Effects of muscarinic receptor stimulation on Ca2+ transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells
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- van Borren, M.M.G.J., Verkerk, A.O., Wilders, R. et al. Basic Res Cardiol (2010) 105: 73. doi:10.1007/s00395-009-0048-9
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We investigated the contribution of the intracellular calcium (Cai2+) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Cai2+ transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE®). Our data show that the Cai2+ transient, like the hyperpolarization-activated “funny current” (If) and the ACh-sensitive potassium current (IK,ACh), is an important determinant of ACh-mediated pacemaker slowing. When If and IK,ACh were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 μM ACh was still able to reduce pacemaker frequency by 72%. In these If and IK,ACh-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Cai2+ transient decay (r2 = 0.98) and slow diastolic Cai2+ rise (r2 = 0.73). Inhibition of the Cai2+ transient by ryanodine (3 μM) or BAPTA-AM (5 μM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Cai2+ transient and reduced the sarcoplasmic reticulum (SR) Ca2+ content, all in a concentration-dependent fashion. At 1 μM ACh, the spontaneous activity and Cai2+ transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 μM) and IK,ACh was inhibited by tertiapin (100 nM). Also, inhibition of the Cai2+ transient by ryanodine (3 μM) or BAPTA-AM (25 μM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Cai2+ affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Cai2+ transient via a cAMP-dependent signaling pathway. Inhibition of the Cai2+ transient contributes to pacemaker slowing and inhibits Cai2+-stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Cai2+ transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.
KeywordsAcetylcholineCa2+ transientcAMPPacemaker cellsSinoatrial node
The mechanisms underlying pacemaker activity of sinoatrial node (SAN) cells are still a matter of debate [14, 17, 19, 20, 24]. Classically, β-adrenergic and muscarinic modulation of SAN pacemaker activity is explained by cAMP and PKA-dependent acceleration or deceleration of the “membrane clock”, which consists of time- and voltage-dependent ion channels located in the plasma membrane of SAN cells . Noradrenaline (NA) increases the inward currents that are active during diastolic depolarization , including the hyperpolarization-activated cation current (If) , L-type Ca2+ current (ICa,L)  and sustained inward current (Ist) [11, 32]. In addition, outward currents are reduced by hastening deactivation of the total delayed rectifier current (IK) . On the other hand, acetylcholine (ACh) slows the diastolic depolarization [4, 52] by decreasing the inward currents, If , ICa,L  and Ist , and activating the outward current IK,ACh .
Only during the last decade, spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR) have been widely recognized as an additional pacemaker mechanism: the “calcium clock” . It was demonstrated that inhibition of the “calcium clock” largely prevented β-adrenergic speeding of the pacemaker rate [25, 36]. Although pacemaker cells lack T-tubules, have sparsely developed SR, and contain little contractile proteins , they are endowed with significant intracellular Ca2+ (Cai2+) transients . β-adrenergic stimulation augments the amplitude of the Cai2+ transients and the local Ca2+ releases (LCRs) from the SR late during the diastolic depolarization . Besides the amplitude, also the frequency at which the LCRs occur increases . The accelerated “calcium clock” dynamically interacts with the “membrane clock” through the LCR-driven activation of an inwardly directed Na+/Ca2+ exchange current (INCX) , which substantially contributes to the diastolic depolarization rate and to the ignition of the action potential (AP) . The augmented Cai2+ transients and the accelerated “calcium clock” are the consequence of β-adrenergic-mediated cAMP production and increased PKA-dependent phosphorylation of Ca2+ handling proteins . The Cai2+ transients and “calcium clock” are interdependent . At every beat, the “calcium clock” is reset by ICa,L that subsequently triggers a Cai2+ transient. Only after the Cai2+ transient has sufficiently declined and enough Ca2+ is restored in the SR, LCRs can again occur. This makes the Cai2+ transient an integral part of the “calcium clock” [25–27].
It has been hypothesized that Cai2+ also indirectly couples to the “membrane clock” via a route involving cAMP, based on the observation that Cai2+ transient inhibition abolished If modulation by β-adrenergic agonists, while a membrane-permeable cAMP analog restored it . Indeed, Mattick et al.  recently identified Ca2+-stimulated adenylyl cyclase in guinea pig SAN cells that could explain this interesting observation.
