Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca(2+) cycling and I(KACh)

Abstract

Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a G(i)-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization-activated current (I(f)) or L-type Ca(2+) currents (I(Ca,L)), respectively; and 2) direct G(i)-coupled activation of ACh-activated potassium current (I(KACh)). More recent studies, however, have indicated that Ca(2+) cycling by the sarcoplasmic reticulum within SANC (referred to as a Ca(2+) clock) generates rhythmic, spontaneous local Ca(2+) releases (LCR) that are AC-PKA dependent. LCRs activate Na(+)-Ca(2+) exchange (NCX) current, which ignites the surface membrane ion channels to effect an AP. The purpose of the present study was to determine how ChR signaling initiated by a cholinergic agonist, carbachol (CCh), affects AC, cAMP, and PKA or sarcolemmal ion channels and LCRs and how these effects become integrated to generate the net response to a given intensity of ChR stimulation in single, isolated rabbit SANC. The threshold CCh concentration ([CCh]) for BRR was approximately 10 nM, half maximal inhibition (IC(50)) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. G(i) inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that I(f) blockade did not affect BRR at any [CCh] and that I(KACh) activation, evidenced by hyperpolarization, first became apparent at [CCh] > 30 nM. At IC(50), CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by approximately 50%. The dose response of BRR to CCh in the presence of I(KACh) blockade by a specific inhibitor, tertiapin Q, mirrored that of CCh to reduced PLB phosphorylation. At IC(50), CCh caused a time-dependent reduction in the number and size of LCRs and a time dependent increase in LCR period that paralleled coincident BRR. The phosphatase inhibitor calyculin A reversed the effect of IC(50) CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that Ca(2+) cycling is integrated into the cholinergic modulation of BRR via LCR-induced activation of NCX current, providing theoretical support for the experimental findings. Thus ChR stimulation-induced BRR is entirely dependent on G(i) activation and the extent of G(i) coupling to Ca(2+) cycling via PKA signaling or to I(KACh): at low [CCh], I(KACh) activation is not evident and BRR is attributable to a suppression of cAMP-mediated, PKA-dependent Ca(2+) signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca(2+) signaling and I(KACh) activation underlies a more pronounced BRR.

Fig. 1.

A: average beating rate reduction (BRR) time-dose effects of 5 different carbachol (CCh) concentrations ([CCh]; control, n = 39; 10 nM, n = 9; 30 nM, n = 6; 100 nM, n = 19; 300 nM, n = 17; 1,000 nM, n = 15). Each cell received only a single dose of CCh. B: average normalized BRR in control cells, cells exposed to CCh for 3 min, cells superfused with atropine alone or with atropine and CCh, and cells pretreated with and superfused with Tyrode's buffer containing 1.5 mg/ml of pertussis toxin (PTX), with and without CCh (control, n = 15; CCh 1 μM, n = 10; 10 μM atropine, n = 4; CCh+atropine, n = 4; PTX 1.5 mg/ml, n = 4; PTX+CCh, n = 4). The times of CCh addition and washout are indicated by arrows, which pertain only to protocols in which CCh was added and washed.

Fig. 2.

A: average current-voltage (I-V) curves of total current density measured at 450 ms of the voltage pulse in the absence or presence of CsCl. Inset: difference current between the traces recorded in the absence and presence of 2 mM CsCl. This difference current is hyperpolarization-activated current (I_f). B: average effects of CCh on the I-V relationship of ACh-activated potassium current (I_KACh) measured during I_f blockade (CsCl+CCh, n = 13) in control experiments (red line) and in experiments in which I_KACh was blocked by 1 μM tertiapin Q (TQ) and I_f was blocked by 2 mM CsCl (TQ+CsCl+CCh, n = 9; blue line).

Fig. 3.

A: average dose-dependent peak effects of CCh on maximum diastolic potential (MDP) and BRR in spontaneously beating sinoatrial nodal cells (SANC) and in SANC with blocked I_f or blocked I_KACh, or block of both currents simultaneously (CCh, n = 43; TQ+CCh, n = 22; CsCl+CCh, n = 22; TQ+CsCl+CCh, n = 12). Each cell received only a single dose of CCh. B: the average change in BRR at each [CCh] for CCh alone and for CCh+TQ has been normalized to the respective average maximum effect. Since each cell received only a single concentration of CCh, individual cells do not have a dose response. Therefore an average dose-response curve was calculated from the average of all cells at all concentrations. This yields a unique normalized dose-response curve, but without x-axis variation. The average normalized curves are the best fit with a nonlinear regression model for the experimental data points. The log[IC₅₀] calculated from this regression model is indicated in the inset. TQ shifts the CCh IC₅₀ toward the lower concentrations of CCh by 2.6-fold.

Fig. 4.

Average dose-time BRR effects in SANC produced by CCh alone (n = 43), with block of I_f by 2 mM CsCl (n = 22), with I_KACh by 1 μM TQ (n = 22), or with block of both I_f and I_KACh (n = 12). Each cell received only a single CCh dose.

Fig. 5.

