alpha1-Adrenoceptor-activated cation currents in neurones acutely isolated from rat cardiac parasympathetic ganglia

doi:10.1113/jphysiol.2002.033100

$alpha$ ₁-Adrenoceptor-activated cation currents in neurones acutely isolated from rat cardiac parasympathetic ganglia

Hitoshi Ishibashi, Mari Umezu, Il-Sung Jang, Yushi Ito and Norio Akaike†

Cellular and System Physiology, and *Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, and †Research Division for Physiology and Pharmacology, Kumamoto Health Science University, Kumamoto 861-5533, Japan

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

The noradrenaline (NA)-induced cation current was investigated in neurones freshly isolated from rat cardiac parasympathetic ganglia using the nystatin-perforated patch recording configuration. Under current-clamp conditions, NA depolarized the membrane, eliciting repetitive action potentials. NA evoked an inward cation current under voltage-clamp conditions at a holding potential of -60 mV. The NA-induced current was inhibited by extracellular Ca²⁺ or Mg²⁺, with a half-maximal concentration of 13 µM for Ca²⁺ and 1.2 mM for Mg²⁺. Cirazoline mimicked the NA response, and prazosin and WB-4101 inhibited the NA-induced current, suggesting the contribution of an $alpha$ ₁-adrenoceptor. The NA-induced current was inhibited by U73122, a phospholipase C (PLC) inhibitor. The membrane-permeable IP₃ receptor blocker xestospongin-C also blocked the NA-induced current. Furthermore, pretreatment with thapsigargin and BAPTA-AM could inhibit the NA response while KN-62, phorbol 12-myristate 13-acetate (PMA) and staurosporine had no effect. These results suggest that NA activates the extracellular Ca²⁺- and Mg²⁺-sensitive cation channels via $alpha$ ₁-adrenoceptors in neurones freshly isolated from rat cardiac parasympathetic ganglia. This activation mechanism also involves phosphoinositide breakdown, release of Ca²⁺ from intracellular Ca²⁺ stores and calmodulin. The cation channels activated by NA may play an important role in neuronal membrane depolarization in rat cardiac ganglia.

(Received 25 September 2002; accepted after revision 22 January 2003; first published online 21 February 2003)
Corresponding author H. Ishibashi: Cellular and System Physiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Email: h-ishi{at}physiol2.med.kyushu-u.ac.jp

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

The mammalian heart is innervated by autonomic nerve fibres from both the parasympathetic and sympathetic nervous systems. The neurones of cardiac parasympathetic ganglia, situated at the outer surface of the atria, receive input from efferent vagal fibres. They innervate the atrial musculature, in particular the sinoatrial and atrioventricular nodes, thus playing a crucial role in the regulation of heart rate (Ardell & Randall, 1986; Burkholder et al. 1992; de Souza et al. 1996). It has also been shown that in vivo stimulation of canine sympathetic stellate ganglia activates atrial parasympathetic ganglion neurones, suggesting that the neurones in cardiac ganglia also receive input from the sympathetic nervous system (Gagliardi et al. 1988). In fact, the sympathetic postganglionic axons are reported to form synapses with somata and short dendrites of parasympathetic neurones within mammalian cardiac ganglia (Ellison & Hibbs, 1976). However, the mechanisms involved in the activation of neurones in cardiac parasympathetic ganglia by sympathetic stimulation are not fully understood.

Noradrenaline (NA) is known to modulate voltage-dependent Ca²⁺ and K⁺ channels via $alpha$ -adrenoceptors in various peripheral and central neurones. Previous studies have shown that activation of $alpha$ -adrenoceptors suppresses voltage-dependent Ca²⁺ channels in rat sympathetic neurones (Bernheim et al. 1991; Chen & Schofield, 1993; Caulfield et al. 1994), parasympathetic neurones (Xu & Adams, 1993) and nucleus tractus solitarii neurones (Ishibashi & Akaike 1995). NA also modulates K⁺ channels in cat vesical parasympathetic neurones (Akasu et al. 1985) and rat locus coeruleus neurones (Arima et al. 1998). In smooth muscle cells, stimulation of $alpha$ ₁-adrenoceptors coupled with G-protein (Gq/11) activates phospholipase C (PLC) and produces diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃), causing a release of Ca²⁺ from intracellular Ca²⁺ stores and an accompanying Ca²⁺ influx into the cell (Yamada et al. 1996; Helliwell & Large, 1997; Inoue et al. 2001). The non-selective cation channel activated by $alpha$ ₁-adrenoceptors is thought to contribute to this Ca²⁺ influx, and activation of this channel also depolarizes the cell membrane and induces Ca²⁺ influx through voltage-dependent Ca²⁺ channels (Helliwell & Large, 1997; Inoue et al. 2001). Recent studies have revealed that the transient receptor potential (TRP) protein and its mammalian homologues are non-selective cation channels activated by Gq/11-coupled receptors and that they are molecular models for the Ca²⁺ influx mechanisms associated with phosphoinositide breakdown and depletion of intracellular Ca²⁺ stores (Mori et al. 2001; Minke & Cook, 2002).

