Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes

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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: [email protected]

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).


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


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).


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.


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


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,


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).


Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes

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