Vol. 290, Issue 2, 641-648, August 1999
Lack of Effect of McN-A-343 on Membrane Current and Contraction
in Guinea Pig Ventricular Myocytes1
Jian-Bing
Shen,
Bin
Jiang and
Achilles J.
Pappano
Department of Pharmacology, University of Connecticut Health
Center, Farmington, Connecticut
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Abstract |
We asked whether agonist occupancy of M1 muscarinic
receptor (mAChR) causes increased L-type Ca2+
[ICa(L)] and contractions in ventricular myocytes.
Voltage-clamp pulses evoked ICa(L) in guinea pig
ventricular myocytes superfused with Tyrode's solution (22-24°C).
The mAChR agonists carbachol (Cch, nonselective), McN-A-343 (McN,
M1-selective), and oxotremorine (Oxo,
M2-selective) were tested at 0.1 mM. None of these agonists affected basal ICa(L). McN did not change
isoproterenol-stimulated ICa(L) in 13 of 15 cells. The
slight decrease in two cells was not muscarinic because atropine (1 µM) did not antagonize it. Carbachol or Oxo decreased
isoproterenol-stimulated ICa(L) by 87 ± 6.7 (n = 8 cells) and 49 ± 9.0%
(n = 4 cells), respectively. Atropine blocked
inhibition by Cch or Oxo. External stimulation evoked contractions of
single myocytes (35°C). McN increased contraction in 1 of 22 cells
stimulated at 0.2 Hz and in 0 of 16 cells stimulated at 1.0 Hz.
Carbachol significantly increased contraction in 10 of 15 cells at 0.2 Hz and in 8 of 10 cells at 1.0 Hz stimulus frequency. Summarily, the
M1-selective agonist McN had a negligible role to regulate
ICa(L). The antiadrenergic effect of mAChR agonists is
attributable to M2 receptor occupancy. That Cch, but not
McN, increased cell shortening excludes participation of M1
mAChR in the stimulant effect of Cch on guinea pig ventricular myocyte contractions.
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Introduction |
The
choline esters acetylcholine (ACh) and carbamylcholine (Cch) can
increase contraction force in the mammalian heart (reviewed in Levy and
Pappano, 1994
). Stimulation of contractions by ACh or Cch has been
detected in atrial muscle (Webb and Pappano, 1995), Purkinje cells
(Gilmour and Zipes, 1985), and ventricular muscle (Korth and
Kühlkamp, 1985; Yang et al., 1996). This phenomenon does not
require antecedent inhibition although examples of "rebound" contraction stimulation by ACh are reported in sinoatrial node (McMorn et al., 1993) and ventricle (Endoh et al., 1970). The choline
esters act at muscarinic receptors (mAChR) to stimulate contractile
force because atropine antagonizes their effect.
The mAChR subtype that initiates ventricular muscle contraction
stimulation is disputed (Sharma et al., 1997). Experiments in guinea
pig ventricular myocytes (Protas et al., 1998) and human heart
ventricular muscle (Du et al., 1995) implicate the
M2 mAChR because the effect of Cch or ACh is more
susceptible to block by
(11[[2-[diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3- 6][1,4]benzodiazepine-6-)one (AF-DX 116), an
M2-selective antagonist, than by pirenzepine, an
M1-selective antagonist. The M1 mAChR reportedly is involved in rat
ventricular myocytes because pirenzepine was more potent than
methoctramine (M2-selective) as an antagonist of
Cch-induced increases in intracellular Ca2+
transients (Sharma et al., 1996). Participation of
M1 mAChR in stimulation of L-type
Ca2+ [ICa(L)] and of the
synthesis of inositol monophosphate also has been presented (Gallo et
al., 1993). These results form the basis of a mechanism by which
agonist occupancy of M1 mAChR increases ICa(L) and, thereby, intracellular
Ca2+ transients (Sharma et al., 1996). Although
mRNA and cell surface expression indicate the abundance of
M2 mAChR in heart (Sharma et al., 1996), the
M1 mAChR is also detected, particularly in ventricle (Gallo et al., 1993; Sharma et al., 1996).
