Ca2+-Induced Ca2+ Release Activates Spontaneous Miniature Outward Currents (SMOCs) in Parasympathetic Cardiac Neurons

Laura A. Merriam, Fabiana S. Scornik, and Rodney L. Parsons

Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, Vermont 05405


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Merriam, Laura A., Fabiana S. Scornik, and Rodney L. Parsons. Ca2+-Induced Ca2+ Release Activates Spontaneous Miniature Outward Currents (SMOCs) in Parasympathetic Cardiac Neurons. J. Neurophysiol. 82: 540-550, 1999. Mudpuppy parasympathetic cardiac neurons exhibit spontaneous miniature outward currents (SMOCs) that are thought to be due to the activation of clusters of large conductance Ca2+-activated K+ channels (BK channels) by localized release of Ca2+ from internal stores close to the plasma membrane. Perforated-patch whole cell recordings were used to determine whether Ca2+-induced Ca2+ release (CICR) is involved in SMOC generation. We confirmed that BK channels are involved by showing that SMOCs are inhibited by 100 nM iberiotoxin or 500 µM tetraethylammonium (TEA), but not by 100 nM apamin. SMOC frequency is decreased in solutions that contain 0 Ca2+/3.6 mM Mg2+, and also in the presence of 1 µM nifedipine and 3 µM omega -conotoxin GVIA, suggesting that SMOC activation is dependent on calcium influx. However, Ca2+ influx alone is not sufficient; SMOC activation is also dependent on Ca2+ release from the caffeine- and ryanodine-sensitive Ca2+ store, because exposure to 2 mM caffeine consistently caused an increase in SMOC frequency, and 10-100 µM ryanodine altered the configuration of SMOCs and eventually inhibited SMOC activity. Depletion of intracellular Ca2+ stores by the Ca-ATPase inhibitor cyclopiazonic acid (10 µM) inhibited SMOC activity, even when Ca2+ influx was not compromised. We also tested the effects of the membrane-permeable Ca2+ chelators, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) and EGTA-AM. EGTA-AM (10 µM) caused no inhibition of SMOC activation, whereas 10 µM BAPTA-AM consistently inhibited SMOCs. After SMOCs were completely inhibited by BAPTA, 3 mM caffeine caused SMOC activity to resume. This effect was reversible on removal of caffeine and suggests that the source of Ca2+ that triggers the internal Ca2+ release channel is different from the source of Ca2+ that activates clusters of BK channels. We propose that influx of Ca2+ through voltage-dependent Ca2+ channels is required for SMOC generation, but that the influx of Ca2+ triggers CICR from intracellular stores, which then activates the BK channels responsible for SMOC generation.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Neurons, like most cell types, contain intracellular pools of Ca2+ that are important in cellular signaling pathways (Berridge 1998; Henzi and MacDermott 1992; Kostyuk and Verkhratsky 1994). The IP3-sensitive pools of Ca2+ are known to be mobilized through activation of a variety of G protein-coupled neurotransmitter receptors (Berridge 1993), but a role for the ryanodine-sensitive Ca2+ stores in neurons is less clear. It has been hypothesized that neuronal ryanodine-sensitive stores of Ca2+ may amplify and prolong intracellular Ca2+ signaling by Ca2+-induced Ca2+ release (CICR) using mechanisms similar to that demonstrated for cardiac and smooth muscle cells (Bolton and Imaizumi 1996; Fabiato and Fabiato 1984; Kuba 1994; Verkhratsky and Shmigol 1996). CICR in neurons was initially well documented using pharmacological interventions (Kuba 1994; Verkhratsky and Shmigol 1996). More recent results demonstrate physiological activation of CICR in neurons (Cohen et al. 1997; Hua et al. 1993; Llano et al. 1994; Moore et al. 1998; Shmigol et al. 1995; Yoshizaki et al. 1995).

A potential role of CICR in neurons could be to produce localized elevations of internal Ca2+, which in turn would activate Ca2+-dependent processes, thereby modulating intracellular signaling. For instance, transient, localized elevations of intracellular Ca2+, which are not easily detected with traditional Ca2+ imaging techniques, could activate Ca2+-dependent channels in the plasma membrane, thus regulating membrane excitability by altering the resting membrane potential. Indeed, the magnitude of the resting membrane potential of many neurons is dependent on the extent of the activation of large conductance Ca2+-activated potassium channels (BK channels) (Marty 1989).

In some neurons, simultaneous activation of BK channels occurs spontaneously under resting conditions (Fletcher and Chiappinelli 1992; Hartzell et al. 1977; Mathers and Barker 1981, 1984; Satin and Adams 1987). This BK channel activation causes spontaneous miniature hyperpolarizations (SMHs), or spontaneous miniature outward currents (SMOCs) when recorded under voltage clamp. These transient membrane events (SMHs or SMOCs) are proposed to be due to the activation of a cluster of BK channels by transient elevations of intracellular Ca2+ near the inner surface of the plasma membrane (Satin and Adams 1987). Thus neuronal SMOCs may be similar to the spontaneous transient outward currents (STOCs) measured in smooth muscle cells (Bolton and Imaizumi 1996; Nelson et al. 1995) and could potentially be involved in controlling neuronal excitability (Berridge 1998).

Given that SMOCs might be caused by the simultaneous activation of clusters of BK channels, SMOCs provide a convenient model system to detect and analyze sources of localized Ca2+ involved in the activation of BK channels in neurons. Therefore, in the present study, we have analyzed SMOC generation in mudpuppy parasympathetic neurons to establish the relative roles of Ca2+ influx and Ca2+ release in activation of BK channels. In particular, our experiments test the hypothesis that the activation of SMOCs in mudpuppy cardiac neurons requires Ca2+-induced Ca2+ release from internal stores and further, that this pool of Ca2+ gates a closely localized cluster of BK channels. The experiments utilize voltage-clamp recordings from acutely isolated mudpuppy parasympathetic neurons and establish that SMOC generation requires Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs), which then triggers Ca2+ release from internal stores. As part of the study, we demonstrate that the internal Ca2+ released to generate SMOCs comes from a caffeine- and ryanodine-sensitive store.