In addition to the voltage-dependent Ca²⁺ channels and ligand-gated cation channels, the Gq/11-coupled receptor-mediated cation channels (tentatively named receptor-operated cation channels) have recently been recognized for their important roles in the regulation of Ca²⁺ entry into various cells (Mori et al. 2001). As for the neuronal receptor-operated cation channels, the cation currents activated by Gq/11-coupled receptors have been reported in cultured rat sympathetic neurones (Beaudet et al. 2000; Delmas et al. 2002). The channel properties of recombinant TRP protein from mammalian neurones have also been studied in cultured cells (Strübing et al. 2001; Peier et al. 2002). However, little information about the receptor-operated cation channels in parasympathetic neurones has been obtained so far. In the present study, we found that NA activates cation currents in native neurones freshly isolated from rat cardiac ganglia. The physiological and pharmacological properties of the NA-activated cation channels were investigated using the nystatin-perforated patch recording configuration (Horn & Marty, 1988).

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

Preparation

This study was conducted under 'Guiding Principles for the Care and Use of Laboratory Animals' approved by The Japanese Pharmacological Society.

The experiments were performed on cardiac parasympathetic ganglion neurones freshly dissociated from 2-week-old Wistar rats. The procedure for obtaining dissociated ganglion neurones was similar to that used in our previous studies (Murai et al. 1998; Ishibashi et al. 2001). Briefly, rats were killed by decapitation under pentobarbital sodium anaesthesia (50 mg kg^-1, I.P.) and the ganglia situated at the outer surface of the atria were rapidly removed (de Souza et al. 1996). Then the ganglia were treated with a standard external solution containing 0.3 % collagenase and 0.3 % trypsin for 40 min at 37 °C. Thereafter, the ganglion neurones were dissociated mechanically by triturating with fire-polished Pasteur pipettes in a culture dish (Primaria 3801, Becton Dickinson, Rutherford, NJ, USA). The dish for intracellular Ca²⁺ concentration measurements had a central hole which was covered with a microcover glass (24 mm times 24 mm, at a thickness of 0.12-0.17 mm; Matsunami Glass Ind. Ltd, Tokyo, Japan) attached to the bottom of the dish with high vacuum silicon grease (Dow Corning, Tokyo, Japan). The dissociated neurones adhered to the bottom of dish within 20 min. We used the isolated neurones 1-8 h after preparation.

Solutions and chemicals

The ionic composition of the standard external solution was (mM): NaCl 150, KCl 5, MgCl₂ 1, CaCl₂ 2, Hepes 10 and glucose 10. The pH was adjusted to 7.4 with tris(hydroxymethyl)aminomethane (Tris-OH). The nominally Ca²⁺-free solution was made by simply omitting Ca²⁺ from the standard external solution. To prepare the Ca²⁺-free external solution, we added 2 mM EGTA to the nominally Ca²⁺-free solution. The effects of cation channel antagonists, Gd³⁺, La³⁺ and Cd²⁺, were examined in the nominally Ca²⁺-free solution by simply adding the chloride salts. When the effect of extracellular Ca²⁺ was investigated, the extracellular Ca²⁺ concentration ([Ca²⁺]_o) was controlled by adding EGTA to the standard external solution, and the free Ca²⁺ concentration was calculated using EQCAL software (Biosoft, Ferguson, MO, USA). To observe the effect of Mg²⁺, MgCl₂ was simply added to a nominally Ca²⁺- and Mg²⁺-free solution. Other experiments were performed in the Ca²⁺-free solution containing 2 mM EGTA and 1 mM Mg²⁺. The composition of the patch-pipette (internal) solution for nystatin-perforated patch recording under voltage-clamp conditions was (mM): CsCl 70, caesium methanesulfonate 80, Hepes 10. The pH was adjusted to 7.2 with Tris-OH. The initial current-clamp experiment was performed by using the internal solution containing (mM): KCl 70, potassium methanesulfonate 80, Hepes 10. Nystatin was dissolved in methanol, resulting in a 10 mg ml^-1 stock solution, and added to the internal solution at a final concentration of 100 µg ml^-1 just before use. Test solutions were topically applied using the so-called 'Y-tube' solution exchange device (Murase et al. 1989).

