removed and transferred to a dissecting chamber filled with chilled, oxygenated physiological saline Žcomposition; mM.: NaCl, 120; NaHCO 3 , 25; NaH 2 PO4 , 1; KCl, 5; MgCl 2 , 2; CaCl 2 , 2.5; and glucose, 11; bubbled with 95% O 2 –5% CO 2 . The right and left vagus nerves were located as high in the neck as possible and dissected back to the heart. The heart and attached vagus nerves were then removed from the thorax. Preparations consisted of both left and right atria with the ventricles removed, and about 2 cm of vagus nerve on each side. Once isolated, the preparations were used for either tension or electrophysiological experiments as described below.

2.1. Tension experiments Preparations were suspended in a jacketed 50 ml organ bath filled with physiological saline solution maintained at 35 8C and continuously gassed with 95% O 2 –5% CO 2 . Solutions were exchanged by draining the bath and replacing it with fresh solution. Drugs were added directly to the bath where continuous oxygenation ensured mixing within the bath within 2–3 min. All drugs were allowed to equilibrate for 30 min before the final set of observations was made. The force and rate of atrial contractions were measured isometrically with a force transducer ŽHarvard Apparatus, South Natick, MA, USA.. Both vagus nerves were passed through a platinum ring electrode and stimulated at a range of frequencies Ž1–50 Hz. with impulses of 0.5-ms duration delivered from a Grass S-8800 stimulator ŽGrass Instruments, Quincy, MA, USA. isolated from earth. The recordings made were digitized and stored on computer for later analysis. 2.2. Electrophysiological experiments The dorsal myocardium of the right atrium was accessed through the atrioventricular aperture. To allow intracellular recordings to be made, an area of myocardium slightly inferior to the point of entry of the superior vena cava, and medial to the crista terminalis, was partially immobilised using 50-mm tungsten pins. The sinoatrial node was then located by impaling different sites within this area. On the basis of the shape of the diastolic depolarization and the maximum rate of rise ŽdVrdt max . of the cardiac action potentials, we divided cells into three groups: pacemaker cells, secondary pacemaker or driven cells and atrial cells. Pacemaker cells were characterised by the presence of a gradual diastolic depolarization leading smoothly to the upstroke of the cardiac action potential, and a maximum rate of rise for the action potential of less than 15 V sy1 . Secondary pacemaker cells were classified as being cells where the diastolic depolarization was interrupted by the upstroke of the action potential, and the action potential had a dVrdt max that lay in the range 15–60 V sy1 . Atrial cells were classified as being cells where the diastolic depolarization was essentially absent and each action potential, which was abruptly initiated, had a dVrdt max of greater than 60 V sy1 . Vagal bradycardia was induced by stimulation of both vagus nerves as described above. In some experiments, the generation of cardiac action potentials was prevented by adding nifedipine Ž1 mM. to the physiological saline. The membrane potential responses to vagal stimulation were recorded from these arrested preparations. Preparations were viewed with an inverted compound microscope, and intracellular recordings made using highresistance glass microelectrodes Ž90–150 M V . filled with 0.5 M KCl. Signals were amplified with an Axoclamp-2A amplifier ŽAxon Instruments, Foster City, CA., lowpass

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filtered Žcut-off frequency 3 kHz., digitized and stored on computer for later analysis. Throughout the experiment, preparations were constantly perfused with physiological saline solution warmed to 35 8C. When used, drugs were added to the perfusing saline solution at the appropriate concentration. Again, a period of 30 min was allowed for the drugs to equilibrate. Drugs used in this study were hyoscine hydrochloride, nifedipine, caesium chloride and barium chloride Žall from Sigma-Aldrich, Castle Hill, NSW Australia.. In all experiments involving nifedipine, solutions were protected from light. 2.3. Statistical analysis Group data are reported as a mean value " S.E.M. Statistical analysis was carried out using repeated measure ANOVA. Where appropriate, a paired t-test was applied to examine the differences between group means.

