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Am J Physiol Regul Integr Comp Physiol 290: R241-R250, 2006. First published September 15, 2005; doi:10.1152/ajpregu.00498.2005
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INFLAMMATION AND CYTOKINES

Ionic mechanisms of histamine-induced responses in guinea pig intracardiac neurons

Jean C. Hardwick, Amy F. Kotarski, and Melanie J. Powers

Biology Department, Ithaca College, Ithaca, New York

Submitted 8 July 2005 ; accepted in final form 30 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Histamine, released from mast cells, can modulate the activity of intrinsic neurons in the guinea pig cardiac plexus. The present study examined the ionic mechanisms underlying the histamine-induced responses in these cells. Histamine evokes a small membrane depolarization and an increase in neuronal excitability. Using intracellular voltage recording from individual intracardiac neurons, we were able to demonstrate that removal of extracellular sodium reduced the membrane depolarization, whereas inhibition of K+ channels by 1 mM Ba2+, 2 mM Cs+, or 5 mM tetraethylammonium had no effect. The depolarization was also not inhibited by either 10 µM Gd3+ or a reduced Cl– solution. The histamine-induced increase in excitability was unaffected by K+ channel inhibitors; however, it was reduced by either blockage of voltage-gated Ca2+ channels with 200 µM Cd2+ or replacement of extracellular Ca2+ with Mg2+. Conversely, alterations in intracellular calcium with thapsigargin or caffeine did not inhibit the histamine-induced effects. However, in cells treated with both thapsigargin and caffeine to deplete internal calcium stores, the histamine-induced increase in excitability was decreased. Treatment with the phospholipase C inhibitor U73122 [GenBank] also prevented both the depolarization and the increase in excitability. From these data, we conclude that histamine, via activation of H1 receptors, activates phospholipase C, which results in 1) the opening of a nonspecific cation channel, such as a transient receptor potential channel 4 or 5; and 2) in combination with either the influx of Ca2+ through voltage-gated channels or the release of internal calcium stores leads to an increase in excitability.

tetraethylammonium; phospholipase C; mast cells; H1 receptor

THE PARASYMPATHETIC CARDIAC PLEXUS integrates information from a variety of inputs, including sensory afferents, descending parasympathetic preganglionic fibers, sympathetic fibers, and interneurons (1, 22). These ganglia consist of a heterogeneous population of cells, including postganglionic neurons and numerous interneurons (1, 6, 20, 22). The convergence of inputs from such diverse sources allows these intracardiac neurons to rapidly respond to both central commands and local reflex circuits to regulate cardiac function.

In many mammalian species, there is also a high density of mast cells located within the heart (24). Previous studies in the guinea pig demonstrated that cardiac mast cells could be found in close proximity to, and even within, the intrinsic cardiac ganglia located within the wall of the atria. Stimulation of the cardiac mast cells, which results in the release of histamine, causes a depolarization of the intracardiac neurons and an increase in excitability via neuronal H1 receptors (19). H1 receptors have also been shown to mediate histaminergic modulation of neuronal activity in the canine intrinsic cardiac plexus (2). Thus histamine modulation of neuronal activity by H1 receptors may be a common phenomenon in the mammalian heart.