Preliminary descriptions of initial aspects of this work have been presented previously (Merriam et al. 1996; Merriam and Parsons 1997; Scornik et al. 1998).


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METHODS
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All experiments were performed on parasympathetic neurons dissociated from mudpuppy (Necturus maculosus) cardiac ganglia. Procedures for euthanasia were approved by the University of Vermont Institutional Animal Care and Use Committee. The methods of dissociation used a combination of collagenase, type I (Sigma, St. Louis, MO) and neutral protease (Sigma) following methods described previously (Merriam and Parsons 1995). All experiments were completed at room temperature (21-22°C) with cells continuously perfused by a gravity-fed bath solution (flow rate 5-10 ml/min) in a recording chamber with a volume of 1 ml (Warner Instrument, Hamden, CT).

Electrophysiological methods

Recordings were made with the perforated-patch configuration of the whole cell recording technique (Horn and Marty 1988). Either nystatin or amphotericin B were used to establish electrical continuity between the patch pipette and cell interior. No qualitative difference was noted between experiments with either of the pore-forming compounds, but as the perforated-patch configuration was more readily established and more stable with amphotericin B than nystatin, most of the experiments used amphotericin B. Voltage-clamp experiments were controlled using either Axopatch-1C/Labmaster DMA or Axopatch 200/Digidata 1200 acquisition systems (Axon Instruments, Foster City, CA). Currents were filtered at 2 kHz, stored on tape using a PCM recorder (A. R. Vetter , Rebersburg, PA), and then digitized (200-µS digitization rate) for further analysis using the SCAN program (Strathclyde Electrophysiology Software, John Dempster, University of Strathclyde, Glasgow, Scotland). Digitized files were edited by eye, and SMOC frequency was determined by counting the number of SMOCs in 60 s of digitized current traces. Peak amplitude and 50% decay time of individual SMOCs were analyzed using a threshold detection utility that was set to detect events 0.5% of full scale. This threshold, which equals 10 pA, was chosen to eliminate SMOCs that were indistinguishable from baseline noise. For determination of amplitudes and 50% decay times, 100-400 SMOCs were analyzed for each condition; except for experiments in which the SMOC frequency was greatly decreased, 20-150 SMOCs were analyzed. Reported voltages were corrected for a junction potential of 10 mV; no corrections were made for voltage-clamp error due to series resistance, except where noted (Fig. 2, C-F). Data were normalized to control values obtained in the same cell under the same conditions and are presented as means ± SE. Statistical differences were determined by Student's t-test, P < 0.05.

Solutions

The bath solution for SMOC recordings contained (in mM) 110 NaCl, 1.8 or 3.6 CaCl2, 2.5 KCl, 10 NaHEPES, and 0.0003 tetrodotoxin (TTX), pH 7.3. The pipette solution for patch recordings was (in mM) 80 Kaspartate, 40 KCl, 5 MgCl2, and 10 K-HEPES, pH 7.2. The patch pipettes were backfilled with 0.2 mg/ml amphotericin B or 0.33 mg/ml nystatin.

Drugs

All drugs used in the present study were obtained from commercial sources: cyclopiazonic acid (CPA), iberiotoxin, and TTX from Alomone Labs (Jerusalem, Israel); tetraethylammonium (TEA), caffeine, 3-isobutyl 1-methylxanthine (IBMX), and nifedipine from Sigma; omega -conotoxin GVIA from Alexis Biochemicals (San Diego, CA; and Alomone Labs); thapsigargin, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) from Research Biochemicals International (Natick, MA); and EGTA-AM from Calbiochem (La Jolla, CA). All drugs were used at concentrations consistent with those used in published studies and were in excess of the established KD of each drug. Thapsigargin, CPA, BAPTA-AM, and EGTA-AM were prepared as 1,000-10,000× concentrated stock solutions in DMSO and frozen until use. Nifedipine was prepared in acetone as a 10-mM stock and stored at -20°C until used. Vehicle controls indicated that neither DMSO nor acetone at the final concentrations had any measurable effects on SMOCs.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SMOCs are potassium currents generated by the simultaneous activation of BK channnels

The initial experiments verified that mudpuppy neuronal SMOCs resulted from the activation of large conductance KCa channels (BK channels) but not small conductance KCa channels (SK channels) by testing whether 100 nM iberiotoxin or 100 nM apamin affected SMOC amplitude. For these experiments, SMOCs were recorded from neurons that were bathed in a solution containing 1.8 mM Ca2+ and voltage clamped to depolarized potentials. SMOC amplitudes, 50% decay times, and frequency were unaffected by the presence of apamin (n = 3, Fig. 1, A1 and B, Table 1); in contrast, SMOCs were eliminated during exposure to iberiotoxin (n = 4, Fig. 1A3). We also tested the effect of TEA on SMOC amplitude to further demonstrate that BK channel activation was responsible for SMOC generation. Example results obtained with 500 µM TEA, which illustrate the TEA-induced decrease in SMOC amplitude, are shown in Fig. 1A2. In five cells exposed to 500 µM TEA, SMOC amplitude was reduced to 56.2 ± 3.6% (mean ± SE) of control amplitudes measured in the same cells (Fig. 1B, Table 1). Results of other experiments with 100 µM -2 mM TEA demonstrated that the TEA-induced decrease in SMOC amplitude was concentration dependent and reversible (data not shown).



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Fig. 1. Spontaneous miniature outward currents (SMOCs) are tetraethylammonium (TEA) and iberiotoxin sensitive. A: current traces demonstrate SMOCs before and during application of K+ channel blockers. Arrows indicate the start of drug application by superfusion. A1: SMOCs are unaffected by 100 nM apamin. Vhold = -10 mV. A2: SMOCs are decreased after addition of 500 µM TEA. Vhold = -10 mV. A3: SMOC activity was inhibited after exposure to 100 nM iberiotoxin. Vhold = -20 mV. B: normalized SMOC amplitudes were averaged and compared with control amplitudes, Vhold = -10 mV; for 100 nM apamin, n = 3; for 500 µM TEA, n = 5.