CCh effects on total cAMP (A; n = 5) and cGMP (B; n = 4) levels in spontaneously beating SANC suspensions in the absence or presence of phosphodiesterase (PDE) inhibition by IBMX. Each cell suspension from a given sinoatrial node was first divided into 2 major groups: no IBMX prior to CCh, or IBMX (100 μM, 20 min pretreatment before CCh). Each group was further subdivided into 3 groups: control, 100 nM CCh, and 1 μM CCh. Total incubation time in all cell groups was 25 min. In PDE-treated groups IBMX was added immediately at the beginning of incubation. In groups with CCh treatment, CCh was added at the 20th minute of incubation. In other words, the duration of exposure to CCh was 5 min. PDE inhibition in the absence of CCh increases both cAMP and cGMP (*P < 0.05). In the presence of PDE inhibition CCh significantly reduces cAMP (#P < 0.05) but does not affect cGMP.

Fig. 6.

A, top: representative Western blots of phosphorylated phospholamban (PLB) at the PKA-dependent phosphorylation site Ser-16 and total PLB immunolabeling in control SANC suspensions and cell suspensions exposed to a wide range of [CCh]. Bottom: average immunoblot data of phosphorylated PLB normalized to total PLB in response to CCh. The number of experiments at each [CCh] is indicated in parentheses. B: the average response at each [CCh] has been normalized to the maximum effect of CCh. The curves represent the best fit nonlinear regression model for the experimental data points. The curve for BRR in the presence of I_KACh inhibition is the same as that was illustrated in Fig. 3B. The log[IC₅₀] calculated from the nonlinear regression for each curve is shown in the inset. In 3 PLB phosphorylation experiments, cell suspensions received the entire range of [CCh]. Thus a final normalized dose-response curve could be calculated for each of these experiments, permitting direct calculation of the average IC₅₀ and the variance around the average IC₅₀. This is indicated by large solid gray point with ±SD on the PLB dose-response curve. Note that the IC₅₀ for BRR by CCh in the presence of I_KACh inhibition is within the SD of the IC₅₀ for PLB dephosphorylation.

Fig. 7.

Confocal Ca²⁺ images (left) and analog Ca²⁺ transients (right), in a representative SANC prior to and during CCh exposure at the IC₅₀ CCh. Both action potential (AP)-triggered Ca²⁺ transients and local Ca²⁺ release (LCR) characteristics are substantially reduced by CCh in spontaneously beating SANC. F, fluorescence during excitation; F₀, background fluorescence.

Fig. 8.

A: average (n = 10) changes in AP-induced Ca²⁺ transient characteristics in the presence of CCh. d(F/F₀)/dt, maximum rate of rise of the Ca²⁺ transient measured as F/F₀ fluo 3-AM fluorescence; T90, transient duration at 90% of transient amplitude; T50, transient duration at 50% of transient amplitude. In control: d(F/F₀)/dt = 0.032 ± 0.002 s⁻¹; amplitude = 1.46 ± 0.08 F/F₀; T90 = 264.4 ± 11 ms; T50 = 100.2 ± 3 ms. B: average (n = 10) change in LCR characteristics induced by CCh. In control: LCR amplitude = 1.1 ± 0.18 F/F₀; LCR number per cycle = 1.02 ± 0.1; LCR size = 6.9 ± 0.7 μm; LCR period = 351 ± 22 ms (*P < 0.05). The method used to define LCR period and LCR cycle length is illustrated in the inset of B. The cycle length is the time between 2 successive AP-induced Ca²⁺ transients, and LCR period is measured as time from the preceding Ca²⁺ transient upstroke to the maximum rate of rise of subsequent LCR (*P < 0.05).

Fig. 9.

Least squares linear regression of concomitantly measured LCR periods and cycle lengths in 10 SANC in control and during a 1- to 3-min exposure to 100 nM (IC₅₀) CCh.

Fig. 10.

A: representative confocal line scan images of the effects of the phosphatase inhibitor calyculin A (100 nM) to reverse the effects of a 3-min 100 nM CCh exposure on cycle length and LCRs. Calyculin A was added for 3 min in the continued presence of CCh. B: average data on cycle length and LCR characteristics in control, during CCh, and during CCh+calyculin A (n = 5), *P < 0.05 vs. control, ‡P < 0.05 vs. CCh.

Fig. 11.

Least square linear regression of LCR period and cycle length in 5 SANC in control, measured during a 3-min exposure to IC₅₀ [CCh], and following a 3-min exposure to the phosphatase inhibitor calyculin A in the continued presence of 100 nM CCh.

Fig. 12.

Experimental data and numerical modeling (see Online Supplement for details) of the component mechanisms for BRR induced by 100 nM CCh. A: model simulations show LCR signaling to membrane potential (Vm, red) via Na⁺-Ca²⁺ exchange (NCX) current (I_NCX, blue) under variety of perturbations (gray labels). Eight arbitrary LCRs are shown by multiple colors (above I_NCX and V_m) in each subpanel (i–vii). B: the numerical model simulations closely predict negative chronotropic effects of IC₅₀ CCh observed experimentally.