Rabbit Atrioventricular Node Research Articles

Properties and substrate of slow pathway exposed with a compact node targeted fast pathway ablation in rabbit atrioventricular node.

The properties and substrates of slow and fast AV nodal pathway remain unclear. This applies particularly to the slow pathway (SP), which is largely concealed by fast pathway (FP) conduction. We designed a new FP ablation approach that exposes the SP over the entire cycle length range and allows for its independent characterization and ablation. Premature stimulation was performed before and after FP ablation with 5.4 +/- 1.9 lesions (300-microm diameter each; overall lesion size 1.4 +/- 0.5 mm) targeting the junction between perinodal and compact node tissues in seven rabbit heart preparations. The resulting SP recovery curve and control curve had the same maximum nodal conduction time (165 +/- 22 msec vs 164 +/- 24 msec; P = NS) and effective refractory period (101 +/- 10 msec vs 100 +/- 9 msec; P = NS). The two curves covered the same cycle length range. However, the SP curve was shifted up with respect to control one at intermediate and long cycle lengths and thus showed a longer minimum nodal conduction time (81 +/- 15 msec vs 66 +/- 10 msec; P < 0.01) and functional refractory period (180 +/- 11 msec vs 170 +/- 12 msec; P < 0.05). The SP curve was continuous and closely fitted by a single exponential function. Small local lesions (2 +/- 1) applied to the posterior nodal extension resulted in third-degree nodal block in all preparations. The posterior nodal extension can sustain effective atrial-His conduction at all cycle lengths and account for both the manifest and concealed portion of SP. Slow and FP conduction primarily arise from the posterior extension and compact node, respectively.

Effects of ajmaline on rate-dependent atrioventricular node properties: Potential role in experimental atrioventricular re-entrant tachycardia

Ajmaline is a well-known atrioventricular (AV) node depressant agent, but its effects on functional properties of the AV node and on experimental AV re-entrant tachycardia have not been explored. The aims of the present study were (1) to determine whether ajmaline administration modifies the rate-dependent properties of the AV node and (2) to correlate these changes with the actions of ajmaline on an in vitro model of AV re-entrant tachycardia. Selective stimulation protocols and mathematical formulations were used to quantify independently AV node recovery, facilitation, and fatigue in 10 isolated rabbit AV nodes. Ajmaline decreased facilitation and fatigue and had no significant effect on AV node recovery. The most important effect of ajmaline was rate-induced prolongation of AV node effective refractory period, resulting in a greater increase in tachycardia cycle length. AV re-entrant tachycardia was sustained when AV effective refractory period divided to tachycardia cycle length was less than 1, ajmaline suppressed AV re-entrant tachycardia by increasing the slope of the AV effective refractory period divided to tachycardia cycle length versus tachycardia rate relation, causing the critical ratio of 1 to be attained at a slower rate. A mathematical model incorporating quantitative descriptors of recovery, facilitation, and fatigue accounted for changes in nodal conduction time, AV effective refractory period, tachycardia cycle length, and AV effective refractory period divided to tachycardia cycle length under all conditions. It can be concluded that (1) ajmaline increases AV conduction time, decreases AV node fatigue, and facilitation, without altering AV node recovery. (2) Ajmaline significantly prolongs AV effective refractory period in a rate-dependent manner. (3) These changes play a role in ajmaline's actions on experimental AV re-entrant tachycardia. Ajmaline's ability to terminate re-entrant supraventricular tachycardia may be due, at least in part, to its ability to amplify the rate-induced prolongation of the nodal refractory period.

