© 1995 American Heart Association, Inc.
Articles |
Acetylcholine Elicits a Rebound Stimulation of Ca2+ Current Mediated by Pertussis Toxin–Sensitive G Protein and cAMP-Dependent Protein Kinase A in Atrial Myocytes
From the Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois.
Correspondence to Stephen L. Lipsius, PhD, Department of Physiology, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract Cholinergic inhibition of atrial contraction is typically followed by a rebound positive inotropic response. In the present study, we used a nystatin–perforated patch whole-cell recording method to determine whether acetylcholine (ACh) elicits a rebound stimulation of L-type Ca2+ current (ICa,L) in cat atrial myocytes. ACh (1 µmol/L) decreased basal ICa,L (-19±2%). Within
30 s of returning to
ACh-free solution, basal ICa,L exhibited a rebound increase
above the control level (+61±7%) that returned to the control level
within 4 to 5 minutes. ACh elicited concomitant changes in cell
shortening, ie, a decrease followed by a rebound increase. The
EC50 and maximal response of ACh-induced inhibition and
rebound stimulation of ICa,L were 1.9x10-9
mol/L and -30%, respectively, and 2.9x10-8 mol/L and
+64%, respectively. All effects of ACh on ICa,L were
blocked by prior exposure to 1 µmol/L atropine or 100 µmol/L
AFDX116 and unaffected by 0.2 µmol/L pirenzepine or 1 µmol/L
propranolol. In the presence of ACh, exposure to atropine elicited
stimulation of ICa,L. ACh-induced inhibition and rebound
stimulation of current were independent of external
Ca2+. Rebound stimulation of ICa,L was
associated with a negative shift in the voltage dependence of
ICa,L activation. Inhibition of protein kinase A by 50
µmol/L Rp-cAMPs decreased basal ICa,L by 36±1% and
abolished the rebound stimulation of ICa,L. Forskolin (0.01
µmol/L) or isoproterenol (0.01 µmol/L) had no effect on basal
ICa,L, but each accentuated the rebound
increase in ICa,L. When adenylate cyclase was maximally
stimulated with 1 µmol/L isoproterenol plus 2 µmol/L forskolin, ACh
decreased ICa,L but failed to elicit rebound stimulation of
ICa,L. Milrinone (10 µmol/L) increased basal
ICa,L by 70±7% and significantly attenuated the rebound
stimulation of ICa,L. Exposure to 1 mmol/L 8-bromo-cGMP
elicited a small decrease in basal ICa,L, attenuated
ACh-induced inhibition, and enhanced the rebound stimulation of
ICa,L. Incubation in pertussis toxin prevented all
ACh-induced changes in ICa,L. Inhibition of nitric oxide
synthase by 100 µmol/L
NG-monomethyl-L-arginine
(L-NMMA) decreased basal ICa,L by -20±5%, prevented
ACh-induced inhibition, and markedly attenuated the rebound stimulation
of ICa,L. We conclude that in cat atrial myocytes ACh acts
via M2 muscarinic receptors and pertussis toxin–sensitive
G protein to inhibit basal ICa,L and that on withdrawal ACh
elicits a rebound stimulation of ICa,L. Rebound stimulation
of ICa,L is mediated via cAMP-dependent protein kinase A
enhanced by ACh-induced inhibition of phosphodiesterase. This mechanism
contributes directly to the positive inotropic response that follows
cholinergic inhibition of atrial contraction.
Key Words: perforated patch • whole-cell patch • isoproterenol • pertussis toxin • phosphodiesterase
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cholinergic stimulation exerts a strong negative inotropic effect on cardiac atrial muscle. This effect can be attributed to both direct and indirect actions of acetylcholine (ACh). Thus, ACh acts via G proteins to directly activate a K+ channel current (IK,ACh),1 which shortens action potential duration and thereby limits Ca2+ influx via L-type Ca2+ current (ICa,L). In addition, ACh can act via G proteins to inhibit adenylate cyclase and cAMP,2 and thereby decrease basal ICa,L.3 4 This mechanism is responsible for the ability of ACh to antagonize ß-adrenergic receptor–mediated stimulation of cAMP and ICa,L.5 In addition, ACh stimulates cGMP, which may in turn modulate various phosphodiesterase (PDE) isozymes to either inhibit or stimulate ICa,L.6 cGMP can also regulate ICa,L via protein kinase G.7 8 Recent reports indicate that nitric oxide (NO) may act via cGMP9 10 to regulate ICa,L.11 12 13 Moreover, cholinergic regulation of cardiac function may be mediated, at least in part, via an NO-signaling pathway.10 13 14
A consistent observation associated with cholinergic inhibition of atrial15 and ventricular16 contraction is that withdrawal of cholinergic agonist elicits a rebound increase in contractile amplitude. In atrial muscle, this rebound increase in contractility is associated with an increased intracellular Ca2+ transient indicating enhanced sarcoplasmic reticulum (SR) Ca2+ release.15 Several possible mechanisms have been proposed for the positive inotropic effects of ACh. Thus, withdrawal of ACh can elicit a rebound decrease in K+ conductance17 that may lengthen action potential duration and thereby enhance Ca2+ influx. In addition, ACh can induce an inward Na+ current18 that raises intracellular Na+ concentration19 and thereby raises intracellular Ca2+, presumably via stimulation of Na+-Ca2+ exchange.20 ACh may also act via M1 muscarinic receptors and the phosphoinositol pathway to increase ICa,L.21
In the present study, we sought to determine whether an ACh-induced increase in ICa,L could contribute to the rebound positive inotropic response that follows cholinergic inhibition of atrial contraction. We used a perforated-patch whole-cell recording method to minimize intracellular dialysis and thereby preserve second-messenger signaling pathways. The present results indicate that after ACh-induced inhibition of basal ICa,L there is a direct rebound stimulation of ICa,L that is mediated via M2 muscarinic receptors and the pertussis toxin (PTX)–sensitive G protein/adenylate cyclase/cAMP-dependent protein kinase A (PKA) signaling pathway. The rebound stimulation of ICa,L may contribute to the restoration of contractile activity and the positive inotropic response that follows cholinergic inhibition of atrial contraction. Portions of this work have been published in abstract form.22
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Details of the isolation and recording methods have been published previously.23 Briefly, adult cats of either sex were anesthetized with sodium pentobarbital (70 mg/kg IP). Hearts were rapidly excised and perfused via a Langendorff apparatus. The initial perfusion was a bicarbonate-buffered Tyrode's solution for
5
minutes, followed by perfusion with a nominally
Ca2+-free Tyrode's solution. After 5 minutes, the
perfusion was switched to a low (36 µmol/L) Ca2+
Tyrode's solution containing 0.06% collagenase (Worthington
Biochemical, type II) for 30 to 40 minutes. After collagenase
perfusion, both atria were cut into small pieces and agitated in fresh
collagenase and 0.01% protease. Experiments were performed on either
right or left atrial cells, with no discernible differences.
Cells used for study were transferred to a small tissue bath on the
stage of an inverted microscope (Nikon Diaphot) and superfused with a
modified Tyrode's solution containing (mmol/L) NaCl 137, KCl 5.4,
MgCl2 1.0, CaCl2 2.5, HEPES 5, and glucose 11
and titrated with NaOH to a pH of 7.4. The solution was perfused
through a small (0.3-mL) chamber by gravity. The system required 30
s to completely exchange the bath contents. All experiments were
performed at 35±1°C. Cells selected for study were elongated and
quiescent. Voltage and ionic currents were recorded using a
nystatin–perforated patch24 whole-cell recording
method.25 This method minimizes dialysis of intracellular
constituents with the internal pipette solution. Nystatin was dissolved
in dimethylsulfoxide at a concentration of 50 mg/mL and then added to
the internal pipette solution to yield a final nystatin concentration
of 150 µg/mL. The nystatin-containing pipette solution was strongly
sonicated so that the nystatin was completely dispersed into solution.
The internal pipette solution contained (mmol/L) cesium glutamate 100,
CsCl 40, MgCl2 1.0, Na2-ATP 4, EGTA 0.5, and
HEPES 5 and was titrated with CsOH to a pH of 7.2. Cs+
replaced K+ in the internal pipette solution, and 20 CsCl
was added to the external solution to block IK,ACh. If ACh
elicited changes in background K+ conductance, the cell was
discarded.
