Post on 22-Feb-2023
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The systemic effects of cholinergic modulation of whole
heart function have been well-characterized (Levy &
Schwartz, 1994; Opie, 1998). However, there is still
considerable uncertainty over the specific molecular
mechanisms by which acetylcholine (ACh) and other
muscarinic agonists, such as carbachol (CCh), exert their
modulatory effects upon cardiac muscle ion channels
(Hartzell, 1988; Campbell & Strauss, 1995; Balligand et al.2000). Depending upon specific cardiac myocyte type,
muscarinic agonists can exert one, or a combination, of the
following effects: (i) inhibition of the hyperpolarization-
activated non-specific cation current IF (primary pace-
making cells); (ii) activation of an inwardly-rectifying K+
current IK,ACh; and/or (iii) inhibition of the L-type Ca 2+
current ICa,L (reviewed in Hartzell, 1988; Campbell &
Strauss, 1995; Ackerman & Clapham, 2000; DiFranceso
et al. 2000; Parker & Fedida, 2001)
In sinoatrial node and atrio-ventricular myocytes evidence
has been presented indicating that cholinergic-mediated
inhibition of ICa,L is obligatorily coupled to the production
of nitric oxide (NO) by Type III NO synthase (eNOS),
leading to increased soluble guanylyl cyclase (sGC) activity
(Hobbs, 1997; Wedel & Garbers, 2001) and subsequent
activation of a cyclic-guanosine monophosphate (cGMP)-
dependent regulatory cascade (Han et al. 1995, 1996,
1998b). Subsequent studies on myocytes isolated from
the hearts of transgenic mice lacking eNOS led Han etal. (1998a) to conclude that cholinergic modulation of
ventricular ICa,L was also obligatorily dependent upon
NO production. However, two independent studies
(Vandecasteele et al. 1999; Belevych & Harvey, 2000)
concluded that muscarinic inhibition of ICa,L still existed
in mouse ventricular myocytes lacking eNOS. These
conflicting results raise questions about the ‘obligatory
Cholinergic modulation of the basal L-type calcium current inferret right ventricular myocytesGlenna C. L. Bett, Shuiping Dai and Donald L. Campbell
Department of Physiology and Biophysics, University at Buffalo, State University of New York, Buffalo, New York 14214, USA
The effects of the cholinergic muscarinic agonist carbachol (CCh) on the basal L-type calcium
current, ICa,L, in ferret right ventricular (RV) myocytes were studied using whole cell patch clamp.
CCh produced two major effects : (i) in all myocytes, extracellular application of CCh inhibited ICa,L
in a reversible concentration-dependent manner; and (ii) in many (but not all) myocytes, upon
washout CCh produced a significant transient stimulation of ICa,L (‘rebound stimulation’).
Inhibitory effects could be observed at 1 w 10_10M CCh. The mean steady-state inhibitory
concentration–response relationship was shallow and could be described with a single Hill equation
(maximum inhibition = 34.5 %, IC50 = 4 w 10_8M, Hill coefficient n = 0.60). Steady-state inhibition
(1 or 10 mM CCh) had no significant effect on ICa,L selectivity or macroscopic (i) activation
characteristics, (ii) inactivation kinetics, (iii) steady-state inactivation or (iv) kinetics of recovery
from inactivation. Maximal inhibition of nitric oxide synthase (NOS) activity (preincubation of
myocytes in 1 mM L-NMMA (NG-monomethyl-L-arginine) + 1 mM L-NNA (NG-nitro-L-arginine)
for 2–3 h plus inclusion of 1 mM L-NMMA + 1 mM L-NNA in the patch pipette solution) produced
no significant attenuation of the CCh-mediated inhibition of ICa,L. Protocols involving (i) the nitric
oxide (NO) scavenger PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide; 200 mM),
(ii) imposition of a ‘cGMP clamp’ (100 mM 8-Bromo-cGMP), and (iii) inhibition of soluble guanylyl
cyclase (ODQ (1H-[1,2,4,]oxadiazolo(4,3,-a)quinoxalin-1-one), 50 mM) all failed to attenuate CCh-
mediated inhibition of Ica,L. While CCh consistently inhibited basal ICa,L in all RV myocytes studied,
not all myocytes displayed rebound stimulation upon CCh washout. However, there was no
difference between CCh-mediated inhibition of ICa,L between these two RV myocyte types, and in
myocytes displaying rebound stimulation neither ODQ nor 8-Bromo-cGMP (8-Br-cGMP) altered
the effect. We conclude that NO production, activation of soluble guanylyl cyclase, or changes in
intracellular cGMP levels are not obligatorily involved in muscarinic-mediated modulation of basal
ICa,L in ferret RV myocytes.
(Received 18 January 2002; accepted after revision 15 April 2002)
Corresponding author D. L. Campbell: Department of Physiology and Biophysics, University at Buffalo, State University of NewYork, 124 Sherman Hall, Buffalo, NY 14214, USA. Email: dc25@acsu.buffalo.edu
Journal of Physiology (2002), 542.1, pp. 107–117 DOI: 10.1113/jphysiol.2002.017335
© The Physiological Society 2002 www.jphysiol.org
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NO production hypothesis’. Recent results from other
laboratories have also raised doubts about the involvement
of NO production in cholinergic-mediated modulation of
various cardiac myocyte ion channel types (e.g. Méry et al.1996; Zacharov et al. 1996; Gallo et al. 1998; Vandecasteele
et al. 1998; Gödecke et al. 2001).