Antagonism of the Positive Dromotropic Effect of Isoproterenol by Adenosine: Role of Nitric Oxide, cGMP-dependent cAMP-phosphodiesterase and Protein Kinase G

A. Zima, A. E. Martynyuk, C. N. Seubert, T. E. Morey, C. Sumners, R. F. Cucchiara and D. M. Dennis. Antagonism of the Positive Dromotropic Effect of Isoproterenol by Adenosine: Role of Nitric Oxide, cGMP-dependent cAMP-phosphodiesterase and Protein Kinase G. Journal of Molecular and Cellular Cardiology (2000) 32, 1609–1619. We hypothesized that nitric oxide (NO) plays an important role in mediating the anti-adrenergic effect of adenosine on atrioventricular (AV) nodal conduction. In guinea-pig hearts instrumented for measurement of AV nodal conduction time (atrium-to-His bundle, A–H, interval), the NO synthase (NOS) inhibitor, l-NMMA (100 μ m), reversibly inhibited 80% (P=0.009, n=6) of adenosine's anti-adrenergic action on the positive dromotropic effect of isoproterenol (0.01 μ m). In parallel studies carried out in rabbit AV nodal myocytes, intracellular mechanisms whereby NO mediates the inhibitory effect of adenosine on isoproterenol-induced A–H interval shortening were studied. Adenosine (3 μ m) inhibited isoproterenol-stimulated (0.1 μ m) ICa,L(β -ICa,L) by 46±6% (P<0.001, n=17). Consistent with isolated heart data, the NOS inhibitors, l -NMMA (100 μ m) and L-NNA (500 μ m) attenuated the effect of adenosine onβ -ICa,Lby 69±8% (P<0.001, n=16) and 69±7% (P<0.001, n=10), respectively. An inhibitor of NO-stimulated guanylyl cyclase LY83538 (40 μ m) reduced the inhibitory effect of adenosine on β -ICa,Lby 97±6% (P=0.004,n =15). Similarly, the non-specific inhibitor of cAMP-phosphodiesterases IBMX (50 μ m) decreased the anti-adrenergic effect of adenosine by 60% (P=0.02, n=6), whereas the extracellular application of the non-hydrolyzeable cAMP analog 8-Br-cAMP (500 μ m) prevented this action of adenosine. Activation of cGMP-dependent protein kinase (PKG) by CPT-cGMP (300 μ m) diminished β -ICa,L, but to a significantly smaller degree (16±4%, P=0.025, n=12) than that caused by adenosine. NO mediates the anti-adrenergic effect of adenosine on AV nodal conduction by a mechanism predominately involving activation of cGMP-dependent cAMP-phosphodiesterase and to a lesser extent activation of PKG.

Cellular electrophysiology of fast pathway ablation of rabbit atrioventricular node

Discrete radiofrequency lesion at the atrial insertion site of the tendon of Todaro in the perfused rabbit preparation lengthens A-H interval, mimicking fast pathway input ablation. This study attempts to define the cellular electrophysiology of the ablation region prior to and after the elimination of fast AV node conduction. In six superfused rabbit AV node preparations, the cellular electrophysiology around the region of the atrial insertion to the tendon of Todaro was recorded using standard microelectrode technique prior to and after ablation. Before ablation, the action potentials recorded in the area of proposed lesion were exclusively from atrial or AN cells. At postablation, the superior margin of the lesion was populated with atrial or AN cells. AN, N, or NH cells bordered the lower part of the lesion. Electrophysiology of surviving cells at the edges of the lesion showed no significant changes in their Vmax, APD50 or APD90 and MDP from preablation values. Fast AV node pathway input ablation in the rabbit heart can be accomplished with a singular lesion around the atrial insertion site of the tendon of Todaro, involving atrial or AN cells. The results of the studies imply that inputs to the compact node may act as a substrate for successful ablation of AV node reentry tachycardia.

An investigation of the role played by the E-4031-sensitive (rapid delayed rectifier) potassium current in isolated rabbit atrioventricular nodal and ventricular myocytes.

