Research ArticleCELLULAR AND MOLECULAR

Carbachol Inhibits the L-Type Ca2+ Current Augmented by 1,2-bis(2-Aminophenoxy)ethane-N,N,N′,N′-Tetraacetic Acid in Guinea Pig Ventricular Myocytes: Calcium-Sensitivity Hypothesis for Muscarinic Inhibition

Jian-Bing Shen and Achilles J. Pappano
Journal of Pharmacology and Experimental Therapeutics August 2001, 298 (2) 857-864;

Abstract

The L-type Ca2+ current [ICa(L)] increases with time after patch rupture in guinea pig ventricular myocytes dialyzed with pipette solutions containing ≥20 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid ([BAPTA]pip). ICa(L)progressively increases because BAPTA chelates subsarcolemmal Ca2+ to disinhibit cardiac adenylyl cyclase (AC) activity. We studied inhibition by carbachol (CCh) of ICa(L)(22–24°C). At 40 mM [BAPTA]pip, 100 μM CCh reversibly suppressed ICa(L) maximally by 42%; half-maximal inhibition (20%) required 1 μM. Atropine antagonized the CCh effect on BAPTA-stimulated ICa(L), as did dialysis with 50 μM guanosine-5′-O-(3-thio)triphosphate. At 20, 30, and 40 mM [BAPTA]pip, ICa(L) increased by 6.7 ± 1.8, 10.1 ± 1.4, and 11.3 ± 1.2 pA/pF, respectively. Inhibition by 100 μM CCh averaged −1.8 ± 0.6, −2.3 ± 0.4, and −4.1 ± 0.4 pA/pF at 20, 30, and 40 mM [BAPTA]pip, respectively. Dialysis of the AC inhibitor 2′-dAMP (100 μM) suppressed ICa(L) run up in 40 mM BAPTA and its inhibition by CCh. Replacing 1.8 mM external Ca2+ with Ba2+, which lacks high-affinity regulatory sites on AC, suppressed CCh-induced inhibition. Neither ICa(L) run up nor its inhibition by CCh occurred when 40 mM EGTA, a slower chelator, replaced BAPTA. Our results support the AC disinhibition hypothesis for BAPTA. We propose that CCh inhibits ICa(L) in BAPTA by increasing either AC sensitivity to inhibition by ambient Ca2+ or the activity of the inhibitory guanine nucleotide binding protein.

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 [IK(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 [ICa(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 ICa(L) to a small extent in ventricular myocytes, the inhibition becomes greater in the presence of sympathetic stimulation. Recently, the properties of ICa(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, ICa(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 ICa(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 ICa(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 IK(ACh) activated because the cells were dialyzed with a Cs+-rich pipette solution.

Therefore, regulation of ventricular ICa(L) by muscarinic agonist can be examined in BAPTA-dialyzed myocytes in the absence of sympathetic stimulation or β-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+(Kd = 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 ICa(L), but not IBa(L), in myocytes dialyzed with BAPTA. Carbachol action is initiated at mAChR and transduced by the inhibitory guanine nucleotide binding protein Giα. Carbachol has no effect on basal ICa(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 Giα, thereby reducing activation of the cAMP/PKA cascade and inhibiting ICa(L). A preliminary account of some of these findings has been presented previously (Pappano and Shen, 2000).

Materials and Methods

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 MΩ. 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 ICa(L) for the data in some of the figures. Series resistance (3–8 MΩ) could be compensated by 60 to 75% without oscillation. The maximum voltage error was ≤8 mV with ICa(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.

The voltage clamp protocol for eliciting ICa(L)consisted of 300-ms jumps to a test potential of 0 to +10 mV from a holding potential of −40 mV; the frequency was 0.1 Hz. The fast Na+ current and the T-type Ca2+ current were inactivated at −40 mV. In some cells, the membrane was held at −80 mV and the voltage stepped to −40 mV for 350 ms, then to +10 mV for 300 ms to evoke ICa(L), and then returned to −40 mV for 200 ms before repolarizing to −80 mV. No difference (see Results) was obtained; ICa(L) is maximal at 0 to +10 mV.

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.

Drugs (carbachol, atropine, propranolol, and nifedipine) were applied by superfusion from a reservoir by gravity at a rate of 2 ml/min. Nifedipine was dissolved in dimethyl sulfoxide and prepared fresh daily from the stock solution. All nifedipine solutions were protected from light during preparation, storage, and use. All other bath-applied agents were prepared daily from aqueous stock solutions. In some experiments, the pipette solution contained 2′-deoxyadenosine 3′-monophosphate (2′-dAMP) at a final concentration of 100 μM. All drugs were from Sigma.