Drugs used in the present experiments were BAPTA-AM, collagenase, nystatin, 1-oleoyl-2-acetyl-glycerol (OAG), prazosin, thapsigargin, trifluoperazine, trypsin, 1-[6-[[(17 $beta$ )-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), 1-[6-[[(17 $beta$ )-3-methoxyestra-1,3,5[10]-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U73343), xestospongin-C (XeC) (Sigma, St Louis, MO, USA), cirazoline, SK&F96365, WB-4101 (Tocris, Cookson, Avonmouth, UK), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (Seikagaku, Tokyo, Japan) and noradrenaline (NA; Tokyo Kasei, Tokyo, Japan).

Electrophysiological recordings

Electrical measurements were performed with the nystatin-perforated patch recording mode (Horn & Marty, 1988). Patch pipettes were made from borosilicate glass tubes in two stages on a vertical pipette puller (PP-83, Narishige, Tokyo, Japan). The resistance between the recording electrode filled with the internal solution and the reference electrode in the normal external solution was 4-8 M $Omega$ .

The cardiac ganglion neurones were visualized on an inverted microscope with phase-contrast equipment (DMIRB, Leica, Nussloch, Germany). The current signals were amplified by a patch-clamp amplifier (EPC-7, List-Medical, Darnstadt, Germany). After stable perforated patch formation, the series resistance ranged from 10 to 22 M $Omega$ and was compensated in the same manner as previously described (Murai et al. 1998). Before digitization (sampling rate 5 kHz), the signals were filtered at 2 kHz with a three-pole low-pass Bessel-type filter. Data were stored on microcomputer hard disk for subsequent analysis with the pClamp system (Axon Instruments, Foster City, CA, USA). All experiments were carried out at room temperature (21-24 °C). Statistical significance was determined by Student's t test for unpaired or paired data, or by one-way ANOVA followed by Student's t test with Bonferroni's correction for multiple comparisons. In all instances, P < 0.05 was considered statistically significant.

Fluorometric analysis of intracellular Ca²⁺concentration

The method for fluoresence measurements was essentially the same as that described elsewhere (Sorimachi et al. 2001). In brief, isolated neurones were incubated in the standard external solution with 5 µM fura-2 acetoxymethyl ester (fura-2 AM) for 30 min at 37 °C. The neurones were then washed several times with fresh standard external solution. The intracellular Ca²⁺ measurements were performed within approximately 1 h of loading using digital video imaging fluorescence microscopy ( times 40 magnification, with Eclipse E600FN, Nikon, Tokyo, Japan). During measurements, images were captured at dual wavelength (340 and 380 nm), every 2 s and analysed using MetaFluor software (Universal Imaging Corporation, Downingtown, PA, USA). All intracellular Ca²⁺ concentration ([Ca²⁺]_i) measurements were carried out at room temperature.

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Acutely isolated neurones from cardiac ganglia exhibited no spontaneous action potentials, and had resting membrane potentials in the range of -55 to - 70 mV when measured with a pipette containing 150 mM K⁺. External application of 10 µM NA depolarized the membrane, eliciting repetitive action potentials under current-clamp conditions in all neurones tested (n = 6, Fig. 1A). The repetitive firing of action potentials ceased gradually after washout of NA. In the presence of tetrodotoxin (1 µM), NA depolarized the membrane without eliciting action potentials and the mean (±S.E.M.) membrane depolarization was 18.3 ± 1.5 mV (n = 8). It is known that the activation of receptors that couple Gq/11 proteins inhibits M-type K⁺ channels (Marrion, 1997). Since NA (10 µM) markedly inhibits the M-current amplitude measured by a hyperpolarizing step pulse from a holding potential of -20 mV to -50 mV in rat cardiac ganglion neurones (authors' unpublished data), the inhibition of M-current may be involved in this membrane depolarization induced by NA. However, since M-current is deactivated at membrane potentials negative to -60 mV (Constanti & Brown, 1981), the NA-induced depolarization seems too large to result only from M-current inhibition. To examine the NA action in the absence of any contribution of M-current, the following experiments were performed under voltage-clamp conditions with a pipette solution containing 150 mM Cs⁺.

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Figure 1. Noradrenaline response in isolated cardiac ganglion neurones
A, depolarization of a rat cardiac ganglion neurone by noradrenaline (NA). Recordings were made under current-clamp conditions. Horizontal filled bar above the trace indicates the application of NA. The figure is representative of 6 reproducible experiments. B, inward currents evoked by NA (I_NA). All current traces were recorded from the same neurone. Holding potential (V_H) was -60 mV. The NA-induced current was sensitive to extracellular divalent cations. Dotted line shows the zero current level. The inset summarizes the effects of Ca²⁺ and Mg²⁺ on the NA-induced current. The current amplitude was normalized to that observed in the normal external solution containing 2 mM Ca²⁺ and 1 mM Mg²⁺. Each column shows the mean of 5-7 experiments. C, current-voltage relationship of the NA (10 µM)-induced current. The amplitude of the NA-induced current at various V_H was normalized to that observed at -60 mV in each neurone. Each point is the mean of 4-6 experiments. Vertical bars indicate ±S.E.M. D, extracellular Na⁺ dependency. The current trace is representative of 4 reproducible experiments.

