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J Physiol (2003), 553.3, pp. 789-802
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.052449
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ABSTRACT |
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Acetylcholine has long been known to excite sympathetic neurons via M1 muscarinic receptors through an inhibition of M-currents. Nevertheless, it remained controversial whether activation of muscarinic receptors is also sufficient to trigger noradrenaline release from sympathetic neurons. In primary cultures of rat superior cervical ganglia, the muscarinic agonist oxotremorine M inhibited M-currents with half-maximal effects at 1 µM and induced the release of previously incorporated [3H]noradrenaline with half-maximal effects at 10 µM. This latter action was not affected by the nicotinic antagonist mecamylamine which, however, abolished currents through nicotinic receptors elicited by high oxotremorine M concentrations. Ablation of the signalling cascades linked to inhibitory G proteins by pertussis toxin potentiated the release stimulating effect of oxotremorine M, and the half-maximal concentration required to stimulate noradrenaline release was decreased to 3 µM. Pirenzepine antagonized the inhibition of M-currents and the induction of release by oxotremorine M with identical apparent affinity, and both effects were abolished by the muscarinic toxin 7. These results indicate that one muscarinic receptor subtype, namely M1, mediates these two effects. Retigabine, which enhances M-currents, abolished the release induced by oxotremorine M, but left electrically induced release unaltered. Moreover, retigabine shifted the voltage-dependent activation of M-currents by about 20 mV to more negative potentials and caused 20 mV hyperpolarisations of the membrane potential. In the absence of retigabine, oxotremorine M depolarised the neurons and elicited action potential discharges in 8 of 23 neurons; in its presence, oxotremorine M still caused equal depolarisations, but always failed to trigger action potentials. Action potential waveforms caused by current injection were not affected by retigabine. These results indicate that the inhibition of M-currents is the basis for the stimulation of transmitter release from sympathetic neurons via M1 muscarinic receptors.
(Received 30 July 2003; accepted after revision 9 October 2003; first published online 10 October 2003)
Corresponding author S. Boehm: Department of Pharmacology, University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria. Email: stefan.boehm{at}univie.ac.at
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INTRODUCTION |
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Acetylcholine has long been recognized as the major ganglionic transmitter in the sympathetic nervous system (Feldberg & Gaddum, 1934). After being released from preganglionic axon terminals, the neurotransmitter depolarises the postganglionic sympathetic neurons in two phases: within milliseconds, nicotinic acetylcholine receptors (nAChRs) are activated to mediate a large depolarisation; thereafter, within a timescale of seconds, muscarinic acetylcholine receptors (mAChRs) are activated, which mediate a minor depolarisation by a few millivolts only (Brown, 1983). Both nAChRs and mAChRs can mediate ganglionic transmission independently of each other (Trendelenburg, 1966; Brown, 1967). The slow cholinergic excitation is mediated by an inhibition of M-type K+ (KM) channels. These ion channels are opened in the subthreshold voltage range for action potentials and become completely activated when neurons are further depolarised. Hence activated KM channels keep neurons polarized, and closure of these ion channels causes depolarisation and increased action potential discharge (Brown, 1983; Marrion, 1997). The inhibition of KM channels by acetylcholine involves M1 mAChRs (Marrion et al. 1989; Bernheim et al. 1992), 
q subunits of heterotrimeric GTP binding proteins (Haley et al. 1998), and an activation of phospholipase C (Suh & Hille, 2002).
In primary cultures of dissociated sympathetic ganglia of various species, acetylcholine (Boehm & Huck, 1995) and other agonists at nAChRs, such as nicotine (Greene & Rein, 1978; Boehm & Huck, 1996) and DMPP (Dolezal et al. 1994) trigger noradrenaline release. With respect to agonists at mAChRs, however, divergent results have been obtained. In cultures derived from chick embryos, muscarinic agonists, including methacholine and arecoline, triggered [3H]noradrenaline release, and the stimulatory action of methacholine was antagonized by nanomolar concentrations of atropine (Greene & Rein, 1978). Bhave et al. (1988), in contrast, were unable to detect transmitter release from chick sympathetic neurons when challenged by an activation of muscarinic receptors, even though this stimulation clearly increased the intracellular levels of inositol phosphates. In cultures of rat and mouse sympathetic neurons, the muscarinic agonist oxotremorine M (OxoM) at 10-100 µM did trigger transmitter release (Nörenberg et al. 2000, 2001). However, in these latter studies, antagonists have not been used, and oxotremorine M is known to quite potently activate nicotinic acetylcholine receptors (Reitstetter et al. 1994). Thus the presence of release stimulating muscarinic receptors in primary cultures of postganglionic sympathetic neurons remained to be confirmed.
Apart from excitatory mAChRs as described above, sympathetic neurons in cell culture are known to express inhibitory mAChRs, activation of which reduces rather than stimulates noradrenaline release. In rat superior cervical ganglion (SCG) neurons (Boehm & Huck, 1995), for instance, atropine at nanomolar concentrations caused a marked increase (instead of a decrease, as above) in acetylcholine-evoked noradrenaline release. Kindred results have been obtained in rabbit hearts (Lindmar et al. 1968) and are indicative of release-inhibiting presynaptic mAChRs. The presence of such receptors in SCG neurons was confirmed by Koh & Hille (1997), who showed that pirenzepine-insensitive (i.e. not M1) muscarinic receptors reduced noradrenaline release. Most recently, mAChRs of the M2 subtype were found to reduce electrically evoked transmitter release in cultured paravertebral sympathetic neurons from neonatal mice (Göbel et al. 2000).
