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Am J Physiol Heart Circ Physiol 283: H615-H630, 2002. First published April 25, 2002; doi:10.1152/ajpheart.00130.2002
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Vol. 283, Issue 2, H615-H630, August 2002

Effects of ACh and adenosine mediated by Kir3.1 and Kir3.4 on ferret ventricular cells

H. Dobrzynski, N. C. Janvier, R. Leach, J. B. C. Findlay, and M. R. Boyett

School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The inotropic effects of ACh and adenosine on ferret ventricular cells were investigated with the action potential-clamp technique. Under current clamp, both agonists resulted in action potential shortening and a decrease in contraction. Under action potential clamp, both agonists failed to decrease contraction substantially. In the absence of agonist, application of the short action potential waveform (recorded previously in the presence of agonist) also resulted in a decrease in contraction. Under action potential clamp, application of ACh resulted in a Ba2+-sensitive outward current with the characteristics of muscarinic K+ current (IK,ACh); the presence of the muscarinic K+ channel was confirmed by PCR and immunocytochemistry. In the absence of agonist, on application of the short ACh action potential waveform, the decrease in contraction was accompanied by loss of the inward Na+/Ca2+ exchange current (INaCa). ACh also inhibited the background inward K+ current (IK,1). It is concluded that ACh activates IK,ACh, inhibits IK,1, and indirectly inhibits INaCa; this results in action potential shortening, decrease in contraction, and, as a result of the inhibition of IK,1, minimum decrease in excitability.

heart; acetylcholine; muscarinic K+ channel


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PARASYMPATHETIC NERVES extensively innervate the ventricles, and parasympathetic stimulation has a negative inotropic effect on the ventricular muscle (29, 36). Previously, the parasympathetic transmitter ACh was thought only to have a negative inotropic effect on ventricular muscle after the muscle had first been potentiated by a beta -agonist (28). However, it is now well established that ACh does have a negative inotropic effect in the absence of beta -stimulation in ferret, rat, and dog ventricular cells (5, 30, 31, 39). In the dog, ACh has a substantial effect on left ventricular subepicardial cells, a smaller effect on midwall cells, and little or no effect on subendocardial cells (39). The negative inotropic effect of ACh has been suggested to be the result of the activation of the muscarinic K+ current (IK,ACh) despite the fact that the muscarinic K+ channel (a heteromultimer of Kir3.1 or GIRK1 and Kir3.4 or GIRK4), although detectable in the atrium, has not been detected in the ventricles (26). It is postulated that the activation of IK,ACh results in a shortening of the action potential and thus a decrease in the contraction. An ACh-induced decrease in the basal L-type Ca2+ current (ICa,L) may also be involved in the ACh-induced changes in the action potential and contraction (27, 39).

The action potential-clamp technique was devised by Starzak and Needle (37) and first used with cardiac tissue by Doerr et al. (13). Whereas the conventional voltage-clamp technique uses square-wave command pulses, the action potential-clamp technique involves the use of an action potential waveform as the voltage-clamp command. The action potential-clamp technique has been used to study the time course and amplitude of ionic currents during the action potential: current recorded during an action potential voltage-clamp command waveform after the application of a blocker has been subtracted from that under control conditions; ICa, the Na+/Ca2+ exchange current (INaCa), the delayed rectifier K+ current (IK), and the background inward rectifier K+ current (IK,1) have been studied in this way (1, 17, 19, 20). The principal aim of this study was to investigate the ACh-induced changes in current during the ventricular action potential. Adenosine was studied in the same way. This is the first study to investigate agonist-induced changes in membrane current during the action potential. Another aim of the study was to investigate the mechanisms underlying the negative inotropic effect of ACh during the action potential. In part, this involved the detection of Kir3.1 mRNA by screening ventricular tissue cDNA using the PCR technique and Kir3.1 and Kir3.4 proteins in ventricular cells by immunocytochemistry.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments on single cells. Experiments were performed on isolated ferret ventricular cells. Ferrets were anesthetized by intraperitoneal injection of 90-150 mg pentobarbitone sodium. The heart was rapidly excised and placed in "isolation solution" (see below) containing 750 µM CaCl2. The aorta was cannulated and the heart retrogradely perfused at ~20-23 ml/min with isolation solution containing 750 µM Ca2+ for sufficient time to clear all the blood (~2-3 min). The perfusate was then switched to Ca2+-free isolation solution (containing 0.1 mM EGTA) for 4 min. Finally, the heart was perfused for 10 min with "enzyme solution": isolation solution containing 1 mg/ml collagenase (Worthington type II, Lorne laboratories), 0.1 mg/ml protease (Sigma; Poole, UK), and 50 µM CaCl2; this solution was recirculated through the heart. The ventricles were cut away from the atria, finely chopped, placed in a conical flask, and digested with the enzyme solution supplemented with 10% (wt/vol) bovine serum albumin for a further 5 min. The tissue was shaken gently during this period. This process was repeated four times, and the cells from each 5-min period were harvested by filtration and pelleted by centrifugation at 400 rpm for 40 s. Cells were washed by resuspending them in isolation solution containing 750 µM Ca2+ and then recentrifuging them. The cells were stored in isolation solution containing 750 µM Ca2+ at 4°C until required. The isolation procedure was performed at 37°C. Isolation solution contained (in mM) 130 NaCl, 5.4 KCl, 1.4 MgCl2, 0.4 NaH2PO4, 10 creatine, 20 taurine, 10 HEPES, and 10 glucose; pH 7.3 at room temperature. This solution was equilibrated with O2.

Cells were pipetted into a small tissue bath (volume, 0.2 ml) attached to the stage of an inverted microscope (Nikon Diaphot). The cells were allowed to settle for several minutes onto the glass bottom of the chamber before being superfused at a rate of ~1.6 ml/min with Tyrode solution of the following composition (in mM): 136.9 NaCl, 5.4 KCl, 2 CaCl2, 0.57 MgCl2, 0.37 NaH2PO4, 5 HEPES, and 5.6 glucose; pH 7.4 at 37°C. In some experiments 5 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM was added to the Tyrode solution to buffer intracellular Ca2+. A 1 mM BAPTA-AM stock solution was made by dissolving the drug in DMSO. Cells were perfused with Tyrode solution containing 5 µM BAPTA-AM (0.5% DMSO) for 5-10 min. During this period the size of the contraction dwindled as a result of the buffering of intracellular Ca2+. When the contraction was almost completely abolished, the BAPTA-AM was washed off. On washoff of BAPTA-AM, the contraction did not recover (BAPTA remained trapped within the cells as a result of the cleavage of the AM group by intracellular esterases). As a control, cells were exposed to Tyrode solution containing 0.5% DMSO only for 10 min; this had no effect (n = 4). Measurements were only made after more than 2 min of washoff of BAPTA-AM. We used the muscarinic and purinergic agonists acetylcholine chloride and adenosine (Sigma). A 10-2 M stock solution of each drug was prepared in double-distilled water; this was added to the Tyrode solution in appropriate quantities. The pH of Tyrode solution containing a drug was checked and readjusted if necessary.