                              
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Table 1. Modulation of SMOC amplitude and 50% decay

SMOC activation characteristics are voltage dependent

Satin and Adams (1987) reported previously that SMOCs could be recorded from cultured bullfrog sympathetic neurons and from mudpuppy parasympathetic neurons in solutions containing cadmium (Cd2+). These authors characterized the voltage-dependent characteristics of SMOC activity in bullfrog sympathetic neurons, and found that SMOC frequency, amplitude, and decay time all increased with depolarization in the presence of 100 µM Cd2+. They also suggested, from experiments with iontophoretically applied Ca2+, that the SMOC decay characteristics were determined by the closing behavior of BK channels. Fewer experiments involved mudpuppy parasympathetic neurons, but Satin and Adams (1987) did observe SMOC activity in the presence of 200 µM Cd2+ in these neurons but saw no evidence that SMOC frequency increased on membrane depolarization.

We tested whether the characteristics of SMOC activation were altered in the dissociated mudpuppy neurons when 200 µM Cd2+ was added to the bath solution. In these neurons, the addition of 200 µM Cd2+ decreased the SMOC frequency markedly and, in some cells, eliminated SMOC activity completely. With 3.6 mM Ca2+ and 200 µM Cd2+ in the bath solution, SMOC activity decreased quickly to a reduced frequency and was maintained at this reduced frequency throughout the rest of the recording period (Fig. 2A). Under these conditions with SMOC frequency reduced, it was possible to resolve characteristics of individual events. Thus we used cells maintained in 3.6 mM Ca2+ and 200 µM Cd2+ to measure the voltage dependence of the frequency, amplitude, and decay of mudpuppy SMOCs. The cells were voltage clamped to potentials between -50 and +20 mV. Figure 2B shows examples of averaged SMOCs recorded at two different voltages. Under these recording conditions, the SMOC frequency-membrane voltage relationship was bell-shaped with the maximum frequency occurring near -20 mV (Fig. 2C). SMOC amplitude and 50% decay also were voltage dependent; both increased as the holding potential was made more positive (Fig. 2, D and E). Even though SMOC frequency was decreased in the presence of 200 µM Cd2+, SMOC amplitudes or decay times were not significantly different from control (see Table 1).



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Fig. 2. SMOC characteristics in 200 µM CdCl2. A1: current trace demonstrates SMOCs before and during application of 200 µM CdCl2. SMOC frequency is decreased in 200 µM CdCl2, but amplitude is not affected (see Table 1). A1a and A1b: currents displayed at an expanded time scale represent SMOCs identified by the letters in A1. Vhold = -10 mV. B: individual SMOCs recorded at 2 different holding potentials were averaged and superimposed. Holding potentials corrected for junction potential and series resistance are printed next to averaged SMOCs. C-E: voltage dependence of SMOC characteristics. Results are shown from one neuron exposed to 200 µM CdCl2, and holding potentials are corrected for junction potential and series resistance. C: SMOC frequency is voltage dependent, and the relationship resembles that of voltage-dependent calcium influx. D: SMOC amplitude increases at membrane potentials further from the K+ equilibrium potential. E: SMOC duration increases as membrane potential is depolarized. D and E: data plotted are means ± SE.

Caffeine stimulates SMOC activity

The possibility that Ca2+ released from an internal store was involved in SMOC generation was tested using 2 mM caffeine. Caffeine stimulates Ca2+ release from neuronal ryanodine-sensitive Ca2+ stores by sensitizing the release channel to Ca2+ (McPherson et al. 1991). For these experiments, the cells were voltage clamped to membrane potentials between -20 and 0 mV and bathed in a solution containing 1.8 mM Ca2+ and 200 µM Cd2+.

The frequency of SMOCs was consistently increased in the presence of 2 mM caffeine (Fig. 3A and D). Caffeine did not affect SMOC amplitude, but in the presence of caffeine, SMOC 50% decay time was increased significantly, to 186 ± 9% of control values (n = 3; Fig. 3, B and C, see Table 1). Also, a decrease in the outward current was consistently observed when caffeine was applied (Fig. 3A). Caffeine has previously been shown to directly block delayed rectifier K+ channels (Reiser et al. 1996), which could account for the observed effect. All of the effects of caffeine reversed quickly on removal of caffeine.



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Fig. 3. Effects of caffeine on SMOC characteristics. A: representative recording at 0 mV shows that SMOCs increase in frequency after superfusion of 2 mM caffeine. Solution contains 200 µM CdCl2. B: amplitude of SMOCs is unchanged in 2 mM caffeine. C: SMOC duration is increased in the presence of 2 mM caffeine. D: SMOC frequency increases in 2 mM caffeine (for B-D, data averaged from 3 cells, amplitudes, 50% decay times, and frequencies are normalized to control values; error bars represent SE). E: current recording at -10 mV demonstrates that repeated applications of 30 mM caffeine cause large transient outward currents due to the dumping of Ca2+ from intracellular stores. Solid bar below the current trace indicates the duration of caffeine application. Stores will refill with calcium in ~2 min in control solutions.

Control experiments were done to test whether the prolongation of SMOC 50% decay time in caffeine was related to the ability of caffeine to inhibit cellular phosphodiesterases (Ashcroft et al. 1972) rather than to its ability to induce Ca2+ release from internal stores. We tested whether 2 mM caffeine increased SMOC 50% decay time in cells that had been pretreated with the potent phosphodiesterase inhibitor IBMX. In three cells, following pretreatment with 500 µM IBMX, 2 mM caffeine significantly increased 50% decay time (200 ± 21% of the value in IBMX alone), but SMOC amplitude was not affected by caffeine in the IBMX-treated cells (92 ± 2%). Thus we suggest that the increase in 50% decay time by caffeine was not related to a caffeine-induced inhibition of phosphodiesterases, but rather represented a direct effect of caffeine on the Ca2+ release channel.