NA-induced cation current

Application of 10 µM NA evoked a small inward current (17.1 ± 3.6 pA, n = 8) in the standard external solution containing 2 mM Ca²⁺ and 1 mM Mg²⁺ at a holding potential (V_H) of -60 mV (Fig. 1B). As shown in Fig. 1B, removal of extracellular Ca²⁺ markedly potentiated the NA-induced current. The current evoked by 10 µM NA in the nominally Ca²⁺-free solution reached a peak amplitude within approximately 4 s, then decayed slowly, and the current returned to control levels rapidly after washout. Removal of Mg²⁺ from the nominally Ca²⁺-free solution further increased the NA-evoked current. During the application of NA, the membrane conductance was increased (data not shown). The removal of divalent cations from the extracellular solution itself induced inward currents with a clear increase in current noise but the properties of this current were not examined in the present study.

The NA-induced current reversed near 0 mV in the Ca²⁺-free solution (Fig. 1C). In addition, replacement of all extracellular Na⁺ with equimolar N-methyl-D-glucamine fully inhibited the NA-induced current (Fig. 1D), but tetrodotoxin (1 µM) had no effect (n = 3; data not shown). These results suggest the involvement of cation channels in the NA response. As shown in Fig. 2, the cation channel blockers La³⁺, Gd³⁺, SK&F96365 and Cd²⁺ inhibited the NA-induced current in a concentration-dependent manner. The half-maximum inhibitory concentrations (IC₅₀) of La³⁺, Gd³⁺, SK&F96395 and Cd²⁺ were 0.54, 1.76, 8.45 and 188 µM, respectively.

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Figure 2. Effects of organic and inorganic cation channel blockers
Experiments were performed in Ca²⁺-free solution containing 1 mM Mg²⁺. Each blocker was applied 1 min before the application of NA. A, effect of La³⁺ on the NA-induced current. B, effect of SK&F96365. C, concentration-dependent inhibition of the NA-induced current by La³⁺, Gd³⁺, SK&F96365 and Cd²⁺. Curves are the best non-linear fits to the Hill equation, 1/1 + (C/K_i)ⁿ^H where C, K_i and n_H denote the concentration of each blocker applied, dissociation constant and Hill coefficient, respectively. Each point is the mean of 6-8 experiments. Vertical bars indicate ± S.E.M.

As shown in Fig. 3A, the amplitude of the NA-induced current was dependent on [Ca²⁺]_o, where the half-maximum [Ca²⁺]_o to suppress the inward current was 13.3 µM. To further characterize the influence of [Ca²⁺]_o, the effect of switching from a Ca²⁺-free solution to one containing 2 mM Ca²⁺ was tested. As shown in Fig. 3B, extracellular Ca²⁺ rapidly inhibited the NA-induced current within 1 s.

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Figure 3. Effects of [Ca²⁺]_o on the NA-induced current
Experiments were performed in the solution containing 1 mM Mg²⁺. A, the effect of [Ca²⁺]_o on NA-induced current. All current traces were obtained from the same neurone. NA was applied every 6 min in various [Ca²⁺]_o. Lower panel shows the [Ca²⁺]_o dependency. Current amplitude was normalized to that observed in the solution containing 30 nM Ca²⁺. Each point is the mean of 4-6 experiments. Vertical bars indicate ±S.E.M. B, rapid inhibition of the NA-induced current by extracellular Ca²⁺. During the application of NA in Ca²⁺-free solution, the switch of extracellular solution to that containing 2 mM Ca²⁺ rapidly inhibited the current within 1 s The figure is representative of 5 reproducible experiments.

The concentration-dependent effect of extracellular Mg²⁺ on the NA-induced current was also examined. As shown in Fig. 4, the NA-induced current was potentiated when extracellular Mg²⁺ concentration was reduced in the nominally Ca²⁺-free solution containing 1 mM Mg²⁺. Conversely, increases in Mg²⁺ concentration inhibited the NA-induced current. The half-maximal effect was at 1.17 mM Mg²⁺.

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Figure 4. Effects of extracellular Mg²⁺ concentration
A, representative current traces induced by NA in the solution containing various concentrations of Mg²⁺. In this experiment, Mg²⁺ was simply added to the nominally divalent cation-free solution. NA was applied every 6 min. B, concentration-inhibition curve for the NA-induced current by Mg²⁺ in divalent cation-free extracellular solution. The amplitude of the NA-induced current in various concentrations of Mg²⁺ was normalized to that in the solution containing 1 mM Mg²⁺. Each point is the mean of 4-5 experiments. Vertical bars indicate ±S.E.M.