In this study, we tested the hypotheses for muscarinic stimulation of
ventricular myocytes with
3-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343) on the favorable assumption that this drug is an
M1-selective agonist (Goyal and Rattan, 1978;
Hammer and Giachetti, 1982; Watson et al., 1983). McN-A-343 is a
"functionally selective" M1 agonist (reviewed
in Eglen et al., 1996), although agonist/antagonist effects at
M2 mAChR have been reported (Christopoulos and
Mitchelson, 1997). Does McN-A-343 increase ICa(L)
in ventricular myocytes in the absence or presence of elevated
intracellular cyclic AMP (cAMP)? Does McN-A-343 increase the extent of
shortening in electrically stimulated myocytes? We compared the actions
of McN-A-343 with those of the nonselective mAChR agonist Cch and with
the M2-selective agonist oxotremorine (Oxo). A
preliminary account of these findings has been reported in abstract
form (Shen et al., 1999).
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Materials and Methods |
Preparation of Single Myocytes.
Ventricular cells were
obtained from the hearts of anesthetized guinea pigs (250-450 g) by an
enzymatic dissociation procedure as described previously (Protas et
al., 1998). After dissociation, the cells were kept in recovery
solution (130 mM K aspartate, 5 mM K2ATP, 5 mM
HEPES, and 20 mM dextrose, pH 7.4, adjusted with KOH) for at least
1 h before conducting experiments.
Electrophysiologic Experiments.
Cells were superfused with
Tyrode's solution at room temperature (22-24°C) because at
this temperature ICa(L) was more stable and the
low basal current allowed easier detection of the effects of
stimulating drugs. 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, and
10 mM dextrose. CsCl (10 mM) was added to the Tyrode's solution to
block potassium currents.
The whole-cell, patch-clamp technique was used for the experiments.
Electrodes had resistances of 2 to 4 M
when filled with Cs+-rich pipette solution: 135 mM aspartic acid,
10 mM NaCl, 5 mM MgATP, 10 mM HEPES, 10 mM EGTA, pH 7.3, adjusted with
CsOH, whose final concentration was 130 to 140 mM. Voltage-clamp
protocols were generated by pClamp software (version 5.5; Axon
Instruments, Foster City, CA). Cell capacitance was obtained
immediately after patch rupture by integration of the current generated
during a 20-ms hyperpolarization of 5 mV from a holding potential of
40 mV. A specific membrane capacitance of 1 mF/cm2 was assumed. The voltage-clamp protocol
consisted of a voltage jump from 80 mV (holding potential) to 40 mV
for 350 ms to inactivate the fast Na+ and T-type
Ca2+ currents followed by a second depolarizing
jump to +10 mV for 300 ms to elicit the L-type
Ca2+ current [ICa(L)] and
a repolarizing step back to 80 mV. The frequency of stimulation was
0.1 Hz. ICa(L) amplitude was taken as the
difference between the peak inward current at +10 mV and zero membrane
current. Drug-containing solutions were applied to the bath (0.5-ml
volume) by a gravity-fed system at a rate of 2 ml/min.
Contraction Experiments.
Cell contraction experiments were
carried out at 35°C; the cells were superfused with Tyrode's
solution without added CsCl. Our previous experience indicated that
stimulation of contractions by carbachol is evident at 35°C but not
at 22°C (Protas et al., 1998). Carbachol or McN-A-343 was applied by
a solenoid-controlled rapid superfusion device (Saeki et al., 1997) via
a thin, polyethylene tube positioned within 100 µm of the cell to be
tested. Exposure time to either Cch or McN-A-343 was 2 to 3 min
followed by washout (WO). At the beginning of each experiment, the
ratio of contraction amplitudes at 0.2 and 1.0 Hz (
0.2/1.0) was
obtained. We selected those cells with a ratio 
0.8 to test the
contraction effect of McN-A-343 or Cch because of our previous
experience with guinea pig ventricular myocytes, which have a positive
frequency-shortening relation (Protas et al., 1998).
Contractions of single myocytes were elicited by external stimuli
applied through a broken-tip (~50-µm tip diameter) microelectrode and were detected by a video edge-detector device. The cell image was
projected on a high-resolution video monitor through a
sequential-scanning video camera attached to the microscope. The camera
was rotated to keep the video detector raster lines parallel with the
long axis of the cell. The video dimension analyzer monitored a
selected raster line for differences in light intensity between the
cell end and the surrounding field. The time constant and apparent spatial resolution of the video analysis system response at 400× magnification were 16.7 ms and 0.15 µm, respectively. The signals were sent to a chart recorder and to a videocassette recorder for
storage and analysis.
Data Analysis.
The amplitudes of
ICa(L) and cell contractions are expressed as
means ± S.E. Student's paired t test was used to
evaluate the statistical significance of the difference between means
of the results. P .05 is taken as statistically significant.