A single suction pipette was used to record voltage (bridge mode) or ionic currents (discontinuous voltage-clamp mode) by use of an Axoclamp 2A amplifier (Axon Instruments, Inc). Computer software (PCLAMP; Axon Instruments, Inc) was used to deliver voltage protocols and to acquire and analyze data. Unloaded cell shortening was monitored with a video-based edge detector (Crescent Electronics). This system uses a single-raster line-scanning technique to detect edge motion at one or both ends of a cell. All signals were digitally recorded on videotape.
Drugs included ACh chloride (Sigma Chemical Co), isoproterenol (ISO, Sigma), forskolin (Sigma), milrinone (Sigma), H-89 (Seikagaku America, Inc), Rp-cAMPs (LC Laboratories), NG-monomethyl-L-arginine (L-NMMA, Sigma), atropine (Sigma), AFDX116 (a generous gift from Boehringer Ingelheim), pirenzepine (Sigma), propranolol (Sigma), and pertussis toxin (Sigma). Cells studied were isolated on the same morning as the experiment. In general, cells were held at -40 mV to inactivate fast Na+ channels and clamped to 0 mV for 200 ms every 10 s to activate ICa,L. Peak ICa,L was measured with respect to zero current and was not compensated for leak currents. Statistical significance of paired and unpaired data was determined by Student's t test at values of P<.05. Data are expressed as mean±SEM.
The procedures employed in the care and use of the animals in the present study were approved by the Institutional Animal Care and Use Committee of Loyola University Medical Center.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Fig 1
shows a typical response of an atrial myocyte
to a 4-minute exposure to 1 µmol/L ACh. Panel A shows original
recordings of ICa,L recorded before, during, and after
exposure to ACh. The graph in panel B shows consecutive measurements of
peak ICa,L amplitude. The letters a through d correspond to
the selected currents shown in panel A. Tracing a shows basal
ICa,L recorded under control conditions. ACh decreased
basal ICa,L by 22% (tracing b). ACh-induced inhibition
of ICa,L remained constant during the entire exposure,
indicating the lack of desensitization. That the holding current
remained constant during the ACh exposure indicates that
IK,ACh was effectively blocked by cesium. Within 60 s of
returning to ACh-free Tyrode's solution, peak ICa,L
exhibited a rebound increase of 48% above control. In addition, the
time course of ICa,L inactivation was prolonged (panel A,
tracing c). ICa,L amplitude returned to the control levels
4.5 minutes after ACh was withdrawn. Panels C and D show the
concomitant changes in cell shortening associated with the ACh-induced
changes in ICa,L. ACh decreased cell shortening by 10%
(b), and withdrawal of ACh elicited a 38% rebound increase in cell
shortening (c). In addition, upon withdrawal of ACh the development of
the rebound increase in cell shortening (panel D) lagged slightly
behind the development of the rebound increase in peak
ICa,L (panel B). Moreover, the time course of the recovery
of cell shortening to control levels (8 minutes) was almost twice as
long as the recovery of peak ICa,L (4.5 minutes). In a
total of 53 cells, 1 µmol/L ACh decreased basal ICa,L by
-19±2%, and withdrawal of ACh increased basal ICa,L by
+61±7% above control. In 5 cells, ACh decreased peak cell shortening
by -12±3%, and withdrawal of ACh increased peak cell shortening by
+20±5%. Qualitatively similar results were obtained with ACh
exposures as short as 30 s. We did not test exposure times shorter than
30 s because our perfusion system requires 30 s to completely change
the bath contents. Moreover, under control conditions ICa,L
inactivation exhibited a biexponential time course: an initial rapid
phase (
1=9.5±1.1 ms) followed by a secondary slower
phase (2=45.7±2.2 ms) (n=7). During the rebound
increase in ICa,L amplitude, the time constant of the
initial phase of ICa,L inactivation was increased
(1=19.6±3.7 ms) compared with control
(P<.01), whereas the secondary phase was not different
(2=43.2±3.6 ms) (n=7). The rebound increase in
ICa,L amplitude ranged from as low as 5% to as large as
232% above control in any one cell (SD=±48.9; n=53). The following
experiments were designed primarily to determine the mechanisms
responsible for the rebound stimulation of ICa,L elicited
by withdrawal of ACh.
|
The graph in Fig 2 shows the dose-response relation of
different ACh concentrations for both the inhibition and the rebound
stimulation of ICa,L. The threshold concentration of ACh
that decreased ICa,L was 10-9 mol/L,
whereas the rebound increase in ICa,L required
10-8 mol/L ACh. In addition, the ACh-induced rebound
stimulation of ICa,L continued to increase at ACh
concentrations that maximally inhibited ICa,L. For example,
at 10-7 mol/L ACh the rebound increase in
ICa,L was 66% of its maximum response, whereas the
ACh-induced decrease of ICa,L was essentially maximal.
Maximal inhibition of ICa,L was 30% below control
compared with maximal rebound stimulation of ICa,L of
64% above control. Thus, the EC50 for ACh-induced
inhibition of ICa,L (1.9x10-9 mol/L) was
about one order of magnitude smaller than the EC50 for the
rebound stimulation of ICa,L (2.9x10-8
mol/L). These results indicate that the concentration dependence, ie,
sensitivity and responsiveness, of the two responses is different and
therefore suggest that the rebound stimulation of ICa,L is
not functionally related to ACh-induced inhibition of
ICa,L. This is consistent with our observations throughout
the present study that the magnitude of ACh-induced inhibition and
rebound stimulation of ICa,L were unrelated in any given
cell.
|
) and rebound increase (
) of the
L-type Ca2+ current (ICa,L). The percent
decrease or increase of ICa,L is in relation to control
basal ICa,L amplitude. Values of ACh-induced decrease and
rebound increase of ICa,L were obtained from the same
cells, and the number of cells studied is indicated in parentheses. The
EC50 values for ACh-induced decrease and rebound increase
of ICa,L were 1.9x10-9 mol/L and
2.9x10-8 mol/L, respectively.
In Fig 3, we determined which type of receptor mediated
the effects of ACh. Panel A shows selected recordings of
ICa,L, which illustrate a typical response to 1
µmol/L ACh, ie, a decrease followed by a rebound increase in peak
ICa,L. ICa,L recovered to control levels after
4 minutes. Panel B shows recordings of ICa,L obtained
from the same cell exposed to 1 µmol/L atropine. Prior exposure to
atropine blocked all effects of ACh on ICa,L. It should be
noted that atropine alone had no effect on basal ICa,L.
Similar results were obtained in a total of six cells. Panel C shows
recordings from another atrial cell in which atropine was administered
after exposure to ACh was begun. In this experiment, 1 µmol/L ACh
elicited a modest inhibition of ICa,L. However, addition of
1 µmol/L atropine to the ACh-containing solution elicited a prominent
stimulation of ICa,L (n=2). In two additional cells, prior
exposure to 100 µmol/L AFDX116, a selective M2 muscarinic
receptor antagonist,26 also blocked all effects of ACh on
ICa,L (not shown). Moreover, ACh-induced inhibition and
rebound stimulation of ICa,L were unaffected by 0.2
µmol/L pirenzepine, an M1 muscarinic receptor antagonist
(n=3), or 1 µmol/L propranolol, a ß-adrenergic receptor antagonist
(n=3) (not shown). These results indicate that both ACh-induced
inhibition and rebound stimulation of ICa,L are mediated
via M2 muscarinic receptors. Moreover, because atropine is
a competitive inhibitor of muscarinic receptors, these findings support
the idea that rebound stimulation of ICa,L results from
removal of ACh from the muscarinic receptor.