Miniature solenoid valves (Lee Products; Gerrards Cross, Buckinghamshire, UK) controlled which of the four solutions flowed to the chamber. The fluid level in the chamber was controlled using the system described by Cannell and Lederer (8). The temperature of the solution was maintained at 37 ± 0.5°C by a heating coil wrapped around the glass inflow tube immediately before the chamber. Solution temperature was monitored by a thermistor mounted in the side of the chamber and controlled by a feedback circuit, which regulated the flow of current to the heating coil.

Ventricular cells were impaled with conventional microelectrodes (15-30 MOmega ) filled with 1 M KCl. The switch clamp technique was used to voltage clamp cells (Dagan 8800 amplifier, Dagan; switching frequency, ~4.5 kHz) to minimize intracellular dialysis and allow stable recordings of up to and over 1 h. The capacitance of the microelectrode was compensated just before impalement. The electrode potential upon switching between current injection and voltage recording was monitored on an oscilloscope throughout an experiment to ensure that the electrode potential settled between current injection pulses. Membrane current was typically filtered at 1 kHz (low-pass filter). Membrane potential and current were also displayed on an oscilloscope (Tektronix 5000 series) to assess the quality of the voltage clamp. Timing units (Hi-Med; Reading, UK) were used to control voltage-clamp protocols and synchronize other recording equipment. A pulse generator (3) was used to drive the action potential-clamp experiments. Briefly, action potentials at a stimulation rate of 1 Hz were recorded from a cell, and an average of five were transferred to the pulse generator as a text file. The pulse generator was then used to apply the action potential voltage-clamp command waveform (via the Dagan 8800 amplifier) repetitively at a rate of 1 Hz.

Cell length was recorded using an optical system based on a photodiode array (7); twitch shortening (the shortening of a cell during a twitch contraction) was measured electronically (6). Action potential duration was measured electronically at -65 mV (22). Membrane current and potential, cell length, twitch shortening, and action potential duration were displayed on a six-channel chart recorder (Gould 2600S or RS 3600) and simultaneously recorded on videotape using a pulse code modulator (Neuro-Corder DR-890, Neuro Data Instruments) and a video recorder. Membrane current and potential and cell length were also digitized using an analog-to-digital converter (1401 plus, Cambridge Electronic Design; Cambridge, UK) and stored on an IBM-compatible computer running the Cambridge Electronic Design voltage-clamp software.

Cells were stimulated to contract by either a 5-ms current pulse (current clamp mode) or application of the action potential clamp, at 1 Hz.

Data are presented as means ± SE (number of cells).

Computer modeling. Computer simulations were carried out using Version 7 of the Oxsoft HEART program (33). The "simplified guinea pig ventricular cell" model was modified to simulate the ferret ventricular action potential. The modifications made were the following: extracellular K+ concentration set to 5.4 mM (same as experiments); conductance for tetrodotoxin-sensitive Na+ current (INa), GNA = 1.05; Ca2+ permeability for L-type Ca2+ current (ICa,L), PCA = 0.07; conductance for transient outward K+ current (Ito), GTO = 0.03; speed of inactivation variable for Ito, SPEED[18] = 6; maximum outward K+ current for IK, IKM = 0.8; conductance for background Na+ current (Ib,Na) GBNA = 0.0012; voltage shift of the voltage dependence of the background IK,1, SHIFTK1 = -10; steepness of the voltage dependence of IK,1, and STEEPK1 = 2; and conductance for IK,1, GK1 = 1.

cDNA cloning and sequencing. Standard molecular biology techniques were carried out as described by Berger and Kimmel (2) and Sambrook et al. (35). Oligonucleotide primers 1 (bases 1240 to 1263) and 2 (bases 1501 to 1533) were designed by comparison with known Kir3.1 sequences (26): sense primer 1, 5'-AAACTCCTGAGGATGAGTTCT-3'; antisense primer 2, 5'-TGAAATAATGCCTCGAGGGGTGTTTTGCTATGT-3'.

Total RNA was purified from the ferret ventricle and atrium (10). Total RNA was also purified from the dog ventricle (obtained at the end of an experiment carried out by a colleague). Poly (A+) RNA, purified from total RNA by oligo(dT)-cellulose affinity chromatography (35), was used to generate cDNA by the rapid amplification of cDNA ends [RACE technique (14)] using a Marathon cDNA amplification kit according to the manufacturer's instructions (Clontech Laboratories; Palo Alto, CA).

PCR amplification of ~500 pg cDNA (55°C annealing temperature, 50 cycles, Taq polymerase), using oligonucleotides 1 and 2 as primers, generated a single reproducible product when visualized under ultraviolet light after electrophoresis on a 1.5% (wt/vol) agarose gel, containing 50 µg/ml ethidium bromide, for 1 h at 5 V/cm in 40 mM Tris-acetate and 1 mM EDTA buffer (see Fig. 12). No products were generated when PCR was performed in the absence of template cDNA (data not shown). The size of the PCR product was estimated by comparison to fragments of DNA of known size (lambda -HindIII markers; GIBCO-BRL; Paisley, UK). The PCR product obtained from the ferret ventricle was cloned into pCR2.1 T/A cloning vector (Invitrogen; Groningen, The Netherlands) and then sequenced on an Applied Biosystems 373 DNA Sequencer (Perkin Elmer) using fluorescent cycle sequencing.

Immunocytochemistry. Immunocytochemistry on ferret ventricular and atrial cells was carried out as previously described (12). After isolation, heart cells were plated on Bunsen burner flame-treated, polysine-coated slides (BDH; Poole, UK) and were single labeled with either anti-Kir3.1 (Alomone Labs; Jerusalem, Israel) or anti-Kir3.4 (gift from Dr. G. B. Krapivinsky, Harvard Medical School, Boston, MA) primary antibodies followed by an anti-rabbit secondary antibody conjugated to FITC and examined by a confocal laser scanning microscope (Leica; Heidelberg, Germany) as previously described (12). The images recorded were single optical sections of 10 atrial cells and 6 ventricular cells for each antibody used. The anti-Kir3.1 antibody was previously characterized by immunocytochemistry on Chinese hamster ovary and single rat heart cells (12), whereas the anti-Kir3.4 antibody was previously characterized by Western blotting on Sf9 cells (25). Various control experiments were carried out with these antibodies and are fully described (12). In this study, no labeling of heart cells was detected when the secondary antibody was applied only (i.e., primary antibodies not applied; not shown).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of ACh on the action potential and contraction under current clamp conditions. Typical effects of 1 µM ACh on a ferret ventricular cell under current-clamp conditions are shown in Fig. 1A. The cell was stimulated at 1 Hz. Slow time-base records of contractions and action potential duration are shown on the left. On application of ACh during the period shown by the bar, there was an abrupt shortening of the action potential by 66% accompanied by a decrease in the contraction of 55%. Fast time-base records of action potentials and contractions under control conditions and at the time of the peak effect of ACh are shown on the right of Fig. 1A. In five cells, contraction was maximally decreased by 75 ± 4% by 1 µM ACh under current-clamp conditions (see Fig. 2, trace i). After a peak was reached, the effects of ACh faded with time during the remainder of the 2-min exposure to ACh; the duration of the action potential and the amplitude of the contraction both increased (Fig. 1A). This is attributed to desensitization to ACh. On washoff of ACh, action potential duration and contraction rapidly increased; action potential duration returned to its control value, whereas there was a small rebound increase in the contraction beyond its control value (Fig. 1A). The contraction declined to its control value over ~2 min (not shown).