In summary, the afore-mentioned data from literature show that β-adrenergic stimulation of SAN cells accelerates both the “membrane clock” and the “calcium clock” and augments the Cai2+ transient via cAMP–PKA-dependent signaling pathways. Moreover, evidence has been provided that Cai2+ stimulates cAMP production. We hypothesized that muscarinic receptor stimulation would lead to the opposite effects. In the present study, we carried out experiments on isolated rabbit SAN cells to determine (1) whether Cai2+ transients, such as If and IK,ACh, are important determinants of ACh-mediated pacemaker slowing, and (2) whether inhibition of Cai2+ transients by muscarinic agonists lowers cAMP production. Our results demonstrate the importance of Cai2+ transients in ACh-induced inhibition of pacemaker frequency and cAMP content in rabbit SAN cells.
Ethical approval and cell preparation
All experiments were carried out in accordance with guidelines of the local institutional animal care and use committee. In addition, the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Single SAN cells were isolated from 42 rabbit hearts by an enzymatic dissociation procedure as previously described . The hearts were obtained from New Zealand White rabbits (body weight: 3.0–3.5 kg) were first sedated with a 1 ml kg−1 intramuscular injection of Hypnorm® (10 mg ml−1 fluanisone and 0.315 mg ml−1 fentanyl citrate; Janssen Pharmaceutics, Tilburg, The Netherlands) and 15 min later anaesthetized by an injection of 3.0–3.5 ml Nembutal® (60 mg ml−1 pentobarbital sodium; Sanofi, Maassluis, The Netherlands) in the marginal ear vein together with 0.3 ml heparin (5,000 IU ml−1 thromboliquine; Organon, Oss, The Netherlands). For all measurements, except cAMP determination, spindle and elongated spindle-like cells displaying regular contractions in normal Tyrode’s solution were selected.
APs were recorded at 36 ± 0.2°C with the amphotericin B perforated patch-clamp technique using an Axopatch 200B patch-clamp amplifier (Molecular Devices Corporation, Sunnyvale, CA, USA). Patch pipettes (2–5 MΩ) were pulled from borosilicate glass, and data acquisition and analysis were accomplished using pCLAMP 8 software (Molecular Devices). Signals were low-pass filtered with a 1 kHz cutoff frequency, and digitized at 2 kHz. Potentials were corrected for the calculated liquid junction potential (13 mV). Cell membrane capacitance was estimated by dividing the decay time constant of the capacitive transient in response to 5-mV hyperpolarizing voltage clamp steps from 0 mV by the series resistance, and amounted to 34 ± 3 pF (mean ± SEM; n = 12).
[Ca2+]i was measured in Indo-1 loaded cells. In brief, cells were loaded with 5 μM Indo-1-AM for 10 min at room temperature, and subsequently superfused with normal Tyrode’s solution for 15 min at 36 ± 0.2°C to remove excess indicator and allow full de-esterification. A rectangular adjustable slit was used to select a single cell and to reduce background fluorescence. Dye-loaded cells were excited with 340 nm light (75 W Xenon arc lamp) and fluorescent light intensities were measured at 405 (I405) and 505 (I505) nm, using photomultiplier tubes. Signals were digitized at 1 kHz, filtered at 100 Hz, and corrected for background fluorescence before the I405/I505 ratio values were calculated.
[Ca2+]i was computed from [Ca2+]i = β × Kd × (R − Rmin)/(Rmax − R), where β represented the ratio of maximal I505 and minimal I505 (2.2), Kd was 250 nM (data sheet from Molecular Probes, Eugene, OR, USA), and Rmin and Rmax were the minimal and maximal ratio, respectively. To determine Rmin and Rmax, the cells were made Ca2+ permeable with a high potassium solution containing 1.7 μM A23187, 5 μM gramicidin and 10 μM nigericin, while Ca2+ re-uptake was inhibited by 5 μM thapsigargin. In the absence of Ca2+, we obtained an Rmin value of 0.31 ± 0.04 (n = 24), whereas in the presence of 10 mM extracellular Ca2+ the Rmax value was 2.21 ± 0.24 (n = 24).