3. Results 3.1. General obserÕations Vagal stimulation with trains of stimuli, either 5 or 10 s duration, caused a bradycardia that increased in magnitude as the frequency of stimulation was increased in the range 2–20 Hz. An example is shown in Fig. 1 where it can be seen that an appreciable bradycardia was produced when the vagi were stimulated at 2 Hz ŽFig. 1B.. When trains of stimuli were applied every few minutes, the responses were reliably reproduced. When the frequency of stimulation was increased, the bradycardia became more pronounced and the onset more rapid ŽFig. 1B.. Invariably, it was found that when a train of stimuli was delivered at a sufficient frequency to reduce the rate of atrial contractions to half of its control rate, maximum slowing was recorded within 2 s Žsee Figs. 1 and 2.. During the period of stimulation, as the rate of atrial contractions fell, the force of each individual atrial contraction occasionally increased. After the end of the stimulation period, a negative inotropic response was invariably detected ŽFig. 1A.. In other preparations, at the start of vagal stimulation, the first few contractions were larger, but during the period of vagal stimulation, a negative inotropic response developed. Again, at the end of the stimulation period, as the rate of atrial contractions recovered, the force of individual contractions briefly decreased further Žsee, for example, Fig. 2A.. Regardless of the pattern of behaviour recorded, the responses to vagal stimulation were reproducible over a period of several hours. These observations indicate that in the guinea-pig heart, quite moderate frequencies of vagal stimulation produce a reliable bradycardia and negative inotropic response.

Fig. 1. The effect of bilateral vagal stimulation on contractions recorded from guinea-pig atria. Trace ŽA. shows the effect of bilateral vagal stimulation, Žhorizontal bar., 5 s at 5 Hz, on atrial contractions. During the stimulation train, the size of individual contractions increased, but after the end of the stimulation period, the force of atrial contraction was reduced. The lower trace ŽB. shows the time courses of bradycardia produced by trains of stimuli delivered at 2, 5 and 10 Hz. The horizontal calibration bar refers to all traces.

3.2. Effect of Cs q and Ba 2 q on the negatiÕe chronotropic and inotropic responses produced by Õagal stimulation The effects of adding Csq, followed by Ba2q, on the responses to vagal stimulation produced by isolated beating preparations of atria, were examined. In each experiment, we selected a frequency of vagal stimulation that, in control conditions, reduced the rate of atrial contractions by approximately half. The mean control atrial rate was 227.9 " 11.4 beats miny1 , and at end of a 5-s period of vagal stimulation, the mean rate was 125.7 " 15.5 beats miny1 Ž n s 7, where each n value was obtained from a separate preparation.. After ensuring that constant responses were obtained, Csq Ž2 mM. was added to the physiological saline. Trains of stimuli were then repeated at 10-min intervals until the rate of atrial contractions was constant and the responses to vagal nerve stimulation were reproducible. The addition of Csq reduced the control atrial rate from 227.9 " 11.4 to 189.3 " 8.3 beats miny1 , but the negative chronotropic response produced by vagal stimulation persisted ŽFig. 2B and D.; the atrial rate during vagal stimulation fell to 117.1 " 6.9 beats miny1 . Subsequently, Ba2q Ž1 mM. was added to the physiological saline. This further reduced the rate of contractions to 152.6 " 10.5 beats miny1 , and increased the amplitude of the individual atrial contractions ŽFig. 2C.. In the presence of both Ba2q and Csq, vagal stimulation continued to cause a bradycardia ŽFig. 2C and D., with atrial rate during

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the atrial rate from 151.4 " 13.0 to 76.2 " 12.7 beats miny1 . After the addition of hyoscine, the same trains of stimuli reduced atrial rate from 153.4 " 12.2 to 149.4 " 12.1 beats miny1 . Hyoscine had no significant effect on atrial rate Ž p s 0.5. and substantially reduced the ability of the vagus to slow the heart Ž p - 0.025.. In the presence of hyoscine, vagal stimulation failed to produce a detectable negative inotropic response. 3.3. Effect of Õagal stimulation on beating and arrested sinoatrial node cells