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Fig. 1.   Effect of 1 µM acetylcholine (ACh) on the action potential (AP) duration (APD) and contraction under current-clamp (A) and AP-clamp (B) conditions. Left, slow time base records of cell length or twitch shortening (top) and APD (bottom). ACh (1 µM) was applied for 2 min as indicated by bar. Right, fast time base records of the AP or AP command waveform (top) and accompanying contractions (bottom) before (trace i) and during (trace ii) the application of ACh. Fast time base records correspond to the times shown in left. Data in A and B were obtained from the same cell. The cell was under current-clamp conditions in A. In B, the cell was under voltage-clamp control and a control AP recorded earlier from the same cell under current-clamp conditions was used as the voltage-clamp command waveform.



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Fig. 2.   Inotropic effects of 1 µM ACh and 100 µM adenosine under different conditions. Filled and hatched bars represent ACh and adenosine data, respectively. Bars i and iv show the minimum amplitude of contraction during an application of 1 µM ACh or 100 µM adenosine under current-clamp conditions. Bars ii and v show the amplitude of contraction when a short action potential recorded in the presence of the agonist was used as the voltage-clamp command waveform. Contraction amplitude was measured after application of the AP command waveform at the same time the minimum contraction occurred in the presence of ACh under current clamp. Bars iii and vi show the minimum amplitude of contraction during an application of 1 µM ACh or 100 µM adenosine under AP clamp (APClamp) conditions. Values are means + SE of the amplitude of contraction (as a percentage of the control) shown. Parentheses represent number of cells.

Effect of ACh under action potential-clamp conditions. Typical effects of 1 µM ACh on the contraction of a ferret ventricular cell under action potential-clamp conditions are shown in Fig. 1B (all data in Fig. 1 were obtained from the same cell). Before the experiment shown, the cell was stimulated at 1 Hz under current-clamp conditions, and the steady-state action potential was recorded. Recording conditions were then switched from current clamp to voltage clamp. The control action potential was used as the voltage-clamp command waveform. This was repetitively applied to the cell at a rate of 1 Hz. Application of ACh under these conditions, of course, did not result in a shortening of the action potential, and the decrease in contraction (16%) was much reduced compared with that observed under current-clamp conditions (cf. Fig. 1, A and B). Figure 2 shows that in five cells, 1 µM ACh maximally decreased contraction by 7 ± 3% under action potential-clamp conditions (Fig. 2, bar iii), whereas under current-clamp conditions it decreased contraction by 75 ± 4% (Fig. 2, bar i; P < 0.001, paired t-test). This much reduced the inotropic effect of ACh under action potential-clamp conditions suggests that the inotropic effect under current-clamp conditions (Fig. 1A) is primarily the result of the action potential shortening caused by ACh. Notice that the small effect of ACh on contraction under action potential-clamp conditions faded with time as a result of desensitization (Fig. 1B).

The changes in the membrane current during the experiment in Fig. 1B are shown in Fig. 3. The voltage-clamp command waveform (the control action potential) is again shown in Fig. 3A. As expected, under control conditions, little or no current was recorded during the action potential command waveform (Fig. 3B). On application of 1 µM ACh, an outward current was activated during the action potential. Four seconds after the start of the application of ACh, the current was ~2 nA in amplitude in this example, but 60 s after the start, the current had declined to ~1 nA as a result of desensitization (Fig. 3B). The ACh-activated outward current explains the ACh-induced shortening of the action potential under current-clamp conditions. The current-voltage relationship of the ACh-activated outward current was obtained by plotting the current against the membrane potential during the action potential command waveform. Figure 3C shows current-voltage relationships 4 and 60 s after the start of the ACh application. The current-voltage relationships are those expected of an inward-rectifying K+ current such as IK,ACh (see DISCUSSION). Similar data were obtained from 14 cells.


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Fig. 3.   Effect of 1 µM ACh on membrane current during the control AP (voltage-clamp command waveform). A: voltage-clamp command waveform. AP used as the voltage-clamp command waveform was recorded from the same cell under current-clamp conditions. B: membrane currents recorded during the control action potential command waveform under control conditions (Con) and after 4 and 60 s exposure to 1 µM ACh. C: current-voltage relationship of the ACh-activated outward current after 4 and 60 s exposure to ACh. Currents in B were plotted against the membrane potential during the AP command waveform. Arrow indicates the time sequence of the data.

Simulation of the effect of ACh. If the inotropic effect of ACh is primarily the result of the action potential shortening, then it should be possible to simulate the inotropic effect of ACh by using the shortened action potential recorded in the presence of ACh as the voltage-clamp command waveform. Figures 1 and 4 were obtained from the same cell. Before the experiment shown in Fig. 4, the experiment in Fig. 1A was carried out: the cell was stimulated at 1 Hz under current-clamp conditions, 1 µM ACh was applied, and the shortest action potential in the presence of ACh was recorded. ACh was washed off, and the preparation was allowed to return to steady state. The experiment in Fig. 4 was then started. Recording conditions were switched from current clamp to voltage clamp, and the short action potential recorded previously in the presence of ACh was used as the voltage-clamp command waveform and repetitively applied to the cell at 1 Hz. Despite the fact that ACh was absent, there was a decrease in contraction comparable to that observed on application of ACh under current-clamp conditions. In five cells, application of the short ACh action potential command waveform resulted in a 74 ± 3% decrease in contraction (Fig. 2, bar ii), which is not significantly different (P = 0.526, paired t-test) from the decrease in contraction (75 ± 4%; Fig. 2, bar i) when ACh was applied under current-clamp conditions. This confirms that the inotropic effect of ACh is primarily the result of the action potential shortening rather than another action such as a decrease of ICa,L. However, the inotropic effect of the ACh action potential command waveform was not identical to that of ACh; during an exposure to ACh under current-clamp conditions, the inotropic effect faded as a result of desensitization (Fig. 1A), whereas during the application of the ACh action potential command waveform, the inotropic effect did not fade (Fig. 4).


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Fig. 4.   Simulation of the inotropic effect of ACh. A: slow time base records of membrane potential (top), cell length (middle), and APD (bottom). Cell was switched from current clamp to AP clamp for 2 min as indicated by the bar. The shortest action potential recorded earlier under current-clamp conditions in the presence of 1 µM ACh (Fig. 1A) was used as the voltage-clamp command waveform. B: fast time base records of the control AP (trace i) and the ACh AP command waveform (trace ii) (top) and accompanying contractions (bottom).