We also analyzed the effects of high concentrations of caffeine on Ca2+ release from internal stores in mudpuppy neurons. The results presented in Fig. 3E demonstrate that repeated 30-s applications of 30 mM caffeine initiated a large outward current typical of a caffeine-induced outward current caused by release of Ca2+ from internal stores (Marrion and Adams 1992). We found that with repeated caffeine applications, the large transient outward currents were comparable if the interval between caffeine challenges was >2 min. This result demonstrates that the caffeine-sensitive stores in the mudpuppy neurons readily refill with Ca2+ after caffeine challenges. The caffeine-induced outward currents recorded when 200 µM Cd2+ was present were similar to those recorded in cells not exposed to Cd2+ (not shown).

SMOC activity is depressed by removal of external Ca2+ and by blockers of VDCCs

The observation that SMOC frequency was decreased in the presence of Cd2+ suggested that Ca2+ influx through surface membrane Ca2+ channels was involved in SMOC generation. To test the role of external Ca2+, we substituted Mg2+ for Ca2+ in the external solution and examined SMOC frequency. SMOCs initially were recorded at holding voltages between -20 and 0 mV in a control solution that contained 3.6 mM Ca2+ and 200 µM Cd2+. The bath solution was then changed to a Ca2+-deficient solution containing 3.6 mM Mg2+ and 200 µM CdCl2. In six cells, SMOCs disappeared quickly after switching to the Ca2+-deficient solution (Fig. 4A). SMOC activity recovered if the cells were returned to the Ca2+-containing solution (n = 3, Fig. 4, A and B3). Alternatively, when SMOC frequency had decreased to zero in the Ca2+-deficient solution, the addition of 3 mM caffeine to the Ca2+-deficient solution reinitiated SMOC activity (n = 3, Fig. 4A2). To ensure that results observed were not related to the presence of 200 µM Cd2+, we repeated the experiments without Cd2+. In these experiments (n = 5), SMOC activity also disappeared when the 3.6 mM Ca2+ was replaced by 3.6 mM Mg2+, and the time course of SMOC disappearance was closely dependent on the speed of the gravity flow system. In three experiments in which we adjusted the the gravity flow to a fast flow rate of 10 ml/min, we observed that SMOC frequency began to decrease quickly after switching to the Ca2+-deficient solution, so that the frequency was decreased to 3% of the control frequency within 30 s of the solution change (Fig. 4B1).



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Fig. 4. SMOC activation is triggered by calcium influx. A: SMOCs are recorded at -10 mV in a solution that contains 200 µM CdCl2. A1: SMOCs are recorded initially in the presence of 3.6 mM CaCl2, then SMOCs disappear when the Ca2+ is replaced by Mg2+ (0 Ca2+). SMOCs recover after 3.6 mM Ca2+ is returned. A2: in the same cell, solution contains 0 CaCl2/3.6 mM MgCl2, application of 3 mM caffeine (duration indicated by solid bar) causes increase in SMOC frequency (accompanied by decrease in outward current). There is a 90-s break between the traces in A1 and A2. B: SMOCs recorded from a different cell at -10 mV, solution does not contain Cd2+. B1: SMOCs are initially recorded in 3.6 mM CaCl2, then SMOCs disappear when Ca2+ is replaced by Mg2+ (0 Ca2+). B2: currents recorded in the same cell as B1, there is a 4-min break in the current trace, the solution still contains 0 CaCl2/3.6 mM MgCl2. Superfusion of 30 mM caffeine (duration indicated by solid bar) activates a large outward current, suggesting caffeine-sensitive intracellular stores still contain Ca2+. B3: SMOC activity fully recovered after restoring 3.6 mM Ca2+. Current traces in B2 and B3 are continuous.

We also observed that in four cells in which SMOCs disappeared in the Ca2+-deficient solution, application of 30 mM caffeine initiated a large transient outward current, which demonstrated that the intracellular stores still contained Ca2+ (Fig. 4B2). However, once Ca2+ stores were emptied by caffeine in the Ca2+-deficient solution, subsequent applications of high caffeine did not activate SMOCs or any outward current (n = 3, data not shown).

The results of these experiments demonstrated that SMOC activity was inhibited by exposure to the Ca2+-deficient solution even though caffeine-sensitive internal Ca2+ stores still contained Ca2+. SMOC activity could be transiently activated by application of caffeine, but once the internal stores of Ca2+ were depleted, SMOC activity could not be reinitiated.

The observation that SMOC frequency was decreased in Ca2+-deficient or Cd2+-containing solutions suggested that Ca2+ influx through surface membrane Ca2+ channels was important for SMOC generation. Additional experiments were done to test whether the channels involved were VDCCs. Previously, Merriam and Parsons (1995) demonstrated that mudpuppy parasympathetic neurons express both dihydropyridine- and omega -conotoxin GVIA (ctx-GVIA)-sensitive Ca2+ channels, but that most of the whole cell Ca2+ current flowed through ctx-GVIA-sensitive channels. We tested whether inhibition of VDCCs affected SMOC generation and, further, whether a specific class of VDCC was preferentially involved. The Ca2+ concentration in the bathing solution was 1.8 mM, and the cells were voltage clamped to -10 or 0 mV. The addition of 1 µM nifedipine decreased SMOC frequency by 18.5 ± 4.3%, whereas 3 µM ctx-GVIA plus 1 µM nifedipine decreased SMOC frequency by 93.1 ± 2.3% relative to the control frequency (n = 5, Fig. 5A). The reverse order of drug application was also tested in an additional experiment. In this cell, 3 µM ctx-GVIA decreased SMOC frequency by 83.3% relative to control, and the addition of 1 µM nifedipine to ctx-GVIA blocked SMOC frequency further, so that the frequency was decreased by 96.7% of control (data not shown). SMOC frequencies were counted 0.5-2 min after exposure to each drug. Although SMOC frequency was consistently decreased by exposure to the combination of ctx-GVIA and nifedipine (Fig. 5A), SMOC amplitudes were not decreased (see Table 1). Thus the decrease in SMOC frequency was not due to a decrease in amplitude of individual SMOCs. SMOCs measured in the presence of the VDCC inhibitors exhibited a small but statistically significant increase in 50% decay time (Table 1), but the explanation for this observed increase is unclear.



