INTRODUCTION

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MUSCARINIC-RECEPTOR (mAChR) agonists inhibit the heart via a signal transduction mechanism that requires pertussis toxin (PTX)-sensitive G proteins (reviewed in Ref. 21). In the presence of PTX, agonist occupancy of the mAChR stimulates the rate and force of heart contractions when applied at concentrations greater than those that cause inhibition (21). However, PTX treatment is not essential to reveal stimulant effects. In guinea pig ventricular muscle, choline ester muscarinic agonists exert a positive inotropic effect in the absence of PTX treatment (10). In single guinea pig ventricular myocytes, carbachol (CCh) induces a tetrodotoxin (TTX)-resistant Na⁺ current (I_Na) (15) and increases the extent of cell contractions (22). These effects of muscarinic agonist on membrane current and cell contraction in ventricular myocytes also do not require PTX treatment.

Muscarinic agonists per se do not increase the L-type Ca²⁺ current [I_Ca(L)] that initiates excitation-contraction coupling in heart cells (reviewed in Ref. 17). In the absence of PTX, CCh increased phasic contractions without increasing I_Ca(L) in electrically stimulated guinea pig ventricular myocytes (22). Therefore, the increase in contractions by muscarinic agonists is achieved by another mechanism. In guinea pig ventricular myocytes, muscarinic agonists increased the intracellular activity of Na⁺ (aⁱ_Na) (10). This effect, attributed to Na⁺ influx through TTX-resistant Na⁺ channels (15), could increase the subsarcolemmal Na⁺ concentration and promote reverse-mode Na/Ca exchange. In quiescent rat ventricular myocytes, CCh increased intracellular Ca²⁺ activity secondary to the increase in aⁱ_Na (11). Although the Na/Ca exchanger has been suggested to participate in the ionic changes caused by CCh (11), there is no direct evidence for this mechanism in stimulated myocytes. If the CCh effect in stimulated myocytes also includes an action through the Na/Ca exchange, it should be possible to detect this by measurement of the exchange current (I_Na/Ca).

The I_Na/Ca has been measured under voltage-clamp conditions in which it has been related to contractions (reviewed in Ref. 20). In our laboratory, I_Na/Ca was identified in guinea pig ventricular myocytes in several ways (4). Depolarization to 40 mV from a more negative holding potential evokes an early inward tail current evident after I_Na decay. The early inward tail current was not a slowly inactivating component of I_Na. Repolarization to 40 mV from more positive potentials evokes a late inward tail current. The early and late inward tail currents require external Na⁺ and internal Ca²⁺ as expected for I_Na/Ca. Furthermore, the early and late tail currents are suppressed by agents (ryanodine, caffeine) that interfere with sarcoplasmic reticulum (SR) Ca²⁺ content and are augmented by inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], a Ca²⁺-mobilizing messenger (4). Altogether, the results are consistent with a model in which entering Na⁺ that presumably accumulates in a subsarcolemmal space (2, 14, 32) is exchanged with extracellular Ca²⁺ that enters the SR and thereby increases its content. The importance of subsarcolemmal Na⁺ for the regulation of SR function via Na/Ca exchange has been described (12, 19). In view of the results obtained by others (10, 11) and Matsumoto and Pappano (15), one might expect that the muscarinic agonist increases I_Na/Ca in heart cells. Furthermore, there is reason to suppose that the depolarization-induced changes in I_Na/Ca involve an interaction between the plasma membrane and the SR membrane. The present experiments were carried out to test the hypotheses that CCh augments the I_Na/Ca in guinea pig ventricular myocytes and that this involves the SR. A preliminary account of some of these data has been presented in abstract form (22).