The SR Ca2+ content of cells was studied by a brief exposure, via a nearby pipette, to 20 mM caffeine and 5 mM nickel to release Ca2+ from the SR and to prevent Ca2+ efflux by Na+/Ca2+ exchange, respectively. The fractional SR Ca2+ release was determined by normalizing the preceding Cai2+ transient amplitude (average of 6) to the caffeine-evoked Cai2+ transient amplitude.
The LANCE cAMP kit (PerkinElmer, Zaventem, Belgium) was used to determine cAMP concentrations (according to the manufacturer’s instruction) using a Victor plate reader (Wallac, PerkinElmer, Zaventem, Belgium). Cell suspension was added to a 384-well OptiPlate (PerkinElmer, Waltham, MA, USA; 5 μl per well) and incubated for 1 h at room temperature with metacholine (MCh) at concentrations ranging from 10−10 to 10−5 M. All experiments were performed in the presence of 0.05% bovine serum albumin (BSA) and 500 μM of the phosphodiesterase inhibitor isobutyl-methyl-xanthine (IBMX) to allow accumulation of cAMP.
For every experiment, the yields of two SAN cell isolations were pooled and divided over different wells. All MCh–cAMP response curve measurements were performed in triplo and averaged. For each intervention (500 nM NA, 3 μM ryanodine, 25 μM BAPTA-AM), a control MCh–cAMP response curve was measured in the presence of the vehicle used. The obtained cAMP values were normalized to the control cAMP value measured in the absence of MCh. To determine the effect of Cai2+ on cAMP content, SAN cells were pre-incubated with BAPTA-AM or ryanodine for 10 min.
Solutions and drugs
Tyrode’s solution contained (mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 5.5 glucose, 5.0 HEPES; pH 7.4 (2.6 NaOH). The pipette solution contained (mM): 120 K-gluconate, 20 KCl, 5 NaCl, 0.22 amphothericin B, 10 NMDG-Cl (N-methyl-d-glucammonium chloride), 10 HEPES; pH 7.2 (5.5 KOH). The high potassium solution contained (mM): 130 KCl, 10 NaCl, 1 MgCl2, 10 HEPES; pH 7.2 (5.5 KOH).
All drugs were obtained from Sigma (Zwijndrecht, The Netherlands), except ryanodine (Calbiochem-Novabiochem, Darmstadt, Germany; 10 mM), noradrenaline (Centrafarm, Etten-Leur, The Netherlands; 6 mM), Indo-1, BAPTA-AM, and thapsigargin (Molecular Probes). Except for noradrenaline, all drugs were prepared as a stock solution in DMSO (amphothericin B (65 mM), Indo-1 (5 mM), A23187 (1.7 mM), thapsigargin (10 mM), BAPTA-AM (25 mM), forskolin, (10 mM)), in ethanol (gramicidin (50 mM), nigericin (10 mM)), or in H2O (ACh (1 mM), ryanodine (3 mM), MCh (10 mM)). The values in parentheses indicate the concentration of the stock. The ryanodine and BAPTA-AM concentrations were such that Cai2+ transients were largely, but not fully, inhibited, whereas the cesium and tertiapin concentrations were selected to maximally inhibit If and IK,ACh, respectively, without exerting major effects on other membrane currents. We chose 3 μM ryanodine since it has often been used, e.g., by Bogdanov et al. , Bucchi et al.  and Vinogradova et al. , and higher concentrations are known to block ICa,L . A sub-maximal NA concentration (500 nM) was selected to study the anti-adrenergic effects of ACh on Cai2+ transients and pacemaker frequency. Ryanodine, BAPTA-AM, MCh, ACh and NA were prepared freshly on the day of the experiment. The effect of ACh, NA, cesium and tertiapin on pacemaker frequency was determined after 2 min exposure time, whereas the effect of ryanodine and BAPTA-AM was determined after 10 min.
Results are expressed as mean ± SEM, with the number of cells or cell suspensions denoted by n. Statistical analysis was performed using SPSS 10 software (SPSS Inc., Chicago, IL, USA). Two-way ANOVA with repeated measurements was used to compare concentration effect curves, whereas the EC50 values (derived from a Hill equation fit) and the pacemaker frequencies of Fig. 2b were compared with a Student’s t test. One-way ANOVA with repeated measurements, followed by a Student–Newman–Keuls test for pairwise comparsions, was used to compare the pacemaker frequencies of Fig. 2f. P values of <0.05 were considered to be significant.