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Fig. 5. SMOC frequency is decreased by blockers of voltage-dependent Ca2+ channels (VDCC). A: current trace demonstrates SMOCs recorded at -10 mV in control solution, followed by SMOCs recorded after applications of 1 µM nifedipine and 3 µM omega -conotoxin GVIA + 1 µM nifedipine. Current traces are continuous. Downward arrows indicate the start of the continuous superfusion of each drug. B: in the same cell, 15 min after the recordings in A, SMOC frequency was still very low. Application of 30 mM caffeine caused activation of a large transient outward current, demonstrating that caffeine-sensitive stores still contain calcium. Solid bar indicates the duration of caffeine application.

We also tested whether the decreased SMOC frequency during exposure to Ca2+ channel blockers was due to depletion of intracellular Ca2+ stores. After SMOC activity had decreased to very low frequencies, application of 3 mM caffeine stimulated additional SMOC activity (n = 2, data not shown). In five inhibitor-treated cells, application of 30 mM caffeine, given when SMOC activity had declined to very low frequencies, elicited a large outward current (Fig. 5B). Thus, when SMOC activity was depressed during exposure to the Ca2+ channel blockers, internal stores had not been depleted of Ca2+. We also noted that even though SMOC activity decreased to very low frequencies after treatment with the Ca2+ channel blockers, repeated caffeine applications initiated large-amplitude, transient outward currents. This result differed from that obtained with cells kept in the Ca2+-deficient solution. In Ca2+-deficient solutions, once stores were depleted of Ca2 by caffeine, subsequent caffeine applications could not induce an outward current because intracellular Ca2+ stores were not replenished.

SMOC activity is progressively diminished during exposure to CPA

Our results with caffeine demonstrated that SMOC activity could be initiated by release of Ca2+ from internal stores. Furthermore, our results suggested that if internal stores became depleted of Ca2+, then SMOC activity was depressed. In another series of experiments, we tested directly the effect of depletion of internal Ca2+ stores on SMOC generation. For these experiments, we analyzed the effect of 10 µM CPA on SMOC frequency in cells voltage clamped to -10 mV and bathed in 1.8 mM Ca2+ solution. CPA inhibits the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), which sequesters Ca2+ into the endoplasmic reticulum (ER); during continued exposure to 10 µM CPA, internal Ca2+ stores gradually become depleted as Ca2+ leaks out of the ER (for review, see Thomas and Hanley 1994).

In the presence of 10 µM CPA, SMOC activity declined gradually, and, by ~15 min, SMOC frequency was decreased to very low levels (n = 3, Fig. 6A and B). SMOC activity returned once CPA was removed (Fig. 6C). At a time when the SMOC frequency in CPA had declined >60% (9 min), analysis of individual events indicated that there was no CPA-induced change in amplitude or half decay time (n = 3; see Table 1). SMOCs also disappeared in two additional cells that were treated with the irreversible SERCA inhibitor, thapsigargin (100 nM, data not shown).



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Fig. 6. Blockade of the store Ca2+-ATPase by cyclopiazonic acid (CPA) decreases SMOC activation. A: SMOCs recorded at -10 mV before and during exposure to 10 µM CPA. A1: current trace shows SMOCs recorded before, and immediately after the application of CPA. Downward arrow indicates the start of the solution containing CPA, and the currents in A2-A4 were recorded after 5-, 9-, and 12-min exposures to CPA, respectively. B: SMOC frequency decreased during prolonged exposure to 10 µM CPA. Frequencies were normalized to the frequency at the time at which CPA was added (time 0). C: SMOC frequency returns to normal after removal of CPA. Data points represent the recovery of SMOC frequency (normalized to control frequency in B) as CPA is washed out. Time 0 was 30 s after control wash started. For B and C, data represent means ± SE from 3 neurons.

Additional experiments were done to verify that after internal Ca2+ stores were depleted by exposure to 10 µM CPA, SMOCs could not be activated. For these experiments, 30 mM caffeine was applied repeatedly to control cells and cells treated with 10 µM CPA. The bathing solution contained 1.8 mM Ca2+, and the cells were voltage clamped to -10 mV. As illustrated in Fig. 3D, repeated applications of 30 mM caffeine to control cells produced large transient outward currents even when the interval between the caffeine applications was only 90 s, because the internal stores readily refill. In contrast, during exposure to CPA, a single 30-s application of caffeine induced a large transient outward current, but subsequent applications of caffeine could not, because stores were depleted of Ca2+ (Fig. 7, A-C). After the first application of caffeine, no SMOCs were recorded, even though Ca2+ influx was not compromised. When CPA was removed, SMOC activity resumed and the application of 30 mM caffeine induced a large outward current (Fig. 7, D and E). The transient outward current could also be initiated by subsequent applications of 30 mM caffeine. Similar results were obtained in three cells.



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Fig. 7. SMOCs are not activated after calcium stores are depleted by CPA. The current traces are continuous except for a 1.5-min break between traces C and D. A: 10 µM CPA was used to block the endoplasmic reticulum (ER) Ca2+-ATPase, then a 30-s application of 30 mM caffeine was used to deplete the store of Ca2+. Arrow indicates the start of the solution containing CPA, and the solid bar indicates the duration of application of caffeine in the CPA containing solution. B: the cell was washed in CPA for 6 min during which no SMOC activity was observed. C: a 2nd application of 30 mM caffeine did not activate an outward current, but the typical decrease in outward current was observed. Control wash was started as indicated by the downward arrow, and SMOCs gradually returned. D: 6 min after the control wash was started, a 30-s application of 30 mM caffeine activated an outward current, and this could be repeated 6 min later, E. Holding potential was -10 mV, bath contained 1.8 mM CaCl2.

SMOC activity and configuration are affected by ryanodine

The effects of caffeine suggested Ca2+ release from ryanodine-sensitive stores was involved in SMOC generation. We tested the involvement of ryanodine-sensitive stores in experiments in which the effect of ryanodine (10, 50, or 100 µM) on SMOC frequency was analyzed in cells voltage clamped between -20 and 0 mV. For most experiments, the bath solution contained 1.8 or 3.6 mM Ca2+, and 200 µM Cd2+ was present to allow resolution of individual SMOCs.