Fig. 2. The effect of Csq and Ba2q on the negative chronotropic and inotropic response produced in guinea-pig atria by vagal stimulation. Trace ŽA. shows the inhibitory chronotropic and inotropic responses produced by bilateral vagal stimulation, 10 s at 5 Hz, recorded in control solution. The same train of stimuli continued to slow the heart and weaken the force of atrial contraction after the addition of Csq Ž2 mM. to the physiological saline ŽB.. Although the further addition of Ba2q Ž1 mM. slowed the rate of generation of atrial contractions, vagal stimulation continued to produce negative chronotropic and inotropic responses ŽC.. The rate responses associated with these three responses are shown in trace ŽD.. The pooled data from seven experiments are shown in part ŽE.. The relationship illustrated with filled circles indicates the rate of atrial contraction in control, Csq-, and Csq plus Ba2q-containing solutions. It can be seen that the rate of atrial contractions was slowed following the addition of Csq, and further slowed by the subsequent addition of Ba2q. The open squares show the heart rate during vagal stimulation in control, Csq-, and Csq plus Ba2q-containing solutions. It can be seen that vagal stimulation slows the rate of atrial contractions in each solution. The horizontal calibration bar refers to traces A, B, C and D.

vagal stimulation falling to 96.4 " 11.1 beats miny1 . The mean responses are shown in Fig. 2E. The addition of first Csq, and then Ba2q, caused significant reductions in the rate of atrial contractions Ž p - 0.01 and p - 0.005, respectively., but vagal stimulation continued to produce a substantial bradycardia. As has been pointed out, periods of vagal stimulation were followed invariably by periods during which the force of individual atrial contractions was depressed. In each experiment, neither Csq nor Ba2q blocked the negative inotropic response Žsee Fig. 2A–C.. In five of these experiments, hyoscine Ž0.1 mM. was added to the physiological saline. Before addition of hyoscine, 5-s trains of stimuli delivered at 5 Hz reduced

Intracellular recordings were made from the endocardial surface of the right atrium in the region between the entrance points of the superior and inferior vena cavae, medial to the crista terminalis Žsee Materials and Methods for more complete description.. The sinoatrial node lay in this broad region, and could only be identified on electrophysiological criteria ŽSteele and Choate, 1994.. On the basis of the shape of the diastolic depolarization and the maximum rate of rise ŽdVrdt max . of the cardiac action potentials, cells were divided into three groups Žsee Materials and Methods.. Pacemaker cells had amplitudes, measured from the peak diastolic potential to the peak overshoot potential, of 86.4 " 1.3 mV Ž n s 8, where each n value was obtained from a separate animal.. These recordings had a peak diastolic potential of y63.5 " 0.8 mV, dVrdt max of 8.5 " 1.4 V sy1 and a mean rate of 202.3 " 16.2 beats miny1 . Secondary pacemaker cells Ž n s 12. had action potentials of amplitude 96.6 " 1.0 mV, peak diastolic potential of y68.8 " 1.6 mV, dVrdt max of 38.2 " 3.8 V sy1 and a mean rate of 191.5 " 10.9 beats miny1 . Atrial cells Ž n s 9. had action potentials of amplitude 98.8 " 1.9 mV, peak diastolic potentials of y75.2 " 0.9 mV, dVrdt max of 70.8 " 5.0 V sy1 and a mean rate of 182.6 " 12.7 beats miny1 . Vagal stimulation, 25 impulses at 5 Hz, slowed the rate of generation of action potentials recorded from pacemaker ŽFig. 3., secondary pacemaker and atrial cells ŽFig. 7.. In each cell type, slowing was associated with small changes in the peak diastolic potential. In the example shown in Fig. 3, the peak diastolic potential increased by about 4 mV. The largest increase in peak diastolic potential detected in this entire sequence of experiments is illustrated in Fig. 4A, where it can be seen that vagal stimulation caused the peak diastolic potential to become some 10–12 mV more negative. During vagal slowing, the shape of individual pacemaker action potentials was little changed ŽFig. 3B and C.. On two occasions during vagal slowing, the pacemaker cells stopped behaving like true pacemaker cells and acquired the characteristics of secondary pacemaker cells; the pacemaker action potential was initiated abruptly during the diastolic depolarization and dVrdt max increased, although in neither case did dVrdtmax exceed 15 V sy1 . These observations support the idea that a shift of the dominant pacemaker centre may follow changes in