Adrenoceptor subtype

To study the adrenoceptor subtype mediating the NA response, the effects of adrenoceptor agonists and antagonists were examined. Cirazoline, an $alpha$ ₁-adrenoceptor agonist, also evoked an inward current. The half-maximum effective concentrations of NA and cirazoline were 0.31 and 0.28 µM, respectively. Cirazoline acted as a partial agonist, and the maximum response evoked by 10 µM cirazoline was 68.5 ± 3.6 % of that induced by 10 µM NA (Fig. 5A). The current evoked by 10 µM NA was inhibited by $alpha$ ₁-adrenoceptor antagonists, such as prazosin and WB-4101 (Fig. 5B). The half-maximum inhibitory concentrations of prazosin and WB-4101 against 10 µM NA were 10.0 and 7.3 nM, respectively. On the other hand, yohimbine, an $alpha$ ₂-adrenoceptor antagonist, showed no effect on the NA (10 µM)-induced inward current even at a high concentration of 300 nM (Fig. 5B). These results indicate the involvement of the $alpha$ ₁-adrenoceptor in the NA response.

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Figure 5. Effects of adrenoceptor agonists and antagonists
A, concentration-response curve for NA and cirazoline. The current evoked by various concentrations of NA and cirazoline was normalized to that induced by 10 µM NA. In this and subsequent figures, electrophysiological experiments were performed at a V_H of -60 mV in the Ca²⁺-free solution containing 2 mM EGTA and 1 mM Mg²⁺. Each point is the mean of 4-5 experiments. Vertical bars indicate ±S.E.M. B, effects of prazosin, WB-4101 and yohimbine on the current induced by 10 µM NA. These antagonists were applied 1 min before the application of NA. Each point is the mean of 4-6 experiments. Vertical bars indicate ±S.E.M.

Involvement of Ca²⁺ released from intracellular Ca²⁺ stores via IP₃ receptors

The $alpha$ ₁-adrenoceptor couples the GTP-binding protein Gq/11, and the activation of Gq/11-coupled receptors is known to activate PLC. In the present study, therefore, the effect of a PLC inhibitor on the NA-induced current was investigated. Pretreatment with 1 µM U73122, an inhibitor of PLC, markedly inhibited the NA response (Fig. 6A). On the other hand, 1 µM U73343, an inactive isomer of U73122, had no effect, indicating the contribution of PLC to the NA response. The activation of PLC increases the intracellular IP₃ concentration and in turn induces the release of Ca²⁺ from intracellular Ca²⁺ stores via IP₃ receptors. The NA-induced current was inhibited by XeC (2 µM), a membrane-permeable blocker of intracellular IP₃ receptors (Gafni et al. 1997; see Fig. 6). These results suggest the involvement of PLC and IP₃ receptors in the NA response.

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Figure 6. Contribution of PLC and IP₃ receptors
A and B, inhibitory effects of PLC inhibitor U73122 (A) and membrane-permeable IP₃ receptor antagonist XeC (B) on the NA-induced current. Each inhibitor was applied 4 min before the application of NA. C, effects of 1 µM U73122, 1 µM U73343 and 2 µM XeC. Current responses in the presence of various inhibitors were normalized to that obtained just before the application of each inhibitor. Each column represents the mean of 4-6 experiments. ** P < 0.01 vs. control. *** P < 0.001 vs. control. N.S., no significant difference from control.

The activation of intracellular IP₃ receptors is known to cause Ca²⁺ release from intracellular Ca²⁺ stores. In order to test the contribution of Ca²⁺ release from intracellular Ca²⁺ stores to the NA response, we next investigated the effect of thapsigargin, an inhibitor of Ca²⁺-ATPase in intracellular Ca²⁺ stores. As shown in Fig. 7A, 1 µM thapsigargin, which did not itself evoke any detectable current, reduced the NA-induced current. Furthermore, the NA-induced current was inhibited by pretreatment with BAPTA-AM (10 µM), a membrane-permeable analogue of the Ca²⁺ chelator BAPTA (Fig. 7B). This observation suggests that Ca²⁺ released from intracellular Ca²⁺ stores via IP₃ receptors is involved in the NA action. To confirm this, we also examined whether [Ca²⁺]_i is increased by NA in the Ca²⁺-free solution. As shown in Fig. 7D, 10 µM NA increased [Ca²⁺]_i even in the absence of extracellular Ca²⁺. Prior to the agonist application, the mean basal fura-2 ratio was 0.59 ± 0.024 (n = 8). Upon application of 10 µM NA, the mean peak ratio was 1.02 ± 0.056 (n = 8). The effect of thapsigargin on the NA-induced increase in [Ca²⁺]_i was also examined in the Ca²⁺-free solution. Thapsigargin (3 µM) initially increased [Ca²⁺]_i, but this returned to the control level within 3 min (n = 4, data not shown). A similar transient increase in [Ca²⁺]_i induced by thapsigargin was also reported in sympathetic neurones (Stemkowski et al. 2002). Application of NA after thapsigargin treatment failed to produce a measurable increase in [Ca²⁺]_i (n = 4, data not shown).