In 10 cells, SMOC frequency declined during ryanodine application (10 µM, 3 cells; 50 µM, 3 cells; 100 µM, 4 cells). We did not observe any obvious concentration-dependent effects, presumably because ryanodine had been added by bath superfusion, and the actual concentration that reached the ryanodine receptor was unknown. In some cells at each concentration, there was an initial increase in SMOC frequency following the ryanodine application (Fig. 8A), but in all cells, with continued exposure to ryanodine, the SMOC frequency and amplitudes decreased progressively until the events became indistinguishable from noise. As the SMOCs became smaller, quantification of individual events was impossible, but in all cells, SMOC activity had disappeared within 30 min. In addition, in 60% of the cells, ryanodine caused an obvious alteration in SMOC configuration (Fig. 8). Before ryanodine exposure, individual SMOCs exhibited a fast rise to peak and then decayed more slowly back to the baseline (Fig. 8B1). In ryanodine-treated cells, the rising phase was not noticeably different from that in control cells, but the decay phase in some cells was greatly exaggerated. In these instances, the SMOCs decayed only part way to a plateau level that remained for many tens of milliseconds (Fig. 8, B2 and B3). This plateau phase appeared similar to what would be expected if the ryanodine had blocked the release channel in an open subconductance state, and the Ca2+ release had been prolonged (see DISCUSSION).



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Fig. 8. Example current traces obtained at various times during a recording from a cell exposed to 50 µM ryanodine. All traces were recorded at -20 mV, in solution containing 200 µM CdCl2. A: top trace demonstrates SMOCs recorded before the application of ryanodine. Subsequent traces show SMOCs recorded after 8 min exposure to 50 µM ryanodine, after 11 min, after 19 min, and after 20 min exposure, respectively. SMOCs were completely eliminated after 25 min exposure to ryanodine in this cell. B: traces 1-3 demonstrate SMOC configuration on an expanded time scale for events identified by the numbers in A.

To verify that SMOC disappearance in the presence of ryanodine was not simply related to the presence of 200 µM Cd2+, we recorded SMOCs at -10 mV in three additional cells without Cd2+ present. In these cells, 100 µM ryanodine also decreased SMOC frequency in a time-dependent manner. As observed in the ryanodine- and Cd2+-treated cells, SMOC activity initially increased, then became more difficult to distinguish as SMOCs merged with the noise. All SMOC activity had disappeared within 30 min.

BAPTA eliminates SMOC activity

We have presented results that support the hypothesis that SMOCs are initiated by Ca2+-induced Ca2+ release from caffeine- and ryanodine-sensitive internal stores. Evidence has been presented demonstrating an involvement of both Ca2+ influx and Ca2+ release from internal stores in SMOC generation. Additional experiments were done to test the effects of the membrane-permeable Ca2+ chelators, BAPTA-AM and EGTA-AM, on SMOC activity. Although the affinity of BAPTA and EGTA for Ca2+ are similar, the association rate for Ca2+ is ~150 times greater for BAPTA than EGTA (Naraghi 1997). Thus it is possible to distinguish between fast and slow Ca2+-stimulated events that occur over short distances by use of these two chelators. For these experiments, the cells were bathed in 1.8 mM Ca2+ and voltage clamped to -10 mV.

Eight cells were loaded with 10 µM BAPTA-AM for time periods ranging from 10 to 37 min, at room temperature in the dark. Whole cell recordings were initiated at different times. In three cells, the recordings were begun before BAPTA-AM exposure and continued during the incubation period. In the remaining cells, the recordings were initiated after the BAPTA-AM loading had ended with the longest time interval being ~90 min. In all cases, SMOC frequency decreased progressively after application of BAPTA-AM. In those cells in which the recording was started before BAPTA-AM application, SMOCs were present, but during the exposure to BAPTA-AM the frequency gradually declined until by ~20 min, no SMOCs were recorded (Fig. 9, A1 and A2). In the remaining cells that were pretreated with BAPTA-AM, SMOC frequency (if SMOCS were present) was very low and continued to decline further over time.



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Fig. 9. SMOC activation is disrupted by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA. A1: SMOCs recorded at -10 mV, shortly after starting solution that contains 10 µM BAPTA-AM. After the membrane-permeable BAPTA-AM enters the cell cytoplasm, intracellular esterases cleave the acetyoxymethyl group to form the membrane-impermeable, but active Ca2+ chelator, BAPTA. A2: SMOC frequency has decreased ~20 min after starting BAPTA-AM solution. A3: after loading cell for 30 min with BAPTA-AM, solution is changed to control wash (active BAPTA should still be in cytoplasm). Approximately 2 min after starting wash, 3 mM caffeine was applied, and SMOCs returned during the exposure to caffeine. This effect with caffeine was reversible. B: in a different cell, SMOCs are recorded at -10 mV, 30 min after addition of 10 µM EGTA-AM to the bath solution. SMOC frequency remained high throughout the duration of recording. Cell was loaded with EGTA-AM for 25 min.

In four BAPTA-AM-loaded cells, after SMOC activity had ceased, 3 mM caffeine was applied. SMOC frequency increased quickly in these cells (Fig. 9A3). The SMOC activity also quickly declined on removal of caffeine (not shown). The induction and reversal of SMOC activity by caffeine could be repeated throughout the length of the recording, which in the case of one cell was longer than 90 min.

We also tested whether loading cells with EGTA-AM had a comparable effect to that of BAPTA on SMOC generation. Four cells were loaded with 10 µM EGTA-AM for ~25 min before initiation of SMOC recording. Recordings were then continued for 40-120 min after loading. It was possible to record SMOCs from EGTA-loaded cells for the duration of recording without any decrease in frequency (Fig. 9B).