Determination of pacemaker frequency by Cai2+ transients
Inhibition of cAMP production and activation of IK,ACh underlie pacemaker slowing
In NA-stimulated SAN cells, pacemaker frequency, as derived from Cai2+ transients, was significantly higher compared to non-stimulated SAN cells (3.94 ± 0.11 Hz, n = 22, vs. 3.31 ± 0.17 Hz, n = 13, respectively) (Fig. 2b). In both groups, pacemaker frequency decreased with increasing ACh concentrations (12.5, 50, 100 and 1,000 nM; Fig. 2c). However, the relative decrease in pacemaker frequency was significantly larger in non-stimulated compared to NA-stimulated cells at ACh concentrations of 12.5, 50 and 100 nM (Fig. 2c, asterisks). Accordingly, the averaged EC50 value of the individual frequency–ACh relations of the 13 non-stimulated SAN cells tested (30 ± 2 nM) was significantly smaller than that for the 22 NA-stimulated cells tested (45 ± 4 nM) (Fig. 2c, inset).
Determination of cAMP accumulation in SAN cell suspensions showed that stimulation of the muscarinic receptor by metacholine (MCh), an ACh analog that cannot be hydrolyzed by acetylcholinesterase possibly present in the cell suspension, inhibits cAMP accumulation concentration dependently in both non-stimulated (n = 8) and NA-stimulated (n = 8) cells (Fig. 2d). In NA-stimulated SAN cells, the accumulated cAMP was significantly higher in basal conditions (no MCh) compared to non-stimulated cells and showed a smaller percent decrease with increasing MCh concentrations (ANOVA, P < 0.05). Accordingly, the averaged EC50 value of the individual cAMP accumulation–MCh relations (81 ± 14 vs. 55 ± 6 nM, Fig. 2d, inset) proved significantly higher in NA-stimulated compared to non-stimulated cells. Interestingly, the EC50 values of the pacemaker frequency–ACh and the cAMP accumulation–MCh relations (Fig. 2c, d, insets) are both increased by ≈50% in NA-stimulated cells compared to non-stimulated cells, suggesting a strong correlation between muscarinic agonist-induced frequency and cAMP changes.
Next, we examined whether activation of IK,ACh and inhibition of cAMP production can explain pacemaker slowing by muscarinic agonists. Therefore, we tested the effects of the IK,ACh blocker tertiapin and the adenylyl cyclase activator forskolin on 1 μM ACh effects in non-stimulated SAN cells (n = 6). Typical Cai2+ transients recorded under these conditions are depicted in Fig. 2e. Spontaneous activity recovered partially by IK,ACh blockade (tertiapin 100 nM) and fully when in addition adenylyl cyclase was activated (forskolin 10 μM) (Fig. 2f; blue and green bars, respectively). From these data, we conclude that inhibition of cAMP production and activation of IK,ACh could explain the effect of muscarinic receptor stimulation on pacemaker frequency.
Contribution of If and IK,ACh to ACh-mediated pacemaker slowing
In non-stimulated SAN cells, the If blocker cesium (2 mM; n = 10) slowed pacemaking only at low ACh concentrations (<50 nM), whereas the IK,ACh blocker tertiapin (100 nM; n = 9) opposed pacemaker slowing at all ACh concentrations tested (Fig. 3b) (ANOVA, P < 0.05). In NA-stimulated cells, pacemaker slowing by the If blocker cesium (n = 8) was visible up to 100 nM ACh (ANOVA, P < 0.05), whereas the IK,ACh blocker tertiapin (n = 4) no longer significantly opposed pacemaker slowing (Fig. 3c). These results demonstrate that If and IK,ACh contribute to ACh-mediated pacemaker slowing. If gains importance and IK,ACh loses importance in ACh-mediated pacemaker slowing under NA-stimulated conditions. However, ACh-mediated pacemaker slowing was still clearly present during If or IK,ACh blockade, indicating the contribution of other mechanisms (see below).
Contribution of Cai2+ transients to ACh-mediated pacemaker slowing
SAN pacemaker activity has been shown to be modulated also by SR Ca2+ loading and release . Therefore, we next examined the role of Cai2+ transients in ACh-induced pacemaker slowing.