Changes in membrane current during the experiment in Fig. 4 are shown in Fig. 5. Figure 5A shows the control action potential (current-clamp conditions) and the ACh action potential command waveform and accompanying contractions. When the ACh action potential command waveform was first applied, there was a slight decrease in the amplitude of the contraction as a result of a decrease in the time to peak of the contraction (Fig. 5A). With time, the contraction then declined to a steady state (Fig. 5A; see also Fig. 4). Figure 5B shows the first and steady-state membrane currents recorded during the ACh action potential command waveform. When the action potential clamp was first applied, a prolonged inward current was recorded. Once a steady state was reached during the application of the action potential clamp, the inward current was much abbreviated. The difference between the two currents is shown in Fig. 5C: there is a small transient outward current followed by a large inward current. A similar result was obtained from 11 cells. A substantial component of the inward current during the ACh action potential command waveform is likely to be inward INaCa triggered by the Ca2+ transient (see DISCUSSION). The decrease in the inward current during the repetitive application of the ACh action potential command waveform in Fig. 5B (responsible for the inward difference current in Fig. 5C) might be the result of a decline in inward INaCa caused by a decline in the underlying Ca2+ transient (presumably responsible for the decrease in the contraction).


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Fig. 5.   Membrane current during the short ACh action potential (voltage-clamp command waveform). A: fast time base records of the control AP under current-clamp conditions (Con) and the ACh AP command waveform under APClamp conditions (top) and accompanying contractions (bottom). Contractions accompanied the control action potential (Con), the first ACh AP command waveform (1st), and the ACh AP command waveform once contraction had reached a steady state (ss). B: currents during the first ACh AP command waveform (1st) and the ACh AP command waveform once contraction had reached a steady state (ss). C: difference (Delta ) in current between the 1st and ss ACh AP command waveforms. Difference current was calculated by subtracting the current at ss from the current during the 1st ACh AP command waveform.

Figure 6 shows how we tested this hypothesis. We have shown that buffering intracellular Ca2+ by BAPTA-AM results in the loss of contraction (and, presumably, the underlying Ca2+ transient) as well as the loss of a Ito during the ferret ventricular action potential; the current displays the characteristics expected of inward INaCa triggered by the Ca2+ transient (20). If the hypothesis above is correct, then buffering intracellular Ca2+ by BAPTA-AM by eliminating the Ca2+ transient should abolish the inward INaCa during the ACh action potential command waveform as well as the difference in the first and steady-state currents. The result in Fig. 6A, obtained under control conditions, is similar to the result in Fig. 5; it shows the membrane current during the ACh action potential command waveform. Once again, the current that flowed during the first ACh action potential command waveform was more inward than the current that flowed under steady-state conditions. This is clear from the difference current shown in Fig. 6A, bottom. After this result was obtained, the cell was exposed to 5 µM BAPTA-AM for 7 min to buffer intracellular Ca2+. After the application and washoff of BAPTA-AM (see MATERIALS AND METHODS), the action potential under current-clamp conditions was shortened (Fig. 6B). This effect has been described previously and has been attributed to the loss of inward INaCa (20). After the application and washoff of BAPTA-AM, the ACh action potential command waveform was again applied (Fig. 6B). As predicted, buffering intracellular Ca2+ reduced the inward current flowing during the ACh action potential command waveform (Fig. 6B, middle). The change in current on repetitive application of the ACh action potential command waveform was also abolished (Fig. 6B, middle), and, as a result, the inward difference current was abolished (Fig. 6B, bottom). Similar results were obtained from four cells. The ACh-activated outward current (Fig. 3B) must overcome the inward current in Figs. 5B and 6A to be able to shorten the action potential.


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Fig. 6.   Effect of buffering intracellular Ca2+ on the inward current during the short ACh AP (voltage-clamp command waveform). Top, fast time base records of an AP during current clamp under Con conditions (A) or after the application of 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA) (B) and the ACh AP command waveform during voltage clamp (APClamp, APClp). Middle, currents during the 1st ACh AP command waveform and the ACh AP command waveform once contraction had reached a steady state or at the equivalent time after the application of BAPTA-AM (ss). Bottom, difference in current between the 1st and ss ACh AP command waveforms. Difference current was calculated by subtracting the current at ss from the current during the first ACh AP command waveform. Records were obtained before (A) and after (B) the buffering of intracellular Ca2+ by the application and washoff of 5 µM BAPTA-AM.

In the experiments shown in Figs. 5 and 6A, repetitive application of the ACh action potential command waveform resulted in the loss of a Ito; this is best seen in the difference currents in Fig. 5C and the bottom panel of Fig. 6A. Figure 6B, bottom, shows that this Ito was reduced after the application of BAPTA-AM (see DISCUSSION for interpretation). Similar results were obtained from two cells.

Activation of IK,1 by ACh. The shortening of the action potential in the presence of ACh is attributable to the ACh-activated outward current shown in Fig. 3B. The current-voltage relationship of this current suggests that it is an IK,1. To test this possibility, we investigated the effects of Ba2+, a potent blocker of inward-rectifying K+ channels. First, the effect of Ba2+ alone was investigated. Figure 7A shows the effect of 2 mM Ba2+ on a cell under current-clamp conditions. Under control conditions, a stimulus resulted in a normal action potential. After the application of Ba2+, there was a large depolarization. The cell was no longer excitable, and instead there were small spontaneous oscillations of the membrane potential (accompanied by contractions; not shown). Figure 7B shows a control action potential command waveform that was repetitively applied to the same cell at a rate of 1 Hz under voltage-clamp control. Application of Ba2+ under these conditions, of course, did not result in a depolarization of the resting membrane. Instead it resulted in the loss of an outward current. The Ba2+-sensitive outward current was obtained by subtracting the current after the application of Ba2+ from the current before the application of Ba2+ and is shown in Fig. 7B, bottom. The current-voltage relationship of this current was obtained by plotting the current against the membrane potential during the control action potential command waveform and is shown in Fig. 7C. The current displays inward rectification more pronounced than that of the ACh-activated current (Fig. 3C). The current is a combination of all Ba2+-sensitive currents but is likely to be primarily the background IK,1. Similar results were obtained from five cells.


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Fig. 7.   Effect of 2 mM Ba2+ on the AP and membrane current. A: AP under control conditions and membrane potential after application of 2 mM Ba2+. B: control AP recorded under current-clamp conditions used as the APClamp waveform (top) and accompanying current during application of 2 mM Ba2+ (bottom; current during the AP clamp in the presence of Ba2+ was subtracted from the current during the AP clamp before the application of Ba2+). C: current-voltage relationship of the Ba2+-sensitive current. Ba2+-sensitive current was plotted against the membrane potential during the APClamp waveform.