In both human and ferret ventricles there is a significant
expression gradient of both eNOS and sGC protein across
the left ventricular (LV) wall, with both enzymes being
highly expressed in LV subepicardium but markedly
reduced to absent in LV subendocardium (Brahmajothi &
Campbell, 1999, 2001). Hence, there may be significant
differences in NO- and muscarinic-mediated mechanisms
of ion channel modulation not only between pace-
making (e.g. Han et al. 1995, 1996, 1998b) versus working
(e.g. Vandecasteele et al. 1999; Belevych & Harvey, 2000)
cardiac myocytes but also between myocytes located in
distinct anatomical regions of the ventricle. Therefore, at
present the exact role of myocyte NO production in
indirect cholinergic-mediated inhibition of ICa,L in different
working ventricular myocyte types is unclear.
In addition to inhibition, recent studies have indicated
that in some working cardiac myocyte types ICa,L can
display a significant rebound stimulation upon washout of
muscarinic agonists (e.g. cat atrial myocytes: Wang &
Lipsius, 1995; Wang et al. 1998; mouse and guinea pig
ventricular myocytes: Belevych & Harvey, 2000; Belevych
et al. 2001). However, arguments both for (Wang et al.1998) and against (Belevych & Harvey, 2000) the
obligatory involvement of NO production in generation of
the rebound stimulation have been presented.
To begin to address these issues in one specific working
ventricular myocyte type we have analysed the effects of
the cholinergic agonist carbachol (CCh) on the basalICa,L (i.e. in the absence of any previous b-adrenergic
stimulation) in ferret right ventricular (RV) myocytes. We
chose to work on basal ICa,L in these myocytes for two main
reasons: (i) CCh significantly inhibits basal ICa,L, thereby
precluding pretreatment with b-agonists (and, hence, any
potential complications associated with such treatment);
and (ii) the majority of ferret RV myocytes express both
eNOS and sGC proteins (Brahmajothi & Campbell, 1999,
2001). Thus, all of the putative components hypothesized
to be involved in the obligatory NO production hypothesis
are present within these myocytes, and muscarinic
responses can be studied under simple basal conditions.
We therefore specifically focused on the following
question: are RV myocyte NO production, activation of
sGC, and/or changes in intracellular cGMP levels
obligatorily involved in muscarinic-mediated modulation
of basal ICa,L?
A preliminary account of this work has appeared in
abstract form (Bett & Campbell, 2001).
METHODS Myocyte isolationAll animal protocols were conducted in accordance with NIHapproved guidelines of the Institutional Animal Care and UseCommittee, University at Buffalo, SUNY, USA (protocol numberPGY16010N). Single myocytes were isolated exactly as previouslydescribed (Qu et al. 1993a,b; Campbell et al. 1996; Brahmajothiet al. 1999; Brahmajothi & Campbell, 1999). Briefly, male ferrets(10–16 weeks old) were injected I.P. with 35 mg kg_1 sodiumpentobarbital. Upon attainment of deep stage 3 anaesthesia(monitored by foot pad reflex) the heart was removed andmounted on a Langendorff apparatus. The heart was thenperfused with low [Ca2+]o enzyme solution (collagenase Type II,(Worthington Biochemical Corporation, Lakewood, NJ, USA),pronase type XIV and elastase type I-A (Sigma ChemicalCompany, St Louis, MO, USA)). After 10–20 min of perfusion theright ventricle (middle one-third region; Brahmajothi et al. 1999)was dissected free, placed in fresh enzyme solution, and gentlyrocked at 37 °C to obtain single myocytes After isolation, myocyteswere immediately stored (20–22 °C) in control (Na+- and Ca 2+-containing) solution (mM): 144 NaCl, 5.4 KCl, 1 MgSO4, 1.8CaCl2, 10 Hepes, pH = 7.40. All measurements were conducted at20–22 °C and within 10–12 h of myocyte isolation.
Recording conditions, solutions, and analysisRecording techniques and equipment were exactly as previouslydescribed (Campbell et al. 1996) with the following slightexception: voltage clamp pulses were generated either using acustom-built optically isolated pulse generator (Campbell et al.1996) or under direct personal computer control using pCLAMP8.0 software (Axon Instruments, Inc., Union City, CA, USA).