The specific mechanisms underlying the histamine-induced responses in the guinea pig intracardiac neurons are unknown. In other neurons, histamine activation of H1 receptors has been shown to produce a depolarization and an increase in firing frequency (4, 5) similar to that seen in the intracardiac neurons. The ionic mechanisms underlying histamine responses are diverse and include the inhibition of a background potassium conductance, stimulation of a chloride current, stimulation of a nonspecific cation current, and/or an increase in intracellular calcium levels (4). H1 receptors are normally coupled to phospholipase C (PLC) activation and the subsequent increase in both diacylglycerol and inositol 1,4,5-trisphosphate (4). The present study investigated the potential cellular mechanisms underlying the histamine-induced changes in intracardiac neurons using a variety of pharmacological agents and ion substitutions. These experiments provide greater insight into the regulation of intracardiac neuron activity by inflammatory signals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
General methods. Guinea pigs (male or female, Charles River), 300–500 g, were euthanized by CO2 inhalation and exsanguination in accordance with procedures approved by the Ithaca College Institutional Animal Care and Use Committee. The heart was removed and placed into ice-cold Krebs-Ringer (in mM: 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, 8 glucose, aerated with 95% O2- 5% CO2 for a pH of 7.4). The cardiac plexus, located in the epicardium of the atria, was dissected as previously described (8). Briefly, the region studied, which is located primarily in the wall of the left atria underlying the area of the coronary sinus, was exposed by opening the atria and removing the overlying muscle and connective tissue. The tissue was pinned to a Sylgard-lined 60-mm Petri dish and continuously superfused (6–8 ml/min) with 35–37°C Krebs-Ringer. Drugs were applied by inclusion in the circulating bath solution. In all cases, bath-applied solutions were allowed to equilibrate for a minimum of 10 min before testing. Histamine (10–4 M in Krebs solution) was applied by local pressure ejection (6–9 psi, Picospritzer, General Valve) through a small diameter (5–10 µm) glass electrode positioned 50–100 µm from the individual neuron. For multiple tests of histamine responses in the same cell, the cells were allowed to wash (via the circulating Krebs solution) for several minutes between histamine applications. This time frame allowed for multiple, repeatable responses, with no apparent desensitization.

Electrophysiological methods. Intracellular voltage recordings from intracardiac neurons were obtained with an AxoClamp 2B amplifier (Axon Instruments) from cells impaled with 2 M KCl-filled microelectrodes (40–80 M{Omega}). Data were collected, digitized, and analyzed using pClamp 8.2 (Axon Instruments). Individual neurons were used for an experiment if the membrane potential was –40 mV or below and they produced action potentials (APs) with an overshoot of at least 20 mV. The neurons were identified as putative parasympathetic postganglionic neurons by their morphology (~30 µm in diameter) and their basic electrical properties relative to previous studies (6, 8, 19).

Single APs were stimulated by positive current injection (0.1–0.7 nA, 5 ms), averaged (5–6 individual recordings), and analyzed to determine the amplitude and duration of the after hyperpolarizing potential (AHP). Neuronal excitability was monitored by observing the response to a series of long depolarizing current pulses (0.1–0.6 nA, 500 ms). Increases in excitability appear as an increase in the number of APs fired at a given stimulus. The AP frequency at each stimulus amplitude was determined, and a least squares linear regression analysis of frequency vs. stimulus amplitude was determined to assess relative changes in excitability. Input resistance was determined by negative current injections (–0.1 to –0.5 nA, 1 s), and the resistance was determined from the plateau of the voltage drop at different stimulus intensities at a common time point (800 ms). The combination of AHP parameters, firing patterns, and input resistance was used to confirm the neurons as parasympathetic postganglionic (6, 8), such that the neurons used in this study had input resistances in the range of 70–140 M{Omega} and AHPs with a duration of 180–300 ms. Due to the insufficient number of neurons with AHPs of either <180 ms [P cells, Edwards et al. (6)] or >300 ms, these cells were excluded from the study. Finally, neurons were categorized as phasic (1–3 APs at the onset of depolarizing stimuli) or tonic (multiple APs throughout depolarizing stimuli).

For each cell, the membrane response to a 1- to 2-s application of histamine was determined. In addition, excitability was determined before and immediately after a 1- to 2-s drug application. For ion channel blockers, control responses were determined in the Krebs solution, and then the inhibitor was perfused over the tissue for a minimum of 5–10 min before subsequent testing.

Drugs and solutions. For ion substitution experiments, extracellular Na+ was replaced with N-methylglucamine (NMG; Sigma, St. Louis, MO) in the following manner: 50% NMG (in mM: 61 NaCl, 61 NMG, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 10 HEPES, 8 glucose, pH of 7.4) and 100% NMG (in mM: 121 NMG; 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 15 HEPES, 8 glucose, pH of 7.4). For the reduced chloride solutions, NaCl was replaced with sodium propionate (ICN Biochemicals) as follows: (in mM) 121 Na propionate; 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 10 HEPES, 8 glucose, pH of 7.4. The following ion channel blockers or enzyme inhibitors were added directly to the circulating bath solution: 5 mM tetraethylammonium (TEA), 1 mM BaCl2, 2 mM CsCl, 1 mM 4-aminopyridine (4-AP), 10 µM GdCl3, 10 mM caffeine, 1 µM thapsigargin, 2 µM U73122 [GenBank] , or 2 µM U73433 [GenBank] (all from Sigma). For some drugs, stock solutions were made up in DMSO, with a final DMSO concentration of ≤1%. Previous studies (9) have shown that this concentration has no effect on the neuronal functions studied.