Data Analysis.

The L-type Ca2+ current was taken as peak inward current relative to zero current. Increments of ICa(L) in BAPTA are measured as the difference between maximum and minimum ICa(L). Minimum ICa(L) is the smallest peak inward current measured after patch rupture and dialysis with Cs+-rich pipette solution. Maximum ICa(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

Carbachol Inhibits ICa(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, ICa(L) diminished slightly upon patch rupture as the cytoplasm was exposed to the pipette solution containing Cs+ and BAPTA. After ∼1.5 min, ICa(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 ICa(L) in BAPTA. Carbachol (0.1 mM) suppressed ICa(L), which reached 2.1 nA at 5 min after addition of the choline ester (Fig.1). Thus, ICa(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).

Figure 1
Figure 1

CCh inhibits ICa(L) in ventricular myocyte dialyzed with 40 mM [BAPTA]pip. Ordinate, ICa(L) (nA); abscissa, time (min). Recording begins att = 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.

During dialysis with 40 mM [BAPTA]pip, the L-type Ca2+ current displayed moderate sensitivity to CCh, with the lowest concentration, 0.3 μM, reducing ICa(L) by 13 ± 2.3% (n = 3 cells). The percentage of reduction of ICa(L)averaged 20 ± 6.3% (n = 7), 32 ± 4.5% (n = 7), and 42 ± 5.6% (n = 30) at 1, 10, and 100 μM CCh, respectively. Inhibition increased rather linearly as a function of log CCh concentration.

Does CCh change ICa(L) kinetics when it suppresses the current? Inactivation of ICa(L)was measured in 19 of the cells used to describe the relation between percent inhibition by 100 μM CCh and the increase of ICa(L) by 40 mM BAPTA. In most of these cells (n = 15), inactivation of ICa(L)was a biexponential process with fast (τ1) and slow (τ2) time constants averaging 37 ± 6.3 and 202 ± 27.7 ms, respectively. The amplitude of the fast ICa(L) component (A1), as a fraction of the total amplitude (A1 + A2), averaged 0.33 ± 0.04. In CCh, τ1 was 27 ± 6.1 ms and τ2 was 181 ± 25.6 ms. Although CCh reduced τ1 (11/15 cells) and t2 (10/15 cells), the tendency for inactivation of ICa(L) to be accelerated in CCh did not attain statistical significance (0.2 > p > 0.1). However, CCh significantly reduced the amplitude of the fast component, and the ratio (A1/A1 + A2) decreased to 0.21 ± 0.05 (p = 0.006).

Role of Adenylyl Cyclase in the Inhibition of ICa(L) by Carbachol.

The stimulation of ICa(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 ICa(L) should not be evident in 40 mM [EGTA]pip because this slower calcium chelator does not disinhibit AC to reveal stimulation of basal ICa(L). With 40 mM [EGTA]pip, ICa(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 ICa(L). However, when 30 nM ISO increased ICa(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 ICa(L) to 1.4 ± 0.31 nA and CCh reduced the current to 0.7 ± 0.2 nA.

Figure 2
Figure 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.

We tested the disinhibition hypothesis by varying [BAPTA]pip and adding 100 μM CCh, the maximum concentration used in the experiments. At 10 mM, BAPTA had no significant effect on ICa(L) (You et al., 1997), nor did CCh suppress it (data not shown). With [BAPTA]pip at 20 mM, ICa(L) increased by 6.7 ± 1.77 pA/pF (183 ± 54%) from an initial value at patch rupture of 3.8 ± 0.25 pA/pF (Fig. 3). Carbachol reduced ICa(L) by 1.8 ± 0.59 pA/pF. At 20 mM, [BAPTA]pip seems just suprathreshold for detecting inhibition by CCh. Increasing [BAPTA]pip to 30 and 40 mM was accompanied by ICa(L) increments of 10.1 ± 1.43 pA/pF (235 ± 32%) and 11.3 ± 1.22 pA/pF (301 ± 48%), respectively. The decrease of ICa(L) by CCh also became greater. Carbachol reduced ICa(L) by 2.3 ± 0.36 pA/pF in 30 mM [BAPTA]pip and by 4.1 ± 0.40 pA/pF in 40 mM [BAPTA]pip(Fig. 3).

Figure 3
Figure 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.