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Results |
Basal L-Type Calcium Current.
Basal
ICa(L) was measured in experiments using a
Cs+-rich pipette solution and 10 mM
Cs+-containing bath solution to block potassium currents.
In eight cells tested with 0.1 mM McN-A-343,
ICa(L) was not changed after 5 to 8 min of
exposure to the drug. The average ICa(L) (pA) in
these eight cells was 682 ± 145 (control), 635 ± 113 in
McN-A-343, and 507 ± 88 after 8 to 10 min of WO. As reported previously (Protas et al., 1998), 0.1 mM Cch had no significant effect
on basal ICa(L) (n = 5). The
results of these experiments indicate that ICa(L)
(pA) simply ran down from 734 ± 103 (control) to 671 ± 100 (Cch) and then to 585 ± 112 in WO. Oxo (0.1 mM) also was tested
in two cells; no change of ICa(L) was detected.
Isoproterenol (ISO)-Stimulated L-Type Calcium
Current.
The effects of McN-A-343
(M1-selective), Cch (nonselective), and Oxo
(M2-selective) were examined on ISO-stimulated
ICa(L) to ascertain whether
M1- and M2-selective
agonists have similar inhibitory actions on this current that triggers contractions.
The records shown in Fig. 1 are
illustrative of the results in 12 of 14 experiments. ISO (3 nM)
increased ICa(L) from 0.9 to 1.4 nA. In the
continued presence of ISO, addition of 0.1 mM McN-A-343 did not change
ICa(L), whereas the subsequent addition of 0.1 mM
Cch decreased this current to ~1.0 nA (80% reduction). The Cch
effect was completely reversed by 1 µM atropine, indicating that Cch
acted through mAChR. Removal of ISO caused ICa(L)
to return toward the initial level. It is noted that the slight initial increase of ICa(L) after the addition of Cch and
the "rebound" increase of ICa(L) when
atropine was added occurred only in this experiment. Whether the effect
of Cch arose from the inositol (1,4,5)-trisphosphate-dependent
mechanism (Gallo et al., 1993) or from an effect of accumulated cGMP
that inhibited cAMP hydrolysis (Shirayama and Pappano, 1996) is not
known.
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Fig. 1.
ISO-stimulated ICa(L) is inhibited by Cch
but not by McN-A-343. Voltage jumps from 40 to +10 mV evoked
ICa(L) (see Materials and Methods). A,
individual current traces (a-g) taken at times indicated in B. Duration of drug-treatment periods are shown by thin, horizontal lines
in B. Addition of 3 nM ISO (b) increased ICa(L) from 0.9 to
~1.4 nA. Exposure to McN-A-343 (c) had no effect whereas subsequent
addition of Cch (d) decreased ICa(L) to ~1.0 nA after an
initial small increase. Atropine (e) completely blocked the Cch effect;
a "rebound" increase of ICa(L) is evident.
ICa(L) returned to ~1.4 nA in the presence of ISO alone
(f), and current was restored to control level upon WO of ISO (g).
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In 2 of 14 experiments in this series, McN-A-343 exerted an atypical
action that is illustrated in the records of Figs.
2 and 3
taken from one of the cells. ISO (3 nM), as expected, increased ICa(L) to 990 pA (Fig. 2A-b). Addition of
McN-A-343 (Fig. 2A-c) reduced this current to 790 pA, and subsequent
addition of Cch reduced ICa(L) to 550 pA (Fig.
2A-d). In the presence of ISO, McN-A-343, and Cch, 1 µM atropine
reversed the inhibition seen in the presence of McN-A-343 plus Cch
(Fig. 2A-e); removal of atropine left ICa(L) at
the level to which ISO had elevated it (Fig. 2A-f). A later test of Cch
alone (Fig. 2A-g) indicated that Cch inhibited
ICa(L) and that this effect was muscarinic in
nature (Fig. 2A-h). Upon removal of atropine and Cch,
ICa(L) returned to the value seen in ISO alone
(Fig. 2A-i). The experiment continues in the records of Fig. 3 with the
cell superfused with 3 nM ISO (Fig. 3A-j). A second test with McN-A-343
(Fig. 3A-k) again resulted in diminished ICa(L),
but this action was not muscarinic because atropine did not affect it
(Fig. 3A-l), yet WO restored the L-type Ca2+ current to the sustained increase produced
by ISO (Fig. 3A-m). Addition of Oxo (0.1 mM; Fig. 3A-n) reduced
ICa(L) through an atropine-sensitive mechanism
(Fig. 3A-o); atropine brought the current to the level seen in ISO
alone (Fig. 3A-p). Addition of 1µM propranolol reduced
ICa(L) to the initial level (Fig. A-r) seen in
the control portion of the experiment; this confirms the reliability of
the recordings in the continued presence of ISO with the several
additions of agonists and antagonist during the experimental period of
slightly more than 2 h.