	METHODS

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Cell isolation. Hearts were rapidly excised from adult guinea pigs of either sex (300-500 g) that had been anticoagulated with heparin (1,000 U) and anesthetized with pentobarbital sodium (30 mg/kg) administered intraperitoneally. The heart was perfused with oxygenated Tyrode solution (37°C) for 5-10 min at a rate of 8-10 ml/min through the aorta in a Langendorff apparatus. The composition of Tyrode solution was (in mM) 135 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.0 MgCl₂, 0.33 NaH₂PO₄, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 20 dextrose (pH 7.4 adjusted with NaOH). The enzymatic dissociation procedure was essentially the same as reported previously (25). Enzyme-containing solution was washed out with a recovery solution composed of (in mM) 130 K aspartate, 5 K₂ATP, 5 HEPES, and 20 dextrose (pH 7.4 adjusted with KOH). After the perfusion was completed and the ventricles were removed, the cells were isolated by gentle mechanical agitation and kept in the recovery solution at 4°C for at least 1 h. An aliquot of the cell suspension was placed in a 500-µl chamber mounted on the stage of an inverted microscope. After a 5- to 10-min settling period, the cells were superfused with Tyrode solution (dextrose was reduced to 10 mM) at 2-4 ml/min. All the experiments were done at 35 ± 1°C.

Electrophysiological techniques. The whole cell patch-clamp technique was used for the experiments. Electrodes, prepared from borosilicate glass capillaries, had resistances of 1-3 M when filled with a pipette solution composed of (in mM) 120 K aspartate, 30 KCl, 5 Na₂ATP, 5 HEPES, and 1 MgCl₂ (pH 7.3 adjusted with KOH). The pipette was connected through an Ag-AgCl wire to the head stage of a List EPC-7 amplifier (Medical Systems, Greenvale, NY). After a gigaohm seal between the membrane and the pipette tip was formed, the cell membrane was ruptured by additional negative pressure. Voltage-clamp protocols were generated by pClamp software (Axon Instruments, Foster City, CA), and membrane currents were stored for later analysis. The protocols are described in RESULTS.

The amplitudes of the early and late inward tail currents were obtained by subtracting the steady current at the end (200 ms) of the test potential (I_control) from that detected at 20 ms after the inactivation of the rapid I_Na or the deactivation of the I_Ca(L), respectively. In some illustrations, the change in the tail current produced by CCh (I_CCh) or other agents is shown by the difference current (I_diff = I_CCh  I_control).

Cell contraction. Contractions of single myocytes were evoked by a train of voltage-clamp pulses (75 to 30 mV for 200 ms) at 1 Hz. Because the cell shortens along its long axis, the displacement of one or both ends of the cell edge is an indicator of the extent of cell contraction. A video edge-detector system (Crescent Electronics, Sandy, UT) tracked cell edge movement. The cell image (magnified ×400) was continuously observed on a high-resolution black-and-white television monitor via a sequential scanning video camera attached to a side port of the microscope. The camera position can be rotated to bring the video monitor raster lines parallel with the long axis of the cell. The temporal resolution of this detector is 16.7 ms, and motion as little as 0.1 µm can be detected. The signal from the detector is sent to a strip-chart recorder (Gould 2000) and to a videocassette recorder for storage and analysis.

Intracellular Ca²⁺ transients. The apparatus for measuring intracellular Ca²⁺ (Ca²⁺_i) is built around a Zeiss inverted microscope with a PTI Deltascan system. For this, ultraviolet light at 340 and 380 nm from a 75-W xenon arc lamp is selected by monochromators and alternately passed via a quartz fiber-optic bundle to the epifluorescence port of the microscope. Excitation light is transmitted through a dichroic mirror (410 nm) and ×40 fluor objective (Nikon) onto a myocyte in the test chamber loaded with fura 2 (50 µM, pentapotassium salt) for 5-10 min by dialysis from the patch pipette. Fluorescence emitted from a cell region passes through the objective and a 510-nm filter to a photomultiplier tube. Signals from the photomultiplier tube are digitized and stored for later analysis. Ca²⁺_i is derived from the ratio of the fluorescence signals at the two excitation wavelengths, and its concentration is estimated from an in vitro calibration of a thin film of buffered Ca²⁺ solutions. The fluorescence ratio is corrected for background autofluorescence, which is recorded with the patch pipette sealed to the membrane but before patch rupture.

