Sympathetic and vagal interaction in the control of cardiac pacemaker rhythm in the guinea-pig heart: Importance of expressing heart rhythm using an appropriate metric.

There are many reports that, through pre- and post-junctional mechanisms, sympathetic and parasympathetic (vagal) nerves can interact in the control of heart rate. The predominant interaction is accentuated antagonism (AA), where the bradycardia produced by vagal stimulation (VNS) is amplified when heart rate has been increased by sympathetic stimulation (SNS) or beta-adrenergic agonists. The acetylcholine-activated potassium current (IK,Ach), is the primary driver of vagal bradycardia. To examine the participation of IK,Ach in AA, a series of experiments was performed on isolated, double innervated, guinea-pig atrial preparations. Vagal bradycardia was elicited by 10-s trains (1, 2, 5 and 7.5 Hz) or single bursts of VNS (3 stimuli at 50 Hz) before and during acceleration of HR by either SNS (1-3 Hz) or isoprenaline (ISO), in both absence and presence of tertiapin-Q (TQ-IK,Ach blocker). When expressed as an absolute change in HR (beats/min), bradycardia produced by VNS trains was amplified (AA) at all frequencies of VNS in ISO, and at 5 and 7.5 Hz during SNS. Bradycardia in response to 1 and 2 Hz VNS was reduced during SNS. In TQ, only the bradycardia produced by 5 and 7.5 Hz VNS in ISO was amplified. The bradycardia produced by a single burst of VNS was amplified in both ISO and SNS. After TQ the bradycardia in response to a VNS burst was unchanged in ISO, while it was reduced during SNS. When these data were adjusted to account for the increase in baseline HR brought about by SNS and ISO, there was no longer evidence of AA. Diminished responses to low frequencies of VNS (1 and 2 Hz) persisted, and were also seen during IK,Ach block by TQ. We applied the same adjustment to data from 20 published studies. In 8 studies all data indicated AA; 3 studies provided no evidence for AA, and in 9 studies evidence was mixed. There is no doubt that AA can occur in the control of heart rhythm during simultaneous SNS and VNS, but conditions which determine its occurrence, and the mechanisms involved in this interaction remain unclear.

Effects of tertiapin-Q and ZD7288 on changes in sinoatrial pacemaker rhythm during vagal stimulation.

Heart rate slowing produced by cardiac parasympathetic (vagal) stimulation is thought to be the result of modulation of the acetylcholine-activated K(+) current (IK,ACh) and the pacemaker current (If) in sinoatrial (SAN) pacemaker cells. However, the contribution of these and other ion currents to vagal slowing is controversial. Here, we examined the contributions of IK,ACh and If to vagal slowing in 15 isolated, vagal-innervated preparations of guinea-pig atria, using 300 nM tertiapin-Q (TQ) and 2 µM ZD7288 to obtain full and substantial block of these currents, respectively. Blocking IK,ACh alone reduced atrial rate responses to 10-s trains of regular vagal stimulation (supramaximal stimulation, 2-ms duration, 1-10 Hz) by ~50% (P<0.01; N=11); blocking If alone had no effect (N=7). Blocking both IK,ACh and If produced ~90% reduction (P<0.01; N=4). Atrial cycle length response to a single burst of vagal stimuli (3 stimuli at 50 Hz), delivered at the optimum phase of the cycle was strongly suppressed by blocking IK,ACh (reduced by 98%; P<0.01; N=9), and modestly reduced by blocking If alone (by ~43%; P=0.20; N=6). The response was abolished by combined block of IK,ACh and If (P=0.04; N=4). Our data show that modulation of IK,ACh and If is sufficient to account for all the vagal slowing observed in this preparation. The vagally-induced negative shift in activation potential for If will be opposed by hyperpolarisation of SAN through activation of IK,ACh. Thus removal of IK,ACh by TQ may have exaggerated the overall contribution of If to vagal slowing.

The muscarinic-activated potassium channel always participates in vagal slowing of the guinea-pig sinoatrial pacemaker.