Figure 8 shows the effect of Ba2+ on the ACh-activated outward current. Under action potential-clamp conditions, application of ACh resulted in the usual development of an outward current during the control action potential command waveform (Fig. 8A, top). The current-voltage relationship of this current is shown in Fig. 8B; the current shows the same degree of inward rectification as the current in Fig. 3C. To investigate whether this current could be blocked by Ba2+, we first had to record the membrane current in the presence of Ba2+ alone (see above) and then in the presence of ACh and Ba2+. The trace in Fig. 8A, bottom, shows the membrane current activated by ACh in the presence of Ba2+. This was calculated by subtracting the current in the presence of Ba2+ alone from the current in the presence of ACh and Ba2+. Figure 8B shows the current-voltage relationship of the ACh-activated current in the presence of Ba2+. Ba2+ caused a large reduction in the ACh-activated current (Fig. 8). Similar results were obtained from five cells. The results support the hypothesis that ACh activates an IK,1.


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Fig. 8.   Effect of 2 mM Ba2+ on the ACh-activated outward current during the control AP (voltage-clamp command waveform). A: control AP recorded under current-clamp conditions used as the voltage-clamp command waveform (top), ACh-activated current (middle; current in the presence of 10 µM ACh was subtracted from the current before the application of ACh), and ACh-activated current in the presence of 2 mM Ba2+ (bottom; current in the presence of Ba2+ only was subtracted from that in the presence of 10 µM ACh and Ba2+). B, current-voltage relationship of the ACh-activated current under normal conditions (ACh) and in the presence of 2 mM Ba2+ (ACh + Ba2+). Currents in A were plotted against the membrane potential during the APClamp waveform.

Inhibition of IK,1 by ACh. To confirm that the ACh-activated outward current is a K+ current, we attempted to measure its reversal potential. This was not possible using the action potential-clamp technique, because the membrane potential never exceeds the K+ equilibrium potential (EK) during the action potential. Instead, we used a ramp-clamp protocol that ramped the membrane potential from +33 to -120 mV (below EK) over 200 ms (roughly similar to the duration of the action potential) at a rate of 1 Hz (holding potential, -80 mV). Current-voltage relationships were obtained by plotting membrane current against the membrane potential during the ramp clamp. Figure 9A shows the current-voltage relationship of total membrane current before and early and late during an application of ACh. ACh had the expected effect on membrane current at potentials more positive than EK; it caused an outward shift in current that faded with time as a result of desensitization as in the previous series of action potential-clamp experiments. ACh also had little effect on current near EK. However, at potentials more negative than EK, early during the application of ACh there was an inward shift in current, but later during the application an outward shift in current occurred. The two different effects of ACh can be more clearly seen in Fig. 9B, which shows current-voltage relationships of the ACh-dependent current. The ACh-dependent current was calculated by subtracting the current under control conditions from the current in the presence of ACh. Early during the application of ACh, the current-voltage relationship of the ACh-dependent current is characteristic of an inward-rectifying K+ current such as IK,1, and the current reversed close to the suspected value of EK (see DISCUSSION). However, later during the application of ACh the current-voltage relationship was greatly altered, and the ACh-dependent current was outward at potentials both more negative and more positive than EK.


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Fig. 9.   Effect of 10 µM ACh on membrane current during ramp clamps. A: current-voltage relationships of total membrane current obtained from ramp clamps before (Con) and early (2 s) and late (>15 s) during an application of 10 µM ACh. B: current-voltage relationships of the ACh-dependent current early and late during the application of ACh. ACh-dependent current was obtained by subtracting current under control conditions from current after the application of ACh. C and D: same as A and B, but from a different cell. In addition, D shows the current-voltage relationship of ACh-dependent current in the presence of 2 mM Ba2+. Current in the presence of Ba2+ only was subtracted from current in the presence of ACh and Ba2+.

In some cells, ACh caused an outward shift of current at potentials more negative than EK both early and late during the ACh application. An example is shown in Fig. 9C. At potentials above EK, ACh caused the expected outward shift in current that faded with time. At potentials below EK, ACh also caused an outward shift in current; the outward shift in current increased with time. Current-voltage relationships for the ACh-dependent current are shown in Fig. 9D; the current-voltage relationship had a U-shape both early and late during the ACh application. Figure 9D also shows a current-voltage relationship for ACh-dependent current in the presence of Ba2+. The cell was exposed to 2 mM Ba2+ and subsequently to ACh and Ba2+. The current shown was obtained by subtracting current in the presence of Ba2+ only from current after the application of ACh and Ba2+. Ba2+ abolished the ACh-dependent outward current at potentials both negative and positive to EK. The U-shape of the current-voltage relationship of ACh-dependent current was observed in four cells. Ba2+ block of the current was observed in four cells.

A possible explanation of the U-shape of the current-voltage relationship of the ACh-dependent current is that ACh, as well as activating IK,ACh, inhibits IK,1. This would explain why ACh has little effect on the membrane current close to EK. According to this hypothesis, the outward shift of current at potentials positive to EK is the activation of IK,ACh, whereas the outward shift at potentials negative to EK is the inhibition of IK,1. Why the activation of IK,ACh should dominate at potentials positive to EK and the inhibition of IK,1 should dominate at potentials negative to EK is considered below.

Computer model of the activation of IK,ACh and inhibition of IK,1 by ACh. The consequences of the simultaneous activation of IK,ACh and inhibition of IK,1 by ACh were investigated in a modified Oxsoft HEART model of the ventricular action potential (see MATERIALS AND METHODS for details). Figure 10A shows calculated current-voltage relationships for IK,ACh, and Fig. 10B shows calculated current-voltage relationships for IK,1; on application of ACh, it was assumed that IK,ACh was activated (maximum current at positive potentials, 1 nA) and IK,1 was inhibited by 75%. Figure 10C shows calculated current-voltage relationships for IK,ACh plus IK,1. These are assumed to be roughly equivalent to the current-voltage relationships in Fig. 9, A and C. As observed experimentally, on application of ACh, the current was shifted in the outwards direction at potentials both positive and negative to EK. The current-voltage relationship of the ACh-dependent current had the characteristic U-shape (cf. Figs. 10D and 9, B and D). The reason why the activation of IK,ACh dominates at potentials positive to EK and the inhibition of IK,1 dominates at potentials negative to EK is further considered in the DISCUSSION.


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Fig. 10.   Computer simulation of the action of ACh on the ferret ventricular AP incorporating activation of muscarinic K+ current (IK,ACh) and inhibition of background inward rectifier K+ current (IK,1). A and B, current-voltage relationships for IK,ACh (A) and IK,1 (B) under control conditions and after the application of ACh. Data were calculated using equations taken from the Oxsoft HEART model of the ventricular AP. ACh was assumed to activate IK,ACh (maximum current, 1 nA) and decrease IK,1 by 75%. C: current-voltage relationships for "total" membrane current (IK,1 plus IK,ACh) under Con conditions and after the application of ACh. Total current was calculated as the sum of the individual currents in A and B. D: current-voltage relationship of the ACh-dependent current. Current under Con conditions was subtracted from current after the application of ACh. E: membrane potential recorded from a ferret ventricular cell before (trace i) and after (traces ii and iii) the application of 1 µM ACh. Stimulus current is shown below. AP are shown in traces i and iii, but the stimulus-evoked depolarization only is shown in trace ii. F: membrane potential from a modified version of the Oxsoft HEART model of the ventricular AP before (trace i) and after (traces ii and iii) the application of ACh. Stimulus current is shown below. Action potentials are shown in traces i and iii, but the stimulus-evoked depolarization only is shown in trace ii. In trace ii IK,ACh was activated only (maximum current, 1 nA), whereas in trace iii IK,1 was inhibited by 75% in addition to the activation of IK,ACh.