Statistical analysis. All experiments were performed on preparations from three or more different animals. Values are expressed as means ± SE. Statistical significance was determined by paired Students t-test, with a P value of <0.05 considered significant. Least squares linear regressions were performed with the program SigmaStat, which provided estimates of the slope and the adjusted R2 values.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Intracellular recordings. The guinea pig cardiac plexus consists of multiple cell types, including parasympathetic postganglionic neurons and interneurons. For these studies, the results were taken from cells that can be generally classified as parasympathetic postganglionic, based on their approximate size (30 µm) and their membrane properties (6, 8, 19). A total of 55 neurons from ~40 different animals was recorded in this study. The average resting membrane potential of these cells was –47 ± 7 mV. Each neuron was also categorized based on the input resistance (cells used in this study had an input resistance of 70–140 M{Omega}), AHP duration (only cells with a duration ranging from 180 to 300 ms were used), and firing patterns.

Histamine application produces two types of responses in these neurons: a small membrane depolarization and an increase in excitability (Fig. 1). These two responses were studied in an attempt to elucidate the underlying ionic mechanisms.



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  		Fig. 1. Histamine-induced responses in intracardiac neurons. A: local application of histamine (1 s, arrow) produces a small depolarization lasting an average of 30–60 s. B: in addition, the number of action potentials (APs) produced in response to a depolarizing stimulus (0.3 nA, 500 ms) is greater after histamine application (right) than under control conditions (left). Resting membrane potential for this cell was –43 mV.

 
Histamine-induced depolarizations. The histamine-induced depolarization of intracardiac neurons was measuring using standard intracellular current clamp recordings. Histamine applied by local pressure ejection (10–4 M, 1 s, 6–9 psi) produced an ~5-mV depolarization of the membrane potential that lasted 30–60 s (Fig. 1, Table 1). This depolarization was not altered by the inclusion of several different inhibitors of potassium channels, such as 5 mM TEA, 1 mM Ba2+, or 2 mM Cs+. In all of these cases, there was no significant change in the amplitude or duration of the histamine response (data not shown). However, when the extracellular sodium concentration was either partially or completely replaced with the impermeant cation NMG, the amplitude of the depolarization was significantly reduced (Table 1). Partial replacement of Na+ with NMG resulted in an ~30% decrease in amplitude (compared with control amplitudes in the same cell) and complete replacement of Na+ with NMG resulted in an average 64% decrease in amplitude (n = 9). Conversely, substitution of most of the extracellular Cl– with propionate had no significant effect (P = 0.97 vs. controls by paired t-test) on the amplitude of the histamine-induced depolarizations. Finally, inclusion of 10 µM GdCl3 in the circulating bath solution to inhibit most nonspecific cation channels also had no effect on the histamine-induced depolarization (P = 1.0 vs. controls by paired t-tests).


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  		Table 1. Inhibition of the histamine-induced depolarization

 
H1 receptors have been shown to work primarily through the activation of PLC (4). Therefore, the effect of the PLC inhibitor U73122 [GenBank] (2 µM) was tested to determine its ability to inhibit the histamine-induced depolarization. The inactive analog U73343 [GenBank] (2 µM) was used as a control. Incubation of the tissue in a bath solution containing U73122 [GenBank] for at least 10 min resulted in the inhibition of the depolarization (Fig. 2, Table 1), whereas incubation with U73343 [GenBank] did not significantly alter the histamine-induced response (Fig. 2, Table 1).