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to the physiological saline. The pacemaker membrane potential settled at a value of about y35 mV Žmean s y33.7 " 1.3 mV., and vagal stimulation evoked an IJP ŽFig. 4C.. The amplitudes of the IJPs evoked by 5-s trains of stimuli, again delivered at 5, 10, 20 and 50 Hz, were determined. As seen with the bradycardia recorded from the beating preparation, IJPs increased in amplitude with increasing stimulation frequency. For each frequency of vagal stimulation, the change in rate of pacemaker action potentials recorded in the preparation before arrest was compared with the IJP in the arrested preparation ŽFig. 4D.. Changes in both variables were calculated from values obtained just before the end of the 5-s period of vagal stimulation. In sets of data from individual preparations, the relationship between the magnitude of the IJP and vagal bradycardia was well described by a linear relationship Žmean R 2 s 0.78, range 0.58–1.0, n s 7..

Fig. 3. The effect of vagal stimulation on pacemaker action potentials recorded from guinea-pig sinoatrial node. Trace ŽA. shows the response to 25 impulses delivered at 5 Hz. The time course of the associated bradycardia is shown in the rate plot below it ŽB.. Expansions of pacemaker action potentials recorded before Ža. and during the train of stimuli Žb. are shown in ŽC.. It can be seen that the peak diastolic potential is a few millivolts more negative and that the major change is restricted to the rate of diastolic depolarization.

activity in the cardiac autonomic nerves ŽBromberg et al., 1995.. Qualitatively similar observations were made from secondary pacemaker cells. In most secondary pacemaker cells, the rate of diastolic depolarization was slowed during and following a period of vagal stimulation, but the shape of the action potential was unchanged. On one occasion, a secondary pacemaker cell took on the characteristics of a pacemaker cell; during vagal stimulation, the diastolic depolarization led smoothly into the upstroke of the action potential. In each of the experiments where a pacemaker cell had been positively identified, the responses to 5-s trains of stimuli delivered at 5, 10, 20 and 50 Hz were obtained. Invariably, each train of stimuli caused a bradycardia, but often even the highest frequency of stimulation did not cause a complete cessation of pacemaking activity. On these occasions, action potentials of small amplitude Žabout 40 mV. and rapid time course Žhalf widths of less than 50 ms. occurred at low frequency. After a complete set of responses to the trains at each stimulation frequency had been obtained, the impalement was maintained and pacemaking activity was arrested by adding nifedipine Ž1 mM.

Fig. 4. Comparison between the responses to vagal stimulation recorded from the same cell in a beating, then arrested guinea-pig sinoatrial node. Traces ŽA. and ŽB. show the effect of vagal stimulation, 50 impulses at 10 Hz, and the associated bradycardia. The generation of pacemaker action potentials was prevented by adding nifedipine Ž1 mM. to the physiological saline, the membrane potential settled at y34 mV, and the same train of stimuli now evoked an IJP ŽC.. Part ŽD. shows the pooled data recorded from beating and arrested pacemaker cells. The relationships between stimulation frequency and decrease in atrial rate Žclosed circles., and between stimulation frequency and IJP amplitude Žopen circles. were very similar.

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3.4. Effect of Cs q and Ba 2 q on IJPs recorded from arrested sinoatrial node cells of the guinea-pig heart The previous observations indicate that the IJPs recorded from arrested sinoatrial node cells correlate with the ability of the vagus to produce a bradycardia. In the next series of experiments, the effects of Csq and Ba2q on the IJPs produced by 5-s trains of stimuli at 5, 10, 20 and 50 Hz, were examined. Part of the results from one experiment is shown in Fig. 5A–C. The upper trace shows that a train of stimuli delivered at 10 Hz evoked an IJP of about 7 mV ŽFig. 5A.. Following the addition of Csq Ž2 mM., the membrane potential was unchanged and the same train of stimuli evoked a similar IJP ŽFig. 5B.. The addition of Ba2q Ž1 mM. depolarized the membrane by about 10 mV Žfrom a mean value of y33.7 " 1.3 to y21.5 " 1.9 mV; n s 7.. The results from this group of experiments are illustrated graphically ŽFig. 5D.. It can be seen that the amplitudes of the responses measured at each frequency were unchanged by the addition of Csq to the physiological saline ŽFig. 5D.. The subsequent addition of Ba2q increased the amplitude of the IJPs measured at 5 and 10