|
There is evidence that ICa,L amplitude can be enhanced
during repetitive stimulation and that this facilitation of
ICa,L depends on Ca2+ entry through
L-type channels.27 To determine whether
Ca2+ influx participates in the rebound stimulation
of ICa,L, ACh was tested in the absence of external
Ca2+. Influx of Na+ through L-type
Ca2+ channels was promoted by lowering external
Mg2+ concentration in the
Ca2+-free solution.28 Fig 4A shows a control ICa,L recording in normal
Tyrode's solution. This cell exhibited a typically slow late component
of ICa,L inactivation, which can be attributed to
Na+-Ca2+ exchange.29
Exposure to Ca2+-free Tyrode's solution containing
1 mmol/L EGTA eliminated ICa,L. In panel B, lowering the
Mg2+ concentration to 250 µmol/L restored a
smaller net inward current that exhibited very slow inactivation
kinetics (control). The slow inactivation can be attributed to the loss
of Ca2+-mediated inactivation30 and the
fact that the inward current is now carried by Na+ through
L-type channels.28 Exposure to 1 µmol/L ACh decreased
the current by 47%, and withdrawal of ACh elicited a rebound
increase of 38%. In a total of four cells, ACh decreased inward
current by 48±9%, and withdrawal of ACh elicited a 61±10% rebound
increase in inward current. These findings are qualitatively similar to
those obtained in normal external Ca2+ (Fig 1
). In
three additional cells, 1 µmol/L ryanodine, an inhibitor of SR
Ca2+ release,31 had no effect on
ACh-induced inhibition or rebound stimulation of ICa,L (not
shown). These results, therefore, indicate that neither the inhibition
nor the rebound stimulation of ICa,L elicited by ACh is
dependent on external Ca2+ influx or intracellular
Ca2+ release.
|
The previous results make it unlikely that currents mediated by
Ca2+ are participating in the rebound stimulation of
total current. The possibility remains that currents that are not
Ca2+-mediated may contribute to the rebound increase
in total current. Therefore, we tested 1 µmol/L ACh in the absence
and presence of 1 µmol/L verapamil to determine whether currents
other than ICa,L may be participating in the rebound
response. Fig 5A shows selected control recordings of
basal ICa,L (left) and ICa,L rebound stimulated
by withdrawal of ACh (right). Panel B shows the same experimental
protocol repeated in the presence of verapamil. Under basal conditions
(left), verapamil abolished all inward current, leaving only a small
residual outward current during the depolarizing step. Exposure to ACh
(not shown) and withdrawal of ACh (right) failed to elicit any change
in current. Subtraction of these verapamil-insensitive currents from
control recordings (panel A) yielded the verapamil-sensitive currents
shown in panel C. Clearly, the peak verapamil-sensitive currents are
essentially the same as those in the control condition. The
verapamil-sensitive inward currents show a background inward current
component that can be accounted for by the residual outward current
seen in the presence of verapamil (panel B). In a total of three
cells, there were no differences between control basal peak
ICa,L (-276±74 pA) and verapamil-sensitive basal peak
ICa,L (-277±75 pA) and between control rebound-stimulated
peak ICa,L (-485±68 pA) and verapamil-sensitive
rebound-stimulated peak ICa,L (-486±69 pA). The
present results indicate that the rebound increase in inward
current elicited on withdrawal of ACh is entirely verapamil-sensitive
ICa,L.
|
In Fig 6, we determined the voltage dependence of
ICa,L activation before, during, and after the post-ACh
rebound period by delivering depolarizing voltage-clamp pulses every 2
s. The graph shows the current-voltage relations of ICa,L
obtained from six cells. Under control conditions, the voltage
dependence of ICa,L activation exhibited typical features
with the maximum peak ICa,L at 0 mV (-268±20 pA). The
same voltage-clamp protocol was performed 30 s after withdrawal of
ACh. During this time, peak ICa,L was increased at all
voltages and maximum peak ICa,L was shifted to -10 mV
(-391±34 pA). To demonstrate that the maximum ICa,L
shifted to the left during the rebound response, we compared the
rebound ICa,L amplitude at 0 mV (the voltage at which
maximum ICa,L occurred in control) with the rebound
ICa,L amplitude at -10 mV. Peak ICa,L was
significantly larger at -10 mV compared with 0 mV (P<.05).
After 5 minutes, ICa,L returned to control levels at all
voltages and maximum peak current returned to 0 mV (-280±18 pA).
These results indicate that the rebound increase in ICa,L
involves both an increase in conductance and a shift in the voltage
dependence of peak ICa,L activation. Similar changes in
ICa,L amplitude and voltage dependence are elicited by
ß-adrenergic stimulation of cAMP.4
|
). Maximum peak ICa,L
was shifted to more negative voltages during the rebound stimulation of
ICa,L.
Therefore, we sought to determine whether the rebound increase in
ICa,L is mediated by stimulation of cAMP-dependent PKA
activity. As shown in Fig 7, a 4-minute exposure to 1
µmol/L ACh elicited a typical decrease in basal ICa,L
(-20%), which, on withdrawal of ACh, was followed by a large rebound
increase in ICa,L above control (+72%). ICa,L
returned to control levels within 5 minutes. Exposure to 50 µmol/L
Rp-cAMPs, a selective PKA antagonist,32 elicited a rapid
decrease in basal ICa,L of 35%, indicating inhibition
of basal PKA-mediated stimulation of ICa,L. In the presence
of Rp-cAMPs, a second 4-minute exposure to 1 µmol/L ACh elicited a
smaller decrease in ICa,L and the rebound increase in
ICa,L was abolished. Washout of Rp-cAMPs for 4 minutes
resulted in an incomplete recovery of basal ICa,L
amplitude. Rp-cAMPs abolished the rebound increase in ICa,L
in all three cells tested. In three additional cells, 2 µmol/L H-89,
another PKA inhibitor,33 also abolished the rebound
increase in ICa,L (not shown). These results provide
evidence that basal ICa,L is mediated by endogenous
cAMP-dependent PKA activity and that rebound stimulation of
ICa,L is mediated by enhanced cAMP-dependent PKA
activity.
|
Low concentrations of forskolin facilitate receptor-mediated
stimulation of adenylate cyclase without directly stimulating adenylate
cyclase activity.34 Fig 8 shows the results
from an experiment in which ACh was tested in the absence and presence
of 0.01 µmol/L forskolin. The top tracings show selected recordings
of ICa,L obtained at different times throughout the
experiment (tracings a through g), and the graph shows consecutive
measurements of peak ICa,L amplitude during the experiment.
Under control conditions, in the absence of forskolin, 1 µmol/L ACh
elicited a decrease followed by a rebound increase in ICa,L
of +29%. After the return of ICa,L to control levels,
exposure to forskolin had no discernible effect on basal
ICa,L amplitude compared with control ICa,L. In
the presence of forskolin, ACh decreased ICa,L, and
the rebound stimulation of ICa,L (+76%) was enhanced
compared with the control rebound response in the absence of forskolin.
In a total of five cells, withdrawal of ACh in the presence of
forskolin elicited a significantly larger rebound increase in
ICa,L (+62±18%) than the control rebound response
(+22±4%) (P<.02). Similar experiments were performed with
low concentrations (0.01 µmol/L) of ISO. In a total of three cells,
ISO had no affect on basal ICa,L but potentiated the
rebound stimulation of ICa,L (control rebound, +39±2%;
ISO rebound, +74±12%). These findings provide additional evidence
that the rebound stimulation of ICa,L is mediated via
adenylate cyclase and that adrenergic stimulation enhances the effect
of ACh to elicit a rebound stimulation of ICa,L.
|
If the cAMP-dependent PKA pathway mediates the rebound stimulation of
ICa,L, then maximal stimulation of the system should
prevent the rebound stimulation of ICa,L. Therefore, we
tested ACh after maximally stimulating adenylate cyclase/cAMP with 1
µmol/L ISO plus 2 µmol/L forskolin. Under control conditions, 1
µmol/L ACh elicited a typical inhibition (-12%), followed by a
rebound stimulation of ICa,L (+37%, n=4) (not shown). The
recordings in Fig 9 show that exposure to ISO plus
forskolin elicited a large increase in peak ICa,L (+170%)
and prolonged ICa,L inactivation. The holding current was
unchanged. Under these conditions, ACh elicited a larger inhibition of
ICa,L (-38%) and accelerated ICa,L
inactivation. Withdrawal of ACh, however, failed to elicit any further
increase in ICa,L compared with that recorded in ISO and
forskolin, although the time course of inactivation was prolonged once
again. Similar results were obtained in a total of four cells.
|
So far, the results indicate that after ACh-induced inhibition there is
a rebound increase in ICa,L mediated by cAMP. We
hypothesized that the rebound stimulation of ICa,L could
result from at least two possible mechanisms: (1) an ACh-induced
rebound stimulation of adenylate cyclase activity and/or (2) an
ACh-induced inhibition of PDE activity and subsequent increase in cAMP.
