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The Journal of Neuroscience, April 15, 2001, 21(8):2553-2560

Calcium-Dependent Inhibition of L, N, and P/Q Ca²⁺ Channels in Chromaffin Cells: Role of Mitochondria

Jesús M. Hernández-Guijo¹, Victoria E. Maneu-Flores¹, Ana Ruiz-Nuño¹, Mercedes Villarroya¹, Antonio G. García^{1, 2}, and Luis Gandía¹

¹ Instituto Teófilo Hernando, Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, 28029 Madrid, Spain, and ² Servicio de Farmacología Clínica e Instituto de Gerontología, Hospital de la Princesa, 28006 Madrid, Spain

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The hypothesis that the buffering of Ca²⁺ by mitochondria could affect the Ca²⁺-dependent inhibition of voltage-activated Ca²⁺ channels, (I_Ca), was tested in voltage-clamped bovine adrenal chromaffin cells. The protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), the blocker of the Ca²⁺ uniporter ruthenium red (RR), and a combination of oligomycin plus rotenone were used to interfere with mitochondrial Ca²⁺ buffering. In cells dialyzed with an EGTA-free solution, peak I_Ca generated by 20 msec pulses to 0 or +10 mV, applied at 15 sec intervals, from a holding potential of 80 mV, decayed rapidly after superfusion of cells with 2 µM CCCP ( = 16.7 ± 3 sec; n = 8). In cells dialyzed with 14 mM EGTA, CCCP did not provoke I_Ca loss. Cell dialysis with 4 µM ruthenium red or cell superfusion with oligomycin (3 µM) plus rotenone (4 µM) also accelerated the decay of I_Ca. After treatment with CCCP, decay of N- and P/Q-type Ca²⁺ channel currents occurred faster than that of L-type Ca²⁺ channel currents. These data are compatible with the idea that the elevation of the bulk cytosolic Ca²⁺ concentration, [Ca²⁺]_c, causes the inhibition of L- and N- as well as P/Q-type Ca²⁺ channels expressed by bovine chromaffin cells. This [Ca²⁺]_c signal appears to be tightly regulated by rapid Ca²⁺ uptake into mitochondria. Thus, it is plausible that mitochondria might efficiently regulate the activity of L, N, and P/Q Ca²⁺ channels under physiological stimulation conditions of the cell.

Key words: mitochondrial Ca²⁺; Ca²⁺ channels; Ca²⁺-dependent inhibition of Ca²⁺ channels; chromaffin cells; L-type Ca²⁺ channels; N-type Ca²⁺ channels; P/Q-type Ca²⁺ channels

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Ca²⁺-dependent inactivation of high-threshold voltage-dependent Ca²⁺ channels was first suggested in the pioneering work of Hagiwara and Nakajima (1966), subsequently proven by Brehm and Eckert (1978) and Tillotson (1979), and later on extended to various excitable cells (Hagiwara and Byerly, 1981). Since then, various Ca²⁺ channel subtypes (L, N, P/Q, R) have been identified (García et al., 2000), but it is unclear whether all of them are equally prone to inactivation by Ca²⁺ entry or the elevation of the cytosolic concentration of Ca²⁺, [Ca²⁺]_c. For instance, the cardiac and smooth muscle L-type Ca²⁺ channel is quickly inactivated after depolarization, with a time constant in the range of 50-100 msec (Yue et al., 1990; Giannattasio et al., 1991; Neely et al., 1994). However, the neuronal L-type Ca²⁺ channel is inactivated more slowly by the increase of [Ca²⁺]_c (von Gersdorff and Matthews, 1996). The bovine adrenal chromaffin cell is a suitable model to reexamine this problem for two reasons: (1) this cell expresses L-, N- and P/Q-types of Ca²⁺ channels (Albillos et al., 1996), and (2) its mitochondria undergo large Ca²⁺ transients and hence its manipulation might produce drastic local changes of [Ca²⁺]_c to cause the inhibition of Ca²⁺ channels.