	RESULTS

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Effects of CCh on plateau currents. In this series of experiments (n = 4), the membrane was clamped at a holding potential of 45 mV that inactivated I_Na and the T-type Ca²⁺ current. The membrane was depolarized to +20 mV for various durations at 0.1 Hz; records of a representative experiment in the absence and presence of CCh are shown in Fig. 1A, top and middle, respectively. Although CCh had no effect on the peak inward current through L-type Ca²⁺ channels, current during the pulse shifted in an outward direction, and the tail current on repolarization to 45 mV increased. The CCh-induced current, obtained by digital subtraction, is a declining outward current during the voltage jump to +20 mV followed by an increased inward tail current elicited on repolarization to 45 mV (Fig. 1A, bottom). This pattern of currents is very similar to that described for I_Na/Ca by others (8). The results indicate that 100 µM CCh promotes both the forward and reverse modes of I_Na/Ca in guinea pig ventricular myocytes. In this regard, ouabain was also tested because its inhibition of the Na-K pump raises subsarcolemmal Na⁺ and increases I_Na/Ca. Results typical of four experiments with ouabain are shown in Fig. 1B. Compared with control experiment (Fig. 1B, top), ouabain (Fig. 1B, middle) induced an outward current during depolarization and increased the amplitude of the inward tail current on repolarization to 45 mV. The difference currents for ouabain (Fig. 1B, bottom) and CCh (Fig. 1A, bottom) are quite similar.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Time dependence of carbachol (CCh)-induced current (A) compared with that of ouabain-induced current (B) recorded in another cell. CCh (100 µM) or ouabain (1 µM) increased amplitude of late inward tail current. Test pulses to +20 mV from a holding potential of 45 mV were applied at durations of 10, 50, 90, 130, and 170 ms (A) and 15, 50, 85, 120, and 155 ms (B). Inset: protocol for clamp pulses (t) that were applied every 10 s. A: top, control; middle, after 2 min in CCh; bottom, difference current (middle  top). B: top, control; middle, after 3 min in ouabain; bottom, difference current (middle  top). Short horizontal lines at left, zero-current level.

The voltage dependences of the plateau current in the absence and presence of CCh are shown in Fig. 2. The membrane was depolarized to various potentials ranging from 40 to +50 mV in 10-mV steps for 25 ms at 0.2 Hz in the absence and presence of 100 µM CCh (Fig. 2A). The CCh-induced difference currect was obtained by digital subtraction (Fig. 2A'). CCh (100 µM) reversibly increased both the outward current during the depolarizing pulse and the inward tail current elicited on repolarization to 45 mV. Similar results were obtained with 1 µM ouabain (Fig. 2, B and B'). Inspection of the current-voltage relationships (n = 4 cells) for the current during the depolarizing test pulse (Fig. 2C,a) and after repolarization (Fig. 2C,b) indicated that their voltage dependence was similar to that of I_Na/Ca (5, 8). The voltage dependence of the CCh-induced difference current during and after the depolarizing pulses is given in Fig. 2C,c. These results were not obtained when the pipette solution contained 10 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) because I_Na/Ca was absent under this condition (n = 3 experiments; data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Voltage dependence of CCh-induced current (A) compared with ouabain-induced current (B) from superimposed records of membrane currents elicited by a 25-ms depolarization to +50 mV from a holding potential of 45 mV in absence and presence of 100 µM CCh and 1 µM ouabain. Inset: pulse protocol that was applied every 5 s. A' and B': difference currents obtained from each experiment. C: voltage dependence of membrane current at end of depolarizing pulse (a), tail current on repolarization to 45 mV (b), and difference current (c) obtained by subtraction of control current () from CCh current (). Amplitude of outward current was measured at end of pulse, and that of inward tail current was obtained by subtraction of current value at 200 ms after repolarization to 45 mV from that at 20 ms. Values are means ± SE; n = 4 experiments.