These two possibilities can be distinguished by testing ACh during
pharmacological inhibition of PDE. If the former hypothesis is correct,
then pharmacological inhibition of PDE should accentuate the rebound
stimulation of ICa,L. If, on the other hand, the latter
hypothesis is correct, then prior inhibition of PDE should attenuate
the stimulatory response to ACh. A likely PDE candidate for ACh-induced
inhibition is cGMP-inhibited cAMP-dependent PDE type III,6
which is sensitive to milrinone.35 Fig 10
shows consecutive measurements of a typical experiment in which 1
µmol/L ACh was administered in the absence and then in the presence
of 10 µmol/L milrinone. In the control condition, ACh elicited a
decrease (-10%) followed by a rebound increase in ICa,L
(+53%) that returned to baseline within 5 minutes. Exposure to
milrinone increased basal ICa,L by 97% above control. In
the presence of milrinone, 1 µmol/L ACh elicited a much larger
inhibition of ICa,L (-68%), and the rebound stimulation
of ICa,L was only 10% above baseline in the presence of
milrinone. In other words, the rebound stimulation of ICa,L
was attenuated by 81% compared with the control rebound response.
During washout of ACh, ICa,L amplitude returned to baseline
within 5 minutes. In a total of six cells, 10 µmol/L milrinone
increased basal ICa,L by 70±7%. In milrinone, the
ACh-induced rebound increase in ICa,L was 25% of the
control rebound response (control, 47±3%; milrinone, 12±3%). If the
magnitude of rebound increase in ICa,L is related to the
extent of PDE inhibition by milrinone, then a lower concentration of
milrinone should exert less inhibition of PDE and less attenuation of
the rebound increase in ICa,L. Therefore, in four
additional cells we tested the effect of 5 µmol/L milrinone. In
milrinone, the ACh-induced rebound increase in ICa,L was
54% of the control rebound response (control, +52±10%; milrinone,
+24±9%). These results suggest that ACh-induced inhibition of PDE may
enhance cAMP-mediated stimulation of ICa,L.
|
Our interpretation of this last series of experiments is based on the
assumption that ICa,L is not maximally stimulated by
milrinone (see Fig 9). To confirm this point, we determined whether ISO
could elicit a further increase in ICa,L after
ICa,L was upregulated by milrinone. In an additional five
cells, 10 µmol/L milrinone increased basal ICa,L by
106±13%. In the presence of milrinone, 1 µmol/L ISO elicited an
additional 101±26% increase in ICa,L amplitude. When
compared with control basal ICa,L, ISO increased
ICa,L by 317±67%. Clearly, ICa,L is not
maximally stimulated by milrinone, and cAMP is capable of further
stimulating ICa,L. Moreover, these findings support our
interpretation that milrinone attenuated the rebound response and that
ACh-induced inhibition of type III PDE is a primary mechanism
underlying the rebound increase in ICa,L.
Because ACh may generate cGMP, we tested the effects of 8-bromo-cGMP
(8-Br-cGMP) on the rebound stimulation of ICa,L elicited by
withdrawal of ACh. 8-Br-cGMP stimulates protein kinase G (PKG)
activity.36 The Table summarizes the
results obtained in four cells. Under control conditions, ACh elicited
a typical inhibition (-15±3%), followed by rebound stimulation of
ICa,L (+43±3%). After steady state recovery from ACh, 1
mmol/L 8-Br-cGMP elicited a small but significant inhibition of basal
ICa,L (-5.4±0.1%; P<.005). In the presence
of 8-Br-cGMP, a second ACh exposure elicited a significantly smaller
inhibition of ICa,L (-7±2%; P<.05), and
withdrawal of ACh elicited a significantly larger rebound stimulation
of ICa,L (+57±6%; P<.05). The percent changes
in ACh-induced inhibition and rebound stimulation of ICa,L
elicited in the presence of 8-Br-cGMP resulted primarily from the
decrease in basal ICa,L induced by 8-Br-cGMP. These results
suggest that cGMP-induced stimulation of PKG may contribute to
ACh-induced inhibition of ICa,L but not to ACh-induced
rebound stimulation of ICa,L (see "Discussion").
|
PTX-sensitive G proteins couple M2 muscarinic receptors to
adenylate cyclase and mediate the inhibitory effects of
ACh.37 To determine whether PTX-sensitive G proteins
mediated the rebound stimulation of ICa,L, we
incubated atrial cells in PTX (3.4 µg/mL for 8 hours at 36°C). Fig 11 shows selected recordings of ICa,L
obtained from a cell pretreated with PTX. ACh (1 µmol/L) failed to
elicit an inhibition, and withdrawal of ACh failed to elicit a rebound
stimulation of ICa,L. Similar results were obtained in a
total of seven atrial cells pretreated with PTX. As shown in the last
panel, PTX-treated cells still responded to stimulation by 1 µmol/L
ISO. In three PTX-treated cells tested, ISO increased peak
ICa,L amplitude by 135±15%. Moreover, in all three
PTX-treated cells ACh failed to inhibit ICa,L previously
stimulated with 1 µmol/L ISO (not shown). These results indicate that
both ACh-induced inhibition and rebound stimulation of
ICa,L are mediated via PTX-sensitive G proteins.
|
NO may be involved in the inhibitory effects of ACh on
ICa,L.13 Therefore, we performed an initial
series of experiments to determine whether NO may be involved in the
rebound stimulation of ICa,L elicited by withdrawal of ACh.
Fig 12 shows an experiment in which ACh was tested in
the absence and presence of 100 µmol/L
NG-monomethyl-L-arginine
(L-NMMA), a selective antagonist of NO synthase.38 In
these experiments, cells were exposed to L-NMMA for 15 to 20 minutes
before the second ACh exposure. In the experiment shown, ACh induced a
typical inhibition (-16%) followed by a rebound stimulation (+33%)
of ICa,L. A 20-minute exposure to L-NMMA decreased basal
ICa,L by 11%. In the presence of L-NMMA, the
ACh-induced inhibition (-3%) and rebound stimulation of
ICa,L (+8%) were both markedly attenuated. In a total of
five cells, the responses to ACh under control conditions were a
decrease (-22±3%) followed by a rebound increase in
ICa,L (+29±3%). L-NMMA decreased basal ICa,L
by 20±5%. In the presence of L-NMMA, ACh failed to inhibit
ICa,L, and the rebound stimulation of
ICa,L (+8±2%) was significantly attenuated by 72%
compared with control responses (P<.05). In other
experiments, when cells were incubated in 100 µmol/L L-NMMA for at
least 1 hour, ACh failed to elicit inhibition or rebound stimulation of
ICa,L (n=4). To help ensure that L-NMMA did not exert a
nonspecific rundown of ICa,L, we performed one
experiment in which 1 µmol/L ISO was administered during inhibition
of ICa,L by 100 µmol/L L-NMMA. In the presence of L-NMMA,
ISO elicited a typical stimulation of ICa,L.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cholinergic inhibition of atrial contraction is followed by a rebound increase in contractility5 15 that is associated with a rebound increase in SR Ca2+ release.15 Because ICa,L triggers SR Ca2+ release, we sought to determine whether cholinergic inhibition of ICa,L is followed by a rebound stimulation of ICa,L and the possible mechanisms that underlie an ACh-induced increase in ICa,L. We used a perforated-patch recording method to minimize alterations in the intracellular milieu and thereby preserve second-messenger signaling mechanisms. The present experiments show that after ACh-induced inhibition of basal ICa,L, withdrawal of ACh elicits a rebound stimulation of ICa,L and contraction. The rebound stimulation of ICa,L is mediated primarily via the M2 muscarinic receptor/PTX-sensitive G protein/adenylate cyclase/cAMP-dependent PKA signaling pathway.