Transmitter release strictly depends on transmembrane Ca2+ influx. In sympathetic neurons, it is primarily voltage-gated Ca2+ channels of the N-type that contribute to this Ca2+ influx, and blockade of these channels prevents transmitter release. Furthermore, the G protein-mediated inhibition of these channels is believed to underlie the inhibition of sympathetic transmitter release via presynaptic receptors (Boehm & Kubista, 2002). In support of this hypothesis, mAChRs were found to inhibit N-type Ca2+ channels (Bernheim et al. 1992) in rat SCG neurons, and the mAChR-mediated inhibition of transmitter release from these neurons was lost when N-type Ca2+ channels were blocked by 
-conotoxin GVIA (Koh & Hille, 1997).
In contrast to Ca2+ channel blockers, blockers of KM channels, such as Ba2+ and linopirdine, were reported to induce instead of inhibit noradrenaline release from rat SCG neurons in a Ca2+-dependent manner (Kristufek et al. 1999). Accordingly, the inhibition of these channels via M1 mAChRs can also be expected to trigger transmitter release. Here, we tested this hypothesis by using the muscarinic receptor agonist OxoM and the KM channel opener retigabine.
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METHODS |
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Primary cultures of rat superior cervical ganglion neurons
Primary cultures of dissociated SCG neurons from neonatal rats were prepared as described previously (Boehm, 1999). Briefly, ganglia were dissected from 2- to 6-day-old Sprague-Dawley rat pups which had been killed by decapitation in accordance with the rules of the university animal welfare committee. Ganglia were cut into three to four pieces and incubated in collagenase (1.5 mg ml-1; Sigma) and dispase (3.0 mg ml-1; Boehringer Mannheim) for 20 min at 36 °C. Subsequently, they were trypsinised (0.25 % trypsin; Worthington) for 15 min at 36 °C, dissociated by trituration, and resuspended in Dulbecco's modified Eagle's medium (Gibco) containing 2.2 g l-1 glucose, 10 mg l-1 insulin, 25 000 IU l-1 penicillin and 25 mg l-1 streptomycin (Gibco), 50 µg l-1 nerve growth factor (Gibco), and 5 % fetal calf serum (Gibco). Thereafter, the cells were plated onto 5 mm discs for superfusion experiments and onto 35 mm culture dishes (Nunc) for electrophysiological experiments, both coated with rat tail collagen (Biomedical Technologies Inc., Stoughton, MA, USA). All cultures were kept in a humidified 5 % CO2 atmosphere at 36 °C for 4-8 days. On day 1 after plating, the medium was completely exchanged, and after 4-5 days, the medium was exchanged again and the serum was omitted.
Electrophysiology
Experiments were performed at room temperature (20-24 °C) on the somata of single SCG neurons using the perforated-patch modification of the patch-clamp technique (Rae et al. 1991) which prevents rundown of IM (see Boehm, 1998). Patch pipettes were pulled (Flaming-Brown puller, Sutter Instruments, Novato, CA, USA) from borosilicate glass capillaries (Science Products, Frankfurt/Main, Germany) and front-filled with a solution consisting of (mM) K2SO4 (75), KCl (55), MgCl2 (8) and Hepes (10), adjusted to pH 7.3 with KOH. Then electrodes were back-filled with the same solution containing 200 µg ml-1 amphotericin B (in 0.8 % DMSO), which yielded tip resistances of 1-3 M
. The bathing solution contained (mM) NaCl (140), KCl (6.0), CaCl2 (2.0), MgCl2 (2.0), glucose (20) and Hepes (10), adjusted to pH 7.4 with NaOH. Tetrodotoxin (TTX; 0.5 µM) was included to suppress voltage-activated Na+ currents. OxoM, retigabine and all other drugs were applied via a DAD-12 drug application device (Adams & List, Westbury, NY, USA), which permits a complete exchange of solutions surrounding the cells under investigation within less than 100 ms (see Boehm, 1999). To investigate IM, cells were held at a potential of -30 mV, and three times per minute 1 s hyperpolarisations to -55 mV were applied to deactivate KM channels; the difference between current amplitudes 20 ms after the onset of hyperpolarisations and 20 ms prior to redepolarisation was taken as a measure for IM. Amplitudes obtained during the application of test drugs (b) were compared with those measured before (a) and after (c) application of these drugs by calculating 200b/(a + c) = % of control or 100 - (200b/(a + c)) = % inhibition (see Boehm, 1998).
Measurement of [3H]noradrenaline release
[3H]Noradrenaline uptake and superfusion were performed as described previously (Boehm, 1999). Culture discs with dissociated neurons were incubated in 0.05 µM [3H]noradrenaline (specific activity 52.0 Ci mmol-1) in culture medium supplemented with 1 mM ascorbic acid at 36 °C for 1 h. After labelling, culture discs were transferred to small chambers and superfused with a buffer containing (mM): NaCl (120), KCl (6.0), CaCl2 (2.0), MgCl2 (2.0), glucose (20), Hepes (10), fumaric acid (0.5), sodium pyruvate (5.0) and ascorbic acid (0.57), adjusted to pH 7.4 with NaOH. Superfusion was performed at 25 °C at a rate of about 1.0 ml min-1. Collection of 4 min superfusate fractions was started after a 60 min washout period to remove excess radioactivity.
To investigate noradrenaline release evoked by OxoM, [3H] overflow was induced by the inclusion of this muscarinic agonist in the superfusion buffer for 2 min, unless indicated otherwise. Tritium overflow was also elicited by the application of sixty monophasic rectangular electrical pulses (0.5 ms, 60 mA, 50 V cm-1) at 1.0 Hz and by the presence of the nicotinic agonist DMPP for 2 min. Modulatory agents such as TTX, CdCl2, mecamylamine or retigabine were added to, or CaCl2 was omitted from, the buffer after 50 min of superfusion (i.e. 10 min prior to the start of sample collection). The buffer then remained unchanged until the end of experiments. The radioactivity remaining in the cells after the completion of experiments was extracted by immersion of the discs in 2 % (v/v) perchloric acid followed by sonication. Radioactivity in extracts and collected fractions was determined by liquid scintillation counting (Packard Tri-Carb 2100 TR). Radioactivity released in response to electrical field stimulation from rat sympathetic neurons after labelling with tritiated noradrenaline under conditions similar to those of the present study had previously been shown to consist predominantly of the authentic transmitter and to contain only small amounts (
15 %) of metabolites (Schwartz & Malik, 1993). Hence the outflow of tritium measured in this study was assumed to reflect the release of noradrenaline and not that of metabolites.