Second, we tested the effects of ryanodine and BAPTA-AM on ACh-induced pacemaker slowing. In non-stimulated SAN cells, BAPTA-AM, but not ryanodine, caused a clear slowing at the low ACh concentration of 12.5 nM (Fig. 4b), whereas in NA-stimulated cells both agents exaggerated pacemaker slowing at ACh concentrations of 12.5, 50 and 100 nM (Fig. 4c) (ANOVA, P < 0.05). Taken together, these data demonstrate that Cai2+ transients can contribute to ACh-induced pacemaker slowing.
Effect of ACh on Cai2+ transients
Cai2+ transients of SAN cells
Effect of ACh on Cai2+ transients
We then analyzed the effects of ACh on the Cai2+ transient characteristics in both non-stimulated (n = 13) and NA-stimulated (n = 22) SAN cells in detail. Figure 5b, c, d, e, f shows that all five phases were affected concentration dependently in both non-stimulated and NA-stimulated cells. The fast systolic [Ca2+]i rise slowed (Fig. 5b), the maximum systolic [Ca2+]i fell (Fig. 5c), the diastolic [Ca2+]i decay slowed (Fig. 5d), the minimum diastolic [Ca2+]i fell (Fig. 5e) and the diastolic [Ca2+]i rise slowed (Fig. 5f). Of note, phase V of the Cai2+ transient proved most sensitive to ACh and was inhibited by 94 and 87% at 100 nM ACh in non-stimulated and NA-stimulated SAN cells, respectively. Moreover, the diastolic [Ca2+]i rise of phase V proved also most sensitive to NA stimulation and increased by 70% at 0 nM ACh (Fig. 5f).
From these data, we conclude that ACh inhibited the Cai2+ transients in both non-stimulated and NA-stimulated SAN cells.
Effect of ACh on SR Ca2+ content
From these data we conclude that ACh lowers SR Ca2+ content, which could explain the inhibitory effect of ACh on Cai2+ transients (Fig. 5).
Phases III and V of the Cai2+ transient correlate with pacemaker slowing
Effect of ryanodine and BAPTA-AM on intracellular cAMP content
In this study, we demonstrate for the first time that muscarinic agonists inhibit Cai2+ transients in SAN cells, and that the pacemaker slowing effects of muscarinic agonists are augmented by Cai2+ transient inhibition. Moreover, we provide evidence that, like muscarinic agonists, Cai2+ transient inhibition reduces cAMP levels in SAN cells. Both inhibition of Cai2+ transients and reduction of cAMP levels will inhibit Cai2+ and cAMP-dependent ionic currents. These data suggest that the negative chronotropic effect of muscarinic agonists is in part obtained via Cai2+ transient inhibition and the subsequent reduction in cAMP.
Classical pathways in muscarinic agonist-induced pacemaker slowing
In our rabbit single SAN cell preparation, we demonstrate that muscarinic agonists inhibit pacemaker frequency (Figs. 1, 2, 3, 4, 7) and cAMP production (Figs. 2d, 8) in a concentration-dependent manner. Our data thus support the classical view that cAMP is an important determinant of pacemaker frequency. The classical view that activation of IK,ACh is a cAMP-independent pathway by which muscarinic agonists exert their negative chronotropic effect  (cf. Fig. 2a) is also supported by our data (Figs. 2e, f, 3). Moreover, we demonstrated that NA stimulation, which is known to increase cAMP production, reduced the contribution of the cAMP-independent pathway IK,ACh to ACh-mediated pacemaker slowing. This explains the limited effect of ACh on the maximum diastolic potential seen in Fig. 1a. In contrast, NA stimulation increased the contribution of the cAMP-dependent pathway If to ACh-mediated pacemaker slowing. This can be explained by the NA-mediated increase in cAMP production.
