Nickel suppresses the PACAP-induced increase in guinea pig cardiac neuron excitability

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent intercellular signaling molecule involved in multiple homeostatic functions. PACAP/PAC₁ receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, making them a unique system to establish mechanisms underlying PACAP modulation of neuronal function. Calcium influx is required for the PACAP-increased cardiac neuron excitability, although the pathway is unknown. This study tested whether PACAP enhancement of calcium influx through either T-type or R-type channels contributed to the modulation of excitability. Real-time quantitative polymerase chain reaction analyses indicated transcripts for Ca_v3.1, Ca_v3.2, and Ca_v3.3 T-type isoforms and R-type Ca_v2.3 in cardiac neurons. These neurons often exhibit a hyperpolarization-induced rebound depolarization that remains when cesium is present to block hyperpolarization-activated nonselective cationic currents (I_h). The T-type calcium channel inhibitors, nickel (Ni²⁺) or mibefradil, suppressed the rebound depolarization, and treatment with both drugs hyperpolarized cardiac neurons by 2–4 mV. Together, these results are consistent with the presence of functional T-type channels, potentially along with R-type channels, in these cardiac neurons. Fifty micromolar Ni²⁺, a concentration that suppresses currents in both T-type and R-type channels, blunted the PACAP-initiated increase in excitability. Ni²⁺ also blunted PACAP enhancement of the hyperpolarization-induced rebound depolarization and reversed the PACAP-mediated increase in excitability, after being initiated, in a subset of cells. Lastly, low voltage-activated currents, measured under perforated patch whole cell recording conditions and potentially flowing through T-type or R-type channels, were enhanced by PACAP. Together, our results suggest that a PACAP-enhanced, Ni²⁺-sensitive current contributes to PACAP-induced modulation of neuronal excitability.

pituitary adenylate cyclase-activating polypeptide (PACAP) peptides (Adcyap1), members of the vasoactive intestinal polypeptide/secretin/glucagon family of neuropeptides, are potent trophic and intercellular signaling molecules that are widely distributed within neural tissues (2, 54). The PACAP peptide amino acid sequence is highly conserved, and the action of PACAP is tissue specific dependent on the activation of the different isoforms of the seven transmembrane G protein-coupled PACAP-selective PAC₁ receptor (Adcyap1r1) and/or the vasoactive intestinal peptide (VPAC) receptors (2, 4, 20, 45, 54). PACAP/PAC₁ receptor signaling modulates synaptic transmission and plasticity via pre- and postsynaptic mechanisms, and these effects have been shown to be critically important in central stress responses, peripheral sensory and autonomic function, and maintenance of physiological homeostasis (9, 18, 19, 21, 25, 31, 32, 40, 41, 47, 49, 54).

We previously identified colocalization of PACAP with acetylcholine in cholinergic parasympathetic preganglionic terminals innervating guinea pig cardiac neurons (5, 6) and demonstrated that both endogenously released and exogenously applied PACAP significantly increases cardiac neuron excitability through PAC₁ receptor activation (5, 22, 35, 49). Cardiac neurons are more readily accessible than CNS neurons for experimental manipulations and accordingly, we have used these cells to further our understanding of cellular PAC₁ receptor signaling mechanisms. The results of our previous work indicated that one mechanism contributing to the PACAP/PAC₁ receptor-induced increase in neuronal excitability is via an adenylyl cyclase/cAMP-mediated enhancement of the hyperpolarization-activated, cyclic nucleotide-sensitive nonselective cationic current, I_h (36, 51). However, results of subsequent studies indicated that application of the potent adenylyl cyclase activator forskolin or exposure to the cell-permeable analog of cAMP 8-bromo-cAMP only partially recapitulated the effect of PACAP on excitability (52), suggesting that PACAP enhancement of other membrane conductances, in addition to I_h, also contributes to the peptide-induced modulation of excitability.

Relatedly, we determined that the PACAP-induced increase in cardiac neuron excitability is dependent on calcium influx, but not calcium release from intracellular stores (50). However, the calcium influx pathway was not identified (50). A voltage-dependent calcium channel (VDCC) was thought not to be the pathway as PACAP decreased whole cell barium currents (50); instead the calcium influx was suggested to occur through receptor-activated nonselective cationic channels since different putative nonselective cationic channel inhibitors could blunt the PACAP-induced increase in cardiac neuron excitability (37). However, many of these putative nonselective cationic channel inhibitors also can block voltage-dependent calcium channels, including T-type channels (43). Most of the macroscopic calcium current in cardiac neurons is carried by N-type calcium channels (26, 56), and consequently, it was considered that while decreasing N-type currents, a PACAP potentiation of another smaller VDCC component may have been obscured. L-type calcium currents contribute a small component to the total calcium current recorded in cardiac neurons (26, 56), and PACAP can enhance L-type calcium channel currents in some cells (3, 13). However, pretreatment with the L-type calcium channel blocker nifedipine failed to blunt the PACAP-induced modulation of excitability (50), eliminating L-type currents as a means of PACAP modulation of excitability.