Carbachol's ability to inhibit ICa(L) declined when this current was maximally activated by adding 30 nM ISO in the presence of various [BAPTA]pip. As illustrated in Fig. 3, ICa(L) increased by 16.1 ± 3.22 to 18.2 ± 2.94 pA/pF in 20 mM [BAPTA]pipplus ISO, by 18.8 ± 2.64 to 21.8 ± 2.42 pA/pF in 30 mM [BAPTA]pip plus ISO, and by 16.4 ± 1.16 to 21.6 ± 1.66 pA/pF in 40 mM [BAPTA]pipplus ISO. The fact that 30 nM ISO plus any [BAPTA]pip yielded about the same change of ICa(L) and the same peak value of ICa(L) indicates that AC activity was maximal and that the effects of ISO and BAPTA are additive. Under these conditions, CCh (100 μM) produced similar net decreases of ICa(L) that amounted to 2.1 ± 0.72, 1.5 ± 0.22, and 2.5 ± 0.89 pA/pF at 20, 30, and 40 mM [BAPTA]pip, respectively (Fig. 3, filled symbols).

In 30 cells dialyzed with 40 mM [BAPTA]pip, the net increase of ICa(L) ranged from 3.5 to 29.3 pA/pF, with an average of 11.3 ± 1.22 pA/pF (Fig. 3). The relation between increase in ICa(L) by 40 mM BAPTA and the inhibition by CCh was examined more closely by grouping the former in 5 pA/pF increments (Fig.4). With the increased magnitude of ICa(L), which is expected if there is greater disinhibition of AC activity, inhibition by CCh progressively increased in proportion to the increase in 40 mM [BAPTA]pip when the change of ICa(L) was ≤20 pA/pF. However, when ICa(L) had increased to between 25 and 30 pA/pF in 40 mM BAPTA, inhibition by CCh decreased (n = 2 cells). This small sample precludes a definitive position concerning biphasic inhibition by CCh. However, the result resembles that observed with BAPTA plus ISO (Fig. 3). Biphasic muscarinic inhibition was detected in myocytes displaying accentuated antagonism in the presence of EGTA (Hescheler et al., 1986; Sakai et al., 1999).

Figure 4
Figure 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.

That increased activity of AC caused the stimulation of ICa(L) in 40 mM BAPTA was tested in another way. In addition to 40 mM BAPTA, the pipette solution included 100 μM 2′-dAMP. This adenosine analog inhibits AC and acts at the P-site of the enzyme (Sunahara et al., 1996). The 100 μM pipette concentration is ≤100-fold greater than required to inhibit AC activity by 50%. During dialysis with BAPTA plus 2′-dAMP, ICa(L)did not display its usual sustained increase in BAPTA. As shown in Fig.5, after the transient decline that occurs as Cs+-rich pipette solution enters the cell, ICa(L) increased to 770 pA at 4 min. However, ICa(L) then diminished over the next 16 min; the addition of 100 μM CCh did not change the rate of ICa(L) rundown, which continued unabated after CCh washout. When 30 nM ISO was added, rundown slowed and slightly reversed as ICa(L) increased from 210 to 245 pA. The failure of 40 mM BAPTA to increase ICa(L) and of CCh to decrease it was seen in three other experiments of this type. Overall, at 8 to 10 min after patch rupture, ICa(L) was 501 ± 160 pA compared with 438 ± 184 pA at the time of patch rupture (n = 4 cells).

Figure 5
Figure 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.

The cardiac AC isoform, although sensitive to suppression by submicromolar Ca2+, is not suppressed by either Ba2+ or Sr2+ until millimolar concentrations at the enzyme are attained (Gu and Cooper, 2000). We asked whether replacing external Ca2+with Ba2+ would support inhibition by CCh with [BAPTA]pip of 40 mM. As shown in Fig.6, IBa(L) increased from a minimum of 0.8 nA soon after patch rupture to 1.6 nA at 8 min. Peak membrane current did not change substantially in the presence of 100 μM CCh, and IBa(L) was well sustained after CCh removal. In 10 experiments of this type, the increase of IBa(L) averaged 19.4 ± 6.71 pA/pF. Although larger than the average increase of ICa(L) (Fig.3), it lies between the largest increments of ICa(L) seen in the data presented in Fig. 4. Inactivation of IBa(L) could be fit by two exponentials with τ1 of 45 ± 7.2 ms and τ2 of 247 ± 32.2 ms. Neither of these time constants differed significantly from the corresponding values for ICa(L). The decrease of IBa(L) by CCh averaged 0.41 ± 1.51 pA/pF (n = 10 cells) and was not statistically significant. The failure of CCh to inhibit IBa(L) compared with ICa(L) is not readily explained by the larger IBa(L) inasmuch as when ICa(L) had increased by 17.1 ± 0.48 pA/pF (Fig. 4), CCh reduced ICa(L) by 6.8 ± 0.94 pA/pF.