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  		Fig. 2. Inhibition of the histamine-induced responses by U73122 [GenBank] . A: membrane responses to a 1-s application of histamine (arrows) were determined in the control solution and in the presence of either U73343 [GenBank] (2 µM, inactive analog) or U73122 [GenBank] (2 µM, inhibitor of PLC) for 10–15 min. U73343 [GenBank] had no effect on the ability of histamine to depolarize the membrane, whereas U73122 [GenBank] treatment resulted in an inhibition of the depolarization. Resting membrane potentials: –64 mV (control), –55 mV (U73343 [GenBank] ), –45 mV (U73122 [GenBank] ). B: ability of histamine to alter neuronal excitability was tested by monitoring the change in AP frequency at different stimulus intensities in control Krebs and then after a 10-min incubation with 2 uM U73122 [GenBank] . A least squares regression fit for each condition was generated using SigmaStat. The slopes of the linear regression curves for each condition were 8.0 ± 0.9 (control), 16.8 ± 1.3 (control with histamine), 9.0 ± 1.1 (U73122 [GenBank] alone), and 11.5 ± 1.2 (U73122 [GenBank] with histamine). The adjusted R2 values for all fits were ≥0.92. Data are means ± SE from 6 cells for each condition. Paired t-tests of control responses vs. responses following histamine at each stimulus level were used to determine significant changes in AP frequency. *Significant increase with histamine vs. paired controls in test solution (P < 0.05). #Significant increase with histamine vs. paired controls in normal Ringer solution (P < 0.05).

 
Histamine-induced changes in excitability. To monitor neuronal excitability, cells were stimulated with a series of depolarizing current pulses (0.1–0.6 nA, 500 ms) and the frequency of APs produced at each stimulus level was recorded. For most neurons, the cells were phasic in nature (43 of 50 cells tested), producing one AP at the onset of the current pulse or, in some cases, a small burst of APs (2–3 in number) at the higher stimulus levels. A small minority of cells (7 cells) was tonic in nature, firing APs throughout a prolonged depolarization that significantly increased in number as the stimulus intensity increased. In all cases, the frequency of APs was determined at each stimulus level under control conditions and then after a 1-s application of histamine. Paired t-tests of the frequency of APs at a each stimulus level before and after histamine application were performed to determine whether histamine results in a significant change in AP frequency. The results show that histamine consistently caused an increase in the number of APs, regardless of the initial firing pattern of the cell (Fig. 3). This change was readily reversible, with the cells returning to control levels of activity within 1–2 min. Although all cell types showed an increase in AP frequency with histamine application, the relative change (vs. control) was greatest for the phasic cells, with an approximate twofold increase in the slope of the change in frequency observed with increasing stimulus intensity. In the tonic cells, histamine application resulted in an increase in firing at the majority of the stimulus levels and caused an increase in the relative slope, but the change was not as great (1.4-fold). For the remaining studies, phasic and tonic cells were grouped together in regression analyses, since both cell types showed similar relative changes in excitability. Additionally, the small number of tonic cells recorded from made separate analysis of their responses for each condition impossible.



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  		Fig. 3. Histamine-induced changes in control excitability responses. The frequency of APs produced in response to depolarizing current steps (0.1–0.6 nA, 500 ms) was determined before and after a 1-s application of histamine. The points are means ± SE from several cells, with statistically significant differences between controls and histamine at each point determined by paired t-test (#P < 0.05). In all cases, histamine application resulted in a significant increase in AP frequency with increasing stimulus amplitude. A least squares regression fit for each condition was generated using SigmaStat. Top: combined data from all cell types. The slope of the control response for all of the cells was 8.6 ± 0.6, and with histamine the slope was 16.7 ± 1.5. Middle: analysis of the phasic cells only gives slope values for the control of 6.7 ± 0.4 and histamine of 15.3 ± 1.2. The adjusted R2 values for all of the fits were ≥0.90. Bottom: analysis of the tonic cells only gives slope values for the control of 16.3 ± 1.2 and histamine of 22.6 ± 3.1.

 
U73122 was used to test whether the change in excitability in response to histamine was also dependent on PLC activation. Histamine responses were determined in control solution and with bath application of either 2 µM U73122 [GenBank] or 2 µM U73343 [GenBank] . No alterations in the histamine-induced increase in excitability were observed in the presence of U73343 [GenBank] (data not shown), whereas treatment with U73122 [GenBank] resulted in a decrease in the histamine-induced excitability at all but the highest stimulus level (Fig. 2).