In the present study, inhibition of PDE by milrinone markedly
increased basal ICa,L, and inhibition of
cAMP-dependent PKA with either Rp-cAMPs or H-89 markedly decreased
basal ICa,L. In cardiac muscle, adenylate cyclase and cAMP
production are thought to be regulated by inhibitory (Gi)
and stimulatory (Gs) G proteins that are coupled to
M2 muscarinic and ß-adrenergic receptors,
respectively.37 39 However, either or both of these
systems can exert tonic or endogenous effects on adenylate cyclase
activity. It appears, therefore, that in cat atrial myocytes there is
significant endogenous Gs-mediated stimulation of adenylate
cyclase that is independent of receptor-mediated activation. Moreover,
this endogenous cAMP production is strongly modulated by endogenous PDE
activity. Thus, inhibition of PDE by milrinone increased basal
ICa,L by 70%. In other experiments, 50 µmol/L
isobutylmethylxanthine (IBMX), a nonspecific PDE
inhibitor,6 increased basal ICa,L by 220%,
which was maximal activation of ICa,L (authors'
unpublished observations). These findings suggest that more than one
type of PDE may modulate stimulation of ICa,L. In addition,
these results suggest that cAMP-mediated stimulation of
ICa,L would be effectively modulated by mechanisms that
regulate cAMP-dependent PDE activity. Generally, this appears to be the
case in mammalian cardiac myocytes in contrast to amphibian cardiac
myocytes.40
Although the inhibitory effects of ACh were not the primary focus of
the present study, the results indicate that ACh inhibited basal
ICa,L. This effect appears to be mediated, at least in
part, through inhibition of endogenous cAMP via the classical
M2 muscarinic receptor/Gi protein/adenylate
cyclase/cAMP-dependent PKA cascade. Thus, ACh-induced inhibition of
ICa,L was (1) abolished by AFDX116, a selective
M2 muscarinic receptor blocking agent, (2) abolished by
pretreatment with PTX, (3) enhanced by the inhibition of PDE with
milrinone, and (4) decreased by the inhibition of cAMP-dependent PKA
activity by Rp-cAMPs. These findings are similar to those described in
another study, which reported that ACh decreases basal
ICa,L via inhibition of endogenous cAMP.3
Although inhibition of PKA with Rp-cAMPs decreased basal
ICa,L, ACh still elicited some inhibition of
ICa,L (Fig 7). It seems likely that this action of ACh is
via other pathways, such as cGMP-stimulated PKG (Table). Moreover,
inhibition of NO synthase with L-NMMA prevented ACh-induced inhibition
of ICa,L. Others have shown that inhibition of NO
production can prevent the inhibitory effects of cholinergic agonist on
the beating rate10 and ICa,L.13
Therefore, these results support the idea that NO is required for
ACh-induced inhibition of ICa,L in atrial myocytes.
The main focus of the present study was to examine the mechanisms underlying the rebound stimulation of ICa,L elicited by the withdrawal of ACh. The present results show that the rebound increase in ICa,L was (1) independent of external Ca2+ influx, (2) unaffected by inhibition of SR Ca2+ release by ryanodine, and (3) entirely verapamil-sensitive. These findings suggest that the rebound increase in current elicited by the withdrawal of ACh was entirely due to ICa,L and not mediated by intracellular Ca2+ events or related to Ca2+-dependent facilitation of ICa,L.27 Several lines of evidence indicate that the primary pathway mediating the rebound stimulation of ICa,L is the Gi protein/adenylate cyclase/cAMP-dependent PKA second-messenger signaling system. Thus, the rebound increase in ICa,L was (1) abolished by pretreatment with PTX, (2) abolished by cAMP-dependent PKA antagonists, (3) potentiated by facilitation of adenylate cyclase with forskolin or ISO, and (4) prevented by maximal stimulation of adenylate cyclase. In addition, the rebound stimulation of ICa,L was associated with a negative shift in the voltage dependence of ICa,L activation and a prolongation in the time course of ICa,L inactivation. Both of these changes are associated with cAMP-dependent PKA phosphorylation of ICa,L.4 28 41 That ACh-induced changes in ICa,L were blocked by atropine or AFDX116 and unaffected by pirenzepine indicates that ACh is not acting via M1 muscarinic receptor subtypes. Therefore, it seems unlikely that the rebound stimulation of ICa,L is mediated by the phosphoinositol signaling pathway, which is coupled to M1 muscarinic receptors.21 26 These findings are consistent, however, with an effect of ACh via M2 muscarinic receptors, which are coupled to the PTX-sensitive G protein/adenylate cyclase/cAMP-dependent PKA system.26 This conclusion is supported by direct measurements of cAMP in embryonic chick heart cells.42 Thus, termination of acute (2- to 5-minute) exposure to cholinergic agonists increased basal and ISO-stimulated cAMP content above control levels. Moreover, this response was abolished in cells pretreated with PTX and was not mediated by the phosphoinositol signaling pathway.
The present results also show that exposure to atropine in the
presence of ACh stimulated ICa,L in a manner similar to
that elicited by the withdrawal of ACh (Fig 3). Because atropine is a
competitive antagonist of the muscarinic receptor, these results
support the view that the rebound stimulation of ICa,L is
due to removal of ACh from the receptor. Atropine, however, has been
shown to directly stimulate ICa,L via the muscarinic
receptor.43 44 These stimulatory effects of atropine on
ICa,L appear to require prior stimulation of adenylate
cyclase. Thus, atropine was unable to stimulate basal ICa,L
in frog myocytes unless previously stimulated by ISO or forskolin but
was able to stimulate basal ICa,L in rat myocytes, which
are thought to exhibit a higher basal adenylate cyclase
activity.44 In the present experiments, however,
atropine had no effect on basal ICa,L, even though
our results clearly indicate that atrial myocytes exhibit significant
basal adenylate cyclase activity. Therefore, it appears that the
stimulatory effects of atropine on ICa,L may not be a
general phenomenon and may be species-dependent. In the present
experiments, it appears that the primary mechanism by which atropine
stimulated ICa,L was to displace ACh from the receptor.
Furthermore, the present findings are consistent with those in
embryonic chick heart cells, where the addition of atropine after acute
preincubation with cholinergic agonist resulted in enhanced
ISO-stimulated accumulation of cAMP.42
As mentioned earlier, based on the pronounced response of
ICa,L to PDE inhibitors, modulation of PDE activity could
effectively modulate ICa,L. A primary mechanism regulating
PDE activity is cGMP. Thus, cGMP can act on various PDE isozymes to
either stimulate or inhibit ICa,L.6 In cardiac
muscle, stimulation of ICa,L can result from cGMP-mediated
inhibition of the type III PDE isozyme.35 Type III PDE
exhibits a high affinity for cAMP and is selectively inhibited by
milrinone. That milrinone markedly increased basal ICa,L
suggests that type III PDE is present in cat atrial myocytes. The
remaining question is how withdrawal of ACh elicits a rebound
stimulation of ICa,L. On the basis of the present
findings, we propose that ACh is acting via M2 muscarinic
receptors and PTX-sensitive G proteins to inhibit adenylate
cyclase/cAMP-dependent PKA and thereby elicit the inhibition of
ICa,L. At the same time, ACh inhibits type III PDE
activity, probably via stimulation of cGMP. Inhibition of PDE would
have little effect on ICa,L as long as cAMP levels are
depressed by ACh. Withdrawal of ACh initiates the recovery from the
ACh-induced inhibition of cAMP and PDE activities. However, the time
course of recovery of each system may differ. Recovery of adenylate
cyclase/cAMP may occur more rapidly than the recovery of PDE activity,
resulting in the return of basal cAMP levels at a time when PDE is
still depressed from the previous ACh exposure. This results in an
increase in the effective basal cAMP and rebound stimulation of
ICa,L. As PDE activity slowly recovers, the effect of cAMP
is diminished and ICa,L returns to control over several
minutes. This scheme suggests a two-component model, in which the
primary mechanism responsible for ACh-induced inhibition of
ICa,L is the inhibition of adenylate cyclase and the
mechanism responsible for rebound stimulation of ICa,L is
the inhibition of PDE activity. This two-component mechanism is
consistent with the fact that the EC50 values and maximum
responses of ACh-induced inhibition and rebound stimulation of
ICa,L are different from one another (Fig 2). In addition,
the fact that milrinone elicited a dose-dependent attenuation rather
than enhancement of the rebound stimulation of ICa,L
suggests that withdrawal of ACh does not elicit a direct rebound
stimulation of adenylate cyclase activity.