Figure 6
Figure 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 ICa(L) arise from suppression of the activity of constitutively active β-adrenoceptors? The ability of [BAPTA]pip to disinhibit AC might allow β-adrenoceptors to stimulate the enzyme in the absence of agonist. This possibility was tested with the nonselective β-adrenoceptor antagonist propranolol. At 10 min after patch rupture with 40 mM [BAPTA]pip, ICa(L) was 2.1 ± 0.30 nA (n = 8). At 5 to 6 min in 1 μM propranolol, ICa(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, ICa(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 ICa(L). There is no evidence for constitutive activity of β-adrenoceptors in myocytes dialyzed with 40 mM BAPTA. Pretreatment with 0.1 to 1 μM propranolol did not prevent the inhibition of ICa(L) by CCh (data not shown).

Muscarinic inhibition of ICa(L) is transduced by Giα (Löffelholz and Pappano, 1985;Hartzell, 1988). The transducer action of Giαcan be suppressed by GTPγS, a nonhydrolyzable guanine nucleotide. We included 50 μM GTPγS in the pipette solution with 40 mM BAPTA or 40 mM EGTA. In cells dialyzed with BAPTA plus GTPγS (n = 10), ICa(L) increased from an initial value of 5.1 ± 1.0 to 16.9 ± 3.19 pA/pF at 8 to 10 min (Fig.7, left). Carbachol had little effect because ICa(L) decreased only to 16.8 ± 3.12 pA/pF, an indication that Giα-dependent function was lacking. In another seven cells subjected to the same pipette solution, ICa(L) rose to 21.7 ± 3.09 pA/pF at 8 to 10 min from an initial magnitude of 5.2 ± 1.39 pA/pF (Fig. 7, right). Isoproterenol (30 nM) increased ICa(L) further to 27.8 ± 4.37 pA/pF, yet CCh (100 μM) had a negligible action and reduced this current insignificantly to 27.5 ± 5.3 pA/pF. After we removed CCh, ICa(L) remained elevated at 28.5 ± 6.0 pA/pF in ISO for 5 min, and after we removed ISO, this current was sustained at 27.5 ± 6.92 pA/pF for 15 to 20 min. This pattern is similar to that reported by Breitwieser and Szabo (1985), where nonhydrolyzable guanine nucleotides uncoupled mAChR and β-adrenoceptor agonists from ion channels in heart. In the experiments with 50 μM GTPγS plus 40 mM EGTA in the pipette solution, CCh was unable to inhibit ICa(L) in the absence (n = 4 cells) or presence (n = 6 cells) of ISO (data not shown).

Figure 7
Figure 7

Summary of experiments with 50 μM GTPγS 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]pipincreases ICa(L) by removing Ca2+-dependent inhibition of AC (You et al., 1997), selective block of ICa(L) could reveal the presence of other cAMP/PKA-dependent ionic currents such as cAMP-induced Cl− current [ICl(cAMP); Harvey et al., 1990]. We tested this in experiments with nifedipine (30 μM), which completely blocked ICa(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 ICl(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 (ΔICTR-CCh) and washout and CCh (ΔIWO-CCh). The intersections of the difference currents had reversal potentials of −29 and −32 mV for ΔICTR-CCh and ΔIWO-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.

Figure 8
Figure 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 (ΔICTR-CCh, black line) and washout and CCh (ΔIWO-CCh, dashed line). See text for details.

In the remaining cells dialyzed with 40 mM [BAPTA]pip and exposed to nifedipine, the end-of-pulse current rectified outwardly but was small, and this limited our testing the effect of CCh. We believe that the low amplitude arises from an action of BAPTA on Cl−channels independent of its effect on AC and not from insensitivity to CCh, because muscarinic agonist suppressed ICa(L)in these cells before the addition of nifedipine. This confirms an earlier observation (You et al., 1998) that dialysis with BAPTA could inhibit ICl (cAMP) independent of its chelation of Ca2+. Our success in detecting the inhibition of ICl (cAMP) by CCh conceivably could stem from a more rapid disinhibition of AC activity than from occlusion of Cl− channels by BAPTA.

Discussion

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

BAPTA and Disinhibition of Adenylyl Cyclase.