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Figure 7. Involvement of Ca²⁺ release from intracellular Ca²⁺ stores in the NA response
A and B, representative current traces showing the inhibitory effects of thapsigargin and BAPTA-AM on the NA response. Thapsigargin and BAPTA-AM were applied 4 min before the application of NA. C, inhibitory effects of thapsigargin and BAPTA-AM on the NA-induced current. Each column represents the mean of 5-6 experiments. ** P < 0.01 vs. Control. *** P < 0.001 vs. Control. D, increase in [Ca²⁺]_i (measured as 340 nm/380 nm fura-2 fluorescence ratio) induced by NA even in the absence of extracellular Ca²⁺. The response is representative of 8 reproducible experiments.

Contribution of calmodulin

To further clarify the signal transduction pathway following the increase in intracellular free Ca²⁺ concentration, the possible contribution of Ca²⁺-calmodulin was investigated. The NA-induced current was inhibited by W-7 in a concentration-dependent way with an IC₅₀ of 10.1 µM (Fig. 8A and B). W-5, which has a similar chemical structure to W-7 but lacks chlorine in the molecule, also inhibited the NA response but its action was weaker than that of W-7 (Fig. 8B): the IC₅₀ of W-5 was 76.3 µM. The difference between the actions of W-7 and W-5 supports the specificity of these drugs for calmodulin (Kanamori et al. 1981). Furthermore, trifluoperazine (3 µM), another calmodulin antagonist, fully inhibited the NA response (n = 4; data not shown). On the other hand, pretreatment (10 min) with 3 µM KN-62, a potent inhibitor of calmodulin-dependent protein kinase II, did not affect the NA-induced current (n = 4; Fig. 8C), thereby indicating the contribution of Ca²⁺-calmodulin to the NA-induced cationic currents.

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Figure 8. Effects of calmodulin inhibitor, KN-62 and PKC modulators on the NA-induced current
A, inhibitory effect of W-7 on the NA-induced current. B, concentration-inhibition curve of W-7 and W-5 for the NA (1 µM)-induced current. W-7 and W-5 were applied 4 min before the application of NA. Each point is the mean of 5 experiments. Vertical bars indicate ±S.E.M. C, KN-62 had no effect on the NA response. D, effects of 3 µM KN-62, 0.3 µM staurosporine and 0.3 µM PMA on the NA-induced current. KN-62, staurosporine and PMA were applied 10 min before the application of NA.

The hydrolysis of phosphoinositide by PLC also produces DAG as a second messenger, which activates protein kinase C (PKC). The $alpha$ ₁-adrenoceptor-activated non-selective cation channel is activated by DAG in vascular smooth muscle cells (Helliwell & Large, 1997). Therefore, the modulatory effect of PKC on the NA-induced current was examined to clarify whether this protein kinase was involved in the NA action. Extracellular application of 0.3 µM PMA (n = 4) and 30 µM OAG (n = 4), PKC activators, failed to activate cation currents in the Ca²⁺-free solution (data not shown). In addition, as shown in Fig. 8D, 0.3 µM staurosporine, a non-specific protein kinase inhibitor, and 0.3 µM PMA had no effect on the NA-induced current.

DISCUSSION

Top
Abstract
Introduction
Methods
Results
Discussion
References

The present study has demonstrated that NA activates the extracellular Ca²⁺- and Mg²⁺-sensitive cation channels in neurones freshly isolated from rat cardiac parasympathetic ganglia. This activation involves the formation of IP₃ by PLC, release of Ca²⁺ through IP₃ receptors and calmodulin.

Cation channels activated by NA

Dependence on [Ca²⁺]_o is an interesting feature of native receptor-operated cation channels and recombinant TRP channels. The $alpha$ ₁-adrenoceptor-activated cation channels in rabbit portal vein smooth muscle cells show a biphasic dependence on [Ca²⁺]_o, i.e. potentiation and inhibition, and the half-maximal [Ca²⁺]_o for the facilitatory and inhibitory actions was 6 and 400 µM, respectively (Helliwell & Large, 1996). In the present study, the inhibitory action of [Ca²⁺]_o on NA-induced current was observed, and the half-maximum [Ca²⁺]_o was 13 µM (Fig. 3). It has been reported that several native and recombinant TRP channel currents depend on [Ca²⁺]_i (Launay et al. 2002). However, it is unlikely that the inhibitory effect of [Ca²⁺]_o observed in the present study resulted from indirect changes in [Ca²⁺]_i, since the action of extracellular Ca²⁺ was rapid in onset and recovery, typically occurring within 1 s (Fig. 3B). The rapid inhibition of cation currents by extracellular Ca²⁺ has also been reported in TRP7-expressing cells (Okada et al. 1999). In addition to the external Ca²⁺ sensitivity, the NA-induced current was sensitive to extracellular Mg²⁺ (Fig. 1B and Fig. 4), although the effect of extracellular Mg²⁺ was less than that of extracellular Ca²⁺. Similar differences between the action of Mg²⁺ and Ca²⁺ on cation channels has also been observed in other native and recombinant systems (Helliwell & Large, 1996; Mubagwa et al. 1997; Mori et al. 2001; Launay et al. 2002).