The aim of this study was to measure and compare the profile of rapid delayed rectifier potassium current (IKr) elicited by action potential (AP) waveforms applied to isolated rabbit atrioventricular nodal (AVN) and ventricular myocytes. All measurements were made using whole-cell patch-clamp recordings at 37 degrees C. In AVN myocytes, IKr during voltage steps and slow ramp depolarisations showed "inward rectification" (characteristic for this channel) at positive potentials. The E-4031-sensitive current showed half-maximal activation at -10.8 +/- 0.86 mV, with a slope factor for the activation relation of 6.5 +/- 0.77 mV (n = 7). During AVN APs, IKr rapidly reached a peak after the AP upstroke and remained at similar amplitude until late in AP repolarisation. At the maximum diastolic potential following the AVN AP, a component of IKr remained which decayed during the pacemaker depolarisation, consistent with a role for the current in generating AVN pacemaker activity. In ventricular myocytes IKr was small at the beginning of the AP, and increased slowly during the AP plateau. Measurement of Ba-sensitive-inward rectifier K current (IK1) in ventricular myocytes revealed that IK1 rapidly increased during the final AP repolarisation phase, whilst IKr declined. It is concluded that IKr may participate in both AP repolarisation and the pacemaker depolarisation in AVN cells, whilst in ventricular myocytes, IKr and IK1 participate in controlling early and final AP repolarisation respectively.

An investigation of the role played by the E-4031-sensitive (rapid delayed rectifier) potassium current in isolated rabbit atrioventricular nodal and ventricular myocytes

An investigation of the role played by the E-4031-sensitive (rapid delayed rectifier) potassium current in isolated rabbit atrioventricular nodal and ventricular myocytes

Characteristics of a transient outward current (sensitive to 4-aminopyridine) in Ca2+-tolerant myocytes isolated from the rabbit atrioventricular node.

A transient outward current (Ito) has been observed in the atrioventricular node (AVN), but its characteristics in Ca-tolerant AVN myocytes have not been investigated previously. In this study, Ito was measured from Ca-tolerant rabbit AVN myocytes at 37 degrees C, using the whole-cell patch-clamp technique. With interfering currents inhibited, 500-ms voltage-clamp pulses applied from -80 mV elicited Ito at potentials positive to -30 mV, which increased in magnitude with test potential amplitude. This current was completely blocked by external application of 5 mM 4-aminopyridine (4-AP). During a command pulse, Ito activated rapidly then inactivated with a bi-exponential time-course. Fast and slow time constants of current inactivation (tauf and taus, respectively) showed voltage dependence. At 0 mV, tauf was 14.5+/-2.7 ms and taus was 112.8+/-21. 2 ms, whilst at +60 mV tauf was 6.7+/-1.1 ms and taus was 63.7+/-9.2 ms (n=25). The steady-state inactivation relationship showed half-maximal inactivation at -33.8 mV (n=8). Re-activation of Ito after an inactivating pre-pulse showed a bi-exponential time-course of recovery: tau1 was 196+/-70 ms, and tau2 was 2707+/-1010 ms (n=6, at -80 mV). Repetitive application of voltage-clamp test pulses showed that Ito inactivation accumulated on repetitive stimulation, but reached a steady state rapidly for a given pulse frequency (0. 2-1.0 Hz). AVN Ito was sensitive to the class 1 anti-arrhythmic flecainide (EC50 for peak current of 24 microM), which showed selectivity for the rapidly inactivating current component. Quinidine also inhibited Ito in a dose-dependent fashion, but did not affect the current time-course. Under voltage-clamp conditions, a simulated diastolic depolarisation from -70 to -45 mV did not significantly reduce Ito amplitude, and under current-clamp conditions 4-AP inhibited spontaneous action potentials. Although this is consistent with a significant role for Ito in shaping AVN activity, under the conditions of this study 4-AP also partially blocked the "rapid" delayed rectifier current, IKr, and so the effects of 4-AP on action potentials could not be attributed exclusively to its effects on Ito.

Differences Between the Delayed Rectifier K+ Currents in Myocytes From Rabbit Sinoatrial and Atrioventricular Nodes

Differences Between the Delayed Rectifier K+ Currents in Myocytes From Rabbit Sinoatrial and Atrioventricular Nodes

Mechanism of atrioventricular nodal facilitation in rabbit heart: role of proximal AV node.