BAPTA is believed to increase ICa(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 ICa(L) in BAPTA. The τ1and the rapidly inactivating ICa(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 ICa(L) did not contribute to increased ICa(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 ICa(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 ICa(L)(Sako et al., 1998). Sensitivities of ICa(L) and IBa(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 ICa(L) inactivation did not explain their observation.

Removing Ca2+ could inhibit phosphodiesterase (PDE) and phosphatase activities. However, these have been excluded as mechanisms for increased cAMP (Yu et al., 1993) and ICa(L) (You et al., 1997). Inhibition of Ca/calmodulin (CaM)-sensitive PDE1 is least effective in increasing ICa(L) when AC is stimulated (Verde et al., 1999). The results with 2′-dAMP bolster the AC disinhibition hypothesis; ICa(L) did not increase when this P-site inhibitor of AC was dialyzed with BAPTA. Neither CCh nor ISO affected ICa(L), indicating that AC activity could not be regulated. The PKA inhibitor H-89 opposed the BAPTA-induced increase of ICa(L) (You et al., 1997). Finally, BAPTA and EGTA chelate Mg2+ with equal Kd values (20 mM; You et al., 1997). Reducing Mg2+ cannot account for the progressive increase of ICa(L) in BAPTA.

Cardiac AC isoforms, types V and VI (ACV, ACVI), are uniquely inhibited by submicromolar concentrations of subsarcolemmal Ca2+([Ca2+]sm) (Cooper et al., 1995; Sunahara et al., 1996; Ishikawa and Homcy, 1997). In canine and chick embryonic heart membranes, increasing Ca2+(10−8–10−4 M) inhibited AC activity stimulated by ISO, forskolin, or guanine nucleotides (Colvin et al., 1991; Yu et al., 1993). Inhibition did not require Giα, because pertussis toxin did not modify calcium's effect. cAMP formation by ISO increased in the presence of ICa(L) antagonists or reduced extracellular Ca2+; cAMP hydrolysis was unchanged (Yu et al., 1993). For entering Ca2+ to inhibit AC, the channel and enzyme should be apposed (You et al., 1997). Rabbit ventricular myocytes displayed full-length α1c subunits of L-type channels and AC molecules colocalized on T-tubule membranes (Gao et al., 1997). In cardiac (You et al., 1997; Sako et al., 1998) and other cells expressing ACV or ACVI isoforms, Ca2+ entry across the plasma membrane, not Ca2+ released from the endoplasmic reticulum, inhibited AC (Cooper et al., 1995).

Signal Transduction for ICa(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).

The signal initiated at atropine-sensitive mAChR is transduced by Giα. GTPγS, which uncouples the mAChR from AC and reduces agonist affinity (Hescheler et al., 1986; Shirayama et al., 1993), prevented muscarinic inhibition. Dialyzed BAPTA did not augment AC activity by constitutively active β-adrenoceptors because propranolol did not interfere with ICa(L)stimulation by BAPTA or with its inhibition by CCh.

Mechanism(s) for Muscarinic Inhibition of ICa(L) in BAPTA-Dialyzed Myocytes.

The most prominent mechanisms for muscarinic inhibition in BAPTA-dialyzed myocytes are suppression of AC activity and/or greater ICa(L) inactivation. Although CCh did not change τ1 or τ2 values of ICa(L)inactivation, it reduced the amplitude of the A1component. 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+(Kd ∼ 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 ICa(L).

Muscarinic agonists inhibit AC and subsequently ICa(L) in ventricular myocytes only after AC has been stimulated and l-type channels are phosphorylated (“accentuated antagonism”). Inhibition by CCh confirms observations with ACh in BAPTA-dialyzed myocytes (You et al., 1997). If CCh acted directly on the channel, it should suppress basal ICa(L) as organic antagonists do. This suppression is not observed. Does CCh reduce the amplitude of the rapidly inactivating A1 component without inhibiting AC? Although we have no direct AC measurements, CCh reversibly suppressed ICl(cAMP) when nifedipine blocked ICa(L). This is consistent with muscarinic inhibition of AC activity; ICl(cAMP)does not require Ca2+ for activation.

