Introduction

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The vagus nerve innervates all regions of the mammalian heart, and muscarinic acetylcholine receptors (mAChRs) are present throughout (Löffelholz and Pappano, 1985; Hartzell, 1988). The distribution of vagal nerve fibers and mAChR follows a parallel pattern, with nodal tissues receiving the greatest, the ventricles the least, and the atria an intermediate density of each. The functional effects of vagus nerve stimulation or muscarinic drug action seem to follow the pattern of nerve and mAChR distribution. The vagal transmitter acetylcholine (ACh) had a negligible effect on the ventricle until or unless sympathetic nerve-induced stimulation, via cAMP, augmented various cardiac functions. Then, ACh or vagus nerve stimulation inhibited the sympathetic stimulation of contractility, glucose metabolism, and various ionic currents, a phenomenon termed "accentuated antagonism" (Levy, 1971). The chemical basis for this was ascribed to changes in phosphorylation state of proteins as a result of variations in the levels of second messengers, cAMP and cGMP (Neumann et al., 1994).

Acetylcholine also has direct actions on the mammalian ventricle in the absence of sympathetic stimulation. For example, ACh reduced the action potential duration and effective refractory period, and it decreased the extent of ventricular myocyte contractions in subepicardial but not subendocardial cells in the canine ventricle (Antzelevitch et al., 1995; Yang et al., 1996). This direct effect stems largely from activation of an inwardly rectifying K⁺ current [I_K(ACh)] in ventricular myocytes from ferret (Boyett et al., 1988), rat (McMorn et al., 1993), dog (Yang et al., 1996), guinea pig, cat, and humans (Koumi and Wasserstrom, 1994). However, a direct effect of ACh on L-type Ca²⁺ current [I_Ca(L)] may also occur (Antzelevitch et al., 1995; Yang et al., 1996). The results indicate a tissue-specific distribution of muscarinic effect rather than of mAChR inasmuch as subendocardial cells display indirect effects of ACh in the presence of sympathetic stimulation (Antzelevitch et al., 1995).

Although ACh can suppress I_Ca(L) to a small extent in ventricular myocytes, the inhibition becomes greater in the presence of sympathetic stimulation. Recently, the properties of I_Ca(L) in ventricular myocytes dialyzed with the Ca²⁺ chelator, BAPTA, have been described (You et al., 1997). In the presence of BAPTA, but not EGTA, I_Ca(L) increased as the concentration of this more rapidly acting buffer in the recording pipette ([BAPTA]_pip) increased from 0.2 to 60 mM. Removal of adenylyl cyclase (AC) inhibition by Ca²⁺ entering through L-type channels could account for the augmentation of I_Ca(L). Earlier experiments with chick embryonic heart cells showed that the stimulation of AC activity by isoproterenol (ISO) increased in the presence of Ca²⁺ channel-blocking drugs or when extracellular Ca²⁺ was reduced (Yu et al., 1993). In BAPTA-dialyzed myocytes, ACh per se inhibited I_Ca(L), as did the protein kinase A (PKA) inhibitor H-89 (You et al., 1997). cAMP-loaded cells did not respond further to BAPTA; the converse experiment gave the same result. There was no I_K(ACh) activated because the cells were dialyzed with a Cs⁺-rich pipette solution.

Therefore, regulation of ventricular I_Ca(L) by muscarinic agonist can be examined in BAPTA-dialyzed myocytes in the absence of sympathetic stimulation or -adrenoceptor agonist. That AC activity is disinhibited as BAPTA chelates Ca²⁺ in the subsarcolemmal space ([Ca²⁺]_sm) also permits the study of a mechanism for muscarinic inhibition. Cardiac AC isoforms (types V and VI) are uniquely sensitive to inhibition by submicromolar Ca²⁺ (Cooper et al., 1995). Furthermore, ACVI has high-affinity regulatory sites for Ca²⁺ (K_d = 0.23 µM) but not for Ba²⁺ or Sr²⁺, which do not inhibit enzyme activity (Gu and Cooper, 2000). Our results indicate that carbachol (CCh) inhibits I_Ca(L), but not I_Ba(L), in myocytes dialyzed with BAPTA. Carbachol action is initiated at mAChR and transduced by the inhibitory guanine nucleotide binding protein G_i. Carbachol has no effect on basal I_Ca(L) in myocytes dialyzed with 40 mM EGTA, a slower Ca²⁺ chelator. We hypothesize that in BAPTA-dialyzed myocytes, muscarinic agonist increases Ca²⁺ sensitivity of either AC or G_i, thereby reducing activation of the cAMP/PKA cascade and inhibiting I_Ca(L). A preliminary account of some of these findings has been presented previously (Pappano and Shen, 2000).