The phenomenon of atrioventricular (AV) nodal "facilitation," described in traditional "black box"-functional studies, implies enhanced AV nodal dromotropic function. We investigated the role of atrial prematurities in the modulation of the nodal cellular responses in the mechanism of AV nodal facilitation. Atrial and His (H) bundle electrograms and microelectrode recordings from proximal AV nodal cells were analyzed in 15 superfused rabbit AV node preparations. The pacing protocol consisted of 30 basic beats (S1; coupling interval S1-S1 = 300 ms) followed by a facilitating prematurity (S2; coupling intervals S1-S2 of 300, 200, 150, and 130 ms) followed by the test beat (S3; coupling interval S2-S3 scanned in 5-ms steps). Conduction curves (S2-H2 vs. S1-S2, S3-H3 vs. S2-S3, and S3-H3 vs. H2-S3) were constructed. Facilitation (i.e., shortening of S3-H3 when S1-S2 was shortened) was demonstrated in all preparations using the H2-S3 (P < 0.001) but not the S2-S3 format. Microelectrode recordings revealed a causal relationship between the improved proximal AV nodal cellular responses in facilitation and the prolonged S2-S3 interval. There was no evidence for enhanced nodal dromotropic function directly resulting from the introduction of the facilitating beats. Thus facilitation is based on inherent cycle-length-dependent properties of the AV node during application of a complex pacing protocol and primarily reflects the uncontrolled modulation of the proximal cellular response.

Modulation by mexiletine of action potentials, L-type Ca current and delayed rectifier K current recorded from isolated rabbit atrioventricular nodal myocytes.

Using whole-cell patch clamp recordings at 37 degrees C, we have examined the effects of externally applied mexiletine (a class 1b antiarrhythmic agent) on action potentials, L-type Ca current (ICa, L) and delayed rectifier K current (IK) in single isolated rabbit atrioventricular nodal (AVN) myocytes. In spontaneously active AVN cells, 30-100 micro;M mexiletine depolarised the maximum diastolic potential and slowed both action potential upstroke and repolarisation. Under selective recording conditions for ICa,L, mexiletine reduced peak ICa,L (at +10 mV) amplitude in a dose-dependent fashion (41.8 +/- 3.0% inhibition by 100 micro;M and 16.4 +/- 1.8% at 30 micro;M). The voltage dependence of ICa,L activation was unaffected by both concentrations of the drug. Under selective recording conditions, IK amplitude was measured as the peak of the deactivating tail current following a depolarising voltage pulse to +20 mV. 30 micro;M mexiletine inhibited IK by 34.3 +/- 5.8%, whilst 100 micro;M mexiletine reduced the current by 52.7 +/- 6.1%. The effects of mexiletine on ICa,L and IK are likely to contribute significantly to the changes in action potentials observed in spontaneously active cells. These findings are also suggestive of key roles for ICa,L and IK in determining the shape and rate of action potentials in this region of the heart.

Amiodarone blocks L-type calcium current in single myocytes isolated from the rabbit atrioventricular node

1. 1. Single myocytes were isolated from the rabbit atrioventricular node (AVN) and whole cell patch clamped, using a Cs +-based internal dialysis solution. Depolarizing voltage clamps from a holding potential of −40 mV were applied to investigate the effects of the antiarrhythmic agent amiodarone on L-type calcium current (I Ca,L). 2. 2. The current-voltage (I–V) relation for I Ca,L was bell shaped in normal Tyrode's solution, with a peak at +10 mV. After a 1-min exposure to 10 μM amiodarone, I Ca,L at all potentials between −30 mV and +60 mV was largely and reversibly blocked ( n=4). In 13 cells, peak I Ca,L was blocked by 85% after amiodarone application. 3. 3. The time course of the blocking effect was monitored during continuous pulsing (at 0.33 Hz) from −40 mV to + 10 mV to observe the time course of I Ca,L blockade. This could be described by a single exponential function with a rate constant of 0.21 per second. In other cells, amiodarone was applied for 30 sec without any stimulation; and, when stimulation was resumed, the I Ca,L amplitude with the first pulse was 22% of the control amplitude ( n=4), indicating that a substantial portion of the blockade of I Ca,L by amiodarone was tonic in nature. 4. 4. In addition to the effect of amiodarone on the I Ca,L channel, when the β adrenergic agonist isoprenaline (1 μM) was applied to cells that had been pretreated with amiodarone, I Ca,L amplitude was increased by 44.5±10.8% ( n=4). In cells that had received no such pretreatment with amiodarone, isoprenaline increased the I Ca,L amplitude by 101.5±5.9% ( n=4). Thus, isoprenaline produced a larger increase in I Ca,L in the absence of amiodarone than in its presence ( P<0.001). 5. 5. It is concluded that, in single AVN cells, amiodarone exerts a direct I Ca,L blocking action. In addition, the attenuated increase in I Ca,L with isoprenaline in amiodarone pretreated cells appears consistent with an antagonism of β adrenergic stimulation of I Ca,L.