A clue concerning the possible physiological importance of the inhibition of IK,1 is provided by the observation in Fig. 10E. Figure 10E shows action potentials recorded from a ferret ventricular cell. Although under control conditions a -1.5 nA stimulus was sufficient to trigger an action potential (Fig. 10E, trace i), after application of 1 µM ACh the stimulus was no longer sufficient and a stimulus-evoked depolarization (Fig. 10E, trace ii) only was seen. When the stimulus was increased to -1.8 nA, an action potential (shortened compared with the control) was again triggered (Fig. 10E, trace iii). This observation, which was made in other cells, demonstrates that in the presence of ACh there was a decrease in excitability. Figure 10F shows simulated action potentials from the modified Oxsoft HEART model. In Fig. 10F, a -1.55 nA stimulus was used to trigger a control action potential (Fig. 10F, trace i). After activation of IK,ACh (maximum current, 1 nA), the stimulus was no longer suprathreshold and a stimulus-evoked depolarization (Fig. 10F, trace ii) only was seen. This demonstrates that, in the model at least, it is the activation of IK,ACh that depresses excitability (in the model, the stimulus threshold was increased from -1.15 to -1.80 nA after activation of IK,ACh). After the activation of IK,ACh, if the stimulus was increased to -1.80 nA, an action potential was again triggered, but it was short (not shown). Figure 10F, trace iii, shows that when IK,1 was decreased by 75% (IK,ACh was still activated), the -1.55 nA stimulus was again able to trigger an action potential. Thus the simultaneous inhibition of IK,1 was able to restore excitability (the stimulus threshold fell to -1.55 nA). In conclusion, it is proposed that the activation of IK,ACh depresses excitability of ventricular cells. This is presumably undesirable. It is suggested that the effect of ACh on excitability is blunted as a result of the twin actions of ACh on IK,ACh and IK,1. By the way, in the model the activation of IK,ACh resulted in the shortening of the action potential (Fig. 10F), and the simultaneous inhibition of IK,1 had little antagonistic effect on the shortening. This proposal is considered further in the DISCUSSION.

Adenosine. In addition to activation by ACh, IK,ACh is known to be activated by other agonists, including adenosine (4). Adenosine has a negative inotropic effect on the ferret ventricle (16). To test whether the conclusions of the present study are restricted to ACh, we investigated the inotropic effect of 100 µM adenosine using the action potential-clamp technique. Figure 11 shows three experiments carried out on the same ferret ventricular cell. Figure 11A shows the typical effect of 100 µM adenosine under current-clamp conditions. On application of adenosine there was a shortening of the action potential accompanied by a decrease in contraction, which can be seen in both the slow and fast time base records. In eight cells, 100 µM adenosine caused a maximal reduction of 42 ± 4% in contraction under current-clamp conditions (Fig. 2, bar iv). Like that of ACh, the effect of adenosine faded with time (Fig. 11A). Figure 11B shows an experiment in which a control action potential, recorded under current-clamp conditions, was used as the voltage-clamp command waveform. In this case, application of adenosine had no observable effect on contraction. In four cells under action potential-clamp conditions, adenosine caused a 5 ± 4% reduction in contraction (Fig. 2, bar vi). The current-voltage relationship of the adenosine-activated current was the same shape as the ACh-activated current (Fig. 3C) and as expected of IK,ACh (not shown). The negative inotropic effect of adenosine was simulated in the same way as before for ACh (Fig. 4). Figure 11C shows that switching from current-clamp to voltage-clamp mode, using the shortest action potential under current-clamp conditions in the presence of adenosine (Fig. 11A) as the voltage-clamp command waveform, caused a decrease in contraction comparable to the decrease in Fig. 11A (once again there was no fade in the effect). In five cells, application of the shortened action potential command waveform caused a 45 ± 5% reduction in contraction (Fig. 2, bar v).


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Fig. 11.   Effect of 100 µM adenosine. Left: slow time base records of cell length (top) and APD (bottom). Right: fast time base records of AP s or action potential command waveforms (top) and accompanying contractions (bottom) at the times indicated in left. In A, the cell was under current clamp and 100 µM adenosine was applied for 2 min as indicated by the bar. In B, the cell was under APClamp control throughout. A control action potential recorded under current-clamp conditions was used as the APClamp command waveform. Adenosine (100 µM) was applied for 2 min as indicated by bar. In C, control was switched from current clamp to voltage clamp for 2 min as indicated by the bar. Shortest AP in the presence of adenosine (under current-clamp conditions, labelled trace ii in A) was used as the APClamp command waveform. All data are from the same cell.

Detection of Kir3.1 mRNA in ventricle. The electrophysiological experiments above suggest that the inotropic effect of ACh (and adenosine) on ferret ventricular cells is principally the result of the activation of IK,ACh. The muscarinic K+ channel is known to be a heteromultimer of Kir 3.1 and Kir 3.4. Northern blot analysis of Kir3.1 mRNA expression in the rat and guinea pig heart has previously shown expression of Kir3.1 in the atria in the two species, but not in the ventricles (26). Because of this conflict, we have reexamined the issue of whether there is expression of Kir3.1 in the ferret ventricle. Total RNA from the ferret atrium, ferret ventricle, and dog ventricle was purified to yield mRNA from which cDNA was generated by the RACE technique. With use of Kir3.1-specific oligonucleotide primers (see MATERIALS AND METHODS), the Kir3.1 sequence was amplified by PCR. Figure 12E shows PCR products subjected to agarose gel electrophoresis and shows that Kir3.1 was detected in the ferret ventricle as well as the atrium. Kir3.1 was also detected in the dog ventricle (Fig. 12E). We have previously obtained electrophysiological evidence of the presence of IK,ACh in the dog ventricle (39). In the case of the ferret ventricle, the PCR product was cloned into pCR2.1 vector, and the sequence was determined to confirm that it was Kir3.1.


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Fig. 12.   Detection of Kir3.1 and Kir3.4 protein by immunocytochemistry and confocal microscopy and detection of Kir3.1 PCR products by agarose gel electrophoresis. A-D: Kir3.1 (A, C) and Kir3.4 (B, D) protein labeling in ventricular (A, B) and atrial (C, D) cells. A-D are at same scale (D, 56 µm wide). E: Kir3.1 sequences amplified by PCR from cDNA obtained from various cardiac tissues: lanes 1 and 2, ferret atrium; lanes 3 and 4, ferret ventricle; lanes 5 and 6, dog ventricle.