Effects of CCh on early and late tail currents. We used another protocol to study the Na/Ca exchanger; the procedures are essentially the same as reported previously by others (1, 12) and Gilbert et al. (4). Eight conditioning pulses (80 mV to +30 mV for 200 ms) were applied at 1 Hz to provide a constant SR Ca²⁺ load before each test pulse (Fig. 3A). The test pulse, delivered 1 s after the conditioning pulse train, consisted of a 200-ms step to 30 mV from a holding potential of 80 mV, a second step to +20 mV for 25 ms and sequential repolarizing steps to 30 and 80 mV. The sequence of conditioning plus test pulses was repeated every 10 s. The control responses of a myocyte to test pulses after the first and sixth conditioning trains are depicted in Fig. 3B (trace 1). An early inward tail current of ~280 pA (open arrow) is evident after the decay of I_Na when a myocyte was depolarized from 80 to 30 mV; this current decreased slowly over the next 80-100 ms. A late inward tail current of ~280 pA (solid arrow) occurred on repolarization from +20 to 30 mV. After the sixth conditioning train, the amplitude of the early and late tail currents increased to ~470 pA. CCh (100 µM) increased the early inward tail current after the first and sixth conditioning trains to ~420 and 1,000 pA, respectively (Fig. 3B, trace 2). The I_Ca(L) did not change substantially in the presence of CCh, whereas there was a tendency toward a small increase in the late inward tail current (Fig. 3, B and C). Ten minutes after the washout of CCh, the amplitude of the early inward tail current returned to the control value (Fig. 3, trace 3). The digitally subtracted difference currents (Fig. 3C) show that the increments of the early and late tail currents caused by CCh are greater after the sixth (right) than after the first conditioning train (left).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of CCh on early (open arrows) and late (solid arrows) inward tail currents. A: voltage-clamp protocol consisting of a conditioning pulse train (80 to +30 mV for 200 ms at 1 Hz for 8 s) followed by test pulses that started from a holding potential of 80 mV, a 200-ms step to 30 mV, a second depolarization jump to +20 mV for 25 ms, and sequential repolarization jumps to 30 and 80 mV. B: representative records of transmembrane currents elicited by test voltage-clamp pulse protocol after 1st and 6th conditioning trains. Traces are control (1), after 2-min exposure to 100 µM CCh (2), and 5 min after washout of CCh (3). C: difference current obtained by digital subtraction (trace 2  trace 1). Short horizontal line at left, zero-current level.

In a series of five experiments, the amplitude of the early inward tail current in the control period increased progressively from the first (188 ± 35 pA) through the fifth train (344 ± 40 pA) where it reached maximum. The late inward tail current also increased with conditioning; it averaged 381 ± 30 and 453 ± 37 pA after the first and sixth conditioning periods, respectively. In CCh, the early inward tail current increased to 443 ± 76 pA at the first test pulse and to 662 ± 102 pA at the sixth conditioning train; the difference (I_CCh  I_control) was maximal after the second train. The CCh-induced increase in the early inward tail current was significant (P  0.02) at each test pulse number. In CCh, the late inward tail current averaged 475 ± 31 pA after the first and 486 ± 49 pA after the sixth conditioning periods; the small increase was not statistically significant. The I_Ca(L) during the test pulse after the first conditioning period (2.3 ± 0.37 nA) was not appreciably changed by the sixth conditioning train (2.3 ± 0.36 nA). There was no significant change in I_Ca(L) by CCh because I_Ca(L) averaged 2.3 ± 0.27 and 2.3 ± 0.26 nA after the first and sixth conditioning trains, respectively. In light of previous reports (1, 4, 12), the early and late inward tail currents are I_Na/Ca operating in the forward mode and dependent on release of Ca²⁺ from the SR (see below). The extra Na⁺ influx caused by CCh secondarily increases intracellular Ca²⁺ concentration ([Ca²⁺]_i) via Na/Ca exchange and eventually increases SR Ca²⁺ content. In this regard, the late inward tail current was not significantly increased by CCh, presumably because the Ca²⁺ store of the SR was not fully reprimed after discharge of the early tail current.