In the same studies, Tompkins et al. (50) also demonstrated that pretreatment with 200 μM cadmium potently blocked the PACAP effect on excitability. At this concentration, cadmium effectively blocks all classes of VDCCs whereas low concentrations of Ni²⁺ preferentially inhibits R- and T-type calcium channels (24, 44, 57). Earlier studies indicated that R-type calcium currents are expressed in rat cardiac neurons, although no evidence of T-type calcium channel currents was noted (26). We determined from real-time quantitative polymerase chain reaction (QPCR) analysis that transcripts for both T-type calcium channel and R-type channel isoforms were present in the guinea pig cardiac neurons. Consequently, we have tested in this study whether Ni²⁺ (at concentrations that preferentially block R- and T-type calcium channels) blunts the ability of PACAP to modulate cardiac neuron excitability.

We report here using intracellular recordings that Ni²⁺ can suppress the PACAP-induced increase in excitability and blunts a PACAP-induced enhancement of a hyperpolarization-induced rebound depolarization. Furthermore, initial whole cell voltage-clamp recordings suggest that guinea pig cardiac neurons exhibit a low voltage-activated current, potentially flowing through T-type or R-type calcium channels, which is enhanced by PACAP and which could participate in the PACAP-induced modulation of excitability.

MATERIALS AND METHODS

Animals.

All experiments were done in vitro using Hartley guinea pigs (either sex, 250–350 g). All animal protocols were approved by the University of Vermont Institutional Animal Care and Use Committee and followed methods described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Guinea pigs were euthanized by isoflurane overdose and exsanguination with the heart quickly removed and placed in cold Krebs solution (in mM: 121 NaCl, 5.9 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 25 NaHCO₃, 1.2 NaH₂PO₄, 8 glucose; pH 7.4 maintained by 95% O₂-5% CO₂ aeration).

Chemicals.

PACAP27 (referred to as PACAP throughout) was obtained from American Peptide (Sunnyvale, CA), and mibefradil [(1S,2S)-2-[2[[3-(2-benzimidazololyl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl methoxyacetate dihydrochloride hydrate)] was obtained from Sigma-Aldrich (St. Louis, MO). All drugs were applied directly to the bath solution from frozen concentrated stocks prepared in either DMSO (mibefradil) or water (PACAP).

Intracellular recordings from neurons in cardiac ganglia whole mount preparations.

Intracellular recordings from cardiac neurons followed methods described previously (5, 22, 35, 49, 50). Cardiac ganglia preparations were superfused continuously (6–7 ml/min) with Krebs solution containing 10 mM NaHEPES (32–35°C), and individual neurons were impaled using 2 M KCl-filled microelectrodes (60–120 MΩ). Membrane voltage was recorded using an Axoclamp-2A amplifier coupled with a Digidata 1322A data acquisition system and pCLAMP 8 software (Axon Instruments, Foster City, CA). Depolarizing current steps (0.1–0.5 nA, 1 s) were applied to characterize neuron excitability. The response of mammalian cardiac neurons to 1-s-long suprathreshold depolarizing current pulses can be classified as a phasic, rapidly accommodating or tonic firing pattern (1). PACAP enhances action potential generation elicited by long depolarizing pulses in all three classes of cardiac neurons. This reflects the PACAP-induced increase in excitability. As phasic firing cells are the most prevalent type in the guinea pig cardiac ganglia, the present study quantified the inhibition by Ni²⁺ on the PACAP effect on phasic neurons. Excitability curves were constructed by plotting the number of action potentials generated by increasing stimulus intensities. Hyperpolarizing current steps (500 ms) of increasing amplitude were used to test for 1) rectification in the current-induced hyperpolarization, which occurs when the hyperpolarization-activated current I_h is initiated and 2) a transient hyperpolarization-induced rebound depolarization, which in some cells was sufficiently large to evoke action potential activity.

Whole cell recordings from dissociated neurons.

Voltage-dependent calcium currents were recorded from dissociated cardiac neurons using the amphotericin B (0.2 mg/ml, Sigma, St. Louis, MO) perforated patch whole cell recording technique (36, 50). External solution contained (in mM) 115 NaCl, 20 tetraethylammonium chloride, 10 HEPES, 5 CsCl, 5 CaCl₂, 8 glucose; pH adjusted to 7.3 with TEA-OH. Tetrodotoxin (0.3 μM) was added to block voltage-activated sodium channels. The pipette solution contained (in mM) 110 Cs aspartate, 30 CsCl, 5 MgCl₂, 10 NaCl, 10 HEPES; pH adjusted to 7.2 with CsOH. Solutions were applied by a gravity flow system (5–8 ml/min) to a 0.5 ml bath chamber with the temperature maintained at 33–36°C by an in-line solution heater (Warner Instruments, Hamden, CT).

Voltage-ramp and voltage-step protocols were generated and currents were recorded with an Axopatch 1-C amplifier in combination with a Digidata 1322A and pCLAMP 9 acquisition system using sample rates ranging from 5 to 50 kHz. Currents were filtered at 2 kHz, and voltage step-evoked currents were leak subtracted using a P/5 subpulse protocol. Reported voltages were not corrected for series resistance or junction potential errors.

Real-time quantitative polymerase chain reaction.