We favor AC inhibition as the mechanism because CCh did not inhibit until ICa(L) had been increased by BAPTA, which disinhibits AC. Carbachol reduces ICa(L) and would not increase [Ca2+]sm to inhibit nearby AC molecules or the amplitude of the rapidly inactivating component of ICa(L). Neither action of CCh could be attributed to increased Ca2+ concentration. Rather, sensitivity to residual Ca2+ would have to increase to permit muscarinic agonist action. Calcium-dependent inactivation and facilitation of ICa(L) require a Ca2+-binding EF hand motif and a CaM-binding isoleucine-glutamine segment; both are found in the carboxyl terminus of the channel (Zühlke et al., 1999). Calcium does not require CaM or EF hand proteins to inhibit ACV at submicromolar concentrations (Gu and Cooper, 2000). If muscarinic agonist regulated Ca2+ sensitivity of AC and of the fast inactivating component, only the latter would be CaM-dependent.

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 Giα 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 C1b region (Scholich et al., 1997; Guillou et al., 1999).

We speculate that CCh acts by increasing AC sensitivity to inhibition by ambient Ca2+. Carbachol, via Giα, could shift equilibrium from the active to the inactive state of AC by increasing the degree of inhibition by residual Ca2+. Carbachol could increase either Ca2+ sensitivity in a microdomain of AC and/or the activity of Giα to inhibit AC. At ≤20 nm from the internal aspect of the Ca2+ channel, estimated [Ca2+]sm was submicromolar to micromolar with 40 mM [BAPTA]pip (Stern, 1992; You et al., 1997). This accords with the range of cardiac AC sensitivity to Ca2+ (Colvin et al., 1991; Yu et al., 1993).

Finding the magnitude of inhibition by CCh directly related to [BAPTA]pip and presumably to the extent of Ca2+ buffering at AC supports this hypothesis. As expected, CCh alone failed to inhibit ICa(L) in EGTA-dialyzed myocytes because EGTA buffers [Ca2+]sm less effectively (Stern, 1992; You et al., 1997). With EGTA, AC inhibition by [Ca2+]sm may be maximal. Detecting muscarinic inhibition in EGTA requires ISO, which might decrease AC sensitivity to Ca2+. This could also explain reduced muscarinic inhibition when ICa(L)is maximally stimulated by [BAPTA]pip or ISO plus [BAPTA]pip. Acetylcholine has reduced inhibitory efficacy on ICa(L) stimulated by ISO plus forskolin (Fischmeister and Shrier, 1989).

This hypothesis is consistent with adrenergic/cholinergic antagonism in the heart. When ACh decreases cardiac sarcoplasmic reticulum Ca2+ release and force, it also increases myofilament Ca2+ sensitivity, which limits the negative inotropic effect (Endoh, 1999). β-Adrenoceptor agonist acts oppositely on all these variables. Calcium sensitization by ACh can occur in the absence of cAMP-elevating agents (McIvor et al., 1988;Pucéat et al., 1990). For ACV/ACVI, the calcium-sensitivity hypothesis reflects the antagonism by Ca2+ of Mg2+-dependent enzyme activation (Guillou et al., 1999). Additional information on the structural requirements for Ca2+-dependent inhibition of ACV and ACVI could test the validity of the Ca2+-sensitivity hypothesis and help unravel the nature of muscarinic inhibition.

Footnotes

  • This work was supported by U.S. Public Health Service Grant HL-13339.

  • Abbreviations:
    mAChR
    muscarinic acetylcholine receptor
    ICa(L)
    L-type calcium current
    IBa(L)
    L-type barium current
    IK(Ach)
    inwardly rectifying K+current
    AC
    adenylyl cyclase
    P-site
    purine site
    BAPTA
    1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
    PKA
    protein kinase A
    GTPγS
    guanosine-5′-O-(3-thio)triphosphate
    Giα
    inhibitory guanine nucleotide binding protein
    [Ca]sm
    subsarcolemmal calcium
    τ1
    time constant of fast inactivating component
    τ2
    time constant of slow inactivating component
    CCh
    carbachol
    ACh
    acetylcholine
    ISO
    isoproterenol
    I-V
    current-voltage
    PDE
    phosphodiesterase
    CaM
    Ca/calmodulin
    ICl(cAMP)
    cAMP-induced Ca− current.
    • Received December 18, 2000.
    • Accepted May 9, 2001.

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Research ArticleCELLULAR AND MOLECULAR

Carbachol Inhibits the L-Type Ca2+ Current Augmented by 1,2-bis(2-Aminophenoxy)ethane-N,N,N′,N′-Tetraacetic Acid in Guinea Pig Ventricular Myocytes: Calcium-Sensitivity Hypothesis for Muscarinic Inhibition

Jian-Bing Shen and Achilles J. Pappano
Journal of Pharmacology and Experimental Therapeutics August 1, 2001, 298 (2) 857-864;
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