Histamine-induced increases in excitability are not affected by K+ channel blockers. Several studies have demonstrated that histamine can alter cellular excitability via the inhibition of K+ channel activity (3, 4, 7). To examine this possibility in the guinea pig intracardiac neurons, histamine's effect on neuronal excitability was tested in the presence of a variety of K+ channel inhibitors included in the circulating bath solution: (in mM) 1 Ba2+, 5 TEA, 1 4-AP [A current to inhibit (IA)] and 2 Cs+ hyper-polarization-activated current to inhibit IH. Excitability levels were determined in the presence of the K+ channel inhibitor alone and after histamine application. Examples of the results observed are shown in Fig. 4. Two of the K+ channel inhibitors, Ba2+ and 4-AP, consistently produced an increase in the basal level of neuronal excitability. However, after application of histamine, a further increase in excitability was consistently observed in the presence of these two inhibitors, as well as in the presence of TEA and Cs+ (Fig. 4).



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  		Fig. 4. Effects of potassium channel blockers on histamine-induced changes in neuronal excitability. Neuronal excitability was monitored with depolarizing current injections (0.5 nA, 500 ms). Bath application of 2 mM CsCl, 1 mM BaCl2, 5 mM tetraethylammonium (TEA), or 1 mM 4-aminopyridine (4-AP) was tested before (control; left) and after histamine application (right). Although Ba and 4-AP did increase the basal activity level, none of these inhibitors prevented the histamine-induced increase in AP production. Resting membrane potentials: Cs+ –40 mV, Ba2+ –46 mV, 4-AP –59 mV, and TEA –42 mV.

 
Effects of calcium on the histamine-induced excitability changes. H1 receptors have been shown to stimulate an increase in intracellular calcium in several cell types (4, 11, 12, 16). To test whether the increase in excitability produced by histamine was mediated via a change in intracellular calcium levels, we examined the ability of histamine to stimulate the cells under a variety of conditions designed to alter calcium availability. Histamine's effects were determined 1) in the presence of 10 mM caffeine to stimulate the release of internal calcium by activation of ryanodine receptors, 2) in the presence of 1 µM thapsigargin to inhibit the replenishment of intracellular stores through the inhibition of endoplasmic Ca-ATPase, 3) after depletion of intracellular calcium stores by a combined treatment of caffeine and thapsigargin, 4) in 0 external Ca2+ solution, and 5) in the presence of 200 µM CdCl2 to inhibit voltage-gated Ca2+ channels.

To examine the involvement of intracellular calcium pools, the control responses to histamine were determined and then compared with the responses observed after a 1-min exposure to 10 mM caffeine. The tissue was then perfused with 1 µM thapsigargin in the control Ringer solution for at least 10 min, followed by a 1-min exposure to 10 mM caffeine in the presence of thapsigargin. Previous studies in guinea pig stellate neurons (13) showed that a combined treatment with caffeine and thapsigargin depletes ryanodine-sensitive intracellular calcium stores for up to 30 min. Similar results, with the use of intracellular calcium imaging techniques, have been observed in isolated guinea pig intracardiac neurons (Parsons RL and Locknar SA, personal communication). Subsequent caffeine applications in the thapsigargin/caffeine-treated cells did not have any effect on membrane responses in our experiments (data not shown). Histamine responses were then determined in these calcium-depleted cells. Representative responses are shown in Fig. 5, the regression analysis of frequency vs. stimulus intensity for each condition is shown in Fig. 6, and the estimated slopes are given in Table 2. Release of intracellular calcium by caffeine exposure had no effect on basal excitability levels. Caffeine treatment did produce a significant increase in AHP duration (control 284 ± 22 ms vs. caffeine 370 ± 35 ms, n = 7, see Fig. 5). Histamine application during the cell's response to caffeine resulted in an increase in excitability, although this increase was slightly attenuated compared with controls. Thapsigargin treatment alone did not alter basal levels of excitability or AHP duration (230 ± 28 ms). Application of histamine in thapsigargin-treated cells resulted in a large increase in excitability. In cells treated with both thapsigargin and caffeine to deplete the internal calcium stores, the basal level of excitability was increased over untreated cells. Histamine produced a further increase in excitability in one of four cells tested (Fig. 5). The remaining three cells showed no significant increase in excitability with histamine application (Fig. 6), with the pooled data showing no significant change vs. paired controls.