	Materials and Methods

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Cell Isolation. Single ventricular myocytes were enzymatically isolated from the hearts of male and female guinea pigs (weighing 250-450 g), anesthetized with sodium pentobarbital (30 mg/kg i.p.), and anticoagulated with heparin (1000 IU i.p.). The heart was retrogradely perfused at 8 to 10 ml/min through an aortic cannula with Tyrode's solution for 5 min according to the Langendorff technique. The Tyrode's solution composition was 135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 0.33 mM NaH₂PO₄, 10 mM HEPES, 10 mM glucose; pH was adjusted to 7.4 with NaOH. After a 2- to 3-min perfusion with Tyrode's solution without added Ca²⁺, the extracellular matrix was disrupted by perfusion with the same solution containing collagenase (Worthington type1, 20 mg/50 ml; Worthington Biochemicals, Freehold, NJ) and protease (Sigma type XIV, 4 mg/50 ml; Sigma Chemical, St. Louis, MO). The enzymes were washed out by perfusion with 50 ml of recovery solution. Recovery solution contained 130 mM potassium aspartate, 5 mM K₂ATP, 5 mM HEPES, 20 mM glucose; pH was adjusted to 7.4 with KOH. The ventricles were removed, and the cells dispersed in recovery solution and were kept at 4°C for at least 1 h. An aliquot of cell suspension was placed in a recording chamber (500-µl volume) on the stage of an inverted microscope. After 10 min, superfusion began with Tyrode's solution (2 ml/min) containing 10 mM glucose. The temperature was 22 to 24°C.

Electrophysiology. An EPC 7 patch-clamp amplifier (List Electronics, Darmstadt, Germany) was used to deliver voltage pulses in whole-cell mode. Voltage commands and current data acquisition were displayed by an IBM-compatible computer equipped with pClamp software (version 5.5, Axon Instruments, Burlingame, CA) and a Labmaster TL-1 interface (Axon Instruments). Pipette solutions (see below) filled glass capillary electrodes (i.d.,1.1 mm; o.d.,1.3 mm); the resistance was 2 to 3 M. An Ag-AgCl wire connected the pipette to the amplifier. After establishing a gigaohm seal and compensating electrode capacitance, the cell membrane was ruptured by additional negative pressure. Cell capacitance (70-240 pF) was used to normalize I_Ca(L) for the data in some of the figures. Series resistance (3-8 M) could be compensated by 60 to 75% without oscillation. The maximum voltage error was 8 mV with I_Ca(L) of 2.5 nA. The liquid junction potential between bath and pipette was nulled. In separate experiments, the junction potential changed by 3 to 6 mV when bath solution containing Cs⁺ replaced Tyrode's solution. We did not compensate for this small potential difference.

Solutions and Drugs. The basic stock pipette solution contained 50 mM CsCl, 110 mM cesium aspartate, 2 mM MgCl₂, 4 mM MgATP, and 10 mM HEPES (pH adjusted to 7.2 with CsOH). Stock calcium buffer pipette solutions with 67 mM either BAPTA or EGTA had cesium aspartate reduced to 11 mM as described previously (You et al., 1997). Mixing proportionate volumes of the base and calcium buffer stock solutions gave desired final concentrations ranging from 20 to 40 mM. The bath solution was modified Tyrode's with 10 mM CsCl added.

Data Analysis. The L-type Ca²⁺ current was taken as peak inward current relative to zero current. Increments of I_Ca(L) in BAPTA are measured as the difference between maximum and minimum I_Ca(L). Minimum I_Ca(L) is the smallest peak inward current measured after patch rupture and dialysis with Cs⁺-rich pipette solution. Maximum I_Ca(L) is the largest peak inward current recorded just before applying drugs such as CCh, usually 8 to 10 min after patch rupture. All measurements are reported as mean ± S.E.M. The statistical significance between means was evaluated with Student's t test; p  0.05 was taken as a significant difference.