Muscarinic agonists slow pacemaker rate by inhibition of Cai2+ transients
The importance of Cai2+ in pacemaking has been demonstrated in non-stimulated and NA-stimulated conditions. It has been shown that blunting Cai2+ transients (by ryanodine) or chelating Cai2+ (by BAPTA) slowed or arrested pacemaking and prevented NA-induced pacemaker acceleration . Although ryanodine and BAPTA both inhibit Cai2+ transients, it should be kept in mind that there are several differences in actions between ryanodine and BAPTA. For instance, BAPTA increases the intracellular Ca2+ buffering power. Consequently, the free Cai2+ concentration will fall and any increase in [Ca2+]i through Ca2+ release (from the SR) or Ca2+ influx (through ICa,L) is blunted and slowed. This will affect the LCRs, Cai2+ transients and all Cai2+-dependent currents including ICa,L. Ryanodine, on the other hand, reduces the open probability of the RyR channels. Consequently, less ryanodine channels are available for SR Ca2+ release. This will reduce fractional SR Ca2+ release and the amplitude of LCRs and Cai2+ transients. However, ryanodine will not blunt any Ca2+ influx and will affect Cai2+-dependent modulation of ICa,L to a lesser extent. These differences in actions between ryanodine and BAPTA may explain their quantitatively different effects on pacemaker frequency.
In the present study, we confirm that Cai2+ transients contribute to the intrinsic pacemaker activity of non-stimulated SAN cells (Fig. 4b) and to pacemaker acceleration of NA-stimulated cells (Fig. 4c). In addition, we provide evidence for the involvement of Cai2+ transients in muscarinic agonist-induced pacemaker slowing.
First, both ryanodine and BAPTA-AM facilitated pacemaker slowing at all ACh concentrations in NA-stimulated SAN cells. In non-stimulated SAN cells, the effects of ryanodine and BAPTA on ACh-mediated pacemaker slowing were less abundant and restricted to low ACh concentrations. The latter is likely explained by the concomitant increase in minimum diastolic Cai2+ concentration (Fig. 4a) and the subsequent activation of INCX. A ryanodine-induced increase in diastolic [Ca2+]i has been previously reported in rabbit SAN cells . The diastolic Cai2+ increase was larger in the presence of ryanodine than in the presence of BAPTA-AM. This could underlie the quantitative differences between the effects of both agents on ACh-mediated pacemaker slowing. Moreover, Bucchi et al.  recently reported that ryanodine (3 μM) depolarizes the take-off potential of the AP, which by itself slows pacemaker frequency.
Second, we show that ACh depresses all five (I–V) distinct phases of the Cai2+ transients (Fig. 5b, c, d, e, f) and lowers the SR Ca2+ content (Fig. 6b) of rabbit SAN cells in a concentration-dependent manner, all of which is compatible with reduced cAMP–PKA-dependent phosphorylation of the Cai2+ handling proteins. In accordance, in NA-stimulated cells, where cAMP–PKA signaling is stimulated, higher ACh concentrations were required to obtain a similar level of the maximum systolic [Ca2+]i (Fig. 5c), slow diastolic [Ca2+]i rise (Fig. 5f) and SR Ca2+ content (Fig. 6b) as compared to non-stimulated SAN cells. Moreover, higher ACh concentrations were required to increase the time constant of Cai2+ transient decay in NA-stimulated cells (Fig. 5d). Of interest, the slow diastolic [Ca2+]i rise that probably reflects the LCRs proved the Cai2+ transient parameter with the highest ACh-sensitivity (Fig. 5f).
Third, ACh was still able to slow pacemaker frequency of non-stimulated SAN cells in a concentration-dependent manner when the important contributors to ACh-mediated pacemaker slowing, If and IK,ACh, were both inhibited (Fig. 7b). Thus, other mechanisms, such as Cai2+ releases , ICa,L [34, 43] and Ist , involved in ACh-mediated pacemaker slowing must be active. Both the Cai2+ transient decay and the slow diastolic Cai2+ rise of SAN cells with inhibited If and IK,ACh showed close correlations with the ACh-induced changes in interbeat interval (Fig. 7c, d), suggesting that the ACh-mediated modulation of SERCA activity and of LCRs, and thus the rate of SR Ca2+ refilling and release, are important determinants of ACh-mediated pacemaker slowing. This is in accordance with previous publications by Lakatta and co-workers who found strong correlations between beating frequency and phospholamban phosphorylation status, and beating frequency and the periodicity of LCRs in rabbit SAN cells [27, 47].
Fourth, Cai2+ transient inhibition by ryanodine and BAPTA-AM facilitated MCh-mediated inhibition of cAMP, an important second messenger that is positively correlated with pacemaker frequency (Figs. 2, 8).