Detection of Kir3.1 and Kir3.4 proteins in ventricular and atrial cells. The muscarinic K+ channel protein Kir3.1 was not detected by immunocytochemistry in tissue sections through the ferret ventricle (Kir3.4 not tested). Immunocytochemistry is more sensitive when applied to isolated cells compared with tissue sections, because in isolated cells there is better access of the antibodies to the antigenic sites. Figure 12, A and B, shows ventricular cells labeled for Kir3.1 or Kir3.4. Both Kir3.1 (Fig. 12A) and Kir3.4 (Fig. 12B) labeling was observed (although it was weak) and was absent from the outer cell membrane and was present in the t tubules. Similar results were obtained from six Kir3.1 and seven Kir3.4 ventricular cells. Kir3.1 was detected in tissue sections through the ferret atrium. Figure 12, C and D, shows atrial cells labeled for Kir3.1 or Kir3.4. Both Kir3.1 (Fig. 12C) and Kir3.4 (Fig. 12D) labeling were strong and present in the outer cell membrane (including the intercalated disks). Similar results were obtained from 10 Kir3.1 and 10 Kir3.4 atrial cells. In summary, the immunocytochemical data show low expression of both Kir3.1 and Kir3.4 proteins in the ventricle and high expression of both Kir3.1 and Kir3.4 proteins in the atrium.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study has proven that the negative inotropic effects of ACh and adenosine are the result of the action potential shortening in the presence of the agonists. It has also suggested that ACh activates IK,ACh and inhibits INaCa and IK,1. The first two effects cause the action potential shortening, whereas the third may preserve cell excitability.

Presence of Kir3.1 in the ventricle. This study and previous studies (18, 24, 31, 39) have provided electrophysiological evidence of IK,ACh in ferret, rat, human, cat, guinea pig, and dog ventricular cells. Previously, a Northern blot by Kubo et al. (26) failed to show the presence of Kir3.1 mRNA in the rat ventricle. However, the PCR technique is a more sensitive test because it amplifies cDNA derived from low-abundance mRNAs. Using the PCR technique, we were able to show the presence of Kir3.1 mRNA in the ferret and dog ventricle (Fig. 12E). However, the presence of Kir3.1 mRNA in the ferret ventricle does not prove that the Kir3.1 protein is present in ferret ventricular cells and for this reason we examined Kir3.1 and Kir3.4 protein expression in ferret ventricular cells using immunocytochemistry. Labeling of Kir3.1 and Kir3.4 was detected in the t tubules of ferret ventricular cells (Fig. 12). It is interesting that other channels (alpha 1C, Task-1, Kv4.2, Kir2.1) are also found in the t tubules of ventricular cells (11, 21, 32, 38).

Agonist-induced changes in membrane current. ACh activated an outward current during a control action potential command waveform (Fig. 3B). This current may be IK,ACh for several reasons. First, it was activated by ACh. Second, the ACh-activated current displayed a typical current-voltage relationship of IK,ACh- (Figs. 3C and 8B). IK,ACh shows inward rectification, but the degree of rectification of IK,ACh is less than that of, for example, IK,1 (34, 41); this is illustrated by the calculated data in Fig. 10, A and B. The degree of rectification of the ACh-activated current (Figs. 3C and 8B) is typical of that of IK,ACh. Third, the ACh-activated current was blocked by 2 mM Ba2+ (Fig. 8). Ba2+ at 2 mM is known to block IK,ACh fully (40). Fourth, during an application of ACh, the ACh-activated outward current faded with time (Fig. 3B). Fade during an exposure to ACh is a characteristic feature of IK,ACh and is the result of desensitization to ACh (e.g., Ref. 41). Finally, the reversal potential of the ACh-activated current was close to EK. Using the ramp clamp, in one cell at least, we were able to observe reversal of the ACh-activated current close to the suspected value of EK early during an application of ACh (Fig. 9B).

In all cells studied, ACh eventually inhibited inward current at potentials negative to EK (Fig. 9). ACh has a similar action in dog left ventricular subepicardial cells (39). This may be the result of inhibition of IK,1. In support of this, the ACh-induced change in current was abolished in the presence of 2 mM Ba2+ (Fig. 9D); IK,1 is known to be abolished under these conditions (9). The voltage dependence of the ACh-induced changes in membrane current (Fig. 9, B and D) can be explained by a dual effect of ACh on IK,ACh and IK,1. At approximately -80 mV, there was little change in membrane current; this is expected, because this potential is close to EK and at this potential both IK,ACh and IK,1 are small and, furthermore, it is possible that at potentials near EK any effects of ACh on IK,ACh and IK,1 are equal and opposite. At potentials positive to IK, the results indicate that activation of IK,ACh was more important than inhibition of IK,1. The likely reason for this is shown by Fig. 10, A-D. IK,1 shows greater inward rectification than IK,ACh over this range of potentials. For example, at plateau potentials at ~0 mV, IK,1 may be negligible, whereas IK,ACh, although it rectifies at more positive potentials, does not decline below a maximum value (34, 40). Therefore, at the more positive potentials a decrease in IK,1 will have little or no effect on total current, whereas the activation of IK,ACh will. At potentials negative to EK, the results indicate that the activation of IK,ACh was less important than the inhibition of IK,1, and the likely reason for this result is again shown by Fig. 10, A-D. At these potentials, IK,1 steeply increases with hyperpolarization, and Fig. 10, A-D, suggests that it is greater in amplitude than IK,ACh. Therefore, the change in total current may be dominated by the decrease in IK,1 at these potentials. Figure 10, E and F, suggests a possible reason for the simultaneous inhibition of IK,1 by ACh: it may blunt the depression of excitability caused by the activation of IK,ACh. Excitability will be determined by the slope conductance at the resting potential (close to EK). Figure 10A shows that the activation of IK,ACh alone will result in a large increase in the slope conductance and thus decrease in excitability. When the slope of the current-voltage relationship of the control IK,1 at IK (this will largely determine the slope conductance under control conditions) is compared with that of IK,ACh (after activation of IK,ACh), the two are comparable, which shows that activation of IK,ACh alone will result in a large fractional increase in the slope conductance at the resting potential. The central panel of Fig. 10F confirms that in the model, at least, activation of IK,ACh does result in a decrease in excitability. On the other hand, Fig. 10C shows that the simultaneous activation of IK,ACh and inhibition of IK,1 results in little change in the slope conductance near EK. In this way, the simultaneous inhibition of IK,1 blunts the depression of excitability; in the model, this is confirmed by Fig. 10F.