Transcript levels were determined for the three different T-type calcium channel isoforms (Ca_v3.1, Ca_v3.2, and Ca_v3.3) from laser captured (PALM Microlaser Technologies, Thornwood, NY) cardiac ganglia (37). In brief, clusters of cardiac ganglion cells (ganglia) were identified on nine atrial cardiac ganglia whole mount preparations. The identified ganglia (121 total) were then laser captured with each ganglion catapulted into tubes and immediately frozen on dry ice. The samples were consolidated for subsequent analysis. Transcript levels for Ca_v2.3 were determined from extracts of cardiac ganglia whole mounts collected under RNase-free conditions (17).

The quantitative PCR standards for all transcripts were prepared with the amplified cDNA products ligated directly into pCR2.1 TOPO vector using the TOPO TA cloning kit (Invitrogen). The nucleotide sequences of the inserts were verified by automated fluorescent dideoxy dye terminator sequencing (Univ. of Vermont Cancer Center DNA Analysis Facility). To estimate the absolute expression of the transcripts, 10-fold serial dilutions of stock plasmids were prepared as quantitative standards. The range of standard concentrations was determined by calculating the number of copies of the plasmid and insert. Complementary DNA templates were assayed using HotStart-IT SYBR Green qPCR Master Mix (USB, Cleveland, OH) and 300 nM of each primer in a final 25 μl reaction volume. The amplified products were subjected to SYBR Green I melting analysis by ramping the temperature of the reaction samples from 60°C to 95°C. A single DNA melting profile was observed under these dissociation assay conditions, demonstrating amplification of a single unique product free of primer dimers or other anomalous products (see Fig. 5, A, B, C, and E). For data analyses, a standard curve was constructed by amplification of serially diluted plasmids containing the target sequence. Data were analyzed at the termination of each assay using sequence detection software (Sequence Detection Software, version 1.3.1; Applied Biosystems, Norwalk, CT). In standard assays, default baseline settings were selected. The increase in SYBR Green I fluorescence intensity (ΔR_n) was plotted as a function of cycle number, and the threshold cycle was determined by the software as the amplification cycle at which the ΔR_n first intersects the established baseline (see Fig. 5, D and F).

Fig. 5.

Real-time quantitative polymerase chain reaction (QPCR) analysis demonstrates the presence of T-type and R-type channel isoforms in guinea pig cardiac ganglia. A, B, C, and E: melting point profiles are indicated for Ca_v3.1 (A), Ca_v3.2 (B), and Ca_v3.3 (C) from extracts of laser-captured cardiac neurons and for Ca_v2.3 (E) from extracts of cardiac ganglia whole mounts. The amplified product for each gene was subjected to SYBR Green I melting point analysis by ramping the temperature of the reaction samples from 60°C to 95°C. A single DNA melting profile was observed under these conditions, demonstrating an amplification of a single unique product, free of primer dimers or other anomalous products. Melting temperatures of the quantitative real-time PCR products were 89.9, 82.9, 83.5, and 87.6°C for Ca_v3.1, Ca_v3.2, Ca_v3.2, and Ca_v2.3, respectively. NTC, no template control. D and F: standard curves generated for the T-type channel isoforms (D: top line Ca_v3.2, middle line Ca_v3.1, bottom line Ca_v3.3) and R-type isoform (F). Ct, threshold cycle.

The following primer pairs were used: Ca_v3.1 upper primer: 5′-CGTGCTGGTCAACGTGGTGAT-3′, Ca_v3.1 lower primer: 5′-AGCGAGTGGGCTGCCTTGTAT-3′; Ca_v3.2 upper primer: 5′-ACGACTTCATCTTTGCCTTC-3′, Ca_v3.2 lower primer: 5′-TACTCCATCATGCCTGCCA-3′; Ca_v3.3 upper primer: 5′-TGTTTCCGCATCCGCAAGA-3′, Ca_v3.3 lower primer: 5′-GACCCGGAACCTGTTCTCT-3′; Ca_v2.3 upper primer: 5′-ACTTAAGGAGGACCAACAGT-3′, Ca_v2.3 lower primer: 5′-CAGTTGCACGTCCCCCAGGA-3′.

Extracts of guinea pig brain served as a positive control and all samples also were run on an ethidium bromide gel.

Statistics.

Statistical analysis was performed using GraphPad Prism statistical software (version 5.4; La Jolla, CA). Data are presented as means ± SE. Differences between means (Fig. 2C) were determined using an unpaired t-test and considered statistically significant at P < 0.05.

Fig. 2.

Hyperpolarization-induced rebound depolarizations in guinea pig cardiac neurons. A: an example of the transient rebound depolarization recorded after termination of a 500-ms hyperpolarizing step. Rectification (“sag”) was evident in the hyperpolarization, indicating the presence of hyperpolarization-activated current (I_h). B: the hyperpolarization-induced rebound depolarization was also seen in preparations pretreated with 2 mM CsCl to block hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Note the lack of rectification in the hyperpolarizing trace. In this example the rebound depolarization was sufficient to elicit an action potential. C: an example in which the rebound depolarization before Ni²⁺ (C₁) was blocked after addition of 50 μM Ni²⁺ (C₂). D: an example in which the rebound depolarization before mibefradil (D₁) was blocked after addition of 5 μM mibefradil (D₂). Note that cesium was not present when traces in C and D were recorded.

RESULTS

Ni²⁺ suppresses the PACAP-induced increase in excitability.

