| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Department of Anatomy and Neurobiology, University of Vermont, Burlington, Vermont; and 2Department of Biology, Ithaca College, Ithaca, New York
Submitted 12 October 2005; accepted in final form 10 December 2005
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
; Vaudry et al. 2000), is co-localized with the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), in the parasympathetic preganglionic fibers innervating the intrinsic cardiac neurons of guinea pigs (Calupca et al. 2000) and rats (Richardson et al. 2003). Braas et al. (1998) determined, using a combination of RT-PCR and immunocytochemical staining, that guinea pig intracardiac neurons express the PACAP-selective PAC1 receptor. More recently, DeHaven and Cuevas (2002) showed using RT-PCR that rat neonatal intracardiac neurons express both PAC1 receptors and VPAC receptors. However, there are multiple isoforms of the PAC1 receptor and the PAC1 receptor isoforms expressed by adult guinea pig and neonatal rat intracardiac neurons are different (Braas et al. 1998; DeHaven and Cuevas 2004).
PACAP markedly increases excitability of both guinea pig and rat intracardiac neurons (Braas et al. 1998; DeHaven and Cuevas 2004). In guinea pig intracardiac neurons, PACAP was much more effective than VIP, suggesting that the increase in excitability apparently requires activation of only PAC1 receptors (Braas et al. 1998). In contrast, DeHaven and Cuevas (2004) reported that the increase in excitability by PACAP in rat neonatal cardiac neurons required co-activation of PAC1 and VPAC receptors. These authors also showed that activation of VPAC receptors expressed on rat neonatal intracardiac neurons mediated a VIP- or PACAP-induced rise in intracellular calcium ([Ca2+]i) and proposed that the PACAP-induced increase in excitability in rat neonatal intracardiac neurons required Ca2+ release from internal stores.
Given that the cardiac neurons in adult guinea pigs and neonatal rats apparently express different combinations of receptors, these studies were undertaken to test whether Ca2+ release from internal stores also was required for the PACAP-induced increase in excitability in adult guinea pig intracardiac neurons. We determined that neither PACAP nor VIP elicits a rise in [Ca2+]i in dissociated adult guinea pig intracardiac neurons. In addition, treatment with a combination of ryanodine and caffeine to inhibit the release of Ca2+ from internal stores does not eliminate the PACAP-induced increase in excitability, whereas removal of external Ca2+ or addition of cadmium (Cd2+) to the bath solution blunts the PACAP-induced increase in excitability. These observations indicate that a PACAP-induced Ca2+ influx through Cd2+-sensitive membrane channels, rather than Ca2+ release from internal stores, is required for the peptide-induced increase in excitability in adult guinea pig intracardiac neurons. Preliminary results of this study were presented at the Seventh International Symposium on VIP, PACAP, and Related Peptides (Hardwick et al. 2005).
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Experiments were performed in vitro on atrial whole mount preparations containing the intrinsic cardiac ganglia from Hartley guinea pigs (either sex; 250–350 g). Guinea pigs were killed by halothane or isoflurane overdose followed by exsanguination using animal protocols approved by the University of Vermont Institutional Animal Care and Use Committee and the Ithaca College Institutional Animal Care and Use Committee and methods described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and their suffering. The heart was quickly removed and placed in cold standard Krebs solution (in mM: 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 8 glucose). The pH was maintained at 7.4 by aeration with 95% O2-5% CO2.
For experiments to quantify barium currents (IBa) through voltage-dependent calcium channels or to measure [Ca2+]i transients with fluo-3 fluorescence, the neurons were dissociated from the cardiac ganglia whole mount preparation using techniques described previously (Merriam et al. 2004). After dissection of the cardiac ganglia, the tissue was dissociated by incubation at 37°C in 10 mg/ml collagenase A (Roche Molecular Biochemicals, Indianapolis, IN) for 35 min and incubated for another 35 min in the same enzyme solution to which 5 mg/ml trypsin XII-S (Sigma, St. Louis, MO) was added. After dissociation, the neurons were placed in Eagle minimal essential medium (HEPES modification) containing 2 mM CaCl2 and supplemented with 10% fetal bovine serum, 0.1% bovine serum albumin, 1 mg/ml DNase I, 100 units/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml gentamicin (All from Sigma, except DNase I from Worthington Biochemical, Lakewood, NJ). The cells were stored overnight in a 37°C incubator before recording.