Gigaseals were initially formed in control Na+- and Ca2+-containing solution. After obtaining the whole-cell configuration(generally by dielectric rupture of the patch using a ‘zap’ circuit ofthe patch clamp amplifier (Axoclamp 2-B or 200-A; AxonInstruments)) myocytes were perfused with an extracellular Na+-and K+-free ICa,L solution (in mM): 144 N-methyl-D-glucamine Cl(NMDG-Cl), 5.4 CsCl, 1 MgSO4, 1.8 CaCl2, 10 Hepes, pH = 7.40.Patch pipettes (2–4 MV, heat polished; TW150F, World PrecisionInstruments, Inc., Sarasota, FL, USA) contained (mM): 120 CsCl,20 TEA-Cl, 1 MgSO4, 5 EGTA, 5 Mg-ATP, 5 Tris-creatinephosphate, 0.2 GTP, 10 Hepes, pH = 7.40. The use of thesesolutions isolated ICa,L from other overlapping currents (Na+, K+,Na+–Ca2+ exchanger), and allowed recording of stable ICa,L
currents with minimal rundown for typical periods of 20–60 min(Qu et al. 1993a,b; Campbell et al. 1996). We wish to emphasizethat due to the relatively slow perfusion rates used in theseexperiments (Campbell et al. 1996; Brahmajothi et al. 1999) nodefinitive quantitative conclusions could be reached on thekinetics of carbachol-mediated ‘on’ and ‘off’ responses.Therefore, only steady-state results were analysed.
After initial formation of the whole-cell configuration, myocyteswere voltage clamped to a holding potential (HP) = _70 mV andan approximate 10 min period was allowed to pass for adequateinternal perfusion and stabilization of current gating parameters(Marty & Neher, 1983). Currents (filtered at 1–2 kHz; digitized5–10 kHz) were recorded on video tape (NR-10 digital datarecorder, Instrutech Corporation, Long Island, NY, USA) andeither directly digitized on-line or subsequently digitized off-lineusing pCLAMP software. Details of specific voltage clampprotocols are described in the appropriate figure captions.
G. C. L. Bett, S. Dai and D. L. Campbell108 J. Physiol. 542.1
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Unless otherwise indicated, the standard holding potential wasHP = _70 mV and voltage clamp pulse protocols were applied at afrequency of 0.1–0.167 Hz. ‘Leakage correction’ was not applied,i.e. all illustrated currents are ‘raw’. Analysis of kinetics and fittingto mean data points was conducted using pCLAMP, Fig.P (Biosoft,Cambridge, UK), or Origin (OriginLab Corp., Northampton, MA,USA) software. In the figures all data points are presented asmeans ± S.E.M..
All salts and associated compounds for isolating myocytes andmaking extracellular and intracellular recording solutions wereobtained from Sigma. L-NMMA (NG-monomethyl-L-arginine),L-NNA (NG-monomethyl-L-arginine), ODQ (1H-[1,2,4,]oxa-diazolo(4,3,-a)quinoxalin-1-one), 8-Br-cGMP, and PTIO(2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide) wereobtained from Calbiochem (La Jolla, CA, USA). ODQ and 8-Br-cGMP were aliquoted and stored as stock solutions at _20 °C untilused. Stock solutions of PTIO were dissolved in ethanol andstored at _20 °C. Previous control measurements (Qu et al.1993a,b; Campbell et al. 1996) indicated that the concentrations ofethanol present during final dilutions of PTIO had no significanteffects on ICa,L.
RESULTSBasic observations: inhibition of basal L-type Ca2+
current (ICa,L) by carbachol and transient reboundstimulation upon washout Carbachol (CCh) produced two significant effects upon
basal ICa,L in ferret right ventricular (RV) myocytes. First, in
all myocytes studied extracellular application of CCh
(1–10 mM) inhibited ICa,L, in a reversible manner. This CCh-
mediated inhibition typically reached a final steady-state
level, and could be prevented by simultaneous application of
1 mM atropine (data not shown). Second, upon subsequent
washout of CCh in many (but not all) myocytes there was a
significant transient rebound stimulation of ICa,L followed by
a slower return back to the basal level (Fig. 1A).
Inhibitory CCh effects Since the inhibition of basal ICa,L by CCh reached steady-
state levels, it was possible to conduct both kinetic and
steady-state analyses.
Concentration–response relationship. We first
determined the concentration–response relationship for
inhibition of peak ICa,L elicited at 0 mV. We did not observe
any significant desensitization over the time courses of our
measurements. Nonetheless, to minimize any complications
due to desensitization, only two consecutive applications
of CCh (the first always at a lower concentration) were
applied to any one myocyte. We observed inhibitory
effects beginning at 10_10M CCh, and saturating inhibition
at ~10 mM. Hence, the overall concentration–response
relationship was shallow, covering an ~100 000-fold CCh
concentration range (Fig. 1B). The mean concentration–
response data could be described by a single Hill equation
with the following parameters: maximum ICa,L inhibition
(0 mV) = 34.5 %, IC50 = 4 w 10_8M, Hill coefficient
n = 0.60.
Current–voltage (ICa,L–V) relationship. The effects of CCh
on the peak ICa,L–V relationship were next determined.
CCh scaled down the peak ICa,L–V without producing any
significant effects on activation threshold (~_30 mV),
peak current potential (0 mV), or apparent reversal
potential Erev (Fig. 2A).