	Results

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Carbachol Inhibits I_Ca(L) Directly in Myocytes Dialyzed with 40 mM BAPTA. The L-type Ca²⁺ current progressively increased in magnitude when the recording pipette solution contained 40 mM BAPTA ([BAPTA]_pip). As illustrated in Fig. 1, I_Ca(L) diminished slightly upon patch rupture as the cytoplasm was exposed to the pipette solution containing Cs⁺ and BAPTA. After ~1.5 min, I_Ca(L) began to increase from a minimum value of 1.3 nA to reach 2.8 nA at ~10 min. The difference between these is taken as the extent of stimulation of I_Ca(L) in BAPTA. Carbachol (0.1 mM) suppressed I_Ca(L), which reached 2.1 nA at 5 min after addition of the choline ester (Fig. 1). Thus, I_Ca(L) diminished by ~47% in CCh [(2.8-2.1)/(2.8-1.3) × 100%]. We used this procedure in all experiments to quantitate the inhibition by CCh. Addition of atropine (1 µM) antagonized the effect of CCh (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   CCh inhibits ICa(L) in ventricular myocyte dialyzed with 40 mM [BAPTA]pip. Ordinate, ICa(L) (nA); abscissa, time (min). Recording begins at t = 0 min, the time of patch rupture. After a small decrease, ICa(L) increases to a maximum of 2.8 nA at 10 min. Then, 0.1 mM CCh gradually reduced ICa(L) to 2.1 nA over the next 5 min. Addition of 1 µM atropine (ATR) reversed the CCh effect. Individual current traces recorded at times indicated by letters A to E are shown above with current calibration on the right-hand ordinate.

Role of Adenylyl Cyclase in the Inhibition of I_Ca(L) by Carbachol. The stimulation of I_Ca(L) in BAPTA results from chelation of Ca²⁺ that disinhibits AC (You et al., 1997). The faster kinetics of BAPTA allow this to occur in the region around AC. Accordingly, inhibition by CCh of I_Ca(L) should not be evident in 40 mM [EGTA]_pip because this slower calcium chelator does not disinhibit AC to reveal stimulation of basal I_Ca(L). With 40 mM [EGTA]_pip, I_Ca(L) simply decreased from 741 ± 88 pA at the time of patch rupture to 536 ± 75 pA at 8 to 10 min after beginning dialysis (n = 7 cells). In three of these cells, 100 µM CCh alone had no substantial effect on I_Ca(L). However, when 30 nM ISO increased I_Ca(L) (from 0.13 to 1.5 nA), 100 µM CCh inhibited this current (~66%) by an action initiated at mAChR, because 1 µM atropine completely reversed the inhibition (Fig. 2). In four such experiments, ISO increased I_Ca(L) to 1.4 ± 0.31 nA and CCh reduced the current to 0.7 ± 0.2 nA.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   CCh inhibits ICa(L) stimulated by ISO in an EGTA-dialyzed myocyte. Ordinate, ICa(L) (nA); abscissa, time (min). A, when dialyzed with 40 mM [EGTA]pip, ICa(L) decreased from the time of patch rupture; B, 30 nM ISO increased ICa(L); C, 0.1 mM CCh opposes ISO effect on ICa(L); and D, 1 µM atropine (ATR) antagonizes CCh effect in the presence of ISO. Individual current traces (A-D) recorded at times indicated by letters are shown above with current calibration on the right-hand ordinate.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of [BAPTA]pip alone and with ISO on ICa(L) and on its inhibition by CCh. Ordinate, reduction of ICa(L) by 0.1 mM CCh (pA/pF); abscissa, increase of ICa(L) by various [BAPTA]pip alone [open symbols: 20 (), 30 (), and 40 mM ()] and with 30 nM ISO (corresponding filled symbols). Data are given as mean ± S.E.M. Number of cells at each condition is shown in parentheses.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Extent of ICa(L) inhibition by CCh depends on magnitude of stimulation with [BAPTA]pip. Ordinate, decrease of ICa(L) by 0.1 mM CCh (pA/pF); abscissa, increase of ICa(L) by 40 mM [BAPTA]pip grouped in 5 pA/pF bins. Measurements reported as mean ± S.E.M.; number of cells in each bin is indicated in parentheses. See text for details.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effect of 2'-dAMP on stimulation of ICa(L) by 40 mM [BAPTA]pip and on inhibition by CCh. Ordinate, ICa(L) (pA); abscissa, time (min). The record begins at the time of patch rupture with 100 µM 2'-dAMP plus 40 mM BAPTA in the pipette solution. Soon after patch rupture, control ICa(L) () reached a minimum and then increased to 770 pA at 4 min. Thereafter, ICa(L) began to decrease; this was not changed by the addition of 0.1 mM CCh at 8 to 13 min. Addition of 30 nM ISO at 23 to 28 min slightly reversed the rundown.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   CCh does not inhibit IBa(L) in ventricular myocyte dialyzed with 40 mM [BAPTA]pip. Ordinate, ICa(L) (nA); abscissa, time (min). Bath solution contained 1.8 mM Ba2+ in place of Ca2+. After the usual decline in current post-patch rupture, IBa(L) increased to a maximum of 1.6 nA at 8 min. Addition of 0.1 mM CCh at 9 to 14 min had a negligible effect on IBa(L), which was sustained through 25 min. Individual current traces (A-D) recorded at times indicated by letters are shown above with current calibration on the right-hand ordinate.