Intracellular recordings from neurons in whole mount preparations
For intracellular recordings, atrial whole mount preparations were pinned in a 2.5-ml Sylgard-lined chamber and superfused continuously (2–3 ml/min) with Krebs solution also containing 10 mM HEPES buffer (33–35°C) (Braas et al. 1998; Hardwick et al. 1995, 1997). A Ca2+-deficient solution was made simply by replacing Ca2+ with Mg2+. In other experiments, strontium (Sr2+) replaced calcium or 200 µM Cd2+ was added to the Ca2+-containing solution. Cardiac ganglia were visualized with an inverted microscope equipped with Hoffman optics, and individual intracardiac neurons were impaled using high-impedance borosilicate microelectrodes (2 M KCl-filled; 60–100 M
). Active and passive membrane properties were recorded from the impaled neurons using an Axoclamp-2A amplifier coupled with a Digidata 1322A data acquisition system and pCLAMP 8 (Axon Instruments, Foster City, CA). In some cells, hyperpolarizing current was injected through the recording electrode to ensure that action potential generation was tested at the same potential before and after PACAP application. When current was applied, the resting membrane potential was maintained between –55 and –65 mV, values within the range of membrane potentials recorded from these cells (Braas et al. 1998; Hardwick et al. 1995, 1997; Merriam et al. 2004; Zhang et al. 2001).
Because PACAP27 was determined previously to be more effective than PACAP38 on guinea pig intracardiac neurons (Braas et al. 1998, 2004), it was used exclusively in this study and is referred to as PACAP throughout the text. PACAP was applied by local pressure application (6 psi; Picospritzer, General Valve, Fairfield, NJ) through 
5 µm-diam "puffer" pipettes containing 50 µM PACAP and positioned 50–100 µm from the neuron. Depolarizing current steps (0.1–0.3 nA, 500 or 1,000 ms) were given to characterize changes in neuron excitability before and after PACAP application. Excitability curves were constructed by plotting the number of action potentials generated by increasing stimulus intensities. When both 500- and 1000-ms steps were used to test excitability in a given experimental condition, the number of action potentials generated during the first 500 ms were used to construct the excitability curve.
Recordings of IBa in dissociated intracardiac neurons
The perforated patch configuration of the whole cell patch-clamp technique (Horn and Marty 1988) was used to record IBa from isolated neurons. The recording solution contained 120 mM N-methyl-D-glucamine, 25 mM tetraethylammonium chloride, 10 mM HEPES, 1.2 mM MgCl2, 3 mM BaCl2, 8 mM glucose, and 0.3 µM TTX. The pH was adjusted to 7.3 with HCl. The pipette solution contained 140 mM Cs aspartate, 30 mM CsCl, 5 mM MgCl2, and 10 mM HEPES. The pH was adjusted to 7.2 with CsOH. For some cells, the Cs aspartate was omitted, and 175 mM CsCl was used. The results were identical with either pipette solution. Patch pipettes were backfilled with 0.2 mg/ml amphotericin B (Sigma, St. Louis, MO).
Bath solutions were applied by a gravity flow system (5–8 ml/min) to a 0.5-ml bath chamber. The temperature was maintained at 33–36°C by an in-line solution heater controlled by a single channel heater controller (Warner Instruments, Hamden, CT). PACAP (100 nM) was added to the bath solution and applied to isolated neurons by gravity flow.
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 were leak subtracted using a P/5 subpulse protocol. The methods followed protocols used previously by our laboratory to record IBa in mudpuppy intracardiac neurons (Merriam and Parsons 1995). Reported voltages were not corrected for series resistance (except the I-V plot in Fig. 6B) or junction potential errors, but only data from cells with uncompensated series resistance of 20 M or less were included in the data analysis.