CCh modulation of ferret RV ICa,LJ. Physiol. 542.1 109
Figure 1. Effect of CCh on ICa,L recordings from RV myocytes A, basic observations: extracellular carbachol (CCh; 1 mM) inhibitsbasal ICa,L (0 mV) upon application (indicated by line) and, inmany myocytes, produces rebound stimulation upon washout.Representative recordings from a single ferret right ventricular(RV) myocyte are shown. Each data point is the peak ICa,L elicitedduring a 500 ms voltage clamp step pulse to 0 mV fromHP = _70 mV. Individual current traces for each condition areshown as indicated. Inset currents correspond to individual datapoints indicated by Roman numerals. Calibration bars: 500 pA,200 ms. B, inhibitory CCh effects: concentration–responserelationship. Steady-state percent inhibition of peak basal ICa,L isplotted as a function of extracellular CCh concentration. ICa,L waselicited during a 500 ms voltage clamp step pulse to 0 mV fromHP = _70 mV. Each data point mean value was obtained from theindicated number of myocytes. See text for further methodologicaldetails. Mean data points were fitted to a single Hill equation withthe following parameters: maximum inhibition (0 mV) = 34.5 %,IC50 = 4 w 10_8
M, Hill coefficient n = 0.60.
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Inactivation: kinetic and steady-state effects. CCh had
no significant effects on the macroscopic biexponential
inactivation kinetics of ICa,L recorded at 0 mV (Fig. 2B).
Mean values (n = 10 myocytes) were as follows: t1:
control 9.9 ± 0.6 ms, CCh 11.7 ± 0.6 ms; t2: control,
75.3 ± 2.0 ms, CCh 75.2 ± 1.4 ms; and ratio of initial
amplitudes A1/(A1 + A2): control, 0.853 ± 0.023, CCh
0.806 ± 0.066. None of these values were significantly
different (paired t test, P > 0.05). To determine whether
a hyperpolarizing shift in steady-state inactivation was
involved, the steady-state inactivation relationship was
determined first under control conditions and then after
application of 1 or 10 mM CCh (paired double pulse
protocol; see schematic inset in Fig. 2C). All three
mean data sets could be fitted to the sum of the same
two Boltzmann relationships ‘f’ + ‘r’ (parameters: f, VÎ =
_26 mV, slope factor k = _4.7 mV; r, VÎ = +32 mV,
k = 8 mV, Amax = 0.26).
G. C. L. Bett, S. Dai and D. L. Campbell110 J. Physiol. 542.1
Figure 2. Effects of CCh on ICa,L macroscopic gating characteristicsA, I–V relationship: steady-state inhibitory effects of 1 and 10 mM CCh on the basal ICa,L peak current-voltage(I–V) relationship (500 ms clamp pulses, HP = _70 mV, 0.167 Hz; filled symbols) and the net currentremaining at 500 ms (open symbols). B,C, inactivation: kinetic and steady -state effects. B, effect of CCh onmacroscopic inactivation kinetics of ICa,L recorded at 0 mV. Representative biexponential fits to inactivationare shown for both control and 1 mM CCh with the following parameters: control, t1 = 9.4 ms, t2 = 78.2 ms,ratio of initial amplitudes A1/(A1 + A2) = 0.891; 1 mM CCh, t1 = 11.4 ms, t2 = 78.2ms, ratio of initialamplitudes A1/(A1 + A2) = 0.832. The bar graph inset compares the mean (± S.E.M.) values of t1, t2, and thepercent initial A1 amplitude under control conditions and after adding 1 mM CCh. C, steady-stateinactivation relationship measured using a paired double pulse protocol (schematic inset; pulse protocolfrequency 0.167 Hz). Individual data points are mean values of n indicated myocytes both under controlconditions (circles) and after application of 1 mM (triangles) and 10 mM (inverted triangles) CCh. D, recoverykinetics. Mean recovery kinetics at HP = _70 mV were measured first under control conditions and thenafter application of 1 mM CCh. Paired double pulse recovery protocol is illustrated in the schematic inset, thefrequency of protocol was 0.167 Hz. Individual data points are mean (± S.E.M.) values obtained from pairedmeasurements made in n = 5 myocytes.
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Recovery. To determine whether a slowing in the kinetics
of recovery from inactivation was contributing to the
inhibitory effects, recovery kinetics (HP = _70 mV) were
determined in the same myocyte first under control
conditions and then after application of 1 mM CCh (paired
double pulse protocol; see schematic inset in Fig. 2D).
CCh had no effect on ICa,L recovery kinetics (controltrec,_70 mV = 91 ± 5 ms; CCh trec,_70 mV = 93 ± 7 ms; n = 5).
The overall mean recovery data for both conditions could
be well-described by the same sigmoidal exponential
relationship (ICa,LP2/ICa,LP1) = (1 _ exp_[t/t])n with the
following parameters: t = 92 ms, n = 1.45.
Obligatory involvement of NO in the inhibitoryresponse?To determine whether CCh-mediated inhibition of basal
ICa,L was obligatorily dependent upon myocyte NO
production, we next conducted a series of experiments
designed to either (i) prevent/minimize NO production
or (ii) minimize indirect NO-related effects produced
through activation of sGC and subsequent increases in
intracellular cGMP.