Fig. 5. Persistence of IJPs in the presence of Csq and Ba2q. The three traces show the responses to vagal stimulation, 50 impulses at 10 Hz, recorded in the presence of nifedipine ŽA.; after the addition of Csq ŽB.; and in the presence of both Csq and Ba2q ŽC.. The calibration bars apply to all three traces. The pooled data from arrested pacemaker cells in these three different solutions are shown in ŽD.. Observations in nifedipine are shown as open circles, in the presence of Csq Ž2 mM. are shown as solid triangles, and in the presence of both Csq Ž2 mM. and Ba2q Ž1 mM. are shown as open squares.

Hz Ž p - 0.05 and p - 0.01 respectively; Fig. 5D., while not affecting the amplitude of IJPs measured at 20 and 50 Hz. IJPs were also recorded from each secondary pacemaker cell examined. As with the recordings from sinoatrial node cells, these signals were little changed by the addition of either Csq or Ba2q. The membrane potential of arrested secondary pacemaker cells Žmean membrane potential s y38.3 " 3.8 mV, n s 5. was unchanged by Csq Ž2 mM., and depolarized following the addition of Ba2q Ž1 mM. to y23.4 " 1.4 mV. Small IJPs could also be recorded from arrested atrial cells; these were also unchanged following the addition of Csq, but were potentiated by Ba2q. As well as increasing the amplitude of IJPs Žsee Fig. 7D., Ba2q depolarized the membrane potential of arrested atrial cells from their resting value of about y75 to y38.3 " 2.5 mV Ž n s 4.. 3.5. Some properties of IJPs recorded from sinoatrial node cells of the guinea-pig heart in Cs q and Ba 2 q The properties of the Csq- and Ba2q-resistant IJPs were further characterised. In each preparation, trains of vagal stimuli initiated a hyperpolarization. However, when a single stimulus was applied, IJPs were only detected in a proportion of preparations. IJPs, with amplitudes greater than 0.1 mV, were observed in 4 of the 7 preparations where recordings were made from sinoatrial node cells and in 3 of the 12 secondary pacemaker cells examined. Since it was clear that the responses from sinoatrial node cells and secondary pacemaker cells were identical, the data from these preparations have been combined. In this group of cells, IJPs initiated by a single stimulus had a peak amplitude in the range 0.2–1.3 mV Žmean s 0.6 " 0.2 mV, n s 7, Fig 6A.. The IJPs had a latency, defined as time between stimulus and 5% rise time, in the range 80–200 ms Žmean s 150 " 20 ms.; a rise time, defined as the time from 5% to 95% peak amplitude, in the range 100–240 ms Žmean s 170 " 20 ms.; a half-width, defined as time above 50% peak amplitude, in the range 340–540 ms Žmean s 380 " 30 ms.. During low-frequency stimulation, successive IJPs summed ŽFig. 6B.. As the frequency of stimulation was increased above 3 Hz, responses to individual stimuli could no longer be discriminated and smooth hyperpolarizations were detected ŽFig. 6B.. Such a pattern of behaviour would lead to the sustained bradycardia detected in beating preparations. 3.6. Effect of Õagal stimulation on action potentials recorded from atrial cells of the guinea-pig heart It has been pointed out that during a vagal bradycardia, the shape of individual pacemaker action potentials in sinoatrial node cells was little changed Žsee Fig. 3.. Similar

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observations were also made on secondary pacemaker cells. However, this was not the case when recordings were made from atrial cells. The half-widths of atrial action potentials were invariably reduced during both the periods of vagal stimulation and during the recovery phase ŽFig. 7A–C.. As the rate of generation of the pacemaker action potentials returned to the control value, the duration of the atrial action potential also returned towards its control value. The period when atrial action potential duration was reduced corresponded well with the period during which the force of atrial contraction was reduced Žsee Figs. 1 and 2.. After arresting the preparations with Ca2q antagonist, the membrane potential in atrial cells was little different from the diastolic potential previously recorded. Trains of vagal stimuli initiated IJPs that persisted in the presence of Csq and Ba2q. As pointed out above, the addition of Ba2q was followed by membrane depolarization and an increase in the amplitude of IJPs ŽFig. 7D.. Single stimuli evoked an IJP in three of nine atrial cells. Characteristics of these IJPs were: peak amplitude, 0.3 " 0.6 mV; latency, 170 " 10 ms; rise time, 280 " 40 ms; and half-width, 720 " 190 ms. The amplitudes of IJPs recorded