|
Changes in [Ca2+]i were determined from variations in the fluorescence intensity of fluo-3 (Locknar et al. 2004). Cells were loaded with 5 µM fluo-3-AM and 5 µM pluronic F-127. Both the loading and AM-ester cleavage steps were performed at room temperature. The cells were plated on cover slips and continuously perfused at a flow rate of 1–1.5 ml/min. Because the dissociated cells occasionally fire action potentials spontaneously, 0.3 µM TTX was included in the bath solution during Ca2+ imaging. Images were acquired on two types of microscopes, a DeltaVision Restoration Microscopy system and a Noran Oz confocal microscope. Most of the Ca2+ imaging experiments were done on the DeltaVision Restoration Microscopy system, although similar results were obtained with both microscopes. Using the DeltaVision microscopy system, different sample rates were tried, varying from 1 to 0.33 Hz. There was no difference in the results obtained at different sample rates. Filter sets appropriate for FITC were used and the microscope stage was surrounded by an incubator (Solent Scientific, Segensworth, UK) to maintain the temperature at 37 ± 1°C. For the few experiments done using the Noran Oz confocal microscope (Middleton, WI), the sample rate was 0.33 Hz with 488-nm laser excitation and an emission filter appropriate for FITC. Temperature of the bath was maintained at 33 ± 1°C with an in-line heater (Warner Instruments). The small difference in temperature (33 vs. 37°C) did not affect the results obtained with the two instruments.
Regions of interest corresponding to the cytoplasm of the neurons were selected from the raw image files and average brightness over time plots were generated. Files generated by the DeltaVision microscope were corrected for variations in lamp intensity (recorded by a diode at the same time as image acquisition). The data sets were corrected for dye bleaching using a single or double exponential decay algorithm and normalized to this decay curve to give the fluo-3 fluorescence ratio (F/Fo). Because the F/Fo responses varied from cell to cell (single vs. multiple peaks), we integrated the area under all peaks. The F/Fo integral reflects the total amount of Ca2+ released from internal stores and is referred to as an increase in [Ca2+]i throughout the text. All corrections and integrations were performed with Microcal Origin 7.0 (Northampton, MA).
Drugs
All drugs were obtained from commercial sources: PACAP27 (American Peptide, Sunnyvale, CA); ryanodine and TTX (Calbiochem, La Jolla, CA); pluronic F-127 and fluo-3-AM (Molecular Probes, Eugene, OR); and caffeine (Sigma Chemical). A ryanodine stock solution was made up each day in dimethylsulfoxide (DMSO). Fluo3-AM and pluronic F-127 were diluted each day from frozen aliquots of concentrated solutions dissolved in DMSO.
Statistical evaluation
Paired and unpaired Student's t-test were used to evaluate differences between means as appropriate. Differences were considered statistically significant if P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
In rat neonatal intracardiac neurons, both PACAP and VIP elevate [Ca2+]i (DeHaven and Cuevas 2004). Furthermore, the peptide-stimulated rise in [Ca2+]i in rat intracardiac neurons is blocked by VPAC receptor antagonists indicating that this effect requires activation of VPAC receptors rather than PAC1 receptors (DeHaven and Cuevas 2004). We tested whether PACAP can initiate a rise in [Ca2+]i in adult guinea pig intracardiac neurons using fluorescence measurements of the Ca2+-sensitive dye fluo-3. PACAP (100 nM or 1 µM) was applied for 1–3 min to dissociated neurons loaded with fluo-3. PACAP (13 cells at 100 nM, 4 cells at 1 µM) produced no change in F/Fo (Fig. 1A). In additional experiments, we determined that VIP also did not elicit a rise in [Ca2+]i (13 cells at 100 nM, 3 cells at 1 µM; Fig. 1B).
|
; Smith and Adams 1999). As is evident from inspection of Fig. 1, caffeine elicited a transient rise in [Ca2+]i even though PACAP did not stimulate a rise in [Ca2+]i. In some cells, caffeine challenge resulted in a single rapid transient elevation of [Ca2+]i, whereas in other cells, multiple Ca2+ transients were seen. PACAP increase in excitability is eliminated in Ca2+-deficient solutions or in solutions containing Cd2+
Previously, we showed that PACAP increases excitability in dissociated guinea pig intracardiac neurons (Merriam et al. 2004; Parsons et al. 2000). In addition, the results of the preceding experiments suggested that a PACAP-induced Ca2+ release from internal stores must not be required for this effect. Consequently, we initiated electrophysiological experiments using neurons in the cardiac ganglia whole mount preparation to establish whether Ca2+ influx, rather than Ca2+ release from internal stores, might be required for the PACAP-induced increase in excitability.