The effect of ACh on Cai2+ transients can be explained by reduced cAMP–PKA-dependent phosphorylation of ryanodine receptors and phospholamban. These phosphorylation changes lead to reduced open probability of the ryanodine receptor and to slower SERCA-mediated Cai2+ re-uptake into the SR, respectively. Moreover, in voltage clamp experiments, ACh inhibits the ICa,L of rabbit SAN cells , which may result in Cai2+ transient inhibition. Further experiments are required to elucidate the exact role of ICa,L in ACh-mediated pacemaker slowing.
Membrane clock and calcium clock
The main mechanism by which Cai2+ is thought to interact with the “membrane clock” is through electrogenic Ca2+ extrusion via the Na+/Ca2+ exchanger . For instance, NA stimulates cAMP–PKA-dependent phosphorylation of Cai2+ handling proteins, thereby increasing the amplitude and frequency of the LCRs and subsequently the contribution of INCX to diastolic depolarization [3, 45]. It should be taken into account, however, that various ion currents can also be modulated by Cai2+ (e.g., ICa,L , If , the T-type calcium current (ICa,T) , chloride currents [1, 44] and the slow delayed rectifier current (IKs) ) and by calmodulin or Ca2+/calmodulin-dependent protein kinase II (CaMKII) (e.g., ICa,L , ICa,T  and IKs ). In the present study, we did not assess each of the ionic currents that are affected by the muscarinic agonist-induced Cai2+ transient changes, nor did we study the effect of muscarinic agonists on LCRs as part of the “calcium clock”. Nevertheless, our Cai2+ transient data are still relevant for understanding the “calcium clock”, since the Cai2+ transient and the “calcium clock” are interdependent . LCRs ignite the AP by activating ICa,L, which resets the “calcium clock” and triggers a Cai2+ transient. Only after the Cai2+ transient has sufficiently declined and enough Ca2+ is restored in the SR, another LCR can occur.
Thus, muscarinic agonists slow SAN cell pacemaker activity by inhibition of Cai2+ transients. However, the relative contribution of Cai2+-dependent mechanisms and “membrane clock” mechanisms, including inhibition of If and activation of IK,ACh, to ACh-mediated pacemaker slowing is difficult to assess because they are so strongly interdependent (see above). Only if we assume that ACh-mediated pacemaker slowing is fully explained by the action of ACh on If, IK,ACh and a Ca2+-dependent mechanism, we can estimate their relative contributions. Pacemaker frequency of uninhibited SAN cells was slowed by 66, 79 and 100% when exposed to 50, 100 and 1,000 nM ACh, respectively (Fig. 3b), whereas pacemaker frequency of SAN cells with inhibited If and IK,ACh was slowed by 51, 58 and 72% when exposed to 50, 100 and 1,000 nM ACh, respectively (Fig. 7b). Thus, one could say that at 50–1,000 nM ACh, the Ca2+-dependent mechanism is responsible for ≈75% pacemaker slowing and the combined inhibition of If and opening of IK,ACh for ≈25% pacemaker slowing. Further experiments are required to elucidate the ionic currents involved in the Ca2+-dependent mechanism of ACh-mediated pacemaker slowing.
Muscarinic agonists slow pacemaker rate by inhibition of Cai2+-stimulated cAMP production
It was recently demonstrated that basal cAMP levels are almost tenfold higher in SAN cells as compared to atrial and ventricular myocytes . The presence of Ca2+-stimulated adenylyl cyclases in SAN cells may account for the relatively high basal cAMP level  and PKA activity . The latter proved essential for the spontaneous rhythmic LCRs and “calcium clock” in SAN cells. The presence of these Ca2+-stimulated adenylyl cyclases in SAN cells  provides a potential indirect mechanism by which Cai2+ influences cAMP-dependent ionic currents and thus pacemaker frequency [5, 30].