The present experiments also examined the potential role of
cGMP-stimulated PKG in the rebound stimulation of ICa,L.
Basal ICa,L was slightly inhibited by 8-Br-cGMP. Others
have reported that in ventricular myocytes 8-Br-cGMP, acting via PKG,
inhibits ICa,L previously stimulated by cAMP or ISO but not
basal ICa,L.7 8 The fact that ACh-induced
inhibition of ICa,L was attenuated by 8-Br-cGMP suggests
that cGMP-stimulated PKG activity may mediate a component of
ACh-induced inhibition of ICa,L. As mentioned earlier, this
may explain the finding that ACh elicited some inhibition of
ICa,L that was not dependent on cAMP-dependent PKA activity
(Fig 7). If the rebound stimulation of ICa,L were due to a
rebound decrease in cGMP-stimulated PKG activity, then continuous
stimulation of PKG by 8-Br-cGMP would be expected to attenuate the
rebound response. However, the present experiments show that the
percent rebound stimulation of ICa,L was somewhat enhanced
by 8-Br-cGMP. Our interpretation of these findings is that the
cGMP-induced stimulation of PKG eliminated a component of ACh-induced
inhibition of ICa,L, and that, therefore, the
stimulation of ICa,L elicited by withdrawal of ACh was
enhanced. These results suggest that cGMP-stimulated PKG may contribute
a small component to ACh-induced inhibition of basal ICa,L
but does not underlie the rebound response elicited by withdrawal of
ACh.
The present findings also indicate that inhibition of NO production by L-NMMA prevented ACh-induced inhibition and significantly attenuated the rebound stimulation of ICa,L. Because both responses to ACh are mediated, at least in part, via cAMP, it is possible that endogenous NO may be required to determine the level of endogenous cAMP-mediated stimulation of ICa,L. This is supported by the fact that L-NMMA decreased basal ICa,L by about the same extent as Rp-cAMPs or ACh, both of which act to inhibit basal ICa,L via inhibition of endogenous cAMP. Because NO stimulates cGMP9 and cGMP modulates PDE,6 we speculate that endogenous NO production acts via cGMP to inhibit PDE and thereby enhance endogenous cAMP-mediated stimulation of basal ICa,L. A similar mechanism has been proposed for NO-induced stimulation of ICa,L in human atrial myocytes.12 Therefore, inhibition of NO production by L-NMMA decreases cGMP and thereby indirectly decreases endogenous cAMP and basal ICa,L. Without endogenous cAMP to stimulate basal ICa,L, the inhibitory and rebound stimulatory effects of ACh on ICa,L are markedly attenuated. Additionally, it is possible that NO production is involved in the ACh-induced activation of cGMP and its various affects on substrates such as PDE and PKG. Although these ideas are consistent with our experimental observations, we emphasize that the results obtained with L-NMMA are preliminary and that further experiments are required to fully elucidate the role of NO in the effects of ACh to elicit a rebound stimulation of ICa,L.
It is well known that the inhibitory effects of ACh on the heart are
accentuated by prior ß-adrenergic receptor stimulation, ie,
accentuated antagonism. The mechanism is related to the ability of ACh
to antagonize adrenergically stimulated cAMP. This mechanism can
explain the present findings that ACh-induced inhibition of
ICa,L was greater after ICa,L was enhanced by
the inhibition of PDE. The present findings, however, also indicate
that background concentrations of ISO enhanced the rebound stimulation
of ICa,L elicited by the withdrawal of ACh. This effect was
similar to that seen with forskolin (Fig 8), which facilitates
receptor-mediated stimulation of adenylate cyclase.34
Therefore, the present results indicate that ß-adrenergic
receptor stimulation not only accentuates ACh-induced inhibition of
ICa,L, ie, accentuated antagonism, but also
accentuates stimulation of ICa,L elicited by the withdrawal
of ACh. This mechanism may, therefore, accentuate the rebound positive
inotropic response elicited by the withdrawal of vagal stimulation. In
addition, accentuated antagonism of ICa,L may mediate
antiarrhythmic effects exerted by vagal nerve stimulation, whereas
accentuated stimulation of ICa,L, as shown in the
present study, may mediate arrhythmogenic effects initiated by the
termination of vagal nerve activity.
The present results indicate that pretreatment with PTX abolished both ACh-induced inhibition and rebound stimulation of ICa,L. The effect of PTX on ACh-induced inhibition can be explained by the well-known fact that PTX-sensitive G proteins mediate muscarinic receptor–induced inhibition of adenylate cyclase.26 However, adenylate cyclase is also stimulated by Gs proteins that are insensitive to PTX. The fact that PTX abolished the rebound stimulation of ICa,L without affecting the response to ISO indicates that although Gs proteins are intact, they do not contribute to the rebound stimulation of ICa,L. If Gs protein–mediated stimulation of adenylate cyclase is intact after incubation in PTX, then according to our hypothesis, ACh-induced inhibition of PDE would be expected to stimulate ICa,L by enhancing endogenous cAMP levels. However, ACh had no effect on ICa,L in PTX-treated cells. This suggests that cholinergic regulation of guanylate cyclase or NO synthase may also be mediated by PTX-sensitive G proteins. Both guanylate cyclase9 and NO synthase38 exhibit particulate fractions that may be coupled to PTX-sensitive G proteins. Moreover, the fact that the rebound stimulation of ICa,L was blocked by PTX provides further evidence that the stimulatory effect of ACh on ICa,L is not mediated via the phosphoinositol system.21 26
In summary, ACh elicits both inhibition and rebound stimulation of ICa,L mediated by the modulation of cAMP. The rebound stimulation of ICa,L provides a direct explanation for the positive inotropic response that follows cholinergic inhibition of atrial contractility. This mechanism would be expected to increase Ca2+ influx, to rapidly restore intracellular Ca2+, and to trigger SR Ca2+ release for rapid restoration of contractile function. Moreover, rebound stimulation of ICa,L may also contribute to the positive chronotropic response that follows vagal-induced inhibition of pacemaker activity, ie, postvagal tachycardia45 as well as dysrhythmic atrial pacemaker activity elicited on the termination of vagal nerve activity. In fact, recent preliminary results show that in both sinoatrial node and latent atrial pacemaker cells isolated from cat right atrium, the withdrawal of ACh elicits a rebound increase in ICa,L that is associated with an increase in the rate of pacemaker activity above control.46 This work is currently in progress in our laboratory.
|
Acknowledgments |
|---|
This study was supported by National Institutes of Health grant HL-27652. We would like to thank Dr Edward Perez-Reyes for his helpful discussions regarding these experiments and C. Rechenmacher for her expert technical assistance.
|
Footnotes |
|---|
Previously published in part in abstract form (Circulation. 1994;90:198).
Received July 25, 1994; accepted December 15, 1994.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Sakmann B, Noma A, Trautwein W. Acetylcholine activation of single muscarinic K+ channels in isolated pacemaker cells of the mammalian heart. Nature. 1983;303:250-253. [Medline] [Order article via Infotrieve]
2. Fleming JW, Strawbridge RA, Watanabe AM. Muscarinic receptor regulation of cardiac adenylate cyclase activity. J Mol Cell Cardiol. 1987;19:47-61. [Medline] [Order article via Infotrieve]
3.
Iijima T, Irisawa H, Kameyama M. Membrane currents and their
modification by acetylcholine in isolated single atrial cells of the
guinea-pig. J Physiol (Lond). 1985;359:485-501.
4.
McDonald TF, Pelzer S, Trautwein W, Pelzer DJ. Regulation and
modulation of calcium channels in cardiac, skeletal, and smooth muscle
cells. Physiol Rev. 1994;74:365-507.
5. Levy MN, Pappano AJ. Vagal control of myocardial contractility. In: Levy MN, Schwartz PJ, eds. Vagal Control of the Heart: Experimental Basis and Clinical Implications. Armonk, NY: Futura Publishing Co Inc; 1994:241-259.