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SMOCs in mudpuppy neurons are spontaneous, transient outward K+ currents that are abolished by iberiotoxin and TEA, but not by apamin; thus confirming the role of BK channels (Satin and Adams 1987) and demonstrating that activation of SK channels did not contribute to SMOC generation.

SMOC frequency was greatly diminished by conditions that eliminated or reduced Ca2+ influx, such as removal of external Ca2+, or addition of Ca2+ channel blockers. Mathers and Barker (1984) also found that spontaneous hyperpolarizations could not be recorded in cultured dorsal root ganglion (DRG) cells exposed to a Ca2+-deficient solution. However, in the DRG cells, the recordings were made after a 15- to 30-min pretreatment in Ca2+-deficient solution, during which time the stores could have been depleted of Ca2+. In our experiments, the decrease in SMOC frequency occurred at times when the Ca2+ stores still contained sufficient Ca2+ to support SMOC activity induced by caffeine. Thus depletion of internal stores was not the cause of the decrease in SMOC activation in our experiments.

Also, only SMOC frequency, but not SMOC amplitude, was affected by conditions that decreased Ca2+ influx through VDCC. If Ca2+ influx directly gated the BK channels contributing to SMOC generation, SMOC amplitude should have fallen as fewer and fewer BK channels were activated. This was not the case, suggesting instead that Ca2+ from VDCC did not activate BK channels directly, but triggered an intermediate step such as Ca2+ release from internal stores that then gated BK channel activation.

SMOC frequency exhibited a bell-shaped dependence on membrane voltage when 200 µM CdCl2 was included in the bath solution (Fig. 2C). A similar voltage dependence was noted by Satin and Adams (1987), who suggested that there might be coupling between the internal Ca2+ release sites and the surface membrane so that the voltage dependence of SMOC frequency reflected a voltage dependence of the Ca2+ release channels themselves. However, the voltage dependence of mudpuppy SMOC frequency is very similar to the voltage dependence of Ca2+ influx through VDCCs in mudpuppy neurons (Merriam and Parsons 1995) and mimics the relationship between Ca2+ influx through VDCCs and Ca2+ release probability in cardiac myocytes (Santana et al. 1996). Even though 200 µM Cd2+ was present in our experiments, Ca2+ influx very likely was not completely blocked, because removal of external Ca2+ resulted in more effective block of SMOC activity than did Cd2+ alone. As described by Santana et al. (1996), even when Ca2+ channel open probability is greatly reduced, the Ca2+ flux through an individual open channel is not compromised and is sufficient to activate ryanodine receptors, particularly at more hyperpolarized potentials where there is a large driving force for Ca2+. Therefore we propose that the SMOC frequency-voltage relationship reflects voltage dependence of Ca2+ influx, and not voltage dependence of the Ca2+ release channel.

Previously, Ca2+ influx through specific classes of VDCC has been correlated with activation of particular Ca2+-activated K+ channels (Davies et al. 1996; Marrion and Tavalin 1998; Wisgirda and Dryer 1994). However, in our study, Ca2+ influx through a specific VDCC type did not selectively activate SMOCs. Preferential blockade of VDCC types decreased SMOC frequency by the same proportion as the inhibition of specific voltage-gated Ca2+ channels that contribute to the whole cell Ca2+ current (Merriam and Parsons 1995). The lack of any specific role of a particular VDCC type suggests that the source of Ca2+ that is involved in triggering release may not be critical; any Ca2+ source that contributes free Ca2+ to the microenvironment surrounding the release channel may be sufficient to stimulate CICR (McPherson and Campbell 1993; Sutko and Airey 1996; Zucchi and Ronca-Testoni 1997). This suggestion is supported by the observation that ryanodine (Fig. 8) can transiently increase SMOC frequency as Ca2+ begins to leak out of intracellular stores. The converse is true also; any experimental perturbation that reduces the free [Ca2+]i in the microenvironment would decrease the probability of Ca2+ release and therefore decrease SMOC frequency.

Low concentrations of caffeine increased SMOC frequency, suggesting that Ca2+ release from caffeine-sensitive Ca2+ stores were involved in SMOC generation in mudpuppy neurons. Results of earlier studies also indicated that caffeine increased the frequency of spontaneous miniature hyperpolarizations in neurons (Fletcher and Chiapinelli 1992; Mathers and Barker 1984). Marrion and Adams (1992) suggested, based on an analysis of current variance, that the caffeine-induced (10 mM) outward current recorded in cultured bullfrog sympathetic neurons arose from the summation of individual SMOCs. We report here, that 2 mM caffeine did not change SMOC amplitude but did increase SMOC 50% decay time. Caffeine increases the open probability (Po) of the release channels by decreasing the closed state dwell time of the channel, thus resulting in more frequent openings. The open time and conductance of the release channel are not altered under these conditions (Sitsapesan and Williams 1990). We propose that the caffeine-induced prolongation of the 50% decay time most likely reflected multiple activations of BK channels in the vicinity of the release channel (DiChiara and Reinhart 1995). However, SMOC amplitude was not increased; thus either Ca2+ buffering is sufficiently fast to prevent accumulation of Ca2+ in the vicinity of the BK channel, or the concentration of Ca2+ released is normally high enough so that BK channel activation is maximal, thus any additional increase in Ca2+ would not affect SMOC amplitude.

Ryanodine binds with high affinity to the caffeine-sensitive Ca2+ release channel and alters its gating behavior and conductance. At lower concentrations, ryanodine can lock the release channel in an open, subconductance state, whereas higher concentrations of ryanodine will block the release channel in a use-dependent manner (Lai et al. 1992; Meissner 1986, 1994; Sutko and Airey 1996; Zucchi and Ronca-Testoni 1997). In all mudpuppy neurons tested, ryanodine decreased SMOC frequency in a time-dependent manner. Also, in some cells, SMOC configuration was markedly altered. In these cells, in the presence of ryanodine, SMOC decay sometimes exhibited a flat and prolonged phase, similar to what would be expected if the Ca2+ level remained elevated, as the release channel were locked in an open, subconductance state. This altered configuration was remarkably similar to the altered configuration of Ca2+ sparks observed in rat cardiac myocytes in the presence of ryanodine (Cheng et al. 1993). The results with ryanodine add further support for the hypothesis that Ca2+ release from ryanodine-sensitive intracellular stores controls activation of the BK channels involved in SMOC generation.