Our data add functional evidence for the presence of a Cai2+-dependent cAMP production in rabbit SAN cells. While the present paper was in preparation, Younes et al.  published their work on Ca2+-stimulated basal adenylyl cyclase activity of rabbit SAN pacemaker cells. In this study, they showed that rabbit SAN cells express two Ca2+-stimulated adenylyl cyclases (AC1 and AC8), located in the lipid rafts, which are largely responsible for the basal adenylyl cyclase activity and account for ≈30% of the high basal cAMP levels in SAN cells. In our non-stimulated SAN cells, Cai2+-dependent cAMP production contributes to 33% to the basal cAMP levels, as demonstrated by the reduction in cAMP production on inhibition by BAPTA (Fig. 8c). From our results and the similar observations by Younes et al. , we conclude that in rabbit SAN cells, Ca2+-stimulated adenylyl cyclases (AC1 and AC8) in part contribute to the basal cAMP levels and that the Ca2+–independent adenylyl cyclases (AC5 and AC6) may provide the remaining cAMP [48, 49].
In addition, from our data we conclude that the Cai2+-dependent cAMP production can provide the increase in cAMP in NA-stimulated SAN cells. Thus, the NA-augmented Cai2+ transients may have stimulated the extra cAMP production required for pacemaker acceleration (Figs. 2d, 8d). This finding contrasts with the observation by Younes et al.  that BAPTA did not affect the cAMP production in SAN cells stimulated with a β-adrenergic agonist, but it is in line with the earlier report of Bucchi et al.  who showed that a membrane-permeable cAMP analog restored If modulation by β-adrenergic agonists in ryanodine treated SAN cells.
Furthermore, we demonstrate that Cai2+ transient inhibition facilitates the ACh-mediated inhibition of cAMP production in non-stimulated SAN cells (Fig. 8a, c). Thus, muscarinic agonist-mediated inhibition of the Cai2+ transient may in part underlie its inhibitory effect on cAMP production and pacemaker slowing (Fig. 2c). The remaining muscarinic agonist-induced cAMP reduction is likely obtained via the classical Gi-mediated inhibition of adenylyl cyclases .
The present study has several limitations. First, it is difficult to draw firm conclusions with respect to the role of Cai2+ transients in muscarinic agonist-induced pacemaker slowing due to the interdependence of Cai2+ transients, cAMP content and pacemaker frequency. Ideally, one should vary one parameter while keeping other parameters constant. Unfortunately, this is impossible in SAN cells, even under AP clamp conditions, where pacemaker frequency, but not ion flow, is controlled. Nevertheless, our observations that muscarinic agonists reduce pacemaker frequency, Cai2+ transients and cAMP content dose dependently, and that Cai2+ transient inhibition (by ryanodine or BAPTA) in turn facilitates muscarinic agonist-mediated pacemaker slowing and cAMP inhibition provides valuable insight into the processes underlying pacemaker frequency modulation. Second, the exact spatial and temporal relation between Cai2+ and cAMP may be highly complex, since local concentration differences of both exist within a cell . Phosphodiesterases, which in our cAMP assays were inhibited to allow cAMP accumulation, underlie these spatial differences and have been recently shown to be constitutively active in SAN cells . Nevertheless, also in PDE-inhibited SAN cells, MCh, NA, ryanodine and BAPTA changed cAMP levels, indicating that cAMP production was still subject to modulation. Third, it should be noted that 2 mM cesium not only largely inhibits If , but also slightly reduces outward potassium current , which may have led to an underestimation of the contribution of If to pacemaker slowing in our experiments. However, “more specific” If channel blockers also affect other ion currents present in SAN cells . In addition, If channel blockers may also directly influence Cai2+ transients, since the If channel has been recently shown to be permeable to calcium ions . A further limitation is that IK,ACh may not have been completely blocked by 100 nM tertiapin, as suggested by the experiments of Kitamura et al. , who found a ≈95% block of IK,ACh by 100 nM tertiapin in rabbit atrial myocytes. Therefore, the actual contribution of IK,ACh to ACh-mediated pacemaker slowing may have been underestimated in the present study. Moreover, the potential contribution of ICa,L to ACh-mediated pacemaker slowing has not been investigated because of the lack of appropriate pharmacological tools to assess the role of ICa,L without stopping pacemaking or affecting the action potential waveform. Finally, MCh is less potent compared to ACh in stimulating muscarinic receptor type 2 . Therefore, a direct quantitative comparison between the cAMP–MCh curves and the frequency–ACh response curves is not allowed.
We provide evidence that muscarinic agonists not only slow pacemaker activity by inhibition of cAMP-dependent ionic currents and activation of IK,ACh, but also by inhibition of Cai2+ transients, which reduces cAMP content through inhibition of Cai2+-stimulated cAMP production.
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