Electrotonic modulation of electrical activity in rabbit atrioventricular node myocytes.

Electrotonic effects of electrically coupling atrioventricular (AV) nodal cells to each other and to real and passive models of atrial and ventricular cells were studied using a technique that does not require functional gap junctions. Membrane potential was measured in each cell using suction pipettes. Mutual entrainment of two spontaneously firing AV nodal cells was achieved with a junctional resistance (Rj) of 500 M omega, which corresponds to only 39 junctional channels, assuming a single-channel conductance of 50 pS. Coupling of AV nodal and atrial cells at Rj of 50 M omega caused hyperpolarization of the nodal cell, decreasing its action potential duration and either slowing or blocking diastolic depolarization in the AV node myocyte. Opposite changes occurred in the atrial action potential. When AV nodal and ventricular cells were coupled at Rj of 50 M omega, nodal diastolic potential was markedly hyperpolarized and diastolic depolarization was completely blocked with little change in ventricular diastolic potential. However, coupling did elicit marked changes in the action potential duration of both cells, with prolongation in the nodal cell and shortening in the ventricular cell. Nodal maximum upstroke velocity was increased by both atrial and ventricular coupling, as expected from the hyperpolarization that occurred. With an Rj of 50 M omega, spontaneous firing was blocked in all single AV nodal pacemaker cells during coupling to a real or passive model of an atrial or ventricular cell. These results demonstrate that action potential formation and waveform in a single AV nodal cell is significantly affected by electrical coupling to other myocytes.

Sodium channel distribution within the rabbit atrioventricular node as analysed by confocal microscopy.

1. Paired 20 microns thick sections of fresh frozen tissue taken from the frontal plane of the rabbit atrioventricular (AV) nodal region were processed for histology and immunohistochemistry. Confocal microscopy was used to image the distribution of sodium channels using IgG (R12) developed against a highly conserved sequence in the interdomain 3-4 region of cloned sodium channels. 2. In ventricular and atrial cells, sodium channel immunofluorescence was localized to lateral membranes and T-tubules. In the open AV node, levels of sodium channel immunofluorescence in the transitional cell zone and in the lower nodal cell tract were comparable to that found in the atrial and ventricular myocardium. 3. In the enclosed AV node a gradation of sodium channel immunofluorescence is present such that peripherally located circumferential transitional cells display high levels of immunofluorescence, comparable to that of atrial and ventricular myocardium, while centrally located midnodal cells display decreased levels of or no immunofluorescence. 4. In order to correlate the distribution of sodium channels with the distribution of gap junctions, we used IgG directed against the carboxyl terminus of connexin43 (CT-360). Ventricular cell immunofluorescence was localized primarily to the intercalated disk region, while in the AV node, the pattern of distribution was found to be similar to that of sodium channels. 5. The reduced levels of and/or absence of immunofluorescence in the midnodal cell region indicates a paucity of sodium channel and connexin43 protein expression in this region of the AV node that would favour slow impulse conduction.

Mechanism of atrioventricular nodal facilitation in the rabbit heart: role of the distal AV node.