SMOC activity was eliminated in the presence of the SERCA inhibitor, CPA. The decrease in SMOC frequency was time dependent and occurred when Ca2+ influx through VDCC was not compromised. In addition, after internal Ca2+ stores were depleted by 30 mM caffeine in the presence of CPA, SMOCs could not be activated. SMOCs quickly returned when CPA was removed and the internal stores were allowed to replenish. These results provided additional support for the view that Ca2+ released from internal stores initiated SMOC activity, and that Ca2+ influx through VDCCs, by itself, was not sufficient for SMOC activation. The observation that the SMOC frequency decreased as the stores became more and more depleted suggested that lumenal concentration of Ca2+ also is a critical factor in determining SMOC frequency. Interestingly, although the Ca2+ stores contained less Ca2+ over time, SMOC amplitude did not change over the same time period. We propose two alternative mechanisms that could potentially account for these observations. First, the Ca2+ release process might be quantized (Cheek et al. 1993), so that a fixed amount of Ca2+ is always released even though total levels in the store diminish. Alternatively, the amount of Ca2+ released exceeds that needed to activate the BK channels located very close to the release channels. Thus the local Ca2+ levels are normally well above threshold concentrations so that the amount released from the stores could decrease greatly before there was any noticeable change in number of activated BK channels. The Ca2+ concentration in the local domain must reach quite high levels given that SMOCs occur at voltages as negative as -50 to -60 mV; potentials at which BK channels require relatively high concentrations of Ca2+ for activation (Barrett et al. 1982).

The experiments with Ca2+ chelators provided important insight into the characteristics of the Ca2+ pathways involved in SMOC generation. These conclusions are based on the fact that BAPTA, under the conditions of these experiments, has the same binding affinity, but faster association kinetics than EGTA (Naraghi 1997). First, the results with BAPTA demonstrated that two distinct Ca2+ pathways are involved: 1) a Ca2+ domain coupling Ca2+ influx to the activation of the ryanodine-sensitive ER Ca2+ release channels and 2) a Ca2+ domain coupling Ca2+ release from the ER release channel to plasma membrane BK channels. BAPTA appeared to chelate the transient increases of intracellular Ca2+ due to Ca2+ influx through plasma membrane VDCCs, but could not effectively buffer the transient increase of Ca2+ in the microdomains coupling the ER release channels to BK channels, as caffeine could reinitiate SMOC activity in BAPTA-loaded cells. EGTA was totally ineffective, indicating that the Ca2+ domain for both pathways are restricted to <1-2 µm (Deisseroth et al. 1996; Neher 1998). In addition, the inability of BAPTA to block SMOC initiation by caffeine indicates that the increase in intracellular Ca2+ concentration following release from the ER release channel must be quite high as well as very localized. Thus we propose that the cytoplasmic face of the plasma membrane BK channels is very likely within 10-20 nm of the ER release channel, and furthermore, that the Ca2+ concentration within this local domain conceivably may reach 100 µM.

SMOCs are short lived, with a half decay time of ~6-7 ms at -10 mV. Ca2+ removal from the vicinity of the BK channels must be very efficient and rapid if the SMOC decay time primarily reflects BK channel closing (Satin and Adams 1987). Furthermore, the fact that SMOC time course was readily altered by conditions, i.e., the presence of caffeine or ryanodine, that change the Po of the release channel suggest that the Ca2+ release event normally is very brief and transient. In neurons, like other cell types, Ca2+ homeostasis is highly regulated, with numerous mechanisms to restore resting Ca2+ levels after a transient rise. Active uptake of Ca2+ into the ER by the SERCA pump might be a critical mechanism (Gomez et al. 1996; Steenbergen and Fay 1996). However, SMOC decay time was not altered in the presence of CPA, suggesting that removal of Ca2+ by the SERCA pump does not determine SMOC time course. Recently Cseresnyes et al. (1997) described a novel, fast Ca2+ uptake mechanism in frog sympathetic neurons, activated by Ca2+ released from internal stores. A similar mechanism could potentially be involved in rapidly removing the Ca2+ that gated SMOC activity, although more study is required to establish determinants of SMOC decay.

Neuronal SMOCs are quite similar to spontaneous transient outward currents (STOCs) recorded from smooth muscle cells (Bolton and Imaizumi 1996; Nelson et al. 1995). The STOCs are thought to represent synchronous activation of BK channels by transient, highly localized elevations of Ca2+ released from ryanodine-sensitive internal stores (Nelson et al. 1995). Nelson and colleagues (1995) have provided evidence that rapid, transient Ca2+ signals, termed "Ca2+ sparks," are responsible for STOC generation in arterial smooth muscle cells. We postulate that a similar highly localized, rapid Ca2+ transient (i.e., neuronal sparks) could be responsible for SMOC activation in mudpuppy neurons. However, given that mudpuppy SMOCs have a faster time course than smooth muscle STOCs, the intracellular Ca2+ transient may be faster and more effectively buffered in neurons than in smooth muscle. A recent study in PC12 cells and hippocampal neurons (Koizumi et al. 1999) has demonstrated Ca2+ release signals that may be important in neuronal signaling pathways. However, these Ca2+ signals have durations >200 ms and thus are unlikely to be similar to the Ca2+ release events that underlie SMOC activity in mudpuppy parasympathetic neurons.


    ACKNOWLEDGMENTS

The authors thank Dr. John Dempster for providing the SCAN program. The authors also thank Drs. Mark Nelson and Joseph Patlak for helpful discussions during the course of this study and Dr. Patlak for critical comments during the preparation of this manuscript.

This work was supported in part by National Institute of Neurological Disorders and Stroke Grant NS-23978 to R. L. Parsons and American Heart Association Grant 9820031T to F. S. Scornik.


    FOOTNOTES

Address reprint requests to R. L. Parsons.

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.

Received 3 November 1998; accepted in final form 1 April 1999.


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