For these experiments, intracellular recordings from neurons in whole mount ganglia preparations were used to quantify the change in membrane excitability initiated by puffer application of PACAP (Braas et al. 1998). After a 500-ms PACAP application, a variable depolarization (4–15 mV), which lasted up to a few minutes, was sometimes produced. Because the extent of the depolarization was variable, we did not quantify the PACAP-induced depolarization in this study. Rather, the emphasis of this study focused on mechanisms involved in the PACAP-induced enhancement of excitability. Excitability was tested once the peptide-induced depolarization subsided, by applying long duration, depolarizing current pulses of increasing intensity to elicit action potentials. An excitability curve was generated by plotting the number of action potentials initiated as a function of the current strength (Braas et al. 1998). The majority of the guinea pig intracardiac neurons in the whole mount preparation are phasic so that long depolarizing current pulses only elicit one to two action potentials regardless of current strength (Hardwick et al. 1995). However, PACAP can enhance excitability in phasic cells so that additional action potentials are generated by the depolarizing current pulses (Braas et al. 1998). Less than 10% of the intracardiac neurons in the whole mount ganglia preparation are tonic and fire multiple action potentials during a long current pulse (Hardwick et al. 1995). Although PACAP also enhanced excitability of tonic cells, the change in action potential generation in tonic cells was not included in the averaged data. PACAP enhances excitability in both phasic and tonic neonatal rat intracardiac neurons as well (DeHaven and Cuevas 2004).
Figure 2A shows the increase in excitability produced after PACAP application to a phasic cell, and the results from nine phasic cells are summarized in Fig. 2B. The PACAP-induced increase in excitability often was long lived, lasting many minutes after a single application. Subsequent applications of PACAP repeatedly enhanced excitability.
|
Previously, Parsons et al. (2000) showed that the addition of 1 mM barium (Ba2+) enhanced the excitability of intracardiac neurons, presumably by inhibiting the voltage-dependent noninactivating potassium conductance IM (Brown 1988). We found that, although the PACAP-induced increase in excitability was eliminated in the Ca2+-deficient solution, exposure to 1 mM Ba2+ still increased action potential firing (data not shown). These observations indicated that cells maintained in the Ca2+-deficient solution remained capable of generating multiple action potentials.
In dissociated neonatal rat intracardiac neurons, removal of extracellular Ca2+ results in a rapid depletion of Ca2+ from internal stores (DeHaven and Cuevas 2004). Additional experiments were done to establish whether exposure to the Ca2+-deficient, Mg2+-substituted solution depleted intracellular Ca2+ stores in the dissociated guinea pig intracardiac neurons. Cells were loaded with fluo-3, and the increase in [Ca2+]i produced by 10 and 20 mM caffeine was determined. The dissociated intracardiac neurons were maintained either in the control Ca2+ containing solution or the Ca2+-deficient solution for 10 min before application of caffeine. For neurons maintained in control solution, 10 and 20 mM caffeine elicited a comparable increase in [Ca2+]i (Fig. 3). When intracardiac neurons were kept in the Ca2+-deficient solution, 10 mM caffeine often did not produce a measurable increase in the fluo-3 fluorescence ratio. In contrast, 20 mM caffeine consistently produced an increase in F/Fo, and the magnitude of the increase in [Ca2+]i was identical to that produced by 20 mM caffeine in cells maintained in the control solution (Fig. 3).
|
|
). We tested the effect of PACAP on excitability in the Cd2+ containing solution after the action potential had broadened and HAP decreased. In five cells, pretreatment with 200 µM Cd2+ greatly diminished the PACAP-induced increase in excitability (Fig. 5, A and B).
|
The inability of PACAP to enhance excitability in cells exposed to the Ca2+-deficient solution or Cd2+-containing solution suggested that Ca2+ influx through a PACAP-activated Ca2+ permeable membrane channel was necessary for the PACAP-induced increase in excitability. Because Cd2+ is an efficient blocker of VDCCs in autonomic neurons, we postulated that PACAP may enhance Ca2+ influx through the VDCCs expressed in intracardiac neurons. Consequently, we completed perforated patch whole cell voltage-clamp experiments on dissociated intracardiac neurons to determine whether PACAP enhanced currents flowing through VDCCs. Barium was used as the charge carrier (IBa) to measure macroscopic current flow in these neurons. To elicit IBa, cells were held at –80 mV, and 200-ms depolarizing voltage steps were applied before and during superfusion with 100 nM PACAP. In addition, the I-V relationship was determined before and during PACAP application. As shown in Fig. 6, PACAP reversibly inhibited peak IBa. In six cells, 100 nM PACAP decreased peak IBa by 46 ± 7%, showing that PACAP inhibits macroscopic IBa through VDCCs.