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  		Fig. 5. Changes in AP frequency with changes in intracellular calcium. Traces show examples of changes in neuronal excitability in control (left) and after histamine application (1 s; right) under conditions that alter intracellular calcium. The tissue was treated with 10 mM caffeine for 1 min, 1 µM thapsigargin for 10–20 min, or 1 µM thapsigargin for 10 min, followed by 10 mM caffeine for 1 min to deplete internal calcium stores (calcium depleted). Traces show the voltage response to a depolarizing current pulse (0.5 nA, 500 ms). Resting membrane potentials: caffeine –59 mV, thapsigargin –50 mV, and calcium depleted –40 mV.

 


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  		Fig. 6. Effects of calcium availability on the histamine-induced increase in excitability. Plots of AP frequency vs. stimulus intensity were determined in the presence and absence of histamine under a variety of conditions that would alter calcium availability. For each condition, a linear regression was performed to generate the relative change in frequency vs. stimulus intensity (for slopes of the regressions, see Table 2). Each plot shows the mean frequency ± SE. Caffeine (10 mM) was perfused over the preparation for 1 min, and excitability changes were determined immediately (n = 9). Thapsigargin (1 uM) was applied in the bath for 10 min before testing the excitability levels (n = 7). Caffeine was applied again in the presence of thapsigargin for 1 min, and the excitability levels were tested again in the calcium-depleted cells 2–5 min after the caffeine application (n = 5). Paired t-tests of control responses vs. responses following histamine were used to determine significant changes in AP frequency. *Significant increase with histamine vs. paired controls in test solution (P < 0.05). #Significant increase with histamine vs. paired controls in normal Ringer solution (P < 0.05).

 

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  		Table 2. Changes in excitability with calcium availability

 
To determine the dependence on extracellular calcium, the histamine responses were determined in a 0 Ca2+ solution (CaCl2 replaced with 4 mM MgCl2) and in the presence of 200 µM Cd2+ to inhibit voltage-gated calcium channels (VGCCs). Both removal of extracellular calcium and inhibition of calcium entry resulted in a significant increase in the basal excitability responses of the cells. Many cells became tonic in nature (firing repetitively throughout the depolarization) in these solutions and demonstrated a significant inhibition in AHP amplitude and duration (control: AHP amp 16.5 ± 0.7 mV, duration 200 ± 20 ms; 0 Ca2+: AHP amp 9.1 ± 0.5 mV, duration 85 ± 8 ms; Cd2+: AHP amp 11.8 ± 1.1 mV, duration 68 ± 38 ms; n = 5). Subsequent application of histamine resulted in no increase in excitability in either solution (Fig. 7, Table 2). To determine whether these cells were still able to produce increases in AP frequency, 1 mM BaCl2 (to inhibit K+ channels) was added to the 0 Ca2+ solution and resulted in an increase in excitability over the 0 Ca2+ alone (n = 3, data not shown).



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  		Fig. 7. Extracellular calcium (A) and excitability (B). To alter extracellular calcium availability, the preparation was superfused with a 0 Ca2+/4 mM Mg2+ solution for 10 min before excitability was tested (n = 4). Cells were also tested in the presence of 200 uM Cd2+ in normal Ringer solution (n = 5). In all cases, the tissue was allowed to equilibrate for 10 min in the test solution before the histamine-induced changes in excitability were examined. Paired t-tests of control responses vs. responses following histamine were used to determine significant changes in AP frequency. #Significant increase with histamine vs. paired controls in normal Ringer solution (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The interaction of the immune system with the nervous system provides a mechanism for rapid adjustment of physiological function in response to inflammatory signals. Cardiac mast cells are located in close proximity to cardiac ganglia, and stimulation of these mast cells can result in rapid and significant changes in cardiac function, both via direct actions on cardiac tissue (24) and by direct modulation of intrinsic neuronal function (2, 9, 19).