The spontaneous (unstimulated) rate of [3H] outflow was obtained by expressing the radioactivity of a collected fraction as a percentage of the total radioactivity in the cultures at the beginning of the corresponding collection period. Stimulation-evoked tritium overflow was calculated as the difference between the total [3H] outflow during and after stimulation and the estimated basal outflow, which was assumed to decline linearly throughout experiments. Therefore, basal outflow during periods of stimulation was assumed to equate to the arithmetic mean of the samples preceding and those following stimulation, respectively. The difference between the total and the estimated basal outflow was expressed as a percentage of the total radioactivity in the cultures at the beginning of the respective stimulation (% of total radioactivity; S %). The amount of OxoM-evoked tritium release may vary considerably between different SCG preparations. Therefore, tritium overflow in the presence of release modulating agents, such TTX, CdCl2 or retigabine, was always compared with that obtained within the same SCG preparation in the absence of these drugs. To directly compare effects of different modulatory agents upon electrically and OxoM-evoked overflow, respectively, the values obtained in the presence of these modulators were expressed as percentage of the corresponding control values within the same preparation.
Statistics
Results are presented as arithmetic means ± standard errors of the mean (S.E.M.), and n is the number of cultures in release experiments and of single cells in electrophysiological experiments. Significances of differences between data points were evaluated by the non-parametric Mann-Whitney test, unless indicated otherwise.
Materials
(-)-[7,8-3H]Noradrenaline was obtained from NEN (Vienna, Austria); amphotericin B, OxoM, pirenzepine, mecamylamine, bethanechol and thapsigargin were from Sigma (Vienna, Austria); TTX was from Latoxan (Rosans, France); muscarinic toxin 7 (MT-7) was from the Peptide Institute Inc. (Osaka, Japan); bulk chemicals were from Merck (Vienna, Austria). Retigabine was kindly donated by Dr B. Pechstein, Viatris, Frankfurt/Main, Germany. Water-insoluble drugs were first dissolved in DMSO and then diluted into buffer to yield final DMSO concentrations of up to 0.1 %, which were also included in control solutions. At these concentrations, DMSO did not affect any of the parameters investigated.
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RESULTS |
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OxoM stimulates noradrenaline release and inhibits IM with different concentration dependences
To evaluate whether activation of muscarinic receptors may stimulate transmitter release from sympathetic neurons, cultures of dissociated rat SCG were loaded with [3H]noradrenaline, superfused with a physiological buffer, and exposed to various concentrations of OxoM for 2 min. Figure 1A shows that 1-100 µM OxoM stimulates the release of increasing amounts of radioactivity. This effect reached a maximum at 30-100 µM where 2.64 ± 0.28 % (n = 9) of the cellular radioactivity was released by the muscarinic agonist. At these concentrations, OxoM also reduced currents through KM channels, as evaluated by the reduction of IM deactivation amplitudes (Fig. 1C). This second effect reached a maximum of 85.3 ± 5.0 % (n = 6) inhibition at 10 µM. The two effects, stimulation of tritium release and inhibition of IM, showed clearly diverging concentration dependences, as half-maximal effects were observed at 10 and 1 µM, respectively (Fig. 1B and D).
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Figure 1. OxoM stimulates noradrenaline release and inhibits IM in rat SCG neurons A, primary cultures of rat SCG neurons were labelled with [3H]noradrenaline and superfused. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected. 1, 10 and 100 µM OxoM was applied as indicated by the arrows, each for 2 min. The graph shows the time course of fractional [3H] outflow per minute; n = 6. B shows the concentration dependence of tritium overflow evoked by the indicated concentrations of OxoM, applied as shown in A. OxoM-induced overflow is depicted as S % (% of total radioactivity; see Methods); n = 9. C, IM was measured using the amphotericin B perforated-patch technique in a SCG neuron. The current traces shown were obtained by clamping the cell at - 30 mV and by applying 1 s hyperpolarizing voltage steps to - 55 mV. The recordings were performed before (control), during (OxoM) and after (washout) the application of 10 or 100 µM OxoM. D, concentration dependence of the inhibition of IM relaxation amplitudes by OxoM in experiments performed as shown in C; n = 6. | ||
The stimulation of noradrenaline release by OxoM does not involve nAChRs
OxoM has been found to activate nAChRs in Xenopus myocytes (Reitstetter et al. 1994) and in guinea-pig coeliac ganglion neurons (Xian et al. 1994). To verify whether nAChRs might contribute to the secretagogue action of OxoM in rat sympathetic neurons, tritium overflow was stimulated by electrical fields, OxoM (100 µM), and DMPP (100 µM) in either the absence or presence of 10 µM of the nicotinic antagonist mecamylamine (Fig. 2A and B). Electrically evoked overflow was not affected by mecamylamine, whereas the overflow due to DMPP was virtually abolished. The release stimulating action of OxoM was somewhat reduced in the presence of mecamylamine, but this effect was not statistically significant (P > 0.05).