When a short ACh action potential command waveform was applied, an inward current was recorded (Fig. 5). During an application of ACh, the ACh-activated outward current in Fig. 3B must be able to overcome this current to be able to shorten the action potential (the two currents are expected to be roughly equal and opposite). Strict comparison of the currents in Figs. 3C and 5C is not possible, because they were recorded from different cells and during different action potential command waveforms, but the two currents are roughly equivalent in amplitude. It is well known that after a short square wave voltage-clamp pulse, an inward tail current is recorded (e.g., Ref. 15). The inward tail current is analogous to the current in Fig. 5B. It is also well known that a substantial fraction of the inward tail current after a square wave voltage-clamp pulse is inward INaCa triggered by the Ca2+ transient. Figure 6 shows that this is also likely to be true for the inward current recorded during the short ACh action potential command waveform: after the suppression of the Ca2+ transient by BAPTA-AM, the inward tail current was greatly diminished. Figures 5B and 6A show that during repetitive application of the ACh action potential command waveform, the inward current diminished and Fig. 6B suggests that it was the result of the expected reduction in the underlying Ca2+ transient.

Figures 5C and 6A show that during repetitive application of the ACh action potential command waveform, there was a reduction in a transient outward current. Figure 6B suggests that this too was a consequence of the expected reduction in the Ca2+ transient. Janvier et al. (20) showed that the suppression of the Ca2+ transient in ferret ventricular cells by BAPTA-AM resulted in the loss of a transient outward current as well as a transient inward current. Whereas the transient inward current exhibited the properties of inward INaCa triggered by the Ca2+ transient, the transient outward current exhibited the properties of Ca2+-activated Cl- current triggered by the Ca2+ transient. The time course and amplitude of the transient outward current in the study of Janvier et al. (20) is very similar to those of the transient outward current in the present study (Figs. 5C and 6A). It is suggested that in the present study during the application of the ACh action potential command waveform, a reduction in the Ca2+ transient resulted in a reduction in the Ca2+-activated Cl- current.

Figure 7 shows a Ba2+-sensitive current during the action potential. Inward rectifying K+ currents are most sensitive to Ba2+ and, therefore, the Ba2+-sensitive current is likely to be IK,1. Furthermore, the current-voltage relationship of the Ba2+-sensitive current shows the marked inward rectification characteristic of IK,1 (Fig. 7C) (34).

Agonist-induced changes in the action potential. The shortening of the ferret ventricular action potential in the presence of ACh and adenosine is likely to be primarily the result of the activation of IK,ACh. The current-voltage relationship of IK,ACh (Figs. 3C, 8B, and 9) indicates that over the range of potentials during the action potential plateau, a significant outward current flows that will tend to shorten the action potential. The fade of the action potential shortening in the presence of both agonists (Figs. 1A and 11A) can be explained by IK,ACh, because IK,ACh faded with time during application of both agonists (Fig. 3 and unpublished observation). It is likely that the density of the muscarinic K+ channel is less in the ventricle than the atrium, and it is interesting that despite this, the effects on the ventricular action potential are still substantial. A reduction in ICa,L may also explain some of the action potential shortening, although in dog ventricular cells 10 µM ACh only caused an 8% reduction in ICa,L at a holding potential of -80 mV (39). The results in Figs. 5 and 6 suggest that on application of ACh the reduction of the Ca2+ transient underlying the reduction of the contraction leads to an indirect reduction in inward INaCa, and this is also expected to contribute to the shortening of the action potential. It is already known that suppression of inward INaCa by buffering intracellular Ca2+ by BAPTA-AM leads to a shortened action potential (20) (see also the control action potential and the action potential after the application of BAPTA-AM in Fig. 6). The inhibition of IK,1 by ACh may be expected to antagonize the shortening of the action potential caused by ACh. However, in the model, the simultaneous inhibition of IK,1 had little effect on the shortening of the action potential caused by IK,ACh (Fig. 10F). This is because at plateau potentials there is little or no IK,1 (Fig. 10B).

Agonist-induced changes in contraction. In ferret ventricular cells, the negative inotropic effect of ACh and adenosine is likely to be primarily the result of the shortening of the action potential. With the use a control action potential waveform as the voltage-clamp command, the negative inotropic effect of ACh and adenosine was almost abolished (Fig. 2). Furthermore, use of the agonist-shortened action potential as the voltage-clamp command waveform caused a reduction in contraction comparable to that observed on application of the agonist under current-clamp conditions (Fig. 2). With both agonists there could be a small reduction in contraction when the cell was under action potential-clamp control (Fig. 2). This may be the result of a small reduction in ICa,L by the agonists. The agonist-induced shortening of the action potential is expected to lead to a reduction in the Ca2+ transient and thus the contraction by reducing the Ca2+ content of the sarcoplasmic reticulum by: 1) promoting Ca2+ efflux on the Na/Ca exchanger and 2) reducing Ca2+ influx via ICa,L (because of the shortened action potential, ICa,L flows for a shorter time). Direct evidence of the stimulation of Ca2+ efflux via the Na/Ca exchanger is the inward current measured on application of the ACh action potential command waveform (Figs. 5B and 6A). The evidence that this is partly composed of inward INaCa (corresponding to Ca2+ efflux) has already been discussed.

During an application of ACh or adenosine, the slow increase in contraction under current-clamp conditions (Figs. 1A and 11A) can be explained by fade of the action potential shortening (primarily the consequence of the fade of IK,ACh as a result of desensitization) or a fade of a decrease in ICa,L (39). The slow increase in contraction during an application of ACh has also been suggested to be the result of a slow increase in intracellular Na+ (23). During action potential clamp the small inotropic effect faded with time (Fig. 1B), and this at least could not have been the result of a change in action potential duration.

In summary, it is concluded that the negative inotropic effects of ACh and adenosine are primarily the result of the activation of IK,ACh and the consequent shortening of the action potential. The reduction in the intracellular Ca2+ transient (underlying the decrease in the contraction) causes a reduction in inward INaCa, and this also contributes to the action potential shortening. A small reduction in ICa,L may also play a role in the negative inotropic effect. ACh inhibits IK,1 and this may blunt a reduction in cell excitability following activation of IK,ACh. Although the present study was carried out in the ferret, the results are unlikely to be specific to the ferret, because, in ventricular cells from a range of species, IK,ACh has been recorded and ACh shown to have a negative inotropic effect in the absence of beta -stimulation (5, 18, 24, 30, 31, 39).


    ACKNOWLEDGEMENTS

We thank Luke Blumler, Andy O'Brien, and Dave Johannson for excellent technical assistance.


    FOOTNOTES

This work was supported by grants from the British Heart Foundation and the Wellcome Trust. N. C. Janvier was a Wellcome Prize student.

Address for reprint requests and other correspondence: M. R. Boyett, School of Biomedical Sciences, Univ. of Leeds, Leeds LS2 9JT, UK (E-mail: m.r.boyett{at}leeds.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

April 25, 2002;10.1152/ajpheart.00130.2002

Received 21 February 2002; accepted in final form 12 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arreola, J, Dirksen RT, Shieh R, Williford DJ, and Sheu S. Ca2+ current and Ca2+ transients under action potential clamp in guinea pig ventricular myocytes. Am J Physiol Cell Physiol 261: C393-C397, 1991[Abstract/Free Full Text].

2.   Berger, SL, and Kimmel AR. Guide to Molecular Cloning: Methods in Enzymology. Orlando, FL: Academic, 1987