The inward tail currents, as expected for I_Na/Ca, depended on [Ca²⁺]_i because neither was observed when 10 mM EGTA was present in the pipette solution (5). Addition of CCh had no effect on the tail currents under this condition (n = 3 experiments; data not shown). We also confirmed that the inward tail current, like I_Na/Ca, depended on the extracellular Na⁺ concentration with experiments in which Li⁺ was substituted for the extracellular Na⁺ (n = 4 experiments; data not shown). Li⁺ permeates voltage-gated Na⁺ channels but is unable to substitute for Na⁺ in the Na/Ca exchanger (1, 4, 5).

Na⁺ influx through fast Na⁺ channels is linked by Na/Ca exchange to SR Ca²⁺ content (27) and/or release (12, 14). Accordingly, the increased Na⁺ entry by CCh through TTX-resistant channels (10, 15, 16) should also share this mechanism. We tested this hypothesis in experiments with ryanodine, which interferes with the SR Ca²⁺-release channel. With the use of the same protocol as in Fig. 3, the initial early I_Na/Ca (Fig. 4, trace 1) increased in the presence of 100 µM CCh (trace 2). Five minutes after the addition of 1 µM ryanodine to the CCh-containing solution (Fig. 4, trace 3), the early I_Na/Ca was less than the initial I_Na/Ca, indicating that not only the CCh effect but also the basal I_Na/Ca had been affected. Similar results were obtained in five other cells. Caffeine, which releases Ca²⁺ from the SR, reversibly eliminated the increases in the early and late I_Na/Ca induced by 100 µM CCh as well as those portions of the currents present before CCh (n = 4 experiments; data not shown). Inhibition of the tail currents by caffeine or ryanodine is consistent with the hypothesis that these tail currents arise from Na⁺ influx through the Na/Ca exchanger and are secondary to release of Ca²⁺ from the SR.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effect of ryanodine (1 µM) on tail currents from conditioning/test voltage-clamp protocol in Fig. 3. Trace 1: control membrane currents in response to clamp protocol illustrating early inward tail current (open arrow) of ~1 nA. Trace 2: after 2 min in 100 µM CCh, early tail current is increased to ~1.4 nA and late tail current (solid arrow) is essentially unchanged. Trace 3: addition of ryanodine on top of CCh reduces early tail current to ~0.4 nA, and late tail current is also decreased.

Concentration dependence and effect of atropine. In the concentration range from 10 to 100 µM, CCh increased the early tail current amplitude by 43 ± 11, 134 ± 17, and 236 ± 32 pA at 10, 30, and 100 µM, respectively (n = 5 cells at each concentration). The pharmacological nature of the CCh effect was evaluated in experiments with atropine. The early inward tail current on the sixth test pulse (Fig. 5A) was increased in the presence of 100 µM CCh (Fig. 5B). The change induced by CCh is shown by the difference current (Fig. 5, B-A) which also indicates an declining outward current during the voltage step to +20 mV and a slightly increased late inward tail current on repolarization. After washout of CCh, 3 µM atropine was applied; the early inward tail current or I_Na/Ca was unaffected by atropine (Fig. 5C). CCh had no effect on either the early or late inward tail current or on the current at +20 mV in the presence of atropine (Fig. 5, D and D-C). Similar results were obtained in five other experiments done with 1-3 µM atropine.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Atropine prevents CCh effect on tail current. Voltage-clamp protocol in Fig. 3 was used; all records were taken from 1 cell. A: control; early (open arrow) and late (solid arrow) tail currents are ~0.5 nA in amplitude. B: CCh (100 µM) increases amplitude of early and late tail currents to ~0.8 and 0.6 nA, respectively. Difference current (B  A) shows magnitude of current changes induced by CCh. C: in presence of 3 µM atropine after washout of CCh, tail currents are at initial control values as in A. D: CCh was added on top of atropine; there is no change produced by CCh as indicated by difference current (D  C).