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Figure 2. The stimulation of noradrenaline release by OxoM does not involve nAChRs A, cultures were labelled with [3H]noradrenaline and superfused. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected. Tritium overflow was evoked by electrical field stimulation (EFS; 60 pulses, 1 Hz) starting at 72 min of superfusion and by application of either 100 µM OxoM or 100 µM DMPP for 2 min starting at 92 min of superfusion. 10 µM Mecamylamine was absent ( | ||
To reveal whether OxoM might at all be able to activate nAChRs in sympathetic neurons of the rat, currents through these receptors were determined. As shown in Fig. 2 (C-E), OxoM did induce currents that were abolished by mecamylamine. However, to activate these currents, concentrations of 
100 µM OxoM were required (Fig. 2D), and currents induced by 100 µM OxoM amounted to only 4.9 ± 3.7 % of the currents evoked by 100 µM DMPP (Fig. 2E). Thus activation of nAChRs by OxoM does not occur at concentrations lower than 100 µM, and even at 100 µM, does not significantly contribute to the release stimulating action of OxoM.
Pertussis toxin potentiates the stimulation of noradrenaline release by OxoM
Muscarinic receptors may be linked to proteins of either the Gi or Gq family (Caulfield & Birdsall, 1998). To investigate which types of G proteins are involved in the stimulation of tritium overflow by OxoM, cultures were treated with pertussis toxin (PTX; 100 ng ml-1 for 24 h), which abolishes signalling via the Gi protein family. In PTX-treated cultures, electrically evoked overflow remained unaltered, but OxoM-induced overflow was enhanced fivefold (Fig. 3A and B). This indicates that OxoM-induced noradrenaline release is restricted by a mechanism that involves PTX-sensitive G proteins. In a separate series of experiments, SCG neurons were subsequently stimulated with 100 µM DMPP and 100 µM OxoM in either untreated or PTX-treated cultures. Due to the PTX treatment, the tritium overflow triggered by OxoM was enhanced from 0.99 ± 0.21 to 5.68 ± 0.69 % of total radioactivity (S %; n = 6; P < 0.01), but the overflow induced by the nicotinic agonist DMPP remained unchanged (0.99 ± 0.12 % of total radioactivity in untreated and 0.86 ± 0.15 % of total radioactivity in PTX-treated cultures; n = 6; P > 0.3). This indicates that noradrenaline release induced by OxoM is restricted by a mechanism that involves PTX-sensitive G proteins, whereas other types of stimulation-evoked release are not.
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Figure 3. Pertussis toxin potentiates the stimulation of noradrenaline release by OxoM A, cultures were treated with 100 ng ml-1 pertussis toxin (PTX) for 24 h ( | ||
Concentration dependence and basic mechanisms of noradrenaline release induced by OxoM
To reveal whether this inhibitory PTX-sensitive mechanism might affect the concentration dependence of OxoM-induced noradrenaline release, SCG cultures were treated with PTX or remained untreated, were superfused with a physiological buffer, and were exposed to various concentrations of OxoM for 2 min. In untreated cultures, the release stimulating effect of OxoM was half-maximal at 10.9 ± 8.2 µM and reached a maximum of 3.1 ± 0.6 % of the total radioactivity (S %). In PTX-treated cultures, the maximal effect of OxoM was markedly enhanced and amounted to 8.0 ± 0.7 % of total radioactivity (S %). Furthermore, the concentration-response curve was shifted to the left, and the half-maximal concentration was 2.1 ± 0.5 µM (Fig. 4A). Thus in PTX-treated neurons OxoM stimulates noradrenaline release in the same range of concentrations as it inhibits IM (see Fig. 1D).
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Figure 4. Concentration dependence and basic mechanisms of noradrenaline release induced by OxoM Cultures were treated with 100 ng ml-1 pertussis toxin (PTX) for 24 h ( | ||
To investigate the mechanisms underlying the release stimulating action of OxoM in isolation, all subsequent radiotracer release experiments were performed in PTX-treated cultures. In the absence of extracellular Ca2+, neither electrical field stimulation nor OxoM were able to induce tritium overflow (Fig. 4B and C). Likewise, in the presence of 0.5 µM TTX to prevent action potential propagation, neither stimulus produced tritium overflow. Finally, the blockade of voltage-gated Ca2+ channels by 100 µM Cd2+ also abolished electrically as well as OxoM-induced [3H] overflow (Fig. 4C). Thus OxoM triggers noradrenaline release via the same mechanisms as electrical field stimulation: generation of action potentials and subsequent transmembrane Ca2+ influx through voltage-gated Ca2+ channels.
One mAChR subtype mediates both,the inhibition of IM and the stimulation of noradrenaline release by OxoM
The muscarinic receptor that mediates the inhibition of IM has been reported to belong to the M1 subtype (Marrion et al. 1989; Bernheim et al. 1992). To investigate whether the induction of transmitter release by OxoM is mediated by the same receptor subtype, concentration-response curves for both types of effect were obtained in the absence and presence of the M1 preferring mAChR antagonist pirenzepine. OxoM inhibited IM (in untreated neurons) and evoked noradrenaline release (in PTX-treated cultures) with half-maximal effects at 0.8 ± 0.5 and 3.2 ± 0.7 µM, respectively. In the presence of pirenzepine (0.1 µM), both concentration-response curves were shifted to the right, and the half-maximal concentrations amounted to 3.9 ± 2.0 and 10.8 ± 14 µM, respectively (Fig. 5A and B). Assuming an underlying competitive mechanism, one can calculate a pA2 value for pirenzepine for these two types of effect by applying the equation log (CR - 1) = pA2 + log [B] (where CR is the ratio of equieffective agonist concentrations in the presence and absence of the antagonist concentration B, respectively; Arunlakshana & Schild, 1959). These pA2 values were 7.6 for the inhibition of IM and 7.4 for the stimulation of noradrenaline release. Thus pirenzepine interfered with the OxoM-dependent inhibition of IM in the same range of concentrations as with the stimulation of noradrenaline release by OxoM.