PACAP increase in excitability does not require L-type calcium channels
Intracardiac neurons express a number of different VDCCs with the predominant type expressed being the N-type (Adams and Cuevas 2004; Jeong and Wurster 1997). Previously, a number of investigators have reported that PACAP enhances Ca2+ currents through L-type VDCCs (Bhattacharya et al. 2004; Dziema and Obrietan 2002). Because current flow through the L-type VDCC contributes a minor component of the macroscopic IBa inward current, it was considered possible that a PACAP-induced augmentation of the current flowing through L-type channels would be masked by the inhibition of current flow through other VDCC types. Therefore we tested whether a PACAP-induced enhancement of the L-type current could be involved in the peptide-induced increase in excitability. If this was true, treatment with the specific L-type VDCC blocker nifedipine should eliminate the PACAP-induced increase in excitability. We found that, in four cells pretreated with 10 µM nifedipine, PACAP consistently increased excitability (Fig. 5, B and C). These observations indicated that the PACAP-induced excitability change did not require Ca2+ influx through L-type VDCCs.
Disruption of Ca2+ release from internal stores does not eliminate the PACAP-induced increase in excitability
Although fluo-3 imaging experiments indicated that PACAP did not elevate global Ca2+ levels, it was considered possible that the change in [Ca2+]i was too localized for detection by our imaging methods. We undertook additional experiments to ensure that Ca2+ release from internal stores was not required for the PACAP-induced increase in excitability in adult guinea pig intracardiac neurons. DeHaven and Cuevas (2004) reported that treatment with ryanodine to block Ca2+ release from internal stores eliminated the PACAP-induced increase in excitability in the dissociated neonatal rat intracardiac neurons. Consequently, we tested the effect of ryanodine treatment on the PACAP-induced excitability change in adult guinea pig intracardiac neurons in the whole mount preparation. In these experiments, whole mount cardiac ganglia preparations were pretreated with a 20 µM ryanodine/10 mM caffeine–containing solution for 5 min and, for most experiments, the preparations were returned to drug-free solution before testing the effect of PACAP on excitability. In a few experiments, the bathing solution contained ryanodine throughout the experiment. The results were similar with both conditions. In six cells following ryanodine/caffeine pretreatment, PACAP consistently increased excitability (Fig. 7), providing direct evidence that a peptide-induced release of Ca2+ from internal stores was not required for the increased excitability.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
). Our results suggest that, for adult guinea pig intracardiac neurons, a PACAP-induced Ca2+ influx rather than a peptide-induced Ca2+ release from internal stores is required for the peptide-induced increase in excitability. We base this conclusion on the following observations. First, neither PACAP nor VIP initiated Ca2+ release from internal stores. Second, the PACAP-induced increase in excitability was eliminated when cells were bathed in a Ca2+-deficient solution or a solution containing Cd2+. Third, ryanodine/caffeine pretreatment eliminated caffeine-induced Ca2+ release, but did not block the PACAP-induced increase in excitability.
DeHaven and Cuevas (2004) reported that exposure of dissociated neonatal rat intracardiac neurons to a Ca2+-free solution rapidly depleted internal Ca2+ stores. We found in this study that for guinea pig neurons kept in the Ca2+-deficient solution, the response to 10 mM caffeine was depressed, but that the response to 20 mM caffeine was similar to that of cells maintained in control solution containing Ca2+. Thus we suggest that for adult guinea pig intracardiac neurons, exposure to the Ca2+-deficient solution did not deplete internal Ca2+ stores, in the time frame that we tested. Rather, we postulate that exposure to a Ca2+-deficient solution for short periods decreased the cytosolic Ca2+ concentration as reported for other autonomic neurons (Wanaverbecq et al. 2003). Thus with a decrease in the ambient Ca2+ surrounding ryanodine receptors, caffeine, at a concentration of 10 mM, was ineffective and the higher concentration was required to activate the ryanodine receptors.