In microelectrode recording from cells in whole mount preparations, PACAP (20 nM) increased the number of action potentials elicited in cardiac neurons by 1-s-long suprathreshold depolarizing steps, indicating that the peptide increased neuronal excitability (5, 35, 49). Pretreatment with 50 μM Ni²⁺ suppressed this PACAP-induced effect. The PACAP-induced increase in excitability was determined in eight cells without Ni²⁺ pretreatment and in seven different cells with a 15-min, 50 μM Ni²⁺ pretreatment before 20 nM PACAP and Ni²⁺ application. For these experiments, recordings from each control cell and each Ni²⁺-treated cell were obtained from different whole mount cardiac ganglia preparations. In all control cells the action potential firing pattern shifted from phasic (Fig. 1A₁) to multiple firing in the presence of PACAP (Fig. 1A₂), whereas Ni²⁺ pretreatment markedly suppressed the PACAP-induced increase in excitability (Fig. 1B_{1, 2}). Figure 1C shows the averaged excitability curves in PACAP for the control cells and the cells pretreated with 50 μM Ni²⁺.

Fig. 1.

Ni²⁺ suppresses the pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increase in excitability. A: prior to PACAP, a 1-s, 0.3-nA depolarizing step elicited two action potentials (A₁), whereas during application of 20 nM PACAP, multiple action potentials were generated (A₂). B: following pretreatment with 50 μM Ni²⁺, the ability of PACAP to increase excitability was suppressed (B_{1, 2}). C: averaged excitability curves showing that 50 μM Ni²⁺ depressed the PACAP-induced increase in excitability. ○, Excitability curve for 8 control cells before PACAP exposure. ▽, Excitability curve for 7 Ni²⁺-pretreated cells before PACAP exposure. Note that the excitability curves for the naive cells and Ni²⁺-pretreated cells (both prior to PACAP) overlap. ▼, Excitability curve for 7 cells pretreated with 50 μM Ni²⁺ and exposed to 20 nM PACAP. ●, Excitability curve for 8 control cells during exposure to 20 nM PACAP. APs, action potentials. *Ni²⁺ significantly decreased the PACAP-enhanced excitability at all current steps (P < 0.05). D: the number of action potentials elicited by a 1-s, 0.4-nA depolarizing step in 8 cells exposed to only 20 nM PACAP and 7 other cells pretreated with 50 μM Ni²⁺ and then exposed to 50 μM Ni²⁺ and 20 nM PACAP. The averaged values are significantly different (*P < 0.001) although action potential generation in 2 Ni²⁺-treated cells overlaps that noted for control cells.

Although the averaged excitability curve was significantly depressed by 50 μM Ni²⁺, the suppression of the PACAP effect was markedly different among cells. As evident from Fig. 1D, the PACAP-induced increase in excitability was completely blocked in three cells and blunted in two other cells following Ni²⁺ pretreatment. In the remaining two cells examined, Ni²⁺ pretreatment had no apparent effect on PACAP-stimulated action potential generation. This observation suggested that the contribution of the Ni²⁺-sensitive currents to the PACAP enhancement of excitability varied between cardiac neurons.

Ni²⁺ blunts a hyperpolarization-induced rebound depolarization and hyperpolarizes cardiac neurons.

When long hyperpolarizing current pulses are delivered to cardiac neurons, often the hyperpolarization reaches a peak value, but then progressively declines (Fig. 2A). This rectification or “sag” in the hyperpolarizing response is due to the activation of I_h, the inward current flowing through a hyperpolarization-activated nonselective cationic conductance (HCN) (10, 15, 36, 51, 55). Following termination of the hyperpolarization, a hyperpolarization-induced rebound depolarization often occurs (Fig. 2, A and C₁), which, if sufficiently large, initiates an action potential (Fig. 2, B and D₁). This rebound depolarization has been attributed to inward currents flowing through HCN channels activated by the hyperpolarizing step, and following return to the resting membrane potential, residual inward current causes the rebound depolarization (36). However, the hyperpolarization-induced rebound depolarization was still present in 6 of 14 cells treated with 2 mM cesium chloride to block I_h. In three of these six cells the depolarization was sufficiently large to initiate an action potential, an example of which is shown in Fig. 2B. These observations suggested that some other current, in addition to I_h, can contribute to the hyperpolarization-induced rebound depolarization.

As a hyperpolarization-induced rebound depolarization is also a signature feature of T-type calcium channels (24, 42, 48), we tested whether the rebound depolarization was sensitive to Ni²⁺ or mibefradil, two well-established blockers of T-type calcium channels (29, 30) (Fig. 2, C and D). Cesium chloride was not included in the bath solution for these experiments. The effect of Ni²⁺ on the hyperpolarization-induced depolarization was determined in nine cells (6 exposed to 50 μM; 3 exposed to 100 μM). Prior to 50 μM Ni²⁺ exposure, all six cells exhibited a hyperpolarization-induced depolarization, and in two of the six cells it was sufficient to elicit post-hyperpolarization-induced action potentials. In three of the six cells, 50 μM Ni²⁺ blunted the hyperpolarization-induced depolarization by 58 ± 13%. Recordings from one of these cells are shown in Fig. 2C_{1, 2}. In the remaining three cells, 50 μM Ni²⁺ had no effect. For the three cells exposed to 100 μM Ni²⁺, there was a hyperpolarization-induced rebound depolarization prior to Ni²⁺ application and in two of the three cells, Ni²⁺ reduced the hyperpolarization-induced depolarization by 33% and 80%, respectively. In the latter case, the hyperpolarization-induced depolarization elicited action potentials prior to Ni²⁺, but this activity was lost in the presence of Ni²⁺. In the third cell, Ni²⁺ had no effect on the hyperpolarization-induced depolarization. It should be noted that in those cells in which Ni²⁺ (50 or 100 μM) reduced the hyperpolarization-induced depolarization, there was no decrease in the input resistance.