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Figure 5. Characterization of the receptor mediating the stimulation of noradrenaline release and the inhibition of IM by OxoM IM was recorded from rat SCG neurons using the amphotericin B perforated-patch technique, and current traces were obtained by clamping the cell at - 30 mV and by applying 1 s hyperpolarizing voltage steps to -55 mV once every 20 s (A, C, E). Alternatively, cultures were labelled with [3H]noradrenaline and superfused. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected, and tritium overflow was evoked by electrical field stimulation (EFS; 60 pulses, 1 Hz) or by 2 min applications of the indicated concentrations of OxoM (B, D, F). A shows the concentration dependence of the inhibition of IM by OxoM applied either alone ( | ||
In order to further characterize the receptor mediating these two types of effect, cultures were incubated in MT-7 (100 nM for 60 min), which selectively and irreversibly blocks M1 mAChRs (Olianas et al. 2000). In cultures treated with MT-7, OxoM (10 µM) failed to cause significant changes in IM and to evoke noradrenaline release. In sister cultures not treated with MT-7, however, OxoM clearly reduced IM relaxation amplitudes and stimulated noradrenaline release (Fig. 5C and D). In an additional set of experiments, the actions of OxoM were compared with those of the muscarinic agonist bethanechol, which has been shown to selectively activate M2 mAChRs in rat SCG neurons (Liu & Rittenhouse, 2003). While OxoM (10 µM) unequivocally reduced IM relaxation amplitudes and stimulated tritium overflow, bethanechol (100 µM) failed to mimic either of these two effects (Fig. 5E and F). Nevertheless, bethanechol (100 µM) did reduce electrically evoked tritium overflow by 18.6 ± 2.2 % (n = 8; P < 0.01). In parallel experiments, 10 µM OxoM inhibited electrically evoked overflow by 25.1 ± 7.9 % (n = 9; P < 0.05).
Retigabine interferes with both the inhibition of IM and the stimulation of noradrenaline release by OxoM
The anticonvulsant retigabine has been shown to selectively enhance IM in SCG neurons by shifting the voltage-dependent gating of KM channels to more negative potentials (Tatulian et al. 2001). When retigabine was used in radiotracer release experiments, it suppressed the release stimulating effect of OxoM in a concentration-dependent manner, but left electrically evoked tritium overflow unaltered (Fig. 6A). The inhibition of OxoM-induced [3H] overflow was half-maximal at 0.1 µM retigabine, whereas the overflow due to electrical stimulation was not affected by retigabine concentrations as high as 10 µM (Fig. 6B).
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Figure 6. Effects of retigabine on noradrenaline release and IM A and B, cultures were labelled with [3H]noradrenaline and superfused. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected. Tritium overflow was evoked by electrical field stimulation (EFS; 60 pulses, 1 Hz) starting at 72 min of superfusion and by the application of 10 µM OxoM for 2 min starting at 92 min of superfusion. From minute 50 of superfusion onward, the buffer contained either the indicated concentrations of or no retigabine. A shows the time course of fractional [3H] outflow per minute in the absence ( | ||
To analyse the mechanisms of the functional antagonism between retigabine and OxoM with respect to noradrenaline release, both agents were also applied in perforated-patch recordings of IM. As seen in the original recordings shown in Fig. 6C, OxoM reduced holding currents determined at a potential of -30 mV from 389.8 ± 68.8 to 146.6 ± 37.2 pA (n = 7; P < 0.01, paired t test) on one hand, and the IM relaxation amplitudes due to hyperpolarisation of the neurons to -55 mV from 120.0 ± 23.9 to 44.0 ± 15.8 pA (n = 7; P < 0.001, paired t test), on the other hand. Retigabine (10 µM) augmented outward holding currents at -30 mV from 389.8 ± 68.8 to 661.1 ± 122.6 pA (n = 7; P < 0.01, paired t test), but reduced IM relaxation amplitudes (120.0 ± 23.9 pA before and 68.6 ± 12.4 pA in the presence of retigabine; n = 7; P < 0.01, paired t test). In the presence of retigabine (10 µM), OxoM (10 µM) still reduced the holding currents at -30 mV from 661.1 ± 122.6 to 385.2 ± 89.6 pA (n = 7; P < 0.01, paired t test), but failed to alter the relaxation amplitudes (68.6 ± 12.4 pA in the presence of retigabine alone and 76.6 ± 25.8 pA in the presence of retigabine plus OxoM; n = 7; P > 0.5, paired t test). In order to resolve these apparent inconsistencies, slow ramp hyper-polarisations from -25 to -100 mV were applied in the absence and presence of retigabine. As shown in Fig. 6D, retigabine shifted the current-voltage relationship of outward currents by about 20 mV to the left. Accordingly, hyperpolarisations from -30 to -55 mV are close to the activation threshold of IM in the absence of retigabine and will thus lead to the closure of the channels. In the presence of retigabine, however, the activation threshold is at around -80 mV, and thus at potentials of -55 mV KM channels are far from closing. Therefore, the interaction between retigabine and OxoM with respect to IM was further evaluated by changes in the holding current at -30 mV. Retigabine enhanced these holding currents in the absence and presence of OxoM with similar concentration dependence, and in the presence of 10 µM retigabine plus 10 µM OxoM, the holding currents were identical to those in the absence of both agents (Fig. 6E). Thus the inhibitory effect of OxoM on IM was counteracted by increasing concentrations of retigabine.