6. Fischmeister R, Hartzell HC. Cyclic AMP phosphodiesterases and Ca2+ current regulation in cardiac cells. Life Sci. 1991;48:2365-2376. [Medline] [Order article via Infotrieve]
7.
Ono K, Trautwein W. Potentiation by cyclic GMP of
ß-adrenergic effects on Ca2+ current in guinea-pig
ventricular cells. J Physiol (Lond). 1991;443:387-404.
8.
Mery P-F, Lohmann SM, Walter U, Fischmeister R.
Ca2+ current is regulated by cyclic GMP-dependent
protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci
U S A. 1991;88:1197-1201.
9. Schmidt H, Lohmann SM, Walter U. The nitric oxide and cGMP signal transduction system: regulation and mechanism of action. Biochim Biophys Acta. 1993;1178:153-175. [Medline] [Order article via Infotrieve]
10.
Balligand J-L, Kelly RA, Marsden PA, Smith TW. Control of
cardiac muscle cell function by an endogenous nitric oxide signaling
system. Proc Natl Acad Sci U S A. 1993;90:347-351.
11.
Mery P-F, Pavoine C, Belhassen L, Pecker F, Fischmeister R.
Nitric oxide regulates cardiac Ca2+ current. J
Biol Chem. 1993;268:26286-26295.
12. Kirstein M, Hatem S, Benardeau A, Mercadier J-J, Fischmeister R. Nitric oxide induces a stimulation of the L-type calcium current in human atrial myocytes. J Physiol (Lond). 1994;477P:16P-17P. Abstract.
13. Han X, Shimoni Y, Giles WR. An obligatory role for nitric oxide in autonomic control of mammalian heart rate. J Physiol (Lond). 1994;476.2:309-314.
14. Levi RC, Alloatti G, Penna C, Gallo MP. Guanylate-cyclase-mediated inhibition of cardiac ICa by carbachol and sodium nitroprusside. Pflugers Arch. 1994;426:419-426. [Medline] [Order article via Infotrieve]
15. Endoh M, Blinks JR. Effects of endogenous neurotransmitters on calcium transients in mammalian atrial muscle. In: Fleming WW, Graefe K-H, Langer SZ, Weiner N, eds. Neuronal and Extraneuronal Events in Autonomic Pharmacology. New York, NY: Raven Press Publishers; 1984:221-230.
16.
McMorn SO, Harrison SM, Zang W, Yu XJ, Boyett MR. A direct
negative inotropic effect of acetylcholine on rat ventricular myocytes.
Am J Physiol. 1993;265:H1393-H1400.
17.
Carmeliet E, Mubagwa K. Desensitization of the
acetylcholine-induced increase of potassium conductance in rabbit
cardiac Purkinje fibers. J Physiol (Lond). 1986;371:239-255.
18.
Matsumoto K, Pappano AJ. Sodium-dependent membrane current
induced by carbachol in single guinea-pig ventricular myocytes.
J Physiol (Lond). 1989;415:487-502.
19. Korth M, Kuhlkamp V. Muscarinic receptor-mediated increase of intracellular Na+ ion activity and force of contraction. Pflugers Arch. 1985;403:266-272. [Medline] [Order article via Infotrieve]
20.
Korth M, Sharma VK, Sheu SS. Stimulation of muscarinic
receptors raises free intracellular Ca2+
concentration in rat ventricular myocytes. Circ
Res. 1988;62:1080-1087.
21.
Gallo MP, Alloatti G, Eva C, Oberto A, Levi RC. M1
muscarinic receptors increase calcium current and phosphoinositide
turnover in guinea-pig ventricular cardiocytes. J Physiol
(Lond). 1993;471:41-60.
22. Wang YG, Lipsius SL. Acetylcholine elicits a rebound stimulation of Ca2+ current mediated by inhibitory G-protein and cAMP-dependent PKA in cat atrial myocytes. Circulation. 1994;90:198. Abstract.
23.
Wu J, Vereecke J, Carmeliet E, Lipsius SL. Ionic currents
activated during hyperpolarization of single right atrial myocytes from
cat heart. Circ Res. 1991;68:1059-1069.
24.
Horn R, Marty A. Muscarinic activation of ionic currents
measured by a new whole-cell recording method. J Gen
Physiol. 1988;92:145-159.
25. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85-100. [Medline] [Order article via Infotrieve]
26. Hulme EC, Birdsall NJM, Buckley NJ. Muscarinic receptor subtypes. Annu Rev Pharmacol Toxicol. 1990;30:633-673. [Medline] [Order article via Infotrieve]
27.
Hryshko LV, Bers DM. Ca current facilitation during postrest
recovery depends on Ca entry. Am J Physiol. 1990;259:H951-H961.
28.
Hryshko LV, Bers DM. Citrate alters Ca channel gating and
selectivity in rabbit ventricular myocytes. Am J Physiol. 1992;262:C191-C198.
29.
Zhou Z, Lipsius SL. Na-Ca exchange current in latent pacemaker
cells isolated from cat right atrium. J Physiol (Lond). 1993;466:263-285.
30.
Lee KS, Marban E, Tsien RW. Inactivation of calcium channels
in mammalian heart cells: joint dependence on membrane potential and
intracellular calcium. J Physiol (Lond). 1985;364:395-411.
31.
Meissner G. Ryanodine activation and inhibition of the
Ca2+ release channel of sarcoplasmic reticulum.
J Biol Chem. 1986;261:6300-6306.
32.
Dostmann WR, Taylor SS, Genieser H-G, Jastorff B, Doskeland
SO, Ogreid D. Probing the cyclic nucleotide binding sites of
cAMP-dependent protein kinase I and II with analogs of adenosine
3',5'-cyclic phosphorothioates. J Biol Chem. 1990;265:10484-10491.
33.
Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T,
Naito K, Toshioka T, Hidaka H. Inhibition of forskolin-induced neurite
outgrowth and protein phosphorylation by a newly synthesized selective
inhibitor of cyclic AMP-dependent protein kinase,
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide
(H-89), of PC12D pheochromocytoma cells. J Biol Chem. 1990;265:5267-5272.
34. Daly JW, Padgett W, Seamon KB. Activation of cyclic AMP-generating systems in brain membranes and slices by the diterpene forskolin: augmentation of receptor-mediated responses. J Neurochem. 1982;38:532-544. [Medline] [Order article via Infotrieve]
35. Harrison SA, Reifsnyder DH, Gallis B, Cadd GG, Beavo JA. Isolation and characterization of bovine cardiac muscle cGMP-inhibited phosphodiesterase: a receptor for new cardiotonic drugs. Mol Pharmacol. 1986;29:506-514. [Abstract]
36.
Corbin JD, Ogreid D, Miller JP, Suva RH, Jastorff B, Doskeland
SO. Studies of cGMP analog specificity and function of the two
intrasubunit binding sites of cGMP-dependent protein kinase.
J Biol Chem. 1986;261:1208-1214.
37. Brown AM. Coupling of G-proteins to cardiac ion channels. In: Levy MN, Schwartz PJ, eds. Vagal Control of the Heart: Experimental Basis and Clinical Implications. Armonk, NY: Futura Publishing Co Inc; 1994:133-146.
38. Knowles RG, Moncada S. Nitric oxide synthase in mammals. Biochem J. 1994;298:249-258.
39. Birnbaumer L, Abramowitz J, Yatani A, Okabe K, Mattera R, Graf R, Sanford J, Codina J, Brown AM. Roles of G proteins in coupling of receptors to ionic channels and other effector systems. Biochem Mol Biol Int. 1990;25:225-244.
40. Fischmeister R, Hartzell HC. Regulation of calcium current by low-Km cyclic AMP phosphodiesterases in cardiac cells. Mol Pharmacol. 1990;38:426-433. [Abstract]
41. Bean BP, Nowycky MC, Tsien RW. Beta-adrenergic modulation of calcium channels in frog ventricular heart cells. Nature. 1984;307:371-375. [Medline] [Order article via Infotrieve]
42. Linden J. Enhanced cAMP accumulation after termination of cholinergic action in the heart. FASEB J. 1987;1:119-124. [Abstract]
43.