NOS inhibitors. To inhibit or minimize potential NOS
activity (Brahmajothi & Campbell, 1999) myocytes were
first preincubated for 2–3 h in 1 mM L-NMMA + 1 mM
L-NNA, and 1 mM L-NNMA + 1 mM L-NNA was also
included in both the patch pipette solution and the
extracellular ICa,L recording solution. Under these putative
maximally NOS-inhibited conditions, 1 mM CCh inhibited
ICa,L (0 mV) by 37.6 ± 5.5 % (n = 5; Fig. 3). The fact that
the mean percentage magnitude of the CCh-mediated
inhibition was the same in both control ICa,L solution and
in the presence of L-NMMA + L-NNA strongly argues
against previous suggestions that such NOS inhibitors are
acting as muscarinic receptor antagonists (e.g. Buxton etal. 1993).
NO scavenger. In an attempt to minimize indirect NO-
related effects (Campbell et al. 1996), in a parallel series
of experiments we determined the effects of the NO-
scavenger compound PTIO (2-phenyl-4,4,5,5,-tetramethyl-
imidazoline-1-oxy-3-oxide). PTIO stoichiometrically reacts
with NO (PTIO/NO = 1, rate constant ~104M
_1 s_1) without
altering NOS activity (e.g. Akaike et al. 1993). Extracellular
application of PTIO (200 mM) alone either produced no
effect (n = 4/7 myocytes) or resulted in a stimulation of
basal ICa,L (10.9 ± 3.7 %; n = 3/7 myocytes). However,
regardless of initial effects in the presence of PTIO,
application of CCh (5 mM) inhibited ICa,L (0 mV) by
46.7 ± 5.4 % (n = 7; Fig. 4).
‘cGMP-clamp’. To minimize alterations in intracellular
cGMP levels a ‘cGMP-clamp’ protocol was applied. This
was achieved by continual perfusion of the membrane
permeant cGMP-analogue 8-Br-cGMP (100 mM). Extra-
cellular application of 8-Br-cGMP alone resulted in a
decrease in basal ICa,L elicited at 0 mV (25.7 ± 3.3 %; n = 6).
However, in a series of double application experiments
where 1 mM CCh was first applied and then washed out to
determine control percentage inhibition, subsequent
reapplication of 1 mM CCh during a simultaneous cGMP-
clamp still resulted in additional inhibition of ICa,L (control
CCh application, 23.9 ± 3.1 %; second CCh application
during cGMP-clamp, 25.4 ± 3.8 %; n = 5; Fig. 5).
sGC inhibition. To prevent or minimize activation of
sGC the effects of the putatively specific sGC inhibitor
ODQ (1H-[1,2,4,]oxadiazolo(4,3,-a)quinoxalin-1-one;
Garthwaite et al. 1995) were next determined. In our
hands, extracellular application of 20–80 mM ODQ alone
produced variable effects upon basal ICa,L, increasing it in
some myocytes, decreasing it in others, and producing no
CCh modulation of ferret RV ICa,LJ. Physiol. 542.1 111
Figure 3. NOS inhibition Mean effects of generalized NOS inhibition (1 mM L-NMMA + 1 mM L-NNA; see text for methodological details) onCCh-mediated (1 mM) inhibition of ICa,L (500 ms voltage clamppulse to 0 mV, HP = _70 mV, 0.167 Hz). Mean (± S.E.M.) datawere obtained from n = 13 myocytes (control, mean values fromFig. 2) and n = 5 myocytes (L-NMMA + L-NNA).
Figure 4. NO scavenger PTIO does not inhibit CCh-mediated inhibition of ICa,L
Representative effects of PTIO (200 mM) on CCh-mediatedinhibition of ICa,L (500 ms voltage clamp pulse to 0 mV,HP = _70 mV). In the continued presence of PTIO CCh (5 mM)still inhibited ICa,L.
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effect in others. Due to this variability we have not
attempted to further analyse these basal ODQ effects.
However, in paired CCh application experiments (first
application of 10 mM CCh to determine control inhibitory
response, second application in the presence of 50 mM
ODQ) ODQ failed to significantly attenuate the CCh-
mediated inhibition of ICa,L (control CCh, 43.2 ± 4.2 %;
CCh + ODQ, 44.3 ± 5.2 %; n = 7; Fig. 6).
Transient stimulatory effects produced upon CChwashout (‘rebound stimulation’)
In many but not all myocytes a transient stimulation of ICa,L
was observed upon CCh washout (Fig. 1A). We therefore
conducted a series of experiments designed to determine
(i) if there were differences in the effects of ODQ and 8-Br-
cGMP among myocytes displaying prominent rebound
stimulation versus those that failed to, and (ii) if NO
production was obligatorily involved in the stimulatory
effect.Two successive applications of CCh (10 mM) were
applied, the first to determine the extent of inhibition of
ICa,L (0 mV) and whether a given myocyte displayed
rebound stimulation, the second to determine the effect of
CCh in the presence of either ODQ or 8-Br-cGMP.
Myocytes displaying rebound stimulation were grouped
and analysed as ‘overshoot control’, while myocytes
lacking a rebound stimulation were grouped and analysed
as ‘undershoot control’.