Effect of CCh on caffeine-induced current. The evidence indicates that Ca²⁺ released from the SR participates in the I_Na/Ca and in the effect of CCh on it. If the action of CCh includes an eventual increase of SR Ca²⁺ stores, the activation of I_Na/Ca would generate a larger current. We tested this hypothesis in experiments with caffeine. Rapid application of this alkaloid with the membrane voltage clamped at the resting potential releases Ca²⁺ from the SR and produces an inward current due to forward-mode Na/Ca exchange (18). The results of an experiment with caffeine are shown in Fig 6. A train of 20 conditioning depolarizing pulses (75 mV holding potential to +30 mV for 200 ms at 1 Hz) was applied every 60 s. One second after the end of the conditioning pulse train, 10 mM caffeine was applied for 1 s. Caffeine induced an inward current of ~500 pA in the absence of CCh (Fig. 6A); this current developed more rapidly and increased to ~740 pA in the presence of CCh (Fig. 6B). Ten minutes after the removal of CCh, the caffeine-induced current returned toward its initial value (Fig. 6C). In eight experiments of this type, the caffeine-induced current averaged 2.5 ± 0.56 pA/pF in control experiments, 3.2 ± 0.65 pA/pF in CCh experiments, and 2.5 ± 0.54 pA/pF after washout. The increase in the caffeine-induced current by CCh was statistically significant (P < 0.01). Integration of the caffeine-induced current gave estimated charge movements of 1.3 ± 0.16 pC/pF in the absence of CCh. The charge movement increased significantly to 1.8 ± 0.24 pC/pF in the presence of CCh (n = 8 experiments; P < 0.01).

View larger version (11K): [in this window] [in a new window]   Fig. 6.   CCh increases amplitude of caffeine-induced current. Protocol consisted of a train of 20 depolarizing voltage-clamp pulses at 1 Hz and a 1-s superfusion with 10 mM caffeine 1 s after last pulse of train (top). A: membrane current during last conditioning pulse followed by caffeine-induced inward current at 75 mV. B: in 100 µM CCh for 2 min. C: 10 min after washout of CCh. Calibrations are 400 pA for caffeine-induced currents and 1 nA for clamp currents.

In another series of experiments, we evaluated the effect of different CCh concentrations on the magnitude of the caffeine-induced current. The increments in the caffeine-induced current averaged 43 ± 11 (n = 5 cells), 125 ± 17 (n = 3 cells), 216 ± 32 (n = 4 cells), and 249 pA (n = 2 cells) in 10, 30, 100, and 300 µM CCh, respectively. No more than two CCh concentrations were tested in one cell. The results illustrate that the effects of the muscarinic agonist on I_Na/Ca, whether induced by caffeine or by depolarizing voltage jumps, occur over the same concentration range.