As illustrated in Fig. 2, the NA-induced current was blocked by the cation channel blocker SK&F96365 in a concentration-dependent way. In addition, other commonly used inorganic cation channel blockers including La³⁺, Gd³⁺ and Cd²⁺ also inhibited the NA-induced current. The IC₅₀ values of these blockers were similar to those observed for $alpha$ ₁-adrenoceptor-activated cation channels of smooth muscle cells and TRP6 channels (Inoue et al. 2001).

Adrenoceptor subtype

The NA-induced current was inhibited by the $alpha$ ₁-adrenoceptor antagonists WB-4101 and prazosin (Fig. 5B) in a concentration-dependent way, suggesting the involvement of the $alpha$ ₁-adrenoceptor in the NA action on cardiac ganglion neurones. On the other hand, the $alpha$ ₁-adrenoceptor agonist cirazoline, which was reported to be a full agonist at the $alpha$ ₁-adrenoceptor (Ruffolo & Waddell, 1982), acted as a partial agonist on the cardiac ganglion neurones (Fig. 5A). Interestingly, in rat brain cortical slices, cirazoline activates $alpha$ ₁-adrenoceptors and causes only 40-60 % of the inositol phospholipid hydrolysis when compared with the NA-induced hydrolysis (Li et al. 1988). As discussed below, the NA-induced cation currents in rat cardiac ganglion neurones depend on the activity of PLC. Thus, the present results also suggest that cirazoline behaves as a partial agonist at $alpha$ ₁-adrenoceptors linked to inositol phospholipid breakdown in rat cardiac ganglia.

Intracellular mechanisms

It is well known that $alpha$ ₁-adrenoceptors couple PLC via a GTP-binding protein (Gq/11). In the present study, the NA-induced current was markedly inhibited by U73122, a PLC inhibitor (Fig. 6). Conversely, U73343, an inactive isomer of U73122, had no effect. These observations indicate that NA activates cation channels via PLC in rat cardiac ganglion neurones. PLC is known to mediate the formation of IP₃ and DAG, and some TRP channels are known to respond to DAG (Hofmann et al. 1999; Okada et al. 1999; Inoue et al. 2001) or IP₃ (Kiselyov et al. 1998). In our experiments, a membrane-permeable DAG analogue, OAG, failed to induce the inward current. On the other hand, the NA-induced current was inhibited by the membrane-permeable IP₃-receptor antagonist XeC (Fig. 6), thereby indicating the contribution of IP₃ receptors.

As for the activation mechanism of receptor-operated cation channels, the activation of non-selective cation channels by pituitary adenylate cyclase-activating polypeptide (PACAP) is mediated by the IP₃ receptor in rat cultured sympathetic neurones, whereas BAPTA-AM had no effect on the PACAP action (Beaudet et al. 2000). Thus, the authors suggested that the IP₃-induced component of the PACAP action resulted from direct coupling of IP₃ receptor activation to a cationic conductance in the plasma membrane. In the present study, on the other hand, the NA-induced current was suppressed by BAPTA-AM, suggesting the involvement of Ca²⁺ release through IP₃ receptors. In fact, NA did increase [Ca²⁺]_i even in the absence of extracellular Ca²⁺ (Fig. 7D). Depletion of Ca²⁺ stores has been reported to activate the cation channels in non-excitable cells (Hoth & Penner, 1992; Mori et al. 2001; Putney et al. 2001). Thapsigargin is known to inhibit the Ca²⁺ pump of internal Ca²⁺ stores leading to depletion of the stores. Furthermore, BAPTA-AM also depletes internal Ca²⁺ stores by chelating intracellular free Ca²⁺ which is passively moving out of the stores and activates the store-operated cation channels (Albert & Large, 2002). In the present study, thapsigargin and BAPTA-AM themselves did not activate the cationic conductances. Therefore, it could be concluded that the activation of cation channels of rat cardiac ganglion neurones depends on Ca²⁺ released from Ca²⁺ stores via IP₃ receptors, but not on the depletion of the Ca²⁺ store per se.