In myocytes lacking rebound stimulation, ODQ (50 mM)
failed to produce any significant effect on the CCh-
mediated inhibition (Fig. 7A; details in caption). Among
myocytes displaying rebound stimulation neither ODQ
(Fig. 7B) nor 8-Br-cGMP (100 mM; Fig. 7C) had any
significant effect on the magnitude of the CCh-mediated
inhibition or subsequent rebound stimulation produced
upon washout.
The mean results of all of experimental manoeuvres
applied are summarized in Fig. 8.
Novel observation: rebound stimulationprogressively declines upon repeated CChapplicationsIn RV myocytes displaying rebound stimulation we
frequently observed that upon repeated applications of
CCh (10 mM) the degree of inhibition of ICa,L (elicited at
0 mV) remained constant while the amplitude of reboundstimulation progressively declined. An example of this
behaviour is illustrated in Fig. 9A. Mean data pooled
from n = 4–6 myocytes in which at least 3 successive
application/wash-off cycles of CCh were applied are
illustrated in Fig. 9B. For all myocytes studied under these
conditions (n = 6 myocytes for 3 CCh applications, n = 4
myocytes for 4 CCh applications) mean inhibition
remained essentially unchanged with repeated CCh
applications (_29 ± 4.9 %, _32.8 ± 4.3 %, _35.2 ± 8.2 %,
and _30.3 ± 4.1 % for CCh applications 1–4, respectively),
while rebound stimulation progressively declined (+148 ±
84 %, +111 ± 88 %, +99 ± 54 %, and +60 ± 34 % for CCh
applications 1–4, respectively).
G. C. L. Bett, S. Dai and D. L. Campbell112 J. Physiol. 542.1
Figure 5. ‘cGMP clamp’Representative results from a RV myocyte first exposed to CCh(10 mM) and then 8-Br-cGMP (100 mM) + CCh (10 mM). While8-Br-cGMP inhibited ICa,L it did not significantly alter thepercentage magnitude of the CCh-mediated inhibitory response.Gaps in the data points correspond to periods during which I–Vrelationships were determined. Here and in Fig. 6, inset currentscorrespond to individual data points indicated by Romannumerals.
Figure 6. Soluble guanylyl cyclase inhibition by ODQ As noted in the text, ODQ produced variable effects on basal ICa,L.However, regardless of initial effects, under steady-state conditionsODQ (50 mM) failed to attenuate the CCh-mediated inhibition ofICa,L elicited at 0 mV.
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DISCUSSIONFerret right ventricular (RV) myocytes possess a
cholinergic-activated ‘background’ K+ current IK,ACh (Ito
et al. 1995; Boyett et al. 1988; D. L. Campbell, unpublished
observations). However, under our recording conditions
IK,ACh would have been eliminated. Similarly, since all
kinetic measurements were conducted using pulses
applied to 0 mV, and ECl was equal to _2.2 mV, the
contribution of any Cl_ current would have been virtually
eliminated in these measurements. Finally, since CCh had
no significant effect on either (i) the apparent ICa,L reversal
potential (Campbell et al. 1988) or (ii) the net current
remaining at 500 ms the contribution of any type of Cl_
current was minimal under our recording conditions. Our
results therefore demonstrate that CCh inhibits basal ICa,L
in ferret RV myocytes in a reversible, concentration-
dependent manner. These inhibitory effects were manifested
as a ‘scaling down’ of peak ICa,L amplitude without any
effects on selectivity or macroscopic gating characteristics.
Inhibition could be observed at 10_10M CCh, suggesting
that very low levels of cholinergic compounds could exert
inhibitory effects upon RV myocyte function via inhibition
of ICa,L. One caveat to this conclusion is the possibility that,
since CCh is resistant to acetylcholine esterase, the
responses we observed may be larger than those produced
under physiological conditions. In addition, the relative
importance of this inhibitory ICa,L pathway versus inhibition
produced via muscarinic activation of IK,ACh awaits
determination. Having stated these reservations, our
conclusions on the lack of obligatory NO-, sGC-, and
cGMP-involvement in CCh-mediated modulation of
basal ICa,L in ferret RV myocytes are valid and have
important physiological relevance.
One interesting feature of the concentration–response
curve for CCh-mediated inhibition of basal ICa,L was its
relative shallowness. Since we could observe a consistent
CCh-mediated inhibition both over periods of several
minutes of application and during multiple successive
applications we believe that the shape of the inhibitory
concentration–response curve is not reflective of a
desensitization process. Whether multiple muscarinic
receptor subtypes and/or multiple regulatory pathways are
involved is presently unclear.