Retigabine prevents action potential discharge, but not depolarisation, in the presence of OxoM
To reveal how the above interactions between OxoM and retigabine at the levels of IM might translate into changes in transmitter release, changes in membrane potentials were determined by perforated-patch current-clamp recordings. OxoM (10 µM) reproducibly depolarised SCG neurons from -54.6 ± 1.5 to -41.5 ± 2.5 mV and triggered action potentials in 8 out of 23 cells (Fig. 7A and B). In the presence of retigabine (10 µM), neurons were hyper-polarized to -76.6 ± 0.7 mV and the addition of OxoM then depolarised the neurons to -67.1 ± 2.5 mV. In the presence of retigabine, however, OxoM always failed to trigger action potentials. Hence retigabine did not abolish the depolarising action of OxoM, but only brought the membrane potential to more hyperpolarised values and thus further away from the action potential threshold.
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Figure 7. Effects of retigabine on the membrane potential Membrane potentials of SCG neurons were recorded in the current-clamp mode using the amphotericin B perforated-patch technique. A shows the time course of the membrane potential of one SCG neuron. OxoM and retigabine (both at 10 µM) were applied as indicated by the horizontal bars. Note the firing of action potentials in the presence of OxoM. B summarizes the effects of 10 µM OxoM and 10 µM retigabine, applied either alone or together, on the membrane potentials of 6-8 SCG neurons; experiments were carried out as shown in A. C shows the time course of the membrane potential. OxoM and retigabine (both at 10 µM) were applied as indicated by the horizontal bars. Subsequently to the application of retigabine, a hyperpolarizing current was injected to bring the membrane to the same potential as 10 µM retigabine did. Note that OxoM failed to cause a significant depolarisation only during current injection. D summarizes the depolarisations caused by 10 µM OxoM applied either alone (control), in the presence of 10 µM retigabine, or during the injection of hyperpolarizing currents, as described in C; n = 5-6; *P < 0.05 vs. the depolarisation under control conditions. | ||
This result raises the question as to whether OxoM depolarised the neurons in the presence of retigabine also by causing a closure of KM channels. Since retigabine hyperpolarized the neurons by about the same value (22 mV, see above) as it shifted the voltage dependence of the outward currents (about -20 mV, see Fig. 6D), one may assume that the conductance of the KM channels in current-clamped cells was the same in the absence and presence of retigabine (see also Tatulian & Brown, 2003). Hence the effect of muscarinic receptor activation on the KM channel conductance, and thus on the membrane potential, should be the same whether retigabine was present or not. In support of this hypothesis, the depolarisation elicited by 10 µM OxoM was almost identical in the absence and presence of 10 µM retigabine (Fig. 7B).
If the depolarising action of OxoM is only due to the closure of KM channels, the muscarinic agonist should lose this action once KM channels are closed a priori. To test for this hypothesis, we first checked the hyperpolarising effect of retigabine (10 µM) and then injected a hyperpolarising current to bring the membrane to exactly the same potential again, but in the absence of retigabine (Fig. 7C). This procedure can be expected to entirely close KM channels (see the current-voltage curve in Fig.6D). When OxoM (10 µM) was applied during the injection of this hyperpolarising current, its depolarising action was largely attenuated. For comparison, in the presence of retigabine OxoM depolarised the neurons by the same extent as in its absence (Fig. 7C and D).
Retigabine does not alter action potential waveforms nor electrically evoked noradrenaline release
The fact that retigabine caused hyperpolarisations by about 20 mV, but failed to alter electrically evoked tritium overflow, appears contradictory. To resolve this issue, 0.5 ms depolarising currents were injected and the arising action potentials were recorded in the absence and presence of 10 µM retigabine. The waveforms of elicited action potentials were not affected by retigabine, even though the neurons were hyperpolarised by about 20 mV (Fig. 8A). However, in the presence of retigabine single SCG neurons were more reluctant to fire action potentials: at current strengths of 10 or 20 nA, more neurons fired action potentials in the absence than in the presence of retigabine (Fig. 8B).
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Figure 8. Retigabine does not alter action potential waveforms nor electrically evoked noradrenaline release Membrane potentials of SCG neurons were recorded in the current-clamp mode using the amphotericin B perforated-patch technique. Action potentials were evoked by 0.5 ms depolarising current injection in the presence and absence of 500 nM TTX. To visualize the pure action potential, traces recorded in the presence of TTX were digitally subtracted from those recorded in its absence. A shows action potentials evoked by the injection of a depolarising 20 nA current in either the absence or presence of 10 µM retigabine. The values of the membrane potentials prior to the action potentials are indicated. B shows the relation between injected currents and the percentage of neurons firing action potentials in response to these currents in either the absence ( | ||
This result leads to the question whether retigabine might also cause a reduction of tritium outflow if electrical fields of different strengths are applied. Accordingly, the current intensities used to trigger transmitter release were reduced from 60 mA (which was routinely used in all other experiments) to 30 mA and subsequently 15 mA (Fig. 8C). The amount of tritium overflow decreased in parallel with these decreased stimulation intensities, but 10 µM retigabine always failed to cause a significant inhibition of transmitter release (Fig. 8D). This lack of direct correlation between the triggering of action potentials and resulting transmitter release is presumably due to the fact that only a small and varying number of action potentials that invade sympathetic varicosities are able to trigger transmitter release (Cunnane & Searl, 1994).
Depletion of intracellular Ca2+ stores does not interfere with the stimulation of noradrenaline release by OxoM
Independently of its inhibitory effect on IM, OxoM induces the formation of inositol trisphophate and triggers Ca2+ release from intracellular stores in SCG neurons (del Rio et al. 1999). The resulting increase in intracellular Ca2+ might contribute to the stimulation of noradrenaline release by OxoM. To test for this hypothesis, radiotracer release experiments were performed in the absence and presence of 0.1 µM thapsigargin, which prevents OxoM-dependent increases in intracellular Ca2+ in SCG neurons (del Rio et al. 1999). However, OxoM-induced tritium overflow in the presence of thapsigargin (1.59 ± 0.43 % of total radioactivity; n = 6) was not different from that in its absence (1.47 ± 0.28 % of total radioactivity; n = 6; P > 0.9). Likewise, electrically evoked tritium overflow was not affected by thapsigargin (not shown) which, however, did abolish the inhibition of IM by bradykinin as a positive control (see Bofill-Cardona et al. 2000).