On the other hand, the concept that mitochondria can act as rapid and reversible Ca²⁺ buffers during depolarization of neurons (Werth and Thayer, 1994; White and Reynolds, 1997; Pivovarova et al., 1999; Colegrove et al., 2000) and chromaffin cells (Herrington et al., 1996; Park et al., 1996; Babcock et al., 1997) is a recent one. Mitochondria can certainly sense the [Ca²⁺]_c transients generated at subplasmalemmal domains (Rizzuto et al., 1994; Budd and Nicholls, 1996; Lawrie et al., 1996; Pivovarova et al., 1999), and reciprocally, Ca²⁺ uptake or release from mitochondria generate changes in local [Ca²⁺]_c that modulate Ca²⁺ entry (Budd and Nicholls, 1996). However, the measured elevations of mitochondrial Ca²⁺ concentrations, [Ca²⁺]_m, were only in the submicromolar or the low micromolar range (Rizzuto et al., 1994; Babcock et al., 1997; Brini et al., 1997). These small [Ca²⁺]_m transients could not account for the efficacy of mitochondria to sense the local [Ca²⁺]_c of 20-50 µM likely occurring in the vicinity of Ca²⁺ channels in bovine chromaffin cells (Neher, 1998). This issue has been clarified recently by using mitochondrially targeted aequorins of low Ca²⁺ affinity; we observed that mitochondria undergo surprising, rapid near-millimolar [Ca²⁺]_m transients on activation of Ca²⁺ entry through Ca²⁺ channels into bovine chromaffin cells stimulated with short pulses of acetylcholine (ACh) or high K⁺ (Montero et al., 2000). These large [Ca²⁺]_m can already account for the large and rapid changes of [Ca²⁺]_c taking place at subplasmalemmal sites during cell depolarization.

In this context, we thought that if we impaired the rapid sequestration of Ca²⁺ by mitochondria of the Ca²⁺ entering the cell during its depolarization, we could provoke larger and long-lasting [Ca²⁺]_c at subplasmalemmal sites; in this manner, we should be able to study how these local [Ca²⁺]_c elevations would affect the Ca²⁺ current through each Ca²⁺ channel subtype (L, N, or P/Q) generated by test depolarizing pulses applied to voltage-clamped bovine chromaffin cells. By following this strategy, we have found that the three Ca²⁺ channel subtypes can undergo full inhibition by local [Ca²⁺]_c elevations, although at different rates. We report here the experiments leading to these conclusions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation and culture of adrenal medulla chromaffin cells. Bovine adrenal medulla chromaffin cells were isolated following standard methods (Livett, 1984) with some modifications (Moro et al., 1990). Cells were suspended in DMEM supplemented with 5% fetal calf serum, 10 µM cytosine arabinoside, 10 µM fluorodeoxyuridine, 50 IU/ml penicillin, and 50 µg/ml streptomycin. They were plated on 1-cm-diameter glass coverslips at low density (5 × 10⁴ cells per coverslip).

Current measurements and analysis. Ca²⁺ (I_Ca), Ba²⁺ (I_Ba), Na⁺ (I_Na), and acetylcholine (I_ACh) currents were recorded using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). Coverslips containing the cells were placed on an experimental chamber mounted on the stage of a Nikon Diaphot inverted microscope. During the preparation of the seal with the patch pipette, the chamber was continuously perfused with a control Tyrode solution containing (in mM): 137 NaCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES/NaOH, 0.005 tetrodotoxin (TTX), pH 7.4 (no TTX was added when measuring I_Na). Once the patch membrane was ruptured and the whole-cell configuration of the patch-clamp technique established, the cell being recorded was locally, rapidly, and continuously superfused with an extracellular solution of a composition similar to the chamber solution, but containing nominally 0 mM Ca²⁺ (no EGTA added; I_Na), 10 mM Ca²⁺ (I_Ca), or 10 mM Ba²⁺ (I_Ba) as charge carriers (see Results for specific experimental protocols). External solutions were rapidly exchanged using electronically driven miniature solenoid valves coupled to a multi-barrel concentration-clamp device, the common outlet of which was placed within 100 µm of the cell to be patched. The flow rate was ~1 ml/min and regulated by gravity. Experiments were performed at room temperature (22-24°C). Cells were dialyzed with an intracellular solution containing (in mM): 10 NaCl, 100 CsCl, 20 TEA.Cl, 5 Mg.ATP, 0.3 Na.GTP, 20 HEPES/CsOH, pH 7.2; in some experiments (see Results) 14 mM EGTA was also included in the pipette solution.