Ca2+ influx seems to play an important role in mediating the PACAP-induced increase in excitability of the guinea pig intracardiac neurons. At 200 µM, Cd2+ effectively blocks essentially all types of VDCCs in autonomic neurons (Adams and Cuevas 2004). Cd2+ blocked the PACAP-induced increase in excitability in the guinea pig intracardiac neurons. This observation suggested a role of Ca2+ influx through VDCCs. However, the results of our voltage-clamp studies showed that PACAP inhibited rather than enhanced macroscopic IBa. Thus as the N and P/Q channels are the predominant type of VDCCs, we concluded that PACAP does not enhance Ca2+ influx through these channels. We had thought it plausible that PACAP might enhance Ca2+ influx through L-type VDCCs, which contribute only a small component of the macroscopic current. However, nifedipine, a potent L-type VDCC blocker, did not affect the PACAP-induced excitability. Thus we concluded that PACAP did not enhance Ca2+ influx through any VDCCs. Rather, we hypothesize that PACAP activates a Cd2+-sensitive receptor operated Ca2+ influx pathway (Clapham 2003) and Ca2+ influx through this channel is required for the peptide-induced increase in excitability. We postulate further that the rise in [Ca2+]i caused by Ca2+ influx must be restricted to areas close to the cell membrane because no PACAP-induced increase in global Ca2+ levels were noted in the fluo-3 studies.
Ca2+ has multiple cellular functions and for some of these actions, Sr2+, but not Mg2+, can substitute for Ca2+. In this study, we found that after replacement of Ca2+ with Sr2+, PACAP enhanced excitability in four of six cells. In the two cells in Sr2+ in which PACAP was ineffective, the peptide increased excitability when the preparation was returned to the Ca2+-containing solution. Thus we suggest that unlike Mg2+, which cannot substitute for Ca2+ in supporting the PACAP-induced increase in excitability, Sr2+ can, but it apparently is less effective than Ca2+.
Merriam et al. (2004) have shown that PACAP, but not VIP, potently enhances the hyperpolarization-activated, cyclic nucleotide-gated conductance, Ih, which is known to regulate excitability in many neurons. A PACAP-enhanced Ih very likely contributes to the increased excitability in guinea pig intracardiac neurons. Ih is modulated by cAMP levels and PACAP activates adenylyl cyclase, leading to the generation of cAMP. Intracellular Ca2+ can promote the modulation of Ih by cAMP (Lüthi and McCormick 1999). Thus we propose that a PACAP-induced Ca2+ influx through receptor operated channels causes a local rise of [Ca2+]i at the inner surface of the plasma membrane near Ih channels, and this local elevation of [Ca2+]i may be a critical co-factor in the cAMP-induced shift in Ih voltage dependence.
After caffeine/ryanodine pretreatment, a second caffeine application did not elicit a rise in [Ca2+]i even though the cells were washed with drug-free solution for 20 min. Caffeine sensitizes the ryanodine receptors to ambient intracellular Ca2+ causing internal Ca2+ stores to be dumped, whereas ryanodine at micromolar concentrations blocks the release channels (McPherson et al. 1991; Meissner 1994). The lack of a second response to caffeine suggests that either the internal Ca2+ stores that were dumped during the first application of caffeine/ryanodine exposure were not adequately refilled or that ryanodine blocked the release channels so that Ca2+ could not be released even though internal stores had begun to refill. Thus the effect of the ryanodine/caffeine pretreatment seems to be irreversible on our time scale and eliminates subsequent release of Ca2+ from internal stores.