We investigated whether atrioventricular (AV) nodal facilitation is the result of distal AV nodal action potential shortening. Atrial and bundle of His (H) electrograms and microelectrode recordings from proximal and distal AV nodal cells were analyzed in eight superfused rabbit AV node preparations in response to two pacing protocols. In the facilitation protocol, an atrial extrastimulus (A3) was preceded by an atrial impulse (A2) introduced 300, 200, 150, or 125 ms after 30 basic beats (A1). The preexcitation protocol differed from the facilitation protocol by the addition of a premature His depolarization (h2) such that the H1-h2 interval was shorter than the H1-H2 interval. Conduction curves (A3-H3 vs. H2-A3, h2-A3, and A2-A3 intervals) were constructed. Facilitation was demonstrated in all preparations when H2-A3 was used (P = 0.02) but not in the A2-A3 format. Compared with facilitation at the same A1-A2 intervals, preexcitation, despite shortening the distal cellular action potential duration, resulted in longer A3-H3 delays (P = 0.002), shorter A2-A3 intervals, and depression of the proximal nodal cellular response. Thus facilitation does not result from altered distal AV nodal characteristics and instead is a manifestation of an uncontrolled pacing protocol-dependent modulation of proximal AV nodal function.

Inhibition of L-type calcium current by propafenone in single myocytes isolated from the rabbit atrioventricular node.

1. The atrioventricular node (AVN) is an important part of the conduction system in the heart and is a significant site of antiarrhythmic drug action. The class 1 antiarrhythmic propafenone is effective in treating a variety of arrhythmias, including those involving the AVN. In this study, we have investigated the effects of propafenone on ionic currents in single rabbit AVN cells, focusing in particular on those on L-type calcium current (ICa,L). 2. With a standard K-based internal dialysis solution, exposure to 5 microM propafenone reduced significantly the amplitude of ICa,L. In spontaneously active AVN myocytes, action potential upstroke velocity was decreased by propafenone exposure, consistent with the observed change in ICa,L. 3. By use of a Cs-based internal dialysis solution to record ICa,L selectively, voltage clamp test pulses were applied from a holding potential of -40 mV to +10 mV (stimulation frequency 0.33 Hz). Propafenone 5 microM reduced mean ICa,L density at +10 mV from -9.58 +/- 1.05 pA/pF to -4.19 +/- 0.60 pA/pF (P < 0.002). A range of propafenone concentrations were applied which reduced ICa,L in a dose-dependent manner (IC50 1.7 microM). When test pulses were applied to a range of potentials, propafenone reduced ICa,L at each potential without significantly affecting the activation curve for this current. Thus, propafenone reduced ICa,L conductance, without affecting the voltage-dependent activation properties of the current. 4. ICa,L block by propafenone exhibited tonic-, use- and frequency-dependent characteristics. 5. In the presence of propafenone, the voltage-dependence of inactivation of ICa,L was shifted 8 mV in the hyperpolarizing direction. Also, the recovery of ICa,L from inactivation was slowed by propafenone. 6. The ICa,L blocking properties of propafenone may mediate some of the antiarrhythmic properties of this agent, particularly in regions of the heart such as the AVN in which ICa,L contributes significantly to the action potential upstroke.

Role of nitric oxide, cyclic GMP and superoxide in inhibition by adenosine of calcium current in rabbit atrioventricular nodal cells.

To study the intracellular pathways which mediate the inhibitory actions of adenosine on isoprenaline-stimulated calcium current (ICa) in atrioventricular (AV) nodal myocytes. The whole-cell patch-clamp technique was used to record ICa from rabbit AV nodal cells, isolated by enzymatic and mechanical dispersion. Isoprenaline, 0.1 microM, increased peak ICa from 0.58 +/- to 1.23 +/- 0.1 nA, and this increase was reversibly inhibited by adenosine, 10 microM (83 +/- 6%), which we have previously shown to be mediated by nitric oxide (NO) production. A membrane-permeable analogue of cyclic GMP, 8-Br-cGMP (300 microM), an inhibitor of cGMP-stimulated phosphodiesterase, prevented the effect of adenosine on ICa-Methylene blue (10 microM), an inhibitor of NO-sensitive guanylyl cyclase and a generator of superoxide (.02-), did not prevent, but increased, the inhibiting action of adenosine (49.5 +/- 6.6%, P < 0.01). Methylene blue (50 microM) caused a reduction of ICa, with further inhibition when combined with adenosine. A .O(2-)-generating system, xanthine oxidase (0.02 U/ml) and purine (2.3 mM), also increased the inhibitory action of adenosine on ICa. Inhibition of ICa by adenosine in the presence of xanthine oxidase was not prevented by 8-Br-cGMP (300 microM) and was not influenced by pre-incubation of cells with a NO synthase inhibitor, L-NAME (0.5 mM). The inhibitory effect of adenosine on ICa in rabbit AV nodal myocytes can be mediated by two mechanisms--stimulation of cGMP-stimulated phosphodiesterase by NO-induced cGMP, and a mechanism which involves interaction with .O2- production.