Fig. 7. Effect of vagal stimulation on action potentials recorded from atrial cells of the guinea-pig heart. Vagal stimulation slowed the rate of generation of atrial action potentials ŽA and B.. When the time courses of individual action potentials were examined, it became clear that during vagal inhibition, individual action potentials had a briefer duration ŽC.. After arresting the preparation with nifedipine ŽD., vagal stimulation evoked hyperpolarizations that persisted in the presence of Csq and Ba2q.

from atrial cells were significantly smaller than those recorded from the pacemaker region Ž p - 0.05..

4. Discussion

Fig. 6. Responses to vagal stimulation recorded from a guinea-pig sinoatrial cell in the presence of Csq and Ba2q. The IJP produced by low-frequency vagal stimulation Ž0.1 Hz. is shown in ŽA.; this trace is the average of eight successive sweeps. ŽB. As the frequency of stimulation was increased, the responses to individual stimuli could no longer be distinguished and the responses summed to give a smooth hyperpolarization.

These experiments have shown that moderate frequencies of vagal stimulation change the frequency and force of guinea-pig atrial muscle contractions. The responses to vagal stimulation persisted after Csq and Ba2q had been added to the physiological saline. However, both Csq and Ba2q caused changes in the rate of atrial contractions. A concentration of Csq, sufficient to block I h ŽDifrancesco and Tromba, 1988., slowed the atria by about 20% of their control rate. This observation suggests that I h makes a definite but limited contribution to pacemaking activity ŽIrisawa et al., 1993.. However, as vagal stimulation continued to slow the atria in the presence of Csq, a change in the activation potential of I h is unlikely to be the sole way in which neurally released ACh causes a bradycardia Žcf. Difrancesco et al., 1989.. The subsequent addition of Ba2q further slowed the rate of contractions, but vagal stimula-

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tion still evoked a substantial bradycardia. It seems unlikely that Ba2q failed to block the responses to vagal stimulation because an inadequate concentration was applied, since the concentration of Ba2q used was much greater than that generally found necessary to block I KA Ch in cardiac tissues ŽMomose et al., 1984; Bramich et al., 1994.. An alternative possibility, that Ba2q could not access channels at the junctions formed by vagal post-ganglionic axons ŽChoate et al., 1993; Hirst et al., 1996. seems unlikely, since muscarinic receptor antagonists readily blocked the responses to vagal stimulation. Together, our observations indicate that in the guinea-pig sinoatrial node, vagally released ACh activates muscarinic receptors distinct from those coupled to I KA Ch . To avoid the complications of interpreting data from preparations where the membrane potential continuously changes owing to the sequential activation of pacemaker currents, and where the baseline atrial rate is altered following the application of Csq and Ba2q, we examined the effects of Csq and Ba2q on IJPs recorded from arrested sinoatrial node cells. Firstly, we established that IJPs reflect the ability of the vagus to slow heart rate. Secondly, we found that neither the membrane potential nor the amplitudes of IJPs in arrested cells were affected when Csq was added to the physiological saline. These observations suggest that little or no I h flows in the arrested preparations, and confirm that IJPs do not result from changes in the activation potential of I h Žsee also Boyett et al., 1995.. The addition of Ba2q depolarized arrested atrial preparations. The depolarization was detected in sinoatrial cells, secondary pacemaker cells and, most markedly, in atrial cells. Presumably, the addition of Ba2q blocked cardiac inwardly rectifying Kq channels Ž I K1 . present in all cardiac cells other than pacemaker cells ŽIrisawa et al., 1993.. In the intact heart, the sinoatrial node is electrically coupled to the atrial cells. Blockade of I K1 will cause a membrane depolarization in the atria and this will be recorded in each cell type, even though I K1 is not generated in sinoatrial node cells Žsee Kodama et al., 1995.. After the addition of Ba2q, IJPs were detected in each cell type. In sinoatrial cells, Ba2q increased the amplitudes of IJPs when the stimulation frequency was 5 or 10 Hz, but did not change IJPs that were evoked with the higher frequency trains of stimuli. Presumably, this reflects the changed electrical properties of the sinoatrial noderatria syncytium resulting from blockade of atrial I K1 by Ba2q. Thus, these observations support the view that the responses to moderate frequencies of vagal stimulation, which produce substantial changes in heart rate, do not involve I KA Ch to any great extent ŽBywater et al., 1989.. If the responses to all frequencies of vagal stimulation involved only Ba2q-insensitive channels, we would have expected the amplitudes of all IJPs recorded from sinoatrial node and atrial cells to be increased by the addition of Ba2q because of the altered electrical properties of the