UNLABELLED: Controversy persists regarding participation of the muscarinic-activated potassium current (c(KACh)) in small and moderate vagal bradycardia. We investigated this by (i) critical examination of earlier experimental data for mechanisms proposed to operate in modest vagal bradycardia (modulation of I(f) and inhibition of a junctional Na(+) current) and (ii) experiments performed on isolated vagally-innervated guinea-pig atria. In 8 superperfused preparations, 10-s trains of vagal stimulation (1 to 20Hz) produced a bradycardia that ranged from 1 to 80%. Hyperpolarisation of sinoatrial cells accompanied bradycardia in 65/67 observations (linear correlation between bradycardia and increase in maximum diastolic potential (mV)=0.076x%; R(2)=0.57; P<0.001). In bath-mounted preparations single supramaximal stimuli to the vagus immediately and briefly increased pacemaker cycle length in 7 of 18 preparations. This response was eliminated by 300nM tertiapin-Q. Trains of 10 single supramaximal vagal stimuli applied at 1-s intervals caused progressive increase in overall cycle length during the train; immediate and brief increases in cycle length occurred following some stimuli. Immediate brief responses and part of the slower response to the stimulus train were removed by 300nM tertiapin-Q. SUMMARY: experimental data shows that small and modest vagal bradycardia is accompanied by hyperpolarisation of the pacemaker cell which is severely attenuated by tertiapin-Q. These observations support the idea that activation of I(KACh) occurs at all levels of vagal bradycardia. Contradictory conclusions from earlier studies may be attributed to the nature of experimental models and experimental design.

The relationship between aortic baroreceptor activity and arterial pressure is not monotonic.

Previous reports indicate that when aortic pressure (AP) falls below the threshold (P (th)) for baroreceptor sensitivity, activity in the aortic depressor nerve (ADN) may increase. To quantify and explain this anomalous behaviour, we analysed curves describing the relationship of baroreceptor fibre activity in rabbit left ADN to AP. Data were obtained in anaesthetised New Zealand White rabbits. Occlusion and release of cuffs around the inferior vena cava and descending aorta generated AP ramps (25-140 mmHg). Response curves were obtained for 173 fibres in 26 animals. Thirty percent of curves had a nadir (J-shaped curve), and in 40% activity was always present. In fibres showing activity below P (th), firing was predominantly diastolic, switching to systolic firing at P (th). The unusual behaviour of a substantial fraction of aortic baroreceptors below P (th) accounts for the J-shaped response curve of the whole ADN. We suggest that fibres that fire during diastole at pressures below P (th) may have sensory endings close to the origin of the left subclavian artery. As a consequence of this anatomical location, low pressures can impose strain on these receptors, which is then relieved by the systolic pulse.

Tertiapin-Q removes a mechanosensitive component of muscarinic control of the sinoatrial pacemaker in the rat.

1. In an isolated right atrial preparation, an increase in right atrial pressure (RAP) produces an increase in atrial rate. This rate response is larger and occurs faster when there is background vagal or muscarinic stimulation. 2. We hypothesized that in the latter situation, an increase in RAP antagonizes the effect of muscarinic stimulation through stretch inactivation of the mechanosensitive muscarinic potassium current I(K,ACh). 3. In two groups of bath-mounted right atria isolated from male Wistar rats (control n = 12; 300 nmol/L tertiapin-Q treated (to block I(K,ACh)) n = 10), we examined the change in atrial rate when RAP was raised from 2 to 8 mmHg; oxotremorine-M (oxo-M; from 10 to 500 nmol/L) was added to incrementally activate muscarinic receptors. 4. In both control and tertiapin-Q-treated groups, oxo-M reduced atrial rate, but its effect was less ( approximately 40-50%) in the latter group (P < 0.001). In control preparations, responses to an increase in RAP became progressively larger and quicker as the concentration of oxo-M was increased, whereas in tertiapin-Q treated preparations oxo-M did not affect either the amplitude or the speed of the response (P < 0.0001 for both). 5. The results support the hypothesis that atrial stretch antagonizes muscarinic slowing by its effect on I(K,ACh). We suggest that through this mechanism, parasympathetic control of heart rate may be modulated continuously by RAP.

Tertiapin-Q removes a large and rapidly acting component of vagal slowing of the guinea-pig cardiac pacemaker.

The participation of acetylcholine-activated potassium current (I(K,ACh)) and hyperpolarization-activated pacemaker current (I(f)) in vagal bradycardia were examined using vagally-innervated preparations of guinea-pig atria. Preparations were maintained in Krebs-Henseleit solution (36 degrees C). Before treatment, trains of vagal stimuli (10 s at 2, 5 and 10 Hz) produced graded bradycardias displaying rapid onset and offset. Tertiapin-Q (300 nM), which blocks I(K,ACh), had no effect on baseline atrial rate. In tertiapin-Q, vagal bradycardia displayed a gradual onset and offset, with a peak response ~50% of that recorded in control conditions. Cumulative addition of 1 mM ZD7288 (blocker of I(f)) caused atrial rate to fall by ~60%, but had no further effect on the amplitude of the vagal bradycardia, while response onset and offset became slightly faster. From these observations, we argue that (i) vagal bradycardia was attributable primarily to activation of I(K,ACh), (ii) vagal modulation of I(f) had a minor influence on the rate of onset and offset of bradycardia, and (iii) removal of the influence of I(K,ACh) unmasked a slow response, of undetermined origin, to vagal stimulation. In a separate set of experiments we compared the effects of 1 mM Ba(2+) and 300 nM tertiapin-Q on vagal bradycardia. Ba(2+) reduced baseline atrial rate and the response to vagal stimulation. Subsequent cumulative addition of tertiapin-Q had no additional effect on baseline atrial rate, but caused further reduction in the amplitude of vagal bradycardia, suggesting that 1 mM Ba(2+) did not achieve a complete block of I(K,ACh) in this preparation.