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Fig. 2.
Atypical suppression of ICa(L) by
McN-A-343. Data were taken from one of two cells in which McN-A-343
reduced ICa(L). Results are presented as in Fig. 1. Control
ICa(L) (a) of 480 pA increased to 990 pA in 3 nM ISO (b)
and then decreased to 790 pA in McN-A-343 (c). Addition of Cch on top
of McN-A-343 (d) reduced ICa(L) further to 550 pA, and this
was reversed by atropine to 1025 pA (e). In ISO alone (f),
ICa(L) was sustained at 1050 pA; Cch reduced this current
to 680 pA (g) and atropine reversed Cch effect (h). ICa(L)
returned to ~1000 pA in ISO alone (i).
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Fig. 3.
Continuation of the experiment shown in Fig. 2.
Results are presented as in Fig. 1. ICa(L) in 3 nM ISO
continues (j), and a second test of McN-A-343 reduced
ICa(L) to 825 pA (k). However, atropine (l) did not oppose
the McN-A-343 effect, which was reversed by its removal in the
continued presence of ISO (m). Oxo caused an atropine-sensitive
suppression of ICa(L) (n and o) like Cch. Current in ISO
alone (p) was suppressed by propranolol (q) to near-control level in
the absence of all drugs (r).
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The results from the data collected in these 14 cells with
ISO-stimulated ICa(L) are shown in Fig.
4. ISO (3-10 nM) increased ICa(L) by 81% from the control value of 699 ± 158 pA (p < .001). McN-A-343 had no effect on
ICa(L) that exceeded rundown of this current
(p = .16). In a subset of these (n = 6), Cch significantly reduced ISO-stimulated
ICa(L) (p < .05), an
effect that was atropine-sensitive. These data indicate not only that
the action of the nonselective agonist Cch was muscarinic but also that
McN-A-343 did not interfere with Cch action.
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Fig. 4.
Summary of the effect of Cch and McN-A-343 on
ISO-stimulated ICa(L). At 4 to 6 min after addition, ISO
(3-10 nM) increased ICa(L) from 699 ± 158 pA at
control to 1267 ± 207 pA (81% increase), and 6-min exposure to
0.1 mM McN-A-343 in the presence of ISO does not change
ICa(L) (1218 ± 192 pA). In 6 of 14 cells, 0.1 mM Cch
was added in the presence of ISO and McN-A-343. At 4 to 6 min after
addition, Cch reduced ICa(L) to 947 ± 87 pA (56%
decrease), and 1 µM atropine restored ICa(L) to 1129 ± 156 pA. Upon WO for 8 to 10 min, ICa(L) was 1055 ± 135 pA. Asterisks indicate a significantly different current
(p < .05) from the immediately preceding
ICa(L). Number of cells tested are shown in parentheses.
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The effect of Oxo, a relatively selective M2
mAChR agonist, was compared with that of Cch on ISO-stimulated
ICa(L). In eight cells, 3 to 10 nM ISO increased
ICa(L) from 623 ± 80 to 1178 ± 111 pA
(p < .05), and 0.1 mM Cch reduced this current to
728 ± 105 pA (p < .05). When the changes from
each cell were averaged, ISO increased ICa(L) by
102 ± 18.8% and Cch reduced the effect of ISO by 87 ± 6.7%. In four other cells (Fig. 5), 3 to
10 nM ISO increased ICa(L) from 726 ± 129 to 1381 ± 145 pA (p < .05), and it declined to
1093 ± 145 pA (p < .05) in 0.1 mM Oxo. From the
individual data, ISO increased ICa(L) by 102 ± 29.9% and Oxo suppressed 49 ± 9.0% of the ISO effect.