Effect of CCh on contractions and Ca²⁺_i transients. A train of 200-ms voltage-clamp pulses (75 to +30 mV) was applied at 1 Hz, with a rest interval of 1 min between trains. The protocol was essentially the same as that used in the experiments with caffeine (see Effect of CCh on caffeine-induced current). A representative experiment is shown in Fig. 7A. The first contraction after the rest period had an amplitude of 2.8 µm, and the succeeding contractions displayed a positive staircase such that the steady-state shortening of 6.1 µm on the last contraction in the train was about twice as large as the initial one (Fig. 7A, control). After 5 min in 100 µM CCh (Fig. 7A, CCh), the first contraction after the rest interval was 4.4 µm, which is 1.57 times the control value. A positive staircase ensued, and the last contraction in the train had a displacement of 8.1 µm. The steady-state contraction in CCh was 1.33 times that in the control experiment. Washout of CCh for 6 min was accompanied by a reduction in the contraction amplitude of corresponding contractions to about initial levels (Fig. 7A, washout). In the five experiments in this series, the steady-state contraction in CCh (4.3 ± 1.13 µm) averaged 1.55 ± 0.25 times the control value (P = 0.03). The initial postrest contraction in CCh (3.0 ± 0.77 µm) was 1.44 ± 0.10 times the control value, but the change was not quite statistically significant (0.06 > P > 0.05). Altogether, the results indicate that the positive staircase seen at 1 Hz was preserved in CCh, albeit with a greater steady-state contraction amplitude, as expected for an agonist that increased SR Ca²⁺ content.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   CCh increases contraction amplitude (A), plateau current (B), and intracelluar Ca2+ transient (C) in guinea pig ventricular myocytes. Voltage-clamp protocol was the same as in Fig. 6. A: records taken from 1 myocyte showing staircase response to 1-Hz stimulation in control, after 5 min in CCh, and 10 min after drug removal (washout). B: records taken from another cell. At steady state, membrane current at +30 mV shifts outward in 100 µM CCh () relative to control (left; 200-pA current calibration). Difference current (right; 70-pA current calibration) shows that CCh induced a slowly declining outward current during 200-ms jump to +30 mV. C: intracellular Ca2+ transients recorded from the same cell at steady state in control, after 5 min in 100 µM CCh, and 5 min after washout of agonist.

CCh also increased Ca²⁺_i transients under conditions where contractions were augmented. An example is shown in Fig. 7, B and C, from a myocyte subjected to a train of 21 depolarizations at 1 Hz. In the steady state, the membrane current during a 200-ms voltage jump to +30 mV from a holding potential of 75 mV shifted in the outward direction in the presence of CCh (Fig. 7B, left). The record in Fig. 7B (right) shows the difference current and indicates that the outward current induced by CCh declined during the voltage jump. The Ca²⁺_i transients from this cell are shown in Fig. 7C. From a basal level of 0.11 µM, the Ca²⁺ transient reached a peak of ~1 µM (Fig. 7C, control). After 5 min in CCh, the Ca²⁺ transient rose to ~1.4 µM from an initial level of 0.13 µM (Fig. 7C, CCh). The Ca²⁺_i transient returned to initial levels 5 min after CCh was removed (Fig. 7C, washout). Similar measurements were made in six additional cells. Basal [Ca²⁺]_i averaged 89 ± 15 nM (n = 7 cells) in the absence and 122 ± 33 nM in the presence of 100 µM CCh. The small increase of 33 ± 20 nM did not attain statistical significance (P = 0.16). However, the peak [Ca²⁺]_i increased significantly by 182 ± 65 nM (from the control level of 591 ± 118 to 773 ± 157 nM in CCh; P = 0.03).

	DISCUSSION

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Carbachol augments I_Na/Ca by an indirect action. Matsumoto and Pappano (15, 16) previously reported that CCh induces a TTX-resistant I_Na in guinea pig ventricular myocytes. Induction of this current confirmed a hypothesis for the mechanism by which choline ester muscarinic agonists increased intracellular Na⁺ in guinea pig papillary muscle (10). In this report, we tested the hypothesis that by virtue of the effect on Na⁺ entry and accumulation, CCh should augment I_Na/Ca and that the SR participates in this action. The hypothesis for CCh action is derived from a general scheme advanced by others (12, 19, 20) and that Gilbert et al. (4) confirmed in a study on I_Na/Ca. Entering Na⁺ accumulates in a restricted subsarcolemmal space and provides substrate to drive reverse-mode Na/Ca exchange. Evidence for restricted distribution of intracellular Na⁺ has come from measurements of Na⁺-pump current (2) and from electron probe microanalysis in guinea pig ventricular myocytes (32). Ca²⁺ entering through reverse-mode Na/Ca exchange or retained by the cell as Na⁺ is extruded is taken up by and increases the SR Ca²⁺ content, from which it is released by I_Ca(L). Our hypothesis neither requires nor excludes that Ca²⁺ entering during reverse-mode Na/Ca exchange triggers SR Ca²⁺ release but simply that it increases the Ca²⁺ content of the SR.