The effect of mibefradil on the hyperpolarization-induced rebound depolarization was determined in six cells (2 cells with 1 μM; 4 cells with 5 μM) that exhibited a hyperpolarization-induced rebound depolarization. In one of the two cells exposed to 1 μM mibefradil, the hyperpolarization-induced rebound depolarization was decreased by 50% whereas in the second cell, there was no change. In two of the four cells exposed to 5 μM mibefradil, the hyperpolarization-induced rebound depolarization prior to drug application elicited action potentials whereas following exposure to mibefradil, the hyperpolarization-induced depolarization decreased progressively so that no action potential was elicited (Fig. 2D_{1, 2}). In a third cell, during exposure to mibefradil, the hyperpolarization-induced depolarization was eliminated. In the fourth cell, which was exposed to 5 μM mibefradil, the rebound depolarization remained similar to control after 4 min in drug, when the impalement was lost. As in the experiments testing the effect of Ni²⁺, when the hyperpolarization-induced depolarization was decreased by mibefradil, this change could not be attributed to a decrease in the input resistance.

The results above showing that treatment with Ni²⁺ or mibefradil suppressed the hyperpolarization-induced depolarization in some cells, but not in other cells, provide further evidence that more than one conductance can contribute to the generation of the hyperpolarization-induced rebound depolarization in the cardiac neurons.

Around the resting membrane potential, T-type calcium channels exhibit a window current that produces a small, sustained depolarization, which is blocked by the addition of T-type calcium channel blockers, leading to cell hyperpolarization (24, 42, 48). Thus, we tested whether the membrane potential was altered during application of Ni²⁺ or mibefradil. During exposure to either inhibitor (50–100 μM Ni²⁺, 5 cells; 1–5 μM mibefradil, 5 cells), cells were hyperpolarized by 2–4 mV (averaged values: Ni²⁺, 2.6 ± 0.6 mV; mibefradil, 2.5 ± 0.4 mV). This hyperpolarization is consistent with a tonic inward current flowing at the resting membrane potential (−55 to −65 mV), which is within the voltage range of the window current for these channels and which is blocked by T-type calcium channel inhibitors (24, 42, 48).

Given these findings, we reviewed data collected from 112 cells to quantify the presence of the hyperpolarization-induced rebound depolarization and determine how often it was correlated with the presence of rectification in the hyperpolarizing step, an indication of I_h activation. A hyperpolarization-induced rebound depolarization occurred in 73% of the cardiac neurons tested. In 21% of the cells, a hyperpolarization-induced rebound depolarization occurred without any rectification in a hyperpolarizing voltage step to ∼−100 mV, an indication of no obvious I_h activation, whereas in 52% of the cells, rectification of the hyperpolarizing step was noted along with the hyperpolarization-induced rebound depolarization. Fifteen percent of the cells exhibited rectification in the hyperpolarizing step without a noticeable rebound depolarization, and 12% of the cells showed neither rectification nor the rebound depolarization. Collectively, these observations suggest that cardiac neurons can express I_h and some other hyperpolarization-modulated current, perhaps a T-type calcium current, either alone or together and both potentially contribute to the generation of a hyperpolarization-induced rebound depolarization.

PACAP enhanced the hyperpolarization-induced rebound depolarization.

The effect of 20 nM PACAP on the hyperpolarization-induced rebound depolarization was determined in eight control cells prior to and during exposure to PACAP. Prior to PACAP, four of the control cells exhibited a hyperpolarization-induced rebound depolarization that was sufficiently large to elicit a single action potential (Fig. 3A₁) whereas in three other cells a smaller amplitude hyperpolarization-induced rebound depolarization was noted. The remaining cell had no discernible hyperpolarization-induced depolarization. In six of the eight cells exposed PACAP, multiple action potentials were elicited by the hyperpolarization-induced rebound depolarization. The recording shown in Fig. 3A₂ was obtained from one of the cells in which a single action potential was elicited by the hyperpolarization-induced rebound depolarization prior to PACAP exposure and in which multiple action potential firing was noted during PACAP treatment. In one of the cells in which the hyperpolarization-induced depolarization did not elicit an action potential before PACAP, the post-hyperpolarization-induced depolarization was increased sufficiently in PACAP to elicit an action potential. The cell, which did not have a discernible rebound depolarization prior to PACAP, did not exhibit a discernible rebound depolarization during exposure to PACAP, even though excitability was markedly increased. These observations indicated that PACAP enhanced the hyperpolarization-induced depolarization in the majority of cells tested.

Fig. 3.