Existence of a low-threshold and sustained inward current in rabbit atrio-ventricular node cells.

Single myocytes were dissociated from the atrio-ventricular (AV) node of rabbit hearts. Spontaneous action potentials of the N or NH type were recorded from the fusiform AV node cells. Under whole-cell voltage clamp, these cells were characterized by the presence of hyperpolarization-activated cation current (I(f)), delayed rectifier K+ current (IK), and L-type Ca2+ current (ICa,L). After blocking I(f) and IK, depolarizing pulses from -80 mV revealed a sustained inward current, which was suppressed by 1 microM nicardipine. The activation threshold of the sustained inward current was around -60 mV and the maximum amplitude was 25.4 +/- 20.1 pA at -40 mV (n = 15). The current was also blocked by 1 microM verapamil. The sustained inward current was not decreased (125 +/- 32% of control, N = 5) by reducing [Ca2+]o from 1.8 to 0.1 mM, while it was nearly suppressed by depleting [Na+]o, differently from ICa,L. We concluded that the nicardipine-sensitive sustained inward current is largely attributable to Ist described in the rabbit sino-atrial node pacemaker cells. I(f) and Ist were not found in rod-shape AV node cells.

Regulation by acetylcholine of Ca2+ current in rabbit atrioventricular node cells.

Effects of acetylcholine (ACh) on L-type Ca2+ current (ICa) were examined in isolated atrioventricular (AV) node cells exhibiting spontaneous contractions and pacemaker current (If). ACh at a saturating concentration of 10 microM reduced basal ICa by 48 +/- 6%. The ACh effect was abolished by dialysis with 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor, or guanosine-5'-O-(2-thiodiphosphate). Dialysis with guanosine 3',5'-cyclic monophosphate (cGMP) or NG-monomethyl-L-arginine (L-NMMA) and application of the cGMP-dependent protein kinase inhibitor KT-5823 (1 microM) did not affect ACh inhibition of ICa. Nitric oxide donor 3-morpholinosydnonimine (100 microM) and type III phosphodiesterase (PDE) inhibitor trequinsin (10 nM) enhanced basal ICa by 10-20%, whereas type IV PDE inhibitor Ro-20-1724 (30 microM) together with trequinsin caused a large ICa stimulation comparable to that by 3-isobutyl-1-methylxanthine (IBMX). These findings indicate that ACh inhibits basal ICa primarily by suppressing cAMP synthesis and that these cells have a potent type III and IV PDE activity to determine the basal cAMP concentration. When ICa was stimulated by IBMX (100 microM), the inhibitory effect of ACh was slightly reduced by L-NMMA, cGMP, and methylene blue but not by KT-5823 or Ro-20-1724. ACh hardly inhibited, or even enhanced, IBMX-stimulated Ica when forskolin (3 microM) was coapplied or the IBMX concentration was increased to 500 microM. These findings suggest that cAMP is degraded in the presence of 100 microM IBMX to some extent. Type II PDE, for which IBMX has a relatively high inhibitor constant, seems to contribute partially to the cAMP degradation.

Actions of the digitalis analogue strophanthidin on action potentials and L-type calcium current in single cells isolated from the rabbit atrioventricular node.