The ionic mechanisms underlying the histamine-induced depolarization and increase in intracardiac neuron firing are mediated through the stimulation of H1 receptors on the neurons (19). The results from the present studies suggest that activation of the H1 receptor results in the activation of PLC and the stimulation of a nonselective cation conductance to produce a depolarization of the neurons. In addition, these experiments suggest that the increase in neuronal excitability is also dependent on the influx of extracellular calcium through VGCCs.

Although the ionic mechanism underlying the histamine-induced depolarization could have resulted from several different potential mechanisms, it appears that, in the intracardiac neurons, histamine is activating a nonspecific cation conductance. Other systems have shown evidence for a histamine-mediated inhibition of potassium leakage channels as the underlying mechanism for depolarization (4, 7). If this were the case in the intracardiac neurons, blockage of these channels with Ba2+ would have prevented the histamine response. However, none of the K+ channel inhibitors tested had any effect on the amplitude of histamine-induced depolarization. Some studies have shown evidence for histamine stimulation of a Ca2+-dependent chloride conductance (4). In the guinea pig cardiac plexus, reduction in extracellular chloride concentrations had no effect on the histamine-induced depolarization. The evidence for stimulation of a nonselective cation conductance derives primarily from the observation that a reduction in the extracellular sodium concentration results in a decrease in the amplitude of the depolarization. Complete replacement of extracellular sodium with the impermeant cation NMG resulted in ~60% inhibition of the histamine-induced depolarization. The fact that this depolarization was not completely inhibited by the removal of Na+ suggests that other cations (such as Ca2+) may be able to utilize the channels activated by histamine. One likely candidate would be the activation of a transient receptor potential channel (TRPC) (18, 23). The TRPC channels are found throughout the nervous systems in multiple isoforms. Conductance through the majority of these channels can be inhibited with lanthanides, such as Gd3+. However, the TRPC4 and TRPC5 isoforms are resistant to inhibition by lanthanides at concentrations that inhibit most nonspecific cation channels (10). Thus the fact that 10 µM Gd3+ failed to inhibit the histamine-induced depolarization would suggest that the TRPC4/5 channels may mediate this response. Additionally TRPC4/5 channels have been shown to couple to H1 receptors via PLC activation in other tissues and show significant conductance to both Na+ and Ca2+ (23).

Histamine also produces an increase in neuronal excitability in the intracardiac neurons. The present study demonstrates that the mechanism underlying this portion of the response also does not appear to involve modulation of potassium channels. Previous studies of the guinea pig cardiac neurons had shown that the Cs+-sensitive IH current can regulate neuronal firing, such that inhibition of IH with 2 mM Cs+ resulted in a decrease in spontaneous activity (6). If histamine were working through a stimulation of this conductance to increase firing, then pretreatment with Cs+ should prevent any change in response to histamine. However, Cs+ treatment did not prevent the histamine-induced increase in excitability. Similarly, inhibition of IA with 4-AP or general inhibition of potassium channels with either TEA or Ba2+ would show similar results if these channels were essential for the histamine response. For example, the large conductance calcium-dependent potassium channels, which are inhibited by TEA, have been shown to regulate neuronal firing in many different cells (17, 25). However, our results provide no evidence for the involvement of any of these channels in the histamine-induced increase in excitability. Treatment of intracardiac neurons with 4-AP or Ba2+ resulted in an increase in the basal activity of the cell, suggesting that Ba2+- and 4-AP-sensitive currents regulate the resting excitability levels of the neurons. Application of histamine in the presence of these inhibitors resulted in a further increase in excitability, thus demonstrating that the histamine response was still functional. Treatment with TEA resulted in no change in basal firing levels but did result in a prolongation of the AP (see Fig. 4). This prolongation was probably due to the inhibition of the delayed rectifier potassium channel that normally mediates rapid repolarization of the cells. Again, histamine was still able to increase the production of APs in the presence of TEA. Thus the results of this study rule out the possibility that histamine, via H1 receptors, is modulating Ba2+-sensitive leakage channels (3, 4, 7), IH channels, IA channels, calcium-dependent potassium channels, or other TEA-sensitive potassium channels.