Atropine (1 µM) fully reversed this effect of Oxo. These results
indicate that activation of M2 mAChR by Oxo plays
an important role in the inhibition of ISO-stimulated
ICa(L) by mAChR. From our experiments at
equimolar concentrations of muscarinic agonist, the inhibition of
ISO-stimulated ICa(L) by Oxo is less than that by
Cch (49 versus 87%, respectively). This difference can be explained by
two possibilities, namely, that Cch is a more potent activator of the
M2 mAChR or that activation of other mAChR
subtypes such as M1 by Cch somehow facilitates the inhibition. However, the evidence from experiments with McN-A-343 and Cch show that activation of the M1 mAChR
alone could not inhibit ISO-stimulated ICa(L),
nor did it affect the response to Cch.
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Fig. 5.
The effect of the M2-selective agonist
Oxo on ISO-stimulated ICa(L). In four cells, 3 to 10 nM ISO
increased ICa(L) from 726 ± 129 to 1381 ± 145 pA (90% increase) after 4 to 6 min. At 6 min after addition, 0.1 mM
Oxo reduced ICa(L) to 1093 ± 145 pA (44% decrease);
this effect was fully reversed by treatment with 1 µM atropine for 4 to 6 min. WO of atropine and Oxo for 8 to 10 min showed no significant
change in the stimulant effect of ISO. Asterisks indicate a
significantly different current (p < .05) from the
immediately preceding ICa(L). Number of cells tested are
shown in parentheses.
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ICa(L) Stimulated by Intrapipette cAMP.
Cch
increased ICa(L) in guinea pig ventricular
myocytes dialyzed with cAMP and a low concentration of the
M1-selective antagonist; pirenzepine opposed this
effect (Gallo et al., 1993). This result supported the conclusion that
the stimulant effect of Cch was initiated at M1
mAChR. We examined this hypothesis by testing the effects of McN-A-343
or Cch on cAMP-stimulated ICa(L). The patch
electrode contained 300 µM cAMP in the pipette solution. Cells were
subjected to this condition from the time of patch rupture to stimulate
ICa(L). Stimulation of
ICa(L) by 300 mM cAMP was maximal inasmuch as we
confirmed in three cells that superfusion of 10 nM ISO could not
further increase ICa(L). In a total of 12 cells
dialyzed with a pipette solution containing 300 µM cAMP, the
ICa(L) density was 20.6 ± 1.62 mA/cm2, which is about three times larger than
that observed in 39 cells without cAMP in the pipette solution
(6.7 ± 3.01 µA/cm2).
The effect of McN-A-343 on cAMP-stimulated ICa(L)
was tested in 8 of 12 cells in this series (Fig.
6). Representative results with McN-A-343
or Cch are shown in Fig. 6A. Neither drug changed ICa(L), which simply ran down during the time of
the experiment. The average ICa(L) in these cells
was 2650 ± 353 pA at control (5 min after membrane rupture),
2188 ± 368 pA at 6 min with 0.1 mM McN-A-343, and 2022 ± 373 pA after 8-min WO (Fig. 6B). In the remaining four cells tested
with Cch, the average ICa(L) was 2668 ± 246 pA at control, 2319 ± 204 pA at 6 min with Cch, and 1843 ± 305 pA after 8-min WO (Fig. 6B). These results indicate that neither
McN-A-343 nor Cch had any additional effect on
ICa(L) when it is maximally activated by a high
concentration of intrapipette cAMP.
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Fig. 6.
Lack of effect of McN-A-343 or Cch on cAMP-stimulated
ICa(L). The pipette solution contained 300 µM cAMP that
stimulates ICa(L) maximally because addition of 10 nM ISO
(n = 3 cells) did not further increase
ICa(L). A, neither McN-A-343 (upper left) nor Cch (upper
right) had any effect that varied from the spontaneous rundown of
ICa(L). The control (CTR) records were taken at 5 min after
patch rupture. The traces in either McN-A-343 (McN) or Cch are at 6 min
in drug-containing solution, and WO records are at 6 min after drug
removal. B, summary of experiments with McN-A-343
(n = 8) and Cch (n = 4). These
results indicate that McN-A-343 and Cch have no effect on
ICa(L) when this current is saturated by a high
concentration of intrapipette cAMP.
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Cell Contractions.
Contractions of single myocytes were
elicited by external stimuli at 35°C; the Tyrode's solution bathing
the cells contained 1.8 mM Ca2+. At the beginning
of each experiment, the ratio of contraction amplitude at 0.2 Hz and
1.0 Hz (0.2/1.0 Hz) was calculated. Our previous experience with
guinea pig ventricular myocytes showed that they had a positive
frequency-shortening relation as expected and that a stimulant effect
of Cch could be detected if the ratio (0.2/1.0 Hz) was 0.8
(Protas et al., 1998). For these reasons, we chose those cells with a
ratio 0.8 to test the effects of McN-A-343 or Cch on contractions.