Pharmacological Properties of Muscarinic Signaling in BAPTA-Dialyzed Myocytes. As shown previously (Fig. 1), atropine antagonized the effect of CCh, an indication that the signal is initiated at mAChR. Does inhibition by CCh of I_Ca(L) arise from suppression of the activity of constitutively active -adrenoceptors? The ability of [BAPTA]_pip to disinhibit AC might allow -adrenoceptors to stimulate the enzyme in the absence of agonist. This possibility was tested with the nonselective -adrenoceptor antagonist propranolol. At 10 min after patch rupture with 40 mM [BAPTA]_pip, I_Ca(L) was 2.1 ± 0.30 nA (n = 8). At 5 to 6 min in 1 µM propranolol, I_Ca(L) was 2.1 ± 0.34 nA (p = N.S.); during washout, the current simply ran down to 1.8 ± 0.24 nA. At a 10-fold lower concentration, I_Ca(L) averaged 2.4 ± 0.24 nA in 0.1 µM propranolol (n = 8); this was not significantly different from the control (2.3 ± 0.14 nA) and washout (2.3 ± 0.25 nA) measurements of I_Ca(L). There is no evidence for constitutive activity of -adrenoceptors in myocytes dialyzed with 40 mM BAPTA. Pretreatment with 0.1 to 1 µM propranolol did not prevent the inhibition of I_Ca(L) by CCh (data not shown).

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View larger version (29K): [in this window] [in a new window]   Fig. 7.   Summary of experiments with 50 µM GTPS together with 40 mM BAPTA in pipette solution. Ordinate, ICa(L) (pA/pF); abscissa, experimental condition. CTR1 and CTR2 are ICa(L) at patch rupture and at 10 min afterward, respectively; CCh is 0.1 mM, WO is washout, and ISO is 30 nM. Panel A, test of CCh alone; panel B, test of CCh in the presence of ISO. Measurements are mean ± S.E.M.; number of cells at each condition is shown in parentheses below.

cAMP-Induced Cl Current in 40 mM [BAPTA]_pip. If [BAPTA]_pip increases I_Ca(L) by removing Ca²⁺-dependent inhibition of AC (You et al., 1997), selective block of I_Ca(L) could reveal the presence of other cAMP/PKA-dependent ionic currents such as cAMP-induced Cl current [I_Cl(cAMP); Harvey et al., 1990]. We tested this in experiments with nifedipine (30 µM), which completely blocked I_Ca(L) within 3 to 4 min (n = 11 cells). Under this condition, CCh predictably had no effect on the peak inward current. However, in four cells, current at the end of 300-ms test pulses to more positive and more negative voltages from the holding potential of 40 mV revealed a time-independent, outwardly rectifying current that could be identified as I_Cl(cAMP). The I-V relationship for this current in one cell bathed in Tyrode's solution with 30 µM nifedipine is shown in Fig. 8A. The current rectified outwardly and had a zero current potential of ~20 mV. Addition of CCh in the presence of nifedipine reversibly reduced end-of-pulse current in inward and outward directions at voltages between 100 and +80 mV (Fig. 8A). Figure 8B illustrates the difference currents between control and CCh (I_CTR-CCh) and washout and CCh (I_WO-CCh). The intersections of the difference currents had reversal potentials of 29 and 32 mV for I_CTR-CCh and I_WO-CCh, respectively. From the four cells displaying this effect, the reversal potential of the CCh-inhibited current averaged 30 ± 2.9 mV. This reasonably approximates the Cl equilibrium potential of 27 mV estimated from the composition of bath and pipette solutions. Thus, another cAMP-regulated current displays characteristic inhibition by CCh.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Carbachol (0.1 mM) inhibits ICl(cAMP) in BAPTA-dialyzed myocyte when ICa(L) is blocked by nifedipine (30 µM). Ordinate, isochronal (300 ms) membrane current (nA); abscissa, membrane test potential (mV). A, I-V relation for end-of-pulse membrane current when nifedipine had abolished ICa(L) before (), in CCh (), and after washout (). B, ICl(cAMP) inhibited by CCh shown as difference currents between control and CCh (ICTR-CCh, black line) and washout and CCh (IWO-CCh, dashed line). See text for details.