PACAP can exert its influence through three G protein–coupled receptors: the PAC1 receptor and two VPAC receptors (Vaudry et al. 2000). The PAC1 receptor is selective for PACAP, with VIP being 1,000-fold less effective. In contrast, the VPAC receptors are equally sensitive to VIP and PACAP. Braas et al. (1998) showed that adult guinea pig intracardiac neurons express the PAC1 receptor. More recently, DeHaven and Cuevas (2002) reported that dissociated neonatal rat intracardiac neurons can express both PAC1 and VPAC receptors. Based on the electrophysiological results, Braas et al. (1998) postulated that the PACAP-induced increase in excitability in adult guinea pig intracardiac neurons is mediated by PAC1 receptors because PACAP was more effective than VIP. In addition, recent studies of other PACAP actions on guinea pig intracardiac neurons consistently suggest that these effects also are mediated through activation of PAC1 receptors rather than VPAC receptors. For instance, PACAP decreases somatostatin expression (Braas et al. 2004) and enhances Ih (Merriam et al. 2004). VIP was considerably less effective than PACAP in both studies. In rat neonatal intracardiac neurons, both VIP and PACAP initiate a rise in [Ca2+]i, an effect mediated through VPAC receptors (DeHaven and Cuevas 2004). In this study, neither PACAP nor VIP elicited a rise in [Ca2+]i, an observation consistent with the absence of VPAC receptor expression in guinea pig intracardiac neurons. Consequently, we tentatively suggest that adult guinea pig intracardiac neurons do not express VPAC receptors.
In conclusion, PACAP increases excitability in adult guinea pig intracardiac neurons and neonatal rat intracardiac neurons (Braas et al. 1998; DeHaven and Cuevas 2004). In both species, the change in excitability occurs in both phasic and tonic type neurons. However, some mechanisms responsible for the increased excitability differ in the intracardiac neurons from these two species. In dissociated rat neonatal intracardiac neurons, a synergistic activation of PAC1 receptors and of VPAC receptors coupled with a rise in [Ca2+]i is required for the enhanced excitability (DeHaven and Cuevas 2004). In contrast, in adult guinea pig intracardiac neurons, the enhanced excitability requires activation of PAC1 receptors alone, followed by Ca2+ influx, presumably through a receptor-operated channel, but does not require Ca2+ release from intracellular stores. Comparison of results from this study and those of DeHaven and Cuevas (2004) showed that the mechanisms of action of specific neuropeptides, such as PACAP, can be quite different in the same class of neurons from different species and/or different ages. Furthermore, comparison of results in these two studies raises the question of whether the differences in neuropeptide effect reflect differences between receptor expression in guinea pig and rat intracardiac neurons or differences between receptor expression in intracardiac neurons from neonatal versus adult animals. In either case, the results show that mechanisms responsible for comparable effects of a given peptide may be species- and/or age-dependent.
|
GRANTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: R. L. Parsons, Dept. of Anatomy and Neurobiology, Univ. of Vermont, Burlington, VT 05405 (E-mail: Rodney.Parsons{at}uvm.edu)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Arimura A. Perspectives on pituitary adenylate cyclase activating polypeptide (PACAP) in the neuroendocrine, endocrine, and nervous systems. Jpn J Physiol 48: 301–331, 1998.[CrossRef][ISI][Medline]
Bhattacharya A, Lakhman SS, and Singh S. Modulation of L-type calcium channels in Drosophila via a pituitary adenylyl cyclase-activating polypeptide (PACAP)-mediated pathway. J Biol Chem 279: 37291–37297, 2004.
Braas KM, May V, Harakall SA, Hardwick JC, and Parsons RL. Pituitary adenylate cyclase-activating polypeptide expression and modulation of neuronal excitability in guinea pig cardiac ganglia. J Neurosci 18: 9766–9779, 1998.
Braas KM, Rossignol TM, Girard BM, May V, and Parsons RL. Pituitary adenylate cyclase activating polypeptide (PACAP) decreases neuronal somatostatin immunoreactivity in cultured guinea-pig parasympathetic cardiac ganglia. Neuroscience 126: 335–346, 2004.[CrossRef][ISI][Medline]
Brown D. M-currents: an update. Trends Neurosci 11: 294–299, 1988.[CrossRef][ISI][Medline]
Calupca MA, Vizzard MA, and Parsons RL. Origin of pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive fibers innervating guinea pig parasympathetic cardiac ganglia. J Comp Neurol 423: 26–39, 2000.[CrossRef][ISI][Medline]
Clapham DE. TRP channels as cellular sensors. Nature 426: 517–524, 2003.[CrossRef][Medline]
DeHaven WI and Cuevas J. Heterogeneity of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide receptors in rat intrinsic cardiac neurons. Neurosci Lett 328: 45–49, 2002.[CrossRef][ISI][Medline]
DeHaven WI and Cuevas J. VPAC receptor modulation of neuroexcitability in intracardiac neurons: dependence on intracellular calcium mobilization and synergistic enhancement by PAC1 receptor activation. J Biol Chem 279: 40609–40621, 2004.