Giles W, Noble SJ. Changes in membrane currents in bullfrog
atrium produced by acetylcholine. J Physiol (Lond). 1976;261:103-123.
44.
Hanf R, Li Y, Szabo G, Fischmeister R. Agonist-independent
effects of muscarinic antagonists on Ca2+ and
K+ currents in frog and rat cardiac cells. J Physiol
(Lond). 1993;461:743-765.
45. Levy MN. Vagal tachycardias. In: Levy MN, Schwartz PJ, eds. Vagal Control of the Heart: Experimental Basis and Clinical Implications. Armonk, NY: Futura Publishing Co Inc; 1994:305-315.
46. Wang YG, Lipsius SL. Withdrawal of acetylcholine elicits a rebound increase in L-type Ca2+ current (ICa,L) and hyperpolarization-activated inward current (If) in atrial pacemaker cells. Biophys J. 1995;68:12. Abstract.
This article has been cited by other articles:
 |
|
 |
|
  R. Lipsky, E. M. Potts, S. T. Tarzami, A. A. Puckerin, J. Stocks, A. D. Schecter, E. A. Sobie, F. G. Akar, and M. A. Diverse-Pierluissi {beta}-Adrenergic Receptor Activation Induces Internalization of Cardiac Cav1.2 Channel Complexes through a {beta}-Arrestin 1-mediated Pathway J. Biol. Chem., June 20, 2008; 283(25): 17221 - 17226. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Molina, H. Gesser, A. Llach, L. Tort, and L. Hove-Madsen Modulation of membrane potential by an acetylcholine-activated potassium current in trout atrial myocytes Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R388 - R395. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Nygren, A. E. Lomax, and W. R. Giles Heterogeneity of action potential durations in isolated mouse left and right atria recorded using voltage-sensitive dye mapping Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2634 - H2643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. H. duBell and T. B. Rogers Protein phosphatase 1 and an opposing protein kinase regulate steady-state L-type Ca2+ current in mouse cardiac myocytes J. Physiol., April 1, 2004; 556(1): 79 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. N. Dedkova, X. Ji, Y. G. Wang, L. A. Blatter, and S. L. Lipsius Signaling Mechanisms That Mediate Nitric Oxide Production Induced by Acetylcholine Exposure and Withdrawal in Cat Atrial Myocytes Circ. Res., December 12, 2003; 93(12): 1233 - 1240. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Liu, R. H. Kennedy, M. H. Creer, and J. McHowat Alterations in Ca2+ cycling by lysoplasmenylcholine in adult rabbit ventricular myocytes Am J Physiol Cell Physiol, April 1, 2003; 284(4): C826 - C838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. N Dedkova, Y. Gao Wang, L. A Blatter, and S. L Lipsius Nitric oxide signalling by selective {beta}2-adrenoceptor stimulation prevents ACh-induced inhibition of {beta}2-stimulated Ca2+ current in cat atrial myocytes J. Physiol., August 1, 2002; 542(3): 711 - 723. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. C L Bett, S. Dai, and D. L Campbell Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes J. Physiol., July 1, 2002; 542(1): 107 - 117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E Belevych, C. Sims, and R. D Harvey ACh-induced rebound stimulation of L-type Ca2+ current in guinea-pig ventricular myocytes, mediated by G{beta}{gamma}-dependent activation of adenylyl cyclase J. Physiol., November 1, 2001; 536(3): 677 - 692. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-B. Shen and A. J. Pappano 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 J. Pharmacol. Exp. Ther., August 1, 2001; 298(2): 857 - 864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wu, R. B. Schuessler, M. D. Rodefeld, J. E. Saffitz, and J. P. Boineau Morphological and membrane characteristics of spider and spindle cells isolated from rabbit sinus node Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1232 - H1240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. G. Wang, W. J. Benedict, J. Huser, A. M. Samarel, L. A. Blatter, and S. L. Lipsius Brief rapid pacing depresses contractile function via Ca2+/PKC-dependent signaling in cat ventricular myocytes Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H90 - H98. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E Belevych and R. D Harvey Muscarinic inhibitory and stimulatory regulation of the L-type Ca2+ current is not altered in cardiac ventricular myocytes from mice lacking endothelial nitric oxide synthase J. Physiol., October 15, 2000; 528(2): 279 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. G. Wang, A. M Samarel, and S. L Lipsius Laminin binding to {beta}1-integrins selectively alters {beta}1- and {beta}2-adrenoceptor signalling in cat atrial myocytes J. Physiol., August 15, 2000; 527(1): 3 - 9. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. G. Wang, A. M Samarel, and S. L Lipsius Laminin acts via {beta}1 integrin signalling to alter cholinergic regulation of L-type Ca2+ current in cat atrial myocytes J. Physiol., July 1, 2000; 526(1): 57 - 68. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-L. Balligand Regulation of cardiac {beta}-adrenergic response by nitric oxide Cardiovasc Res, August 15, 1999; 43(3): 607 - 620. [Full Text] [PDF]
|
|
|
|
|
|
E. P. Anyukhovsky and L. V. Rosenshtraukh Electrophysiological responses of canine atrial endocardium and epicardium to acetylcholine and 4-aminopyridine Cardiovasc Res, August 1, 1999; 43(2): 364 - 370. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M Bers and E. Perez-Reyes Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release Cardiovasc Res, May 1, 1999; 42(2): 339 - 360. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. G. Wang and S. L. Lipsius Genistein elicits biphasic effects on L-type Ca2+ current in feline atrial myocytes Am J Physiol Heart Circ Physiol, July 1, 1998; 275(1): H204 - H212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Han, I. Kubota, O. Feron, D. J. Opel, M. A. Arstall, Y.-Y. Zhao, P. Huang, M. C. Fishman, T. Michel, and R. A. Kelly Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase PNAS, May 26, 1998; 95(11): 6510 - 6515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Gallo, D. Ghigo, A. Bosia, G. Alloatti, C. Costamagna, C. Penna, and R. C Levi Modulation of guinea-pig cardiac L-type calcium current by nitric oxide synthase inhibitors J. Physiol., February 1, 1998; 506(3): 639 - 651. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Vandecasteele, T. Eschenhagen, and R. Fischmeister Role of the NO-cGMP pathway in the muscarinic regulation of the L-type Ca2+ current in human atrial myocytes J. Physiol., February 1, 1998; 506(3): 653 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. G. Wang, C. E. Rechenmacher, and S. L. Lipsius Nitric Oxide Signaling Mediates Stimulation of L-Type Ca2+ Current Elicited by Withdrawal of Acetylcholine in Cat Atrial Myocytes J. Gen. Physiol., January 1, 1998; 111(1): 113 - 125. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-L. Balligand and P. J. Cannon Nitric Oxide Synthases and Cardiac Muscle : Autocrine and Paracrine Influences Arterioscler Thromb Vasc Biol, October 1, 1997; 17(10): 1846 - 1858. [Abstract] [Full Text]
|
|
|
|
 |
|
 Y.G. Wang, J. Huser, L.A. Blatter, and S.L. Lipsius Withdrawal of Acetylcholine Elicits Ca2+-Induced Delayed Afterdepolarizations in Cat Atrial Myocytes Circulation, August 19, 1997; 96(4): 1275 - 1281. [Abstract] [Full Text]
|
|
|
|
|
|
R. A. Kelly, J.-L. Balligand, and T. W. Smith Nitric Oxide and Cardiac Function Circ. Res., September 1, 1996; 79(3): 363 - 380. [Full Text]
|
|
|
|
|
|
Y. G. Wang and S. L. Lipsius A Cellular Mechanism Contributing to Postvagal Tachycardia Studied in Isolated Pacemaker Cells From Cat Right Atrium Circ. Res., July 1, 1996; 79(1): 109 - 114. [Abstract] [Full Text]
|
|
|
|
|
|
X. Han, L. Kobzik, J.-L. Balligand, R.A. Kelly, and T.W. Smith Nitric Oxide Synthase (NOS3)–Mediated Cholinergic Modulation of Ca2+ Current in Adult Rabbit Atrioventricular Nodal Cells Circ. Res., June 1, 1996; 78(6): 998 - 1008. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||