The channel activity of several TRP proteins is affected by [Ca²⁺]_i, and a lot of Ca²⁺-dependent neuronal functions are controlled by calmodulin. In the present study, the NA-induced current was blocked by calmodulin antagonists. It has been reported that all members of 'canonical' TRP channels have calmodulin-binding sites at their C-terminus (Tang et al. 2001). Interestingly, a recent study suggested that the TRP4 protein has two amino acid residues that were identified as being able to interact with calmodulin, and the binding of calmodulin to both domains occurs only in the presence of Ca²⁺ concentrations above 10 µM (Trost et al. 2001). The detailed mechanism underlying calmodulin-dependent activation of cation channels in rat cardiac ganglion neurones is still unclear and remains for future studies.

Physiological implications

It is well documented that sympathetic and parasympathetic nervous systems have antagonistic actions on the heart, and interactions between the two systems are also well known (Levy, 1971; Manabe et al. 1991). For example, extracellular recordings revealed that the canine atrial ganglion neurones can be activated by stimulating the sympathetic postganglionic fibres (Gagliardi et al. 1988). The present study demonstrates that NA induces a depolarization of rat cardiac parasympathetic neurones which is accompanied by repetitive action potentials (Fig. 1A). Thus, it is likely that the stimulation of sympathetic nerves to the heart increases the excitability of cardiac parasympathetic ganglion neurones, which would produce inhibitory effects on the heart, in addition to the direct cardiostimulatory actions of the sympathetic nervous system. Interestingly, it has been reported that there is a period of cardioinhibition after the cessation of stimulation of the sympathetic nerves and that this poststimulation response is inhibited by atropine (Leaders, 1963). From the present and previous (Leaders, 1963) observations, it seems reasonable to postulate that the activation of the sympathetic nervous system triggers a 'braking' system through the parasympathetic nervous system, which induces sufficient deceleration of cardiostimulatory action with a short delay after the sympathetic activity. The activation of cation channels by NA, which produces depolarization of cardiac parasympathetic neurones followed by repetitive firings, may play an important 'braking' role in the neuronal control of the mammalian heart. In the parasympathetic nervous system, a 'braking' system was documented in relation to cholinergic bronchoconstriction (Hakoda & Ito, 1990; Waniishi et al. 1998).

On the other hand, it should also be pointed out that NA inhibits voltage-dependent Ca²⁺ channels in the somata of cardiac ganglion neurones through $alpha$ -adrenoceptors (Xu & Adams, 1993). Therefore, NA might reduce the ACh release from parasympathetic nerve terminals assuming that the postganglionic parasymapthetic nerve terminals possess adrenoceptors and calcium channels similar to those reported in the neuronal somata. In fact, some investigators have reported that NA decreases the amount of ACh released from cardiac parasympathetic nerve terminals (Loiacono & Story, 1986; McDonough et al. 1986). Thus, the suppression of ACh release from the parasympathetic nerve terminals would enhance the cardiostimulatory actions of the sympathetic nervous system, when both systems act on the heart at the same time.

In the present experiments, we found that the depolarizing action of NA on cardiac ganglion neurones ceased gradually after washout of NA (Fig. 1A), thereby indicating that the action of NA on the somata may last for several minutes. Conversely, the inhibition of voltage-dependent Ca²⁺ channels by NA might last for only a few seconds after washout, since the recovery upon rinsing occured rapidly within 9 s (Ishibashi & Akaike, 1995). These observations suggest that the activation of parasympathetic ganglion neurones induced by activation of sympathetic nerves lasts longer than the inhibitory action on the voltage-dependent Ca²⁺ channels, which might regulate the ACh release from parasympathetic nerve terminals. Therefore, it seems reasonable to assume that the ACh release from parasympathetic nerve terminals may increase, thereby causing cardioinhibitory actions with a delay after the cessation of sympathetic activity.

In conclusion, electrophysiological evidence obtained in the present study shows that NA activates cation channels and depolarizes the neurones followed by repetitive firing in rat cardiac parasympathetic ganglia. These excitatory actions of NA would facilitate the parasympathetic inhibitory drive to cardiac tissue, thereby acting as a 'braking' system for the cardiostimulatory action of the sympathetic nervous system.

REFERENCES

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Abstract
Introduction
Methods
Results
Discussion
References

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Acknowledgements

We thank Dr J. Nabekura and Dr Y. Mizoguchi for their technical support with intracellular Ca²⁺ measurements. We also thank Dr R. Inoue for his valuable comments and Dr K. E. Creed for correcting the English. I.-S. Jang is a postdoctoral fellow (P02235) of the Japan Society for the Promotion of Science. This study was supported by Grants-in-Aid for Scientific Research (13307003 to N.A. and 14002235 to Y.I.) from the Japan Society for the Promotion of Science.

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