atrial syncytium. However, the IJPs produced by higher frequency vagal stimulation were unchanged; presumably, this reflects the presence of a Ba2q-sensitive component in these responses Žsee also Boyett et al., 1995.. In summary, IJPs produced at low frequencies of vagal stimulation do not involve either activation of I KA Ch or modification of I h . Presumably, under these conditions, neurally released ACh activates a pathway that reduces I bNa ŽEdwards et al., 1993; Demir et al., 1999.. However, at higher frequencies of vagal stimulation, vagally released ACh may also activate I KA Ch ŽBoyett et al., 1995; Demir et al., 1999.. Clearly, the difference between our conclusions and those based on data obtained from rabbit sinoatrial node cells, which suggest that activation of I KA Ch is of prime importance Žsee Boyett et al., 1995., reflects either species variation or the different experimental approaches taken. In the studies on the rabbit, only high frequencies of stimulation were used to trigger IJPs, and under these conditions, the contribution of I KA Ch may be more marked. Indeed, the recent analysis of vagal effects in the heart predicts that this would be the case ŽDemir et al., 1999.. Furthermore, the finding that reflex vagal responses in mutant mice, devoid of the GIRK-4 subunit, are modified, may indicate that during many physiological responses, bursts of high-frequency vagal traffic occur ŽWickman et al., 1998.. During the negative inotropic responses produced by vagal stimulation, the duration of atrial action potentials is shortened ŽKodama et al., 1995; Fig. 7.; presumably, this reflects a reduced inflow of Ca2q leading to a reduced contraction. A simple explanation for this might be that, in atrial muscle, neurally released ACh activates the cardiac muscarinic receptors that are linked to Kq channels, and the resulting outward current shortens the duration of individual atrial action potentials. This would reduce the time that voltage-dependent calcium channels are open. However, it seems unlikely that this is occurring; negative inotropic responses continued to occur after the addition of Ba2q Žsee Fig. 2C.. Another way by which vagally released ACh could shorten the duration of atrial action potentials would be through an alteration of the opening characteristics of delayed rectifier Kq channels, but this has never been reported. Alternatively, vagally released ACh could alter the functioning of L-type Ca2q channels; this has been shown to occur, but only after b-adrenoceptor stimulation ŽJalife and Moe, 1979.. In our experiments, only the vagi were stimulated; clearly, the preparations were not being concurrently activated by catecholamines. Furthermore, similar observations have been made while recording from rabbit atrial cells in the presence of badrenoceptor blockers ŽKodama et al., 1995.. In summary, we have no explanation as to how neurally released ACh causes a negative inotropic response in guinea-pig atria, except to note that it does not appear to involve I KA Ch . This study has shown that in the guinea-pig heart, the inhibitory chronotropic and inotropic responses produced

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by moderate frequencies of vagal stimulation are little changed when I h and I KACh are blocked. These observations on the negative chronotropic response are very similar to those made on amphibian hearts, and suggest that in the guinea-pig heart, neurally released ACh activates a population of muscarinic receptors coupled to I bNa .

Acknowledgements The project was supported by a research grant from the Australian NH and MRC whom we thank for financial support.

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