Participation of SR in CCh effect on I_Na/Ca. There are several reasons to implicate SR Ca²⁺ stores in the reaction mechanism that is influenced by CCh in addition to the evidence obtained with ryanodine or caffeine. The effect of conditioning with depolarizing voltage-clamp pulses also agrees with the capacity of the SR to increase its store (4, 6, 12). Rapid application of 10 mM caffeine induced an inward current at a holding potential of 75 mV. This current is attributed to forward-mode I_Na/Ca by virtue of Ca²⁺ release from the SR (18). The size of the SR Ca²⁺ store can be estimated from the caffeine-induced charge movement (28). In the absence of CCh, the caffeine-induced charge movement amounted to 1.3 ± 0.16 pC/pF in cells in which the average capacitance was 180 pF (n = 8). If a specific cell capacitance of 10⁶ F/cm² and a surface-to-volume ratio of 5 × 10³ cm¹ are assumed (9), the charge transported by the Na/Ca exchanger involves a Ca²⁺-release flux from the SR of 67 µM. In the presence of CCh, the caffeine-induced charge movement rose to 1.8 ± 0.24 pC/pF, giving an estimated Ca²⁺ flux of 98 µM (an increase of 31 ± 7 µM; P < 0.01). These are underestimates of SR Ca²⁺ content because the calculations neglect contributions of the sarcolemmal Ca²⁺ pump to Ca²⁺ extrusion. The caffeine-induced current of 2.5 ± 0.56 pA/pF is ~36% greater than that reported in guinea pig ventricular myocytes by others (23). The experimental conditions are rather different (pipette Na⁺, temperature, bath composition) so that a strict comparison is not warranted.

Does Ins(1,4,5)P₃ participate in CCh action on I_Na/Ca? Muscarinic agonists increase the synthesis of Ins(1,4,5)P₃, which is able to release Ca²⁺ from the SR in heart cells (4, 29). Ca²⁺ entering through reverse-mode Na/Ca exchange is said to trigger SR Ca²⁺ release (5, 12, 13, 31). Our experiments were not designed to evaluate the function of either messenger, which might serve an ancillary role in the positive inotropic action of CCh because of the interaction between Ins(1,4,5)P₃ and Ca²⁺_i (30). Ca²⁺ entering through reverse-mode Na/Ca exchange, alone or together with Ins(1,4,5)P₃, could sensitize SR Ca²⁺ release and increase the amplitude of I_Na/Ca that occurs with conditioning. If the fraction of SR Ca²⁺ released by Ins(1,4,5)P₃ was smaller than the increment caused by CCh through reverse-mode Na/Ca exchange, SR Ca²⁺ content could still increase but to a lesser extent than expected. This possibility could be tested by including an Ins(1,4,5)P₃-receptor antagonist in the pipette solution. There is evidence against a direct stimulant action of Ins(1,4,5)P₃ on I_Na/Ca. In giant excised membrane patches from guinea pig ventricular myocytes, phosphatidylinositol-4,5-bisphosphate (PIP₂), the precursor of Ins(1,4,5)P₃, promoted I_Na/Ca (7). When PIP₂ was metabolized by its specific phospholipase C to Ins(1,4,5)P₃ or when phospholipase C was activated by elevation of myoplasmic Ca²⁺, I_Na/Ca diminished. Although this does not necessarily mean that Ins(1,4,5)P₃ inhibits I_Na/Ca, it indicates that the preservation of PIP₂ is associated with a larger exchange current.