H1 receptor activation can also lead to increases in intracellular calcium concentrations. H1 responses have been shown to be dependent on the influx of extracellular calcium (11) or coupled to the stimulation of inositol 1,4,5-trisphosphate-sensitive calcium stores (4, 11, 12, 16). To test the requirement for calcium in mediating the histamine-induced responses in the intracardiac neurons, we examined the ability of histamine to increase excitability under a variety of altered calcium conditions. Caffeine was used to stimulate the release of calcium from ryanodine-sensitive intracellular pools. Caffeine treatment resulted in a significant increase in the AHP duration, suggesting that the primary effect of the caffeine-induced increase in calcium was the stimulation of calcium-dependent potassium channels. Histamine was still able to increase neuronal excitability in these cells; however, the relative increase appeared to be somewhat attenuated. This is most likely due to the increase in calcium-activated potassium channel function. Treatment with thapsigargin, which inhibits intracellular Ca-ATPases, thus preventing the removal of cytosolic calcium into intracellular stores, resulted in no change in basal excitability levels but did increase the response to histamine. These results suggest that inhibition of cytosolic calcium removal by intracellular Ca-ATPase may result in prolongation of any effects mediated by a histamine-induced increase in intracellular calcium by preventing one of the mechanisms for calcium removal. Finally, cells that had been depleted of their readily releasable intracellular calcium stores through a combined treatment of thapsigargin and caffeine showed an increase in the basal excitability levels and did not demonstrate a further increase in response to histamine. These cells showed no functional responses to subsequent caffeine applications (i.e., no change in AHP characteristics). These results suggest that internal calcium stores may play a role in regulating neuronal excitability levels, but the involvement of these stores in mediating the histamine response is still unclear. However, since release of the stores by caffeine application resulted in a measurable change in AHP parameters, and previous studies demonstrated that histamine application does not alter the duration of the AHP in these neurons (19), it appears unlikely that a histamine-induced release of intracellular calcium is the primary mechanism underlying the increase in excitability. Future studies can address this hypothesis more directly by using calcium imaging techniques.

Removal of extracellular calcium from the circulating bath solution inhibited the histamine-induced increase in neuronal excitability. Both removal of extracellular calcium and inhibition of calcium entry through VGCCs resulted in a significant increase in basal excitability levels. This is most likely due to the lack of calcium needed for the opening of calcium-gated potassium channels. This was clearly seen in the significant reduction in AHP amplitude and duration in these cells. Application of histamine to these cells showed no change in AP frequency. This lack of response was not due to an inability of these cells to produce higher frequencies, since the addition of Ba2+ to the 0 Ca2+ solution did increase AP frequency. Rather, it appears that the influx of Ca2+ through VGCCs during the initial AP depolarization is a necessary component of the histamine-induced response. This Ca2+ influx may interact with second messengers generated by H1 receptor activation to increase neuronal excitability.

The second messenger system coupling the H1 receptors to the changes in ion channel function is most likely via the activation of PLC. Previous studies have shown that H1 receptors are usually coupled to Gq/11 and the activation of PLC (4). The present studies suggest that, in the intracardiac neurons, PLC activation is the most logical mechanism, based on the ability of U73122 [GenBank] to inhibit the histamine responses.

The results from this study show that histamine, via activation of H1 receptors, can regulate neuronal function of intracardiac neurons through the activation of a nonselective cation conductance and a subsequent influx of extracellular calcium to increase excitability. These results do not indicate the specific mechanism that causes the increased excitability. However, these data provide further background on the mechanisms underlying the regulation of cardiac ganglion function by immune mediators. Given the abundance of mast cells within the heart, their increase with disease (14, 15), and their potential to drastically alter cardiac function (21, 24), understanding the mechanisms whereby these cells can alter intracardiac neuronal function is essential to fully understanding the regulation of the heart by inflammatory stimuli.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by National Heart, Lung, and Blood Institute Grant R15 HL-60619 to J. Hardwick.


   ACKNOWLEDGMENTS
 
The authors thank Kristen Sager, Jeremy Dobson, Abigail Wilkes, Jay Sellers, and Danielle Federico for excellent technical assistance.

Present address of M. J. Powers: Dept. of Physiology and Functional Genomics, College of Medicine, Univ. of Florida, Gainesville, FL 32610.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. C. Hardwick, Biology Dept., Ithaca College, Ithaca, NY 14850 (e-mail: jhardwick{at}ithaca.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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