CCh modulation of ferret RV ICa,LJ. Physiol. 542.1 113
Figure 7. ‘Undershoot’ and ‘overshoot’ controls See text for methodological details. Dashed lines in all panelscorrespond to mean initial basal ICa,L (defined as 100 %).Mean values were obtained from paired CCh (10 mM)application experiments: first application (‘CCh on’), CChapplied alone to determine whether a given myocytedisplayed or lacked rebound stimulation upon washout(‘CCh off’); second application, CCh applied afterpretreatment with and in the continual presence of eitherODQ, (50 mM; ‘CCh on ODQ’) or 8-Br-cGMP (100 mM;‘CCh on BROMO’). After steady-state inhibitory effectswere reached CCh was washed off in the continual presenceof ODQ (‘CCh off ODQ’) or 8-Br-cGMP (‘CCh offBROMO’). A, undershoot control: mean ODQ results frommyocytes lacking rebound stimulation upon washout of firstCCh application. ODQ had no significant effect uponinhibition or return to the new baseline level upon washout.B and C, overshoot control. B, mean ODQ effects onmyocytes displaying a prominent rebound stimulation uponwashout of first CCh application. ODQ had no significanteffect on either inhibition or rebound stimulation. C, mean8-Br-cGMP effects in myocytes displaying a prominentrebound stimulation. 8-Br-cGMP had no significant effecteither upon inhibition or rebound stimulation. Mean values(± S.E.M.) obtained from n = 3 (A), n = 4 (B), and n = 10 (C)myocytes.
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Under all of the experimental conditions which we applied
(NOS inhibition, NO scavenging, cGMP clamp, sGC
inhibition) CCh still inhibited basal ICa,L elicited at 0 mV
to an extent similar to that observed under control
conditions. While a negative result obtained from any
one given pharmacological manoeuvre does not allow
definitive rejection of the obligatory NO-hypothesis, the
fact that all of the pharmacological manoeuvres which we
applied failed to produce any significant effects on CCh-
mediated inhibition of ICa,L is strong evidence against the
hypothesis. We therefore conclude that muscarinic-
mediated inhibition of ferret RV myocyte basal ICa, L is notobligatorily dependent upon NO production, alterations
G. C. L. Bett, S. Dai and D. L. Campbell114 J. Physiol. 542.1
Figure 9. Rebound stimulation declines withsuccessive CCh applications while inhibition doesnotA, RV myocyte displaying rebound stimulation:representative differential responses of CCh-mediatedeffects on ICa,L (0 mV) elicited upon repeated CCh (10 mM)applications. The upper panel illustrates the time course ofCCh-mediated inhibition and rebound stimulation duringsix consecutive CCh wash on/wash off cycles, while the lowerpanel illustrates the relative percentage change in both theinhibitory and rebound stimulatory effects. Note that boththe baseline ICa,L and the magnitude of the CCh-mediatedinhibition remained essentially constant, while themagnitude of the rebound stimulation progressivelydeclined with each subsequent CCh application. For clearerillustration of these effects, the inset displays on an expandedscale the data indicated by the thin line over CCh applicationnumbers 2 and 3. B, mean percentage inhibition andrebound stimulation produced during successive CChapplication/wash-off cycles. Data pooled from n = 6myocytes (3 CCh applications) and n = 4 myocytes (4 CChapplications). See text for further details.
Figure 8. Summary and comparison ofresults of experimental manoeuvresMean percent inhibition of ferret basal RVmyocyte ICa,L (elicited at 0 mV) by CCh undercontrol conditions (concentration–response;Fig. 1A), putative maximal NOS inhibition(L-NNMA + L-NNA; Fig. 3), in the presence ofNO scavenger (PTIO; Fig. 4), ‘cGMP clamp’conditions (Fig. 5), and after sGC inhibition(ODQ; Fig. 6).
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in sGC activity or changes in intracellular cGMP levels.
This is in agreement with recent studies conducted on cat
atrial (Wang et al. 1998) and mouse ventricular myocytes
(Belevych & Harvey, 2000; Gödecke et al. 2001).
With regard to rebound stimulation, we observed two
functional RV myocyte types: those displaying a prominent
rebound stimulation, and those displaying little to no
rebound stimulation. In both myocyte types we could find
no compelling evidence in support of the obligatory
NO production hypothesis. Furthermore, in myocytes
displaying prominent rebound stimulation upon CCh
washout, neither ODQ nor 8-Br-cGMP produced any
significant effects. We therefore conclude that, while these
two effects (inhibition, rebound stimulation) are clearly
related through the common mechanism(s) of muscarinic
receptor(s) activation, neither is obligatorily dependent upon
endogenous myocyte NO production. Our conclusions are
therefore in disagreement with those of Wang et al. (1998),
but are in agreement with those of Belevych & Harvey
(2000).
Using immunofluourescent localization (IF) we have
previously demonstrated that Type III eNOS is present in
the majority of ferret RV myocytes, while it is hetero-
geneously expressed across the wall of the left ventricle
(LV), being high in LV subepicardial myocytes but low to
absent in LV subendocardial myocytes (Brahmajothi &
Campbell, 1999). We have now obtained additional IF
data indicating that a virtually identical heterogeneous
expression pattern exists in the ferret heart for NO-
activated soluble guanylyl cyclase (i.e. high sGC expression
in RV and LV subepicardial myocytes, low sGC expression
in LV subendocardial myocytes; Brahmajothi & Campbell,
2001). Hence, there is a clear correlation between the
expression of eNOS and sGC proteins among distinct
anatomical regions of the ventricle. However, eNOS
and sGC expression levels do not appear to simply
correlate with functional CCh-mediated responses. While
our results do not allow us to address the issue of NO
and sGC involvement in CCh-mediated effects underb-adrenergically stimulated conditions, they do strongly
indicate that there are NO-independent pathways linked
to muscarinic receptor activation that can produce both
inhibition and stimulation of RV ICa,L under basal
conditions.