McN-A-343 did not change the cell contractions in most of the cells
tested, either at 0.2 Hz (16 unchanged, 5 decreased, and 1 increased)
or at 1.0 Hz (15 unchanged and 1 decreased). However, we confirmed that
Cch increased cell contractions in 10 of 15 cells at 0.2 Hz (5 showed
no change) and in 8 of 10 cells at 1.0 Hz (1 unchanged and 1 decreased). In some cells, both McN-A-343 and Cch were tested in random
sequence. An example of the typical results obtained is given in Fig.
7; cell shortening was 5.7 µm before
drug addition (Fig. 7A-a). Addition of 0.1 mM McN-A-343 did not change
the extent of contraction (Fig. 7A-b), but Cch increased cell
contraction from 5.7 to 7.9 µm reversibly (Fig. 7A-d). As shown in
Fig. 7B, McN-A-343 decreased cell contractions by only 0.17 ± 0.11 µm at 0.2 Hz (n = 22) and by 0.06 ± 0.16 µm (n = 16) at 1.0 Hz. McN-A-343 had no significant
effect on cell contractions when the rundown of contractions in the WO
period was taken into account. In contrast, Cch increased cell
contractions by 1.0 ± 0.29 µm at 0.2 Hz (n = 15; p < .004) and by 0.8 ± 0.27 µm at 1.0 Hz
(n = 10; p < .03). The increases of
contractions by Cch were reversed upon washout.
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Fig. 7.
Cch but not McN-A-343 increases isotonic shortening
in ventricular myocytes. A, individual contraction traces (a-e) taken
from the continuous record below it of a myocyte stimulated at 1 Hz and
superfused with Tyrode's solution (35°C). At 0.1 mM, McN-A-343 did
not change the extent of cell contractions (b), but Cch (d) increased
cell contraction reversibly. B, on average, when compared with control,
McN-A-343 had no significant effect on cell contractions when the
rundown of contractions is considered. However, Cch increased cell
contractions significantly at 0.2 Hz (n = 15;
p < .004) and at 1.0 Hz (n = 10; p < .03).
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Discussion |
Our experiments report two principal findings: namely, that
McN-A-343 had no effect either on ICa(L) in the
absence or presence of cAMP (ISO, intrapipette cAMP) or on contractions
in guinea pig ventricular myocytes. When Cch or Oxo inhibited
ICa(L), McN-A-343 had no effect on this current
that triggers contractions. In a few instances (see Figs. 2 and 3),
ISO-stimulated ICa(L) diminished in the presence
of McN-A-343. However, atropine did not antagonize this effect;
therefore, it is not muscarinic in nature. We conclude that McN-A-343
does not regulate ICa(L) in guinea pig
ventricular myocytes. Does McN-A-343 affect contractions by acting at a
site distal to the L-type Ca2+
channel? Attempts to detect such an effect yielded negative results. Our evidence is inconsistent with the hypotheses that agonist occupancy
of M1 mAChR stimulates
ICa(L) and contractions in guinea pig ventricular
myocytes. This conclusion assumes that guinea pig ventricular myocytes
express M1 mAChR and that McN-A-343 is an agonist
at such receptors.
M1 mAChR and the Heart.
The
M2 subtype is the principal mAChR in heart muscle
cells. In rat heart, the M1 subtype accounts for
~3% of total mAChR (Watson et al., 1983). Recent experiments
document the presence of mRNA for M1 mAChR in
guinea pig and rat heart (Gallo et al., 1993; Sharma et al., 1996).
M1 mAChR expression on the surface of guinea pig
and rat ventricular myocytes has contributed to the hypothesis that the
M1 mAChR initiates the novel stimulant effect of
muscarinic agonist in mammalian ventricle (Gallo et al., 1993; Sharma
et al., 1996). The presence of two cell surface mAChR subtypes
initiating opposite effects is not unprecedented. Muscarine causes
depolarization (M1) after hyperpolarization
(M2) in rat parasympathetic ganglion cells (Allen
and Burnstock, 1990).