The effects of tertiapin-Q on responses of the sinoatrial pacemaker of the guinea-pig heart to vagal nerve stimulation and muscarinic agonists.

Using Langendorff preparations of the guinea-pig heart, we have examined the participation of the acetylcholine (ACh)-activated potassium channel, IK,ACh, in the bradycardia produced by electrical stimulation of the vagus (parasympathetic) nerve and muscarinic agonists (ACh and bethanecol, bolus i.a.). Hearts from young animals (160-250 g) were perfused with Krebs-Henseleit solution, and pacemaker frequency was determined from the P wave of an ECG. Tertiapin-Q was used to block IK,ACh. Vagal stimulation (10 s trains at 2, 5 and 10 Hz) produced graded reductions in atrial rate that were substantially attenuated, and to a similar extent, by 300 nm and 1 microm tertiapin-Q (to 0.42 +/- 0.12, mean +/- s.d., of the control values; P < 0.001). Acetylcholine (3 nmol) produced brief graded bradycardias that were also attenuated by tertiapin-Q (0.24 +/- 0.24; P = 0.006). Similar results were obtained when experiments were repeated in 2 mm Cs+ (to block the hyperpolarization-activated pacemaker current). Bethanecol (30, 50 and 70 nmol), a muscarinic agonist with no appreciable nicotinic activity, produced sustained bradycardias that were attenuated by 300 nm tertiapin-Q (0.36 +/- 0.21; P < 0.0001). The responses to vagal stimulation and ACh developed more slowly in tertiapin-Q, indicating that a rapidly acting mechanism had been blocked. Responses to vagal stimulation were faster in 2 mm Cs+. Together, these observations show that ACh released from parasympathetic nerve varicosities exerts a considerable part of its effect on the pacemaker by activating IK,ACh and acts in a manner not readily distinguishable from that of directly applied muscarinic agonists.

Are the carotid bodies of the guinea-pig functional?

We have previously observed that the guinea-pig appears to have a relatively poor ventilatory (V (E)) response to hypoxia, compared to other mammals. Therefore, in this study, we questioned the ability of the carotid bodies (primary peripheral chemoreceptors) in the guinea-pig to detect hypoxia. The ventilatory responses to poikilocapnic hypoxia (8% O(2)), poikilooxic hypercapnia (8% CO(2)), hyperoxia (100% O(2)) and cyanide (NaCN - 200 mug/kg, i.v.) were assessed before and after carotid body denervation (CBD) in anaesthetized guinea-pigs. Although CBD attenuated the V (E) responses to hypercapnia and cyanide, it had no effect on normoxic breathing or the V (E) responses to hypoxia or hyperoxia. In a separate group of guinea-pigs, nerve activity was recorded from single or few-fibre preparations of the carotid sinus nerve (CSN). Basal chemoreceptor activity could not be detected from any of the nerve preparations. NaCN and hypercapnia consistently provoked an increase in neural activity. In contrast, hypoxia never clearly increased activity in any of the single or few-fibre preparations isolated from the CSN. In conclusion, although the carotid bodies of the guinea-pig, like those of other mammals, are able to detect hypercapnia and histotoxic hypoxia and elicit a reflex increase in V (E), they are essentially hypoxia-insensitive. The latter may explain, at least in part, the relatively poor V (E) response to hypoxia shown by the guinea-pig.

The influence of heart rate on baroreceptor fibre activity in the carotid sinus and aortic depressor nerves of the rabbit.