The fact that in some RV myocytes both PTIO (NO-
scavenger) and ODQ (sGC inhibitor) caused a stimulation
of ICa,L may indicate that endogenous eNOS activity may
play a role in maintenance of basal ICa,L (e.g. Gallo et al.1998). However, these stimulatory effects were not
consistently observed. The hypothesis that eNOS activity
may be modulating basal ICa,L amplitude in ferret RV
myocytes therefore requires further experimentation. The
fact that the inhibitory effects of CCh were somewhat
greater in the presence of both PTIO and ODQ may be
indicative of a modulatory role of basal NO production in
these effects.
While our results indicate that NO- and sGC-activity are
not obligatorily coupled to CCh-mediated effects on RV
basal ICa,L, they in turn raise the obvious question: just what
mechanisms are responsible? One possibility (and we wish
to emphasize that at present the following model is only
speculative) could be ‘cross talking’ interactions of
activated G-protein subunits with multiple adenylyl
cyclase (AC) isoforms (Tang & Gilman, 1991; Taussig &
Gilman, 1995; Schulman & Hyman, 1999; Hanoune &
Defer, 2001). For example, if either a Type V and/or Type
VI AC isoform is active under basal conditions in ferret RV
myocytes (Campbell et al. 1996), then CCh-mediated
activation of M2-muscarinic receptors coupled to Gi would
lead to release of activated ai and bg subunits (Fig. 10).
Activated ai would then inhibit basally active Type V/VI
AC, thereby inhibiting basal ICa,L. Since in most cell types Gi
is much more highly expressed than Gs, upon washout of
CCh residually activated bg subunits would transiently
activate Type II and/or Type IV AC isoforms, thus
producing a transient rebound stimulation. However, in
the light of present models of G-protein interactions on
AC isoforms, for this scenario to be viable an activated as
subunit would also have to be involved, i.e. both as and bg(released from Gi) subunits are believed to be required for
activation of Type II/IV AC isoforms (Taussig & Gilman,
CCh modulation of ferret RV ICa,LJ. Physiol. 542.1 115
Figure 10. Speculation: involvement of multiple adenylylcyclase (AC) isoforms?One interpretative scenario for our results would be the proposalthat ferret RV myocytes possess a basally active Type V and/or TypeVI AC isoform which contributes to maintenance of basal ICa,L.Activation of muscarinic receptors (multiple subtypes?) coupled toinhibitory heterotrimeric Gi protein(s) would release ai and bgsubunits, leading to inhibition of Type V/VI AC activity and thusinhibition of basal ICa,L. Assuming Gi is more highly expressed thanGs , upon washout of muscarinic agents residually activated bgsubunits would transiently activate Type II and/or Type IV ACisoforms, thus producing a transient rebound stimulation. See textfor further discussion.
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1995; Schulman & Hyman, 1999). The source of as in
ferret RV myocytes under basal conditions is unclear.
An alternative to the above scenario would be that CCh-
mediated effects under basal conditions may be independent
of AC isoform activity. For example, a very recent study has
indicated that cGMP can inhibit myocardial contractility in
mice lacking cGMP-dependent protein kinase I (cGKI) both
in the absence and presence of forskolin, an AC activator
(Wegener et al. 2002). It was thus concluded that NO, cGMP,
and cGKI are not obligatorily involved in CCh-mediated
inhibition of murine myocardial contractility. If applicable
to ferret RV myocytes, these results would argue against the
‘AC isoform scenario’ outlined above.
In RV myocytes displaying rebound stimulation we
observed that upon repeated applications of CCh the
degree of inhibition remained constant while the amplitude
of rebound stimulation progressively declined with each
successive CCh application (Fig. 9). Whether this represents
a specific ‘rundown’ process of an intracellular regulatory
factor(s) or some form of desensitization is unclear.
Nonetheless, the fact that the two processes could be
differentially modulated suggests involvement of multiple
NO- and sGC-independent pathways and/or ‘crosstalk’
between regulatory pathways (Gallo et al. 1998; Belevych etal. 2001). This phenomenon may therefore provide an
explanation for the discrepancy between our conclusions
and those previously reached for cat atrial myocytes
(Wang et al. 1995,1998).
In conclusion, our results indicate that muscarinic
modulation of basal ICa,L in ferret RV myocytes involves
NO- and sGC-independent pathways and/or crosstalk
between such pathways. To paraphrase Hare & Stamler
(1999), in RV myocytes NO is not an obligatory mediator
of muscarinic responses but is most likely to be a
modulator of such responses under varying conditions.
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Acknowledgements This work was supported by grants to D. L. Campbell from theNational Institutes of Health (R01 HL58913) and the AmericanHeart Association (National Center, Established InvestigatorAward).
CCh modulation of ferret RV ICa,LJ. Physiol. 542.1 117