In the human heart, pirenzepine-sensitive M1
mAChR are autoreceptors on postganglionic parasympathetic nerves
(Pitschner and Wellstein, 1988; Brodde et al., 1998). In cat atrial
myocytes, the M1 mAChR participated in the
ACh-induced potentiation of a glibenclamide-sensitive
K+ current (Wang and Lipsius, 1995). In the same
preparation, M2 mAChR activation by ACh caused an
inward Na+ current, as we reported in guinea pig
ventricular myocytes (Matsumoto and Pappano, 1991). Thus,
M1 and M2 mAChR can
function on different tissues within the heart or on the same cell.
Other variations include having M1 mAChR on
postganglionic cholinergic nerves and on cardiac myocytes in the
chicken heart (Jeck et al., 1988) and M2 mAChR on
postganglionic cholinergic nerves and on cardiac muscle cells in rat
and guinea pig hearts (Jeck et al., 1988; Bognar et al., 1990).
McN-A-343 and M1 mAChR.
Early experimental
evidence designated the receptor activated by McN-A-343 as
M1 (see the introduction). McN-A-343 bound to mAChR in heart and displaced N-methylscopolamine (Birdsall
et al., 1983). This compound has been termed a "functionally
selective" M1 agonist (reviewed in Eglen et
al., 1996). McN-A-343 had negative chronotropic (Pappano and Rembish,
1971) and inotropic effects (Fozard and Muscholl, 1972; Lambrecht et
al., 1993) on the guinea pig sinoatrial node and right atrium of
the rabbit heart. These cholinomimetic actions were prevented by
atropine; however, the mechanisms of action may have been to promote
ACh release from postganglionic parasympathetic nerves as well as a
direct effect on cardiac muscle cells.
Others have reported no functional effects of McN-A-343. In guinea pig
isolated ventricular myocytes, Cch, ACh, and Oxo, but not McN-A-343,
inhibited ISO-stimulated phosphorylation of phospholamban (Gupta et
al., 1994). Carbachol was more effective than Oxo in suppressing
ISO-stimulated phospholamban phosphorylation (Gupta et al., 1994). We
observed the same order of effectiveness for inhibition of
ISO-stimulated ICa(L). Unlike ACh, McN-A-343 did not stimulate 6-keto-PGF1
synthesis in acutely
dissociated rabbit ventricular myocytes; the M2
mAChR was implicated as the site of ACh action (Kan et al., 1996). In
propranolol-pretreated human ventricular muscle, the functionally
selective M1 agonist McN-A-343 had no effect on
contraction force at concentrations up to 1 mM (Du et al., 1995). At 1 mM, ACh maximally increased contraction force by 53 ± 17%. The
M2 mAChR initiated the stimulant effect of
muscarinic agonists in human ventricle because AF-DX 116 (M2-selective) was a more potent antagonist than
pirenzepine (M1-selective). Our observations on
guinea pig ventricular myocytes are very similar because McN-A-343 did
not affect contractions (present report) and AF-DX 116 was a more
potent antagonist than pirenzepine for opposing the stimulation of
contractions (Protas et al., 1998) and the induction of
Na+ current by Cch (Matsumoto and Pappano, 1991).
McN-A-343 did not increase contraction force in human atrium but it
overcame the negative inotropic effect of ACh, prompting the conclusion
that the M1 mAChR agonist stimulated human atrial
tissue (Du et al., 1995). However, McN-A-343 simply restored
contractions inhibited by ACh to, but not above, control levels. Thus,
McN-A-343 may be an antagonist at M2 mAChR, where
ACh acts as a negative inotropic agent. McN-A-343 did not induce a
Na+ current in ventricular myocytes but prevented
Cch from doing so at M2 mAChR (Matsumoto and
Pappano, 1991). Similarly, McN-A-343 actions on rabbit atrium were
attributed to M2 mAChR antagonism (Lambrecht
et al., 1993; Christopoulos and Mitchelson, 1997).
Limitations.
Our results (Matsumoto and Pappano, 1991; Protas
et al., 1998; present report) indicate negligible participation of
M1 mAChR in the stimulant effect of muscarinic
agonist in guinea pig ventricular myocytes. How can one explain the
discrepancies inasmuch as pirenzepine prevented stimulation of
ICa(L) in guinea pig ventricular myocytes (Gallo
et al., 1993) and was more potent than methoctramine
(M2-selective) as an antagonist of increased
intracellular Ca2+ transients by Cch in rat
ventricular myocytes (Sharma et al., 1996)?