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DISCUSSION |
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Acetylcholine has long been known to excite sympathetic neurons via muscarinic receptors by causing a slowly developing depolarisation. This effect is mediated by the closure of KM channels (Brown, 1983). The present results show that this very mechanism is involved in noradrenaline release from rat SCG neurons triggered by the muscarinic agonist OxoM.
Identification of the receptor mediating the secretagogue action of OxoM
OxoM is generally considered a pure muscarinic receptor agonist with some preference for the M2 subtype (Caulfield, 1993). Nevertheless, evidence has been presented that OxoM may also activate nAChRs in either Xenopus myocytes (Reitstetter et al. 1994) or guinea-pig neurons (Xian et al. 1994). In rat SCG neurons, as shown here, OxoM was able to induce inward currents at negative membrane potentials at concentrations > 100 µM, and these currents were abolished by the nAChR antagonist mecamylamine. Hence OxoM did activate nAChRs, but this effect did not contribute to its release stimulating effect since OxoM-induced noradrenaline release reached its maximum at 10-30 µM and was not significantly reduced by mecamylamine. Thus OxoM triggered transmitter release exclusively via one or more subtypes of muscarinic receptors.
Amongst the five known subtypes of mAChRs, two (M2 and M4) are linked to PTX-sensitive G proteins, whereas M1, M3 and M5 are coupled to toxin-insensitive G proteins (Caulfield & Birdsall, 1998; Eglen et al. 2001). PTX enhanced rather than prevented the secretagogue action of OxoM. Therefore, the release stimulating effect cannot be mediated by M2 or M4 receptors. This conclusion is supported by the fact that bethanechol failed to trigger transmitter release, but only inhibited electrically evoked release. In rat SCG neurons, this mAChR agonist selectively activates M2 receptors (Liu & Rittenhouse, 2003). This result corroborates the idea that M2 and/or M4 mAChRs are located at postganglionic sympathetic axon terminals and mediate an inhibition of noradrenaline release (Boehm & Kubista, 2002; Trendelenburg et al. 2003). Accordingly, the inactivation of their associated signalling cascades by PTX is likely to be the reason for the enhancement of OxoM-induced transmitter release by PTX as observed in the present study.
As outlined above, the secretagogue action of OxoM was mediated by M1, M3 and/or M5 receptors. One can differentiate between these receptor subtypes by using, for instance, pirenzepine which displays an affinity of about 10 nM at M1 receptors and at least tenfold lower affinities at M3 and M5 receptors (Caulfield, 1993; Caulfield & Birdsall, 1998). Pirenzepine caused a rightward shift in the concentration response curve for OxoM-induced noradrenaline release, which indicates an underlying competitive mechanism, and the calculated pA2 value (Arunlakshana & Schild, 1959) of 7.4 suggests an antagonist affinity of about 40 nM. To verify whether the secretagogue effect was indeed mediated by M1 mAChRs, cultures were incubated in MT-7, which selectively and for up to 8 h irreversibly blocks M1 receptors (Caulfield & Birdsall, 1998; Olianas et al. 2000). In MT-7-treated cultures, the secretagogue action of OxoM was entirely lost. We therefore conclude that OxoM triggered transmitter release from rat SCG neurons via M1 mAChRs.
The M1 mAChR mediates both the inhibition of IM and the stimulation of noradrenaline release
It is well established that the muscarinic inhibition of IM in SCG neurons is mediated by the M1 receptor subtype (Marrion et al. 1989; Bernheim et al. 1992; Shapiro et al. 2001). This is the same receptor subtype as the one that was found to mediate the secretagogue action of OxoM. Therefore, one might expect that OxoM evokes noradrenaline release and reduces IM in the same range of concentrations. In contrast to this expectation, more than tenfold higher concentrations of OxoM were required to trigger transmitter release than to elicit an inhibition of IM (cf. Fig. 1B and D). However, as soon as the release inhibiting mechanisms of mAChRs were inactivated by PTX (see above), these two effects displayed similar values for half-maximal concentrations of OxoM: 1 µM for the inhibition of IM and 3 µM for the stimulation of transmitter release. Furthermore, both effects were antagonised by pirenzepine in an apparently competitive manner. The calculation of pA2 values (Arunlakshana & Schild, 1959) from the rightward shifts of the two concentration- response curves in the presence of pirenzepine yielded 7.4 for the stimulation of release and 7.6 for the inhibition of IM. Thus pirenzepine antagonised both effects with apparently identical receptor affinity, which indicates that only one receptor subtype was involved. Previously, a pA2 value of 7.5 has been reported for the antagonism between pirenzepine and muscarine in the inhibition of IM in rat SCG neurons (Marrion et al. 1989). Furthermore, both effects were abolished by the M1 receptor-selective toxin MT-7. Thus the M1 receptor mediates both the inhibition of IM and the stimulation of transmitter release.
The inhibition of IM is involved in the stimulation of noradrenaline release by OxoM
The above results suggest that the inhibition of IM might be involved in OxoM-induced noradrenaline release. This idea is also supported by the previous observation that agents that block IM in SCG neurons independently of neurotransmitter receptors also trigger transmitter release: the KM channel blockers linopirdine and Ba2+ evoked noradrenaline release in an entirely Cd2+- and TTX-sensitive manner (Kristufek et al. 1999), as shown in the present study for the mAChR agonist OxoM (Fig. 4C). Thus M1 receptor activation as well as direct KM channel blockade elicit action potentials with ensuing transmembrane Ca2+ entry and concomitant vesicle exocytosis.