The arterial baroreceptors and their afferent fibres provide the sensory arm of the reflex that regulates systemic arterial pressure. We have examined whether the relationship between mean baroreceptor discharge and mean arterial pressure is altered when heart rate changes. Experiments were performed on pentobarbitone-anaesthetized rabbits. We recorded the activity of single and multifibre preparations of the carotid sinus (CSN) and aortic depressor nerves (ADN). Data were collected under control conditions and while heart rate was increased by approximately 30-35% by right atrial pacing. Baroreceptor regions were exposed to ramps of pressure (from approximately 25 to 140 mmHg, at approximately 0.5-1 mmHg s(-1)), generated by inflation and deflation of cuffs placed around the inferior vena cava and descending thoracic aorta. Response curves relating baroreceptor discharge to mean pressure were constructed and fitted with third-order polynomial expressions. To provide a measure of an effect of an increase in heart rate on the response curve in the region of the normal operating pressure, we calculated the position of the test response curve relative to the position of the control curve at 90 mmHg (deltaBP(90)). For the ADN, the activity of single fibres (presumptive myelinated fibres) was unaffected by increasing heart rate (deltaBP(90) = +0.1 +/- 1.0 mmHg), while single fibres in the CSN showed a small increase in activity (deltaBP(90) = -1.5 +/- 0.3 mmHg). In multifibre preparations there was a small increase in activity that may be attributable to additional activity in unmyelinated fibres (ADN, deltaBP(90) = -3.4 +/- 1.2 mmHg; CSN, deltaBP(90) = -5.2 +/- 0.9 mmHg). We conclude that the mean discharge of arterial baroreceptors remains a reliable index of mean arterial pressure in the presence of substantial changes in heart rate.

Respiratory sinus arrhythmia and the heart rate response to carotid baroreceptor activation after hard dynamic exercise in humans.

The aim of this study was to examine the influence of 20 min of hard exercise (HR>160 beats min(-1)) on the efficacy of the cardiac parasympathetic nervous control of heart rate in humans (20-31 years; of either sex). This intensity of exercise was chosen to produce strong activation of the cardiac sympathetic nerves. Using well-controlled stimulus parameters, the efficacy of cardiac parasympathetic control of heart rate was assessed by recording the heart rate response to carotid baroreceptor activation (CBR) and the amplitude of respiratory sinus arrhythmia (RSA). Measurements were made while the subject performed light exercise (100-135 beats min (-1)) before (Control 1) and after very brief (Control 2) and prolonged (20 min; post) periods of hard exercise. There was no difference in the CBR in the three different measurement periods; 0.33 +/- 0.17, 0.38 +/- 0.18 and 0.39 +/- 0.18 beats min(-1) mm Hg(-1) (mean +/- S.D., N=6) for Control 1, Control 2 and post, respectively. At a heart rate of 120 beats min (-1), amplitude of the RSA was 6.1 +/- 2.4, 5.6 +/- 2.4 and 3.3 +/- 2.1 beats min(-1) for Control 1, Control 2 and post, respectively (P<0.001 post vs. Control 1 and Control 2, N=8). The decrease in RSA amplitude following hard exercise may be attributable to an exercise-induced reduction in airway resistance and work of breathing. Overall, these results do not support the hypothesis that sustained hard exercise that produces strong activation of cardiac sympathetic nerves reduces cardiac parasympathetic efficacy.

Do cardiac neurons play a role in the intrinsic control of heart rate in the rat?

Three experiments were performed to see whether cardiac neurons contribute to the intrinsic control of heart rate in right atria of adult rats. The intrinsic heart rate response (IRR) was examined by raising right atrial pressure from 2 to 8 mmHg for 3 min. In isolated preparations of the right atrium, the IRR was not significantly altered by the addition of either 1 microM atropine (n =6; control +19+/- 3 min(-1) ; atropine+18+/-3 min(-1); (mean /+/-S.E.M.)) or 1 microM propranolol (n = 5; control +22+/- 4 min(-1); ; propranolol +21+/-3 min(-1); ). Tetrodotoxin (0.5 microm) had no effect on the IRR (n = 6; control +37+/-5 min(-1); tetrodotoxin 38+/-5 min(-1); ). In another experiment, 2-day-old rat pups were injected with capsaicin (50 mg kg(-1); treated) or with vehicle(control). There was no difference in the IRR of right atrial preparations taken from control and treated animals after they reached adulthood (control (n = 7) and treated (n = 8): +30+/- 4 and +32+/- 4 min(-1)). The influence of right atrial pressure on the efficacy of vagal stimulation was examined. The rate response to vagal stimulation was reduced similarly in control and treated preparations when pressure was elevated from 2 to 4 mmHg (control and treated: -34+/- 5% and -33+/- 3%). The effectiveness of the capsaicin treatment was confirmed by the depletion of substance P-immunoreactive nerve fibres in cardiac tissues. Together, these results strongly suggest that cardiac neurons are not involved in intrinsic heart rate control.