Ni²⁺ suppresses the PACAP-induced increase in the hyperpolarization-induced rebound depolarization. A: prior to PACAP, a hyperpolarizing step to ∼−100 mV evoked a rebound depolarization sufficiently large to evoke an action potential (A₁), whereas in 20 nM PACAP the rebound depolarization elicited a train of action potentials (A₂). Note also that in PACAP, the rectification in the hyperpolarization was noticeably enhanced. B: pretreatment with 50 μM Ni²⁺ blunted the ability of PACAP to enhance the rebound depolarization (B_{1, 2}). The very small increase in the rebound depolarization is attributed to the PACAP-enhanced I_h current as indicated from the increased rectification in the hyperpolarization. Traces obtained from cells in Fig. 2.

We also determined in seven other cells whether 50 μM Ni²⁺ pretreatment affected the PACAP-induced enhancement of the hyperpolarization-induced rebound depolarization. In the Ni²⁺-pretreated cells prior to PACAP exposure, five cells had no discernible or only a small hyperpolarization-induced rebound depolarization, which did not elicit an action potential (Fig. 3B₁). Two other Ni²⁺-pretreated cells had a hyperpolarization-induced rebound depolarization, which elicited a single potential. When the Ni²⁺-pretreated cells were exposed to a Ni²⁺ and PACAP-containing solution, three of the cells, which had exhibited no or a very small rebound depolarization prior to PACAP, still exhibited no or a similarly small rebound depolarization (Fig. 3B₂). In two other Ni²⁺-pretreated cells in which a small rebound depolarization was evident prior to PACAP, the rebound depolarization increased sufficiently during exposure to PACAP and Ni²⁺ to elicit a single action potential. In the two remaining Ni²⁺-pretreated cells in which the depolarization was sufficient to elicit a single action potential prior to PACAP, the rebound depolarization continued to elicit only a single action potential during exposure to PACAP and Ni²⁺. These results indicate that following Ni²⁺ pretreatment, the ability of PACAP to enhance the hyperpolarization-induced rebound depolarization was suppressed in most cells.

Ni²⁺ has cell-specific effects when applied after the PACAP-induced increase in excitability is established.

The experiments summarized in Fig. 1 demonstrate that Ni²⁺ suppressed the ability of PACAP to increase excitability in the majority of cardiac neurons tested. In another series of experiments, we tested whether Ni²⁺ could reverse the PACAP-induced increase in excitability once initiated. A PACAP (20 nM)-containing solution was applied to 5 cardiac neurons in 5 different whole mount preparations and the peptide-induced increase in excitability allowed to develop. Once the PACAP-induced multiple firing was established, the PACAP-containing solution was replaced by one containing 20 nM PACAP and 50 μM Ni²⁺. Excitability was then retested multiple times over a subsequent 6-min period (Fig. 4). The effect of Ni²⁺ on the PACAP enhanced excitability differed between cells. In three of the five cells the peptide-induced increase in excitability remained unchanged after the addition of Ni²⁺ whereas in the two other cells, the PACAP-induced increase in excitability was blunted by 65% and 64%, following a 6-min exposure to Ni²⁺ along with PACAP. This reversal of the PACAP effect was likely due to the addition of Ni²⁺ as we showed in earlier studies that the PACAP-induced increase in excitability was maintained for much longer periods than reported here (35). When the PACAP-induced increase in excitability had developed fully, all five cells exhibited marked rectification in the hyperpolarization produced by current steps indicative of I_h activation. Ni²⁺ had no effect on the rectification noted in all of the cells.

Fig. 4.

Ni²⁺ reversed an established PACAP increase in excitability in a subset of cardiac neurons. Results are summarized for 5 different cells in which Ni²⁺ was added after the PACAP-induced increase in action potential generation had developed fully. Note that the number of action potentials generated by a 1-s, 0.4-nA depolarizing current step is plotted initially for each cell exposed to 20 nM PACAP alone (left, PACAP) and then for additional excitability trials after 50 μM Ni²⁺ was added to the PACAP-containing solution (right, PACAP + Nickel). Each symbol represents PACAP responses from a single cell before and after Ni²⁺ treatment. In 2 cells (+ symbol and ■), the number of action potentials generated decreased progressively during exposure to Ni²⁺, whereas in 3 cells, Ni²⁺ had no effect on the PACAP-enhanced action potential generation.

In four of the five cells a hyperpolarization-induced rebound depolarization, which initiated action potentials, was present during PACAP exposure alone. In the fifth cell, there was no hyperpolarization-induced rebound depolarization noted in PACAP. In the case of the two cells in which Ni²⁺ reversed the PACAP-induced increase in excitability, Ni²⁺ also decreased the hyperpolarization-induced rebound depolarization such that action potentials were no longer elicited. No change in the hyperpolarization-induced rebound depolarization and action potential initiation was noted in two other cells in which Ni²⁺ did not reverse the PACAP enhanced excitability.

These results provide further evidence that the contribution of a Ni²⁺-sensitive current to the PACAP enhancement of excitability varies between cardiac neurons and also provides support for the view that more than one PACAP-enhanced current underlies the peptide modulation of excitability.

Guinea pig cardiac neurons express transcripts for T-type and R-type calcium channels.