Dziema H and Obrietan K. PACAP potentiates L-type calcium channel conductance in suprachiasmatic nucleus neurons by activating the MAPK pathway. J Neurophysiol 88: 1374–1386, 2002.
Hardwick JC, Mawe GM, and Parsons RL. Evidence for afferent fiber innervation of parasympathetic neurons of the guinea-pig cardiac ganglion. J Auton Nerv Syst 53: 166–174, 1995.[CrossRef][ISI][Medline]
Hardwick JC, Mawe GM, and Parsons RL. Tachykinin-induced activation of non-specific cation conductance via NK3 neurokinin receptors in guinea-pig intracardiac neurones. J Physiol 504: 65–74, 1997.[CrossRef][ISI][Medline]
Hardwick JC, Tompkins JD, Locknar SA, Merriam LA, Young BA, and Parsons RL. Ca2+-influx through channels other than voltage-dependent Ca2+ channels is critical to the PACAP-induced increase in excitability in guinea pig cardiac neurons. Regul Pept 130: 163–164, 2005.
Horn R and Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92: 145–159, 1988.
Jeong SW and Wurster RD. Calcium channel currents in acutely dissociated intracardiac neurons from adult rats. J Neurophysiol 77: 1769–1778, 1997.
Locknar SA, Barstow KL, Tompkins JD, Merriam LA, and Parsons RL. Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones. J Physiol 555: 627–635, 2004.
Lüthi A and McCormick DA. Modulation of a pacemaker current through Ca2+-induced stimulation of cAMP production. Nat Neurosci 2: 634–641, 1999.[CrossRef][ISI][Medline]
McPherson PS, Kim YK, Valdivia H, Knudson CM, Takekura H, Franzini-Armstrong C, Coronado R, and Campbell KP. The brain ryanodine receptor: a caffeine-sensitive calcium release channel. Neuron 7: 17–25, 1991.[CrossRef][ISI][Medline]
Meissner G. Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors. Annu Rev Physiol 56: 485–508, 1994.[CrossRef][ISI][Medline]
Merriam LA, Barstow KL, and Parsons RL. Pituitary adenylate cyclase-activating polypeptide enhances the hyperpolarization-activated nonselective cationic conductance, Ih, in dissociated guinea pig intracardiac neurons. Regul Pept 123: 123–133, 2004.[CrossRef][ISI][Medline]
Merriam LA and Parsons RL. Neuropeptide galanin inhibits omega-conotoxin GVIA-sensitive calcium channels in parasympathetic neurons. J Neurophysiol 73: 1374–1382, 1995.
Parsons RL, Rossignol TM, Calupca MA, Hardwick JC, and Brass KM. PACAP peptides modulate guinea pig cardiac neuron membrane excitability and neuropeptide expression. Ann NY Acad Sci 921: 202–210, 2000.
Richardson RJ, Grkovic I, and Anderson CR. Immunohistochemical analysis of intracardiac ganglia of the rat heart. Cell Tissue Res 314: 337–350, 2003.[CrossRef][ISI][Medline]
Smith AB and Adams DJ. Met-enkephalin-induced mobilization of intracellular Ca2+ in rat intracardiac ganglion neurones. Neurosci Lett 264: 105–108, 1999.[CrossRef][ISI][Medline]
Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, and Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 52: 269–324, 2000.
Wanaverbecq N, Marsh SJ, Al-Qatari M, and Brown DA. The plasma membrane calcium-ATPase as a major mechanism for intracellular calcium regulation in neurones from the rat superior cervical ganglion. J Physiol 550: 83–101, 2003.
Zhang L, Tompkins JD, Hancock JC, and Hoover DB. Substance P modulates nicotinic response of intracardiac neurons to acetylcholine in the guinea pig. Am J Physiol 281: R1792–R1800, 2001.
This article has been cited by other articles:
|
|
 |
|
  J. D. Tompkins, J. L. Ardell, D. B. Hoover, and R. L. Parsons Neurally released pituitary adenylate cyclase-activating polypeptide enhances guinea pig intrinsic cardiac neurone excitability J. Physiol., July 1, 2007; 582(1): 87 - 93. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