We did not detect an increase of ICa(L) by Cch,
Oxo, or McN-A-343 in the presence of saturating concentrations of
intrapipette cAMP. Others have obtained the same result (reviewed in
Méry et al., 1997), contrary to the reported increase of the
L-type Ca2+ current by Cch via
M1 receptor activation under this condition (Gallo et al., 1993). Although increases of
ICa(L) are easier to detect at 22-24°C than at
35°C, the temperature used by Gallo et al. (1993), the activity of
M1 mAChR may be highly temperature dependent.
Also, we did not treat cells with pertussis toxin. We found that
pertussis toxin treatment is not essential to detect the Cch-induced
Na+ current (Matsumoto and Pappano, 1991), the
stimulation of Na/Ca-exchange current (Saeki et al., 1997), and the
increase of intracellular Ca2+ transients and
cell contractions (Saeki et al., 1997; Protas et al., 1998).
Conceivably, we could miss the effects reported by others because
pertussis toxin treatment either allows (Gallo et al., 1993) or
increases the likelihood (Sharma et al., 1996) of
M1-mediated stimulation. With respect to the
possibility that the products of phosphoinositide metabolism could be
involved in muscarinic stimulation of ICa(L),
only a partial answer can be given. We find that inositol
(1,4,5)-trisphosphate (1-300 µM) had no effect on
ICa(L) in guinea pig ventricular myocytes (see also Shuba et al., 1990; Saeki et al., 1999). Activation of protein kinase C (PKC) by diacylglycerol, the other product of phospholipase C
stimulation, might have promoted L-type Ca channel
phosphorylation in the experiments of Gallo et al. (1993). Although
such an effect has been reported for L-type Ca channels
expressed in Xenopus oocytes (Bourinet et al., 1992),
cardiac PKC requires Ca2+, which was buffered by
EGTA in the pipette solution. If so, the free myoplasmic
Ca2+ needed to activate PKC would have to be less
than that required to permit contraction in our experiments.
Could species differences explain the discrepancy? The
M1-mediated increase of intracellular
Ca2+ transients by Cch occurred in rat
ventricular myocytes (Sharma et al., 1996) that differ from guinea pig
ventricular myocytes in electrophysiology and excitation-contraction
coupling (reviewed in Bers, 1991). Such differences also may extend to
neurotransmitter regulation of ICa(L). However,
ACh did not increase ICa(L) in rat ventricular
myocytes (McMorn et al., 1993). When ACh was washed out of the bath
solution, a rebound stimulation of contractions and an increase in the
amplitude of the late depolarization phase of the rat ventricular
action potential occurred (McMorn et al., 1993). This action potential
phase is generated by the Na/Ca-exchange current in rat ventricular
myocytes rather than ICa(L) (reviewed in Noble,
1995).
In general, muscarinic agonists are ineffective against basal
ICa(L) in ventricle (reviewed in Pappano, 1995);
however, there are reports to the contrary. In frog and rat ventricular
myocytes, atropine increased ICa(L) in the
presence and, occasionally, in the absence of ISO (Hanf et al., 1993).
If mAChR are constitutively active in guinea pig ventricular myocytes,
we could not detect an effect of muscarinic agonists at 22-24°C.
The findings with the M1-selective agonist
McN-A-343 together with our previous observations with selective
antagonists are consistent with the hypothesis that the stimulant
effect of muscarinic agonists in guinea pig ventricular myocytes is
initiated at M2 mAChR rather than at
M1 mAChR. The lack of effect of McN-A-343 does
not exclude the presence of M1 mAChR because
low-receptor reserve or inefficient receptor-effector coupling could
account for negligible effect even though M1
mAChR are present (Eglen et al., 1996).
| |
Footnotes |
Accepted for publication March 31, 1999.
Received for publication November 18, 1998.
1
This work was supported by United States Public Health
Service Grant HL-13339.
Send reprint requests to: Achilles J. Pappano, Department
of Pharmacology, MC-6125, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030. E-mail: Pappano{at}nso1.uchc.edu
| |
Abbreviations |
Ach, acetylcholine;
ICa(L), L-type
calcium current;
McN-A-343, 3-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium;
AF-DX 116, (11[[2-[diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido
[2,3- 6][1,4]benzodiazepine-6-)one;
PKC, protein kinase C;
Oxo, oxotremorine;
ISO, isoproterenol;
Cch, carbamylcholine;
mAChR, muscarinic receptor(s).
| |
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Abstract
Introduction