The Ni²⁺- and mibefradil-induced hyperpolarization is consistent with the inhibition of a sustained inward current at resting membrane potentials between −55 and −65 mV, a voltage range where T-type calcium channels exhibit an inward window current that can produce a small depolarization (24, 48). Further, the hyperpolarization-induced rebound depolarization is a common response in cells expressing T-type channels (24). Together, these observations suggest that the guinea pig cardiac neurons might express functional T-type calcium channels. Consequently, using QPCR, we tested whether guinea pig cardiac neurons express transcripts for T-type calcium channel isoforms. QPCR analysis on laser-captured neuronal clusters demonstrated that cardiac neurons expressed Ca_v3.1 and Ca_v3.2 transcripts with much less Ca_v3.3 mRNA (Fig. 5, A–C). In addition, from comparison to standard curves, the level of Ca_v3.2 transcript was ∼5-fold greater than for Ca_v3.1. Additional QPCR analyses on cardiac ganglia extracts indicated that these neurons also contain transcripts for Ca_v2.3, suggesting that guinea pig cardiac neurons also express R-type calcium channels (Fig. 5E). Following QPCR analysis, all samples were run on ethidium bromide gels to compare products from either the laser-captured cardiac ganglia (Fig. 6A) or the whole ganglia extract (Fig. 6B) to whole brain samples. When run on the ethidium bromide gel, Ca_v3.3 was not detected, presumably because this method is less sensitive than QPCR.

Fig. 6.

A further demonstration using ethidium bromide gels that T-type and R-type calcium channel transcripts are expressed by guinea pig cardiac neurons and that PACAP enhances a low voltage-activated current. A: PCR products viewed on an ethidium bromide gel after amplification of brain cDNA and laser-captured cardiac neuron cDNA. Extracts of laser-captured neurons had measureable levels of transcripts for Ca_v3.1 and Ca_v3.2, but essentially undetectable transcripts for Ca_v3.3, when viewed on an ethidium bromide gel. B: extracts of cardiac ganglia whole mounts had transcripts for Ca_v2.3. C: low voltage-activated currents can be elicited in dissociated guinea pig cardiac neurons. A voltage step from −90 mV to −30 mV elicited an inward current (trace 1) which was enhanced during application of 25 nM PACAP (trace 2).

PACAP enhances low voltage-activated calcium currents.

Perforated patch whole cell recordings were initiated to investigate whether low voltage-activated calcium currents could be identified in dissociated guinea pig cardiac neurons. In these initial experiments, attempts to dissociate cardiac neurons from 11 cardiac ganglia whole mount preparations were made using standard enzymatic dissociation procedures (36, 50). Whole cell voltage-clamp recordings were obtained from 9 neurons from 7 of the 11 cardiac ganglia dissociations that yielded potentially patchable cells. In seven of the nine cells, voltage ramps from −100 to +50 mV elicited a very small, low voltage-activated inward current followed by a much larger, high voltage-activated calcium current. The low voltage-activated current was consistently enhanced by 25 nM PACAP (data not shown). In three of the seven cells, currents were also elicited by voltage steps from holding potentials between −85 to −100 mV to more positive voltages (−50 to −30 mV in 5-mV increments). Inward currents were evident with steps to potentials more positive than −45 mV and were enhanced in all three cells by 25 nM PACAP (72 ± 19% with steps to −30 mV). Figure 6C shows example currents elicited by a voltage step from −90 mV to −30 mV before (trace 1) and during 25 nM PACAP exposure (trace 2).

DISCUSSION

The present studies demonstrate that PACAP enhancement of an inward current through Ni²⁺-sensitive, low voltage-activated T-type or R-type calcium channels contributes to the peptide-induced increase in excitability in guinea pig cardiac neurons. Ni²⁺ blocked both the PACAP-induced increase in excitability and the peptide-induced enhancement of the hyperpolarization-induced rebound depolarization. However, Ni²⁺ did not block these PACAP effects equally in all cells and was only able to reverse the PACAP increased excitability, once initiated, in a subset of cells. These observations are consistent with multiple conductances, some sensitive to Ni²⁺ and others not, contributing to the PACAP-induced increase in excitability. We already have shown that an enhancement of I_h is one potential, Ni²⁺-insensitive mechanism, underlying the PACAP modulation of excitability. In addition, our QPCR data suggest that transcripts for Ca_v3.1, Ca_v3.2, and Ca_v3.3 isoforms are present in guinea pig cardiac neurons, although the amount of Ca_v3.3 is much less than that of the other two isoforms. Since Ni²⁺ at 50 μM is a much less effective blocker of Ca_v3.1 than Ca_v3.2 or Ca_v3.3 channels (28, 29), any contribution due to a PACAP-enhanced current through Ca_v3.1 and Ca_v3.3 channels would not be markedly affected by Ni²⁺ at this concentration. To explore this latter possibility, we tried to test the effectiveness of mibefradil, which should block all Ca_v3 isoforms, on the PACAP-induced increase in excitability. However, attempts to test whether mibefradil (1–5 μM) pretreatment blunted the PACAP-induced increase in excitability were not successful because during drug treatment, most cells demonstrated a progressive decline in action potential amplitude, consistent with the ability for mibefradil to block voltage-dependent sodium channels (34, 46). Thus, the actions of mibefradil on other voltage-dependent channels precluded our ability to test the effect of this drug on the PACAP alteration of cardiac neuron excitability.