To verify whether KM channels are involved in the secretagogue action of OxoM, the effects of the anticonvulsant retigabine were investigated here. Retigabine has been shown to enhance currents through channels formed by KCNQ2 and -3 heteromultimers (Main et al. 2000; Wickenden et al. 2000) and to selectively enhance IM in rat SCG neurons (Tatulian et al. 2001). Accordingly, in the presence of retigabine, non-inactivating outward currents at -30 mV were enhanced, but IM relaxation amplitudes caused by hyperpolarisations to -55 mV were reduced. These complex changes arose due to a shift in the voltage dependence of KM channels by about -20 mV, as reported before (Main et al. 2000; Tatulian et al. 2001). In parallel with these changes in the voltage dependence of IM, retigabine hyperpolarized the membrane potential of SCG neurons also by 20 mV. Similar retigabine-induced changes in membrane potential have been observed in Xenopus oocytes expressing only KCNQ2/3 channels (Main et al. 2000). The congruence of these results suggests that IM is a major determinant of the membrane potential in SCG neurons.
This conclusion is also supported by the finding that OxoM depolarised the neurons in parallel with the blockade of KM channels. In at least one-third of the neurons, OxoM also triggered action potential discharge, which finally led to transmitter release. In the presence of retigabine, OxoM still depolarised the neurons, but always failed to trigger action potentials. Thus retigabine did not interfere with the activation of M1 receptor-associated signalling cascades by OxoM, but only neutralised its effect on outward currents and on the membrane potential (Fig. 6E and Fig. 7B). Nevertheless, retigabine did abolish noradrenaline release evoked by the muscarinic agonist in a concentration-dependent manner. Electrically evoked release, in contrast, remained largely unchanged in the presence of retigabine. This lack of effect of retigabine might have been attributable to supramaximal electrical field stimulation intensities used in radiotracer release experiments, since the KM channel opener rendered single SCG neurons less likely to fire action potentials in response to depolarising current injections. Nevertheless, when the strengths of electrical fields were reduced, [3H]noradrenaline release declined, but retigabine still failed to cause a significant reduction. Furthermore, retigabine left action potential waveforms unaltered. Thus retigabine did not interfere with some general mechanism of action potential-dependent vesicle exocytosis. Accordingly, the OxoM-induced noradrenaline release must have been abolished by retigabine because of the change in the voltage dependence of KM channels, which indicates that the inhibition of KM channels is involved in the stimulation of transmitter release.
Noradrenaline release from dissociated sympathetic neurons in primary cell culture occurs only at axons or axon terminals, but not at neuronal somata (Boehm & Huck, 1997; Boehm, 1999). The fact that OxoM-induced release was entirely abolished by TTX indicates that the receptors involved were remote from the release sites. Thus the release stimulating M1 receptors are not presynaptic ones, but rather located at the somatodendritic region of SCG neurons. In addition to the inhibition of KM channels, M1 receptors of SCG neurons are linked to other intracellular signalling cascades, in particular to the inositol trisphosphate-dependent liberation of Ca2+ from intracellular stores (del Rio et al. 1999). Considering that the release stimulating M1 receptors are not directly located at axon terminals, it appears unlikely that OxoM-induced increases in intracellular Ca2+ might contribute to the stimulation of noradrenaline release. This assumption was corroborated by the finding that depletion of intracellular Ca2+ stores by thapsigargin did not alter OxoM-induced transmitter release. Previously, this Ca2+-ATPase inhibitor was found not to affect the inhibition of KM channels via M1 receptors (del Rio et al. 1999; Bofill-Cardona et al. 2000). Thus stimulation of transmitter release and inhibition of IM by OxoM are both independent of Ca2+ release from intracellular stores.
In conclusion, the present results demonstrate that activation of M1 mAChRs triggers action potential discharges and concomitant transmitter release from SCG neurons. The link between receptor activation and action potentials is probably provided by the inhibition of KM channels.
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Acknowledgements
The study was supported by the 'Virologiefonds' of the University of Vienna, the Jubiläumsfonds of the Austrian National Bank (8377), the Austrian Science Fund (FWF; P14951 and P15797), and the EC grant QLRT-2000-00929.
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) or present (
) from minute 50 of superfusion. The graph shows the time course of fractional [3H] outflow per minute; n = 3. B shows the amount of tritium overflow (S %) induced by electrical field stimulation (EFS), 100 µM DMPP, or 100 µM OxoM in the absence (open bars) or presence (filled bars) of 10 µM mecamylamine; n = 6-9. P values for the significance of difference between the results obtained in the absence and presence of mecamylamine are indicated above the bars; n.s. indicates no significant difference. C, the current traces shown were recorded in a SCG neuron using the amphotericin B perforated-patch technique. The cells were clamped at - 70 mV and currents were elicited by 1.5 s applications of either OxoM or DMPP (as indicated by the horizontal bars) in the absence (control) or presence of 10 µM mecamylamine as indicated by the arrows. D shows the concentration dependence of time integrals of currents elicited by increasing concentrations of either OxoM or DMPP, recorded as described in C. Values obtained with different agonist concentrations were normalized to those obtained in the very same neuron with 100 µM DMPP (normalized time integral); n = 6. E shows the normalized time integral (determined as in D) of currents evoked by 100 µM DMPP or 100 µM OxoM in the absence (open bars) or presence (filled bars) of 10 µM mecamylamine; n = 5.
in A and all data in B and C) or remained untreated (
in A). Thereafter, the cultures were labelled with [3H]noradrenaline and superfused. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected. A shows the concentration dependence of tritium overflow (S %) evoked by the indicated concentrations of OxoM (each applied for 2 min as shown in Fig. 1A) in cultures treated with PTX for 24 h (