	Discussion

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Carbachol reversibly inhibits I_Ca(L) in guinea pig ventricular myocytes dialyzed with 20 mM BAPTA. Carbachol action on I_Ca(L) conforms to the "accentuated antagonism" hypothesis (Levy, 1971), with the proviso that inhibition of the cAMP/PKA cascade occurs without -adrenoceptor stimulation.

BAPTA and Disinhibition of Adenylyl Cyclase. BAPTA is believed to increase I_Ca(L) by disinhibiting AC activity to augment cAMP/PKA-dependent phosphorylation of L-type channels (You et al., 1997). Neither increased Ca²⁺ driving force nor diminished inactivation adequately explained augmented I_Ca(L) in BAPTA. The ₁ and the rapidly inactivating I_Ca(L) fraction did not differ in the presence of EGTA (40-67 mM) and BAPTA (40-50 mM), an indication that suppressing fast inactivation of I_Ca(L) did not contribute to increased I_Ca(L) (You et al., 1997). That Ca²⁺ entry through L-type channels inhibits AC activity accords with the result that equivalent concentrations of the slower chelator EGTA do not cause I_Ca(L) to increase progressively, as seen in this and previous studies (You et al., 1997). Dialysis with 10 mM BAPTA, but not EGTA, increased ISO sensitivity and its maximal effect on I_Ca(L) (Sako et al., 1998). Sensitivities of I_Ca(L) and I_Ba(L) to ISO were equal in BAPTA even though 10 mM BAPTA did not eliminate Ca²⁺-dependent inactivation (Sako et al., 1998). These authors concluded that BAPTA removed Ca²⁺-dependent suppression of AC activity; diminished I_Ca(L) inactivation did not explain their observation.

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Signal Transduction for I_Ca(L) Inhibition by Carbachol. The signaling mechanism for CCh in 40 mM BAPTA (EC₅₀ of ~1 µM) is less sensitive to ACh or CCh (EC₅₀ of <0.1 µM) than in sinoatrial node (Petit-Jacques et al., 1993) and atrial myocytes (Iijima et al., 1985; Wang and Lipsius, 1995). Maximum inhibition (42%) occurred at 100 µM and lies between the values in mammalian sinoatrial node (56%; Petit-Jacques et al., 1993) and atrial cells (26-32%; Iijima et al., 1985; Wang and Lipsius, 1995).

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Mechanism(s) for Muscarinic Inhibition of I_Ca(L) in BAPTA-Dialyzed Myocytes. The most prominent mechanisms for muscarinic inhibition in BAPTA-dialyzed myocytes are suppression of AC activity and/or greater I_Ca(L) inactivation. Although CCh did not change ₁ or ₂ values of I_Ca(L) inactivation, it reduced the amplitude of the A₁ component. This component is Ca²⁺-sensitive; Ba²⁺ does not replace Ca²⁺ in this function (McDonald et al., 1994). Barium does not mimic Ca²⁺ in inhibiting ACVI because this isoform has a high-affinity regulatory site for Ca²⁺ (K_d ~ 0.23 µM) but not for Ba²⁺ (Gu and Cooper, 2000). The Ba²⁺ results do not distinguish AC suppression from greater inactivation as mechanisms for muscarinic inhibition of I_Ca(L).

Calcium-Sensitivity Hypothesis for Muscarinic Inhibition. Adenylyl cyclases are composed of a ~40 kDa cytoplasmic loop, C₁, that links two hexahelical membrane components, and a carboxyl terminus, C₂ (Sunahara et al., 1996). The C₁ domain has a G_i binding site (Dessauer et al., 1998) and a 20-amino acid peptide that inhibits ACV activity (Kawabe et al., 1994). Catalysis requires interaction between C₁ and C₂ domains. Inhibition of ACV activity by Ca²⁺ is attributed to a cytosolic factor (Cooper et al., 1995) or a Ca²⁺ binding site(s) on the C_1b region (Scholich et al., 1997; Guillou et al., 1999).

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