1. The atrioventricular node (AVN) of the heart is vital to normal cardiac function and is a major site of antiarrhythmic drug action. This study describes the effects of the cardiac glycoside analogue strophanthidin on spontaneous action potentials and L-type calcium current recorded from single AVN cells isolated from the rabbit heart. 2. With a standard KCl-based internal dialysis solution, exposure to 50 microM strophanthidin produced a progressive depolarization of the maximum diastolic potential and a reduction in action potential amplitude and upstroke velocity. Sustained application resulted in the loss of action potentials and occurrence of spontaneous 'bell-shaped' depolarizations. 3. Cells were whole-cell voltage clamped at -40 mV and depolarizing voltage clamps applied. With a standard KCl-based internal dialysis solution, exposure to 50 microM strophanthidin caused a large reduction of ICa,L at all potentials between -30 and +40 mV (n = 4). At + 10 mV, the mean ICa,L amplitude was reduced from -232 +/- 65 pA to -48 +/- 26 pA (P < 0.05; 1 test; n = 5 cells). 4. To record ICa,L more selectively, cells were dialysed with a Cs-based pipette solution. A short strophanthidin exposure reduced ICa,L amplitude from -250 +/- 31 pA to -88 +/- 19 pA (P < 0.001; n = 8 cells). For both KCl and CsCl-based solutions it was observed that sustained exposure to strophanthidin for several minutes caused spontaneous inward fluctuations in the membrane current record similar to the 'ITI' (arrhythmogenic oscillatory transient inward) current shown for other cardiac cells. 5. When the calcium chelator BAPTA was added to the pipette solution (10 mM), the reduction in ICa,L by strophanthidin was largely eliminated (P > 0.1), and no spontaneous inward current fluctuations were observed after sustained exposure to strophanthidin (n = 8 cells). 6. When external Ca in the perfusate was replaced with Ba, strophanthidin did not significantly reduce the Ba current through L-type calcium channels (n = 5 cells). 7. We conclude that strophanthidin reduces ICa,L by an indirect action, mediated by the rise in intracellular calcium (Cai) which follows inhibition of the Na/K pump caused by cardiac glycosides. The appearance of spontaneous ITI with strophanthidin would also seem to be mediated by a rise in Cai, and may contribute to the spontaneous oscillations in membrane potential observed after prolonged strophanthidin exposure.

Electrophysiological properties of morphologically distinct cells isolated from the rabbit atrioventricular node.

1. Experiments were conducted using the whole-cell patch clamp technique to determine the electrophysiological properties and ionic currents of ovoid and rod-shaped single isolated calcium-tolerant rabbit atrioventricular (AV) nodal cells. 2. Action potential morphologies observed in these cells were similar to those obtained previously from intracellular recordings of intact atrioventricular nodal preparations: ovoid cells had N- or NH-like action potential configurations (see below), whereas rod-shaped cells had AN-like configurations. 3. Action potential restitution in AV nodal cells was characterized by a progressive increase in overshoot potential, maximal upstroke velocity (Vmax) and action potential duration, as well as a decrease in latency from stimulus to Vmax. In rod-shaped cells, premature stimuli could induce regenerative membrane responses before full action potential repolarization, whereas ovoid cells showed only post-repolarization refractoriness. In ovoid cells stimulated at the low stimulus intensities there was no shortening of the action potential duration and the most premature action potentials were often prolonged. 4. The quasi-steady-state current-voltage relationship of ovoid cells was significantly steeper, at both depolarized and hyperpolarized potentials, than that of either the rod-shaped AV nodal cells or atrial cells. The rod-shaped AV nodal cells and the atrial cells had similar current-voltage (I-V) relationships in the positive potential range, but the I-V curves crossed over at potentials of about-90 mV. 5. A hyperpolarization-activated inward current (I(f)) was apparent in the range between -60 and -90 mV in 95% of the ovoid cells (n = 75), whereas in 88% of rod-shaped cells (n = 16) I(f) was activated at more negative potentials. The magnitude of I(f) in ovoid cells, measured at -100 mV, was approximately 25 times that in rod-shaped cells. 6. A rapid inward current (INa) greater than 1 nA was found in all rod-shaped cells (n = 16) but in only 30% of ovoid cells (n = 75). A transient outward current (I(to)) was found in 93% of rod-shaped cells (n = 14) and in 42% of ovoid cells (n = 54). The combination of I(to) and INa was found in 93% of rod-shaped cells but in only 24% of ovoid cells. 7. These results suggest that there are at least two populations of isolated AV nodal cells with distinct action potentials and ionic current profiles that may contribute to the complex electrophysiological properties observed in the intact AV node.