Recently, PACAP has been shown to increase T-type calcium channel currents in mouse chromaffin cells (21, 27). In these cells, the PACAP-enhanced calcium influx facilitates catecholamine release during the acute sympathetic stress response. This PACAP effect on catecholamine release is blunted by Ni²⁺ (21). We showed that a PACAP-induced calcium influx is a critical step in the peptide modulation of cardiac neuron excitability (50). Previously, calcium influx through a PACAP-enhanced, receptor-operated nonselective cationic channel was suggested (37). However, as described above, the present results indicate that a PACAP-enhanced inward current through a Ni²⁺-sensitive channel such as T-type or R-type calcium channels more likely contributes to the peptide-induced increase in excitability. A role for T-type calcium channels in guinea pig cardiac neurons was suggested from QPCR analysis demonstrating the presence of Ca_v3.1, Ca_v3.2, and Ca_v3.3 transcripts, the suppression of the hyperpolarization-induced rebound depolarization by T-type calcium channel blockers, and the ability for Ni²⁺ and mibefradil to hyperpolarize neurons with resting membrane potentials ranging from −55 to −65 mV. Previously, we noted in dissociated cardiac neurons pretreated with a potent I_h blocker that PACAP consistently increased the holding current at −50 mV (36). This observation is consistent with a PACAP enhancement of a small, sustained inward current, other than I_h, activated at the resting membrane potential. In addition to increasing neuronal excitability, the present results demonstrate that PACAP can enhance the hyperpolarization-induced rebound depolarization and spike generation. Although limited in scope, we obtained evidence that PACAP enhanced a low voltage-activated calcium current. However, a more extensive voltage-clamp analysis of VDCC profile in the guinea pig cardiac neurons will be needed in future studies to establish the relative expression and characteristics of T-type and/or R-type channel currents.

Although T-type calcium currents were not recorded in dissociated rat cardiac ganglion neurons (26), T-type calcium channels are present in sympathetic neurons dissociated from the rat pelvic ganglia (30, 58). These pelvic neurons also generate a hyperpolarization-induced rebound depolarization with superimposed action potential activity that is sensitive to 50–100 μM Ni²⁺ (30), observations similar to what we report here for the guinea pig cardiac neurons, especially when cells are exposed to PACAP. Furthermore, the two-component whole cell current profile that we noted with voltage ramps, again particularly in PACAP, is very reminiscent of the initial hump followed by a larger inward current induced by voltage ramps in the pelvic sympathetic neurons (30). The authors conclude that the Ca_v3.2 isoform is the predominant isoform in the pelvic sympathetic neurons, consistent with the high sensitivity to Ni²⁺. Our data in aggregate are consistent with that interpretation although as indicated above, our present results do not allow us to rule out the contributions of Ca_v3.1.

Assuming that T-type channels are expressed in the guinea pig cardiac neurons, then how might PACAP enhance currents through these channels? T-type calcium currents can be modulated by multiple intracellular signaling cascades including PKA and PKC (8, 21, 24, 27, 39). PAC₁ receptor activation can stimulate both the adenylyl cyclase/cAMP/PKA and PLC/DAG/PKC transduction pathways (4, 54) and through PAC₁ receptor internalization/endosome formation can recruit the MEK/ERK signaling cascade (33). Although MAPK phosphorylation of Ca_v has not been identified (23), modulation of T-type calcium function and expression requiring activation of MEK/ERK signaling has been demonstrated in a variety of cell types (11, 16, 38). Thus, PACAP could enhance T-type calcium currents in cardiac neurons through multiple signaling cascades, and determining which may be involved is an important question that warrants further study.

In conclusion, neurons within the parasympathetic cardiac ganglia provide an inhibitory input to cardiac pacemaker tissues, a critical mechanism in the regulation of heart rate. Thus, it is imperative to understand what ionic conductances regulate cardiac neuron excitability. Evidence presented previously indicated that a PACAP-enhanced I_h was one mechanism contributing to the PACAP-induced increase in cardiac neuron excitability (36, 51). A PACAP-induced calcium influx also was found to be a critical step in the peptide modulation of excitability (50). The present results suggest that an enhanced calcium influx through low voltage-activated VDCCs, either T-type or R-type channels, likely contributes to the peptide-induced increase in excitability. We tentatively postulate the potential role of T-type calcium currents, because collectively our results suggest their presence in guinea pig cardiac neurons. Furthermore, T-type calcium currents can provide subthreshold depolarizing currents contributing to the regulation of firing patterns, including pacemaker activity and repetitive firing, in multiple neuron types (7, 12, 14, 24, 53), and T-type calcium channel function can be modulated by G protein-coupled receptor activation (7, 24).

GRANTS

This work was supported in part by National Institutes of Health National Institute of General Medical Sciences Grant P30 GM-103498 and National Center for Research Resources Grant P30 RR-032135 (R. L. Parsons).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.D.T., L.A.M., and B.M.G. performed experiments; J.D.T., L.A.M., and B.M.G. analyzed data; J.D.T., L.A.M., and B.M.G. prepared figures; J.D.T., L.A.M., B.M.G., V.M., and R.L.P. edited and revised manuscript; V.M. and R.L.P. conception and design of research; V.M. and R.L.P. interpreted results of experiments; R.L.P. drafted manuscript; J.D.T., L.A.M., B.M.G., V.M., and R.L.P. approved final version of manuscript.