Whole-cell recordings were made with fire-polished electrodes (resistance 2-5 M when filled with the standard Cs⁺/TEA intracellular solution) mounted on the headstage of a DAGAN 8900 patch-clamp amplifier, allowing cancellation of capacitative transient and compensation of series resistance. A Labmaster data acquisition and analysis board and a 386-based microcomputer with pCLAMP software (Axon Instruments, Foster City, CA) were used to acquire and analyze the data.

Cells were clamped at 80 mV holding potential (HP). Step depolarization to 0, +10, or +20 mV from this HP of different duration, were applied at various time intervals (see Results). Cells with pronounced rundown of I_Ca or I_Ba were discarded (Fenwick et al., 1982). Leak and capacitative currents were subtracted by using currents elicited by small hyperpolarizing pulses.

Measurements of changes of [Ca²⁺]_c in fura-2-loaded chromaffin cells. Chromaffin cells were loaded with fura-2 by incubating them with fura-2 AM (4 µM) for 30 min at room temperature in Krebs-HEPES solution, pH 7.4, containing (in mM): 145 NaCl, 5.9 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 10 Na-HEPES, 10 glucose. The loading incubation was terminated by washing several times the coverslip containing the attached cells, using Krebs-HEPES. The cells were then kept at 37°C in the incubator for 15-30 min. The fluorescence of fura-2 in single cells was measured with the photomultiplier-based system described by Neher (1989), which produces a spatially averaged measure of the [Ca²⁺]_c. Fura-2 was excited with light alternating between 360 and 390 nm, using a Nikon 40× fluorite objective. Emitted light was transmitted through a 425 nm dichroic mirror and 500-545 nm barrier filter before being detected by the photomultiplier. [Ca²⁺]_c was calculated from the ratios of the light emitted when the dye was excited by the two alternating excitation wavelengths (Grynkiewicz et al., 1985). Experiments were performed at room temperature (22-24°C).

Chemicals. Collagenase type A was purchased from Boehringer Mannheim (Madrid, Spain). DMEM, fetal calf serum, penicillin, and streptomycin were purchased from Life Technologies (Madrid, Spain). BSA, TTX, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), ruthenium red, oligomycin, and rotenone were purchased from Sigma (Madrid, Spain). Other chemicals were obtained from either Sigma or Merck (Madrid, Spain).

Statistical analysis. Results are expressed as means ± SEM. The statistical differences between means of two experimental results were assessed by Student's t test or one-factor ANOVA by Scheffe F test. A value of p  0.05 was taken as the limit of significance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effects of CCCP on I_Ca in cells dialyzed with intracellular solutions with or without EGTA

Experiments were designed to test how interference with the ability of mitochondria to sequester Ca²⁺ during cell activation modified the amplitude of peak inward Ca²⁺ channel currents. I_Ca was measured as indicated in Materials and Methods. In cells dialyzed with an EGTA-free intracellular solution, the current declined slowly and stabilized after 3-4 min. In eight cells, the current loss amounted to 35 ± 3% and had a time constant () of 78 ± 11 sec. Cells with initial current decline that was not stabilized after 3 min were discarded. The cell of Figure 1A shows an already stabilized initial I_Ca of 420 pA (peak current). Superfusion of the cell with CCCP caused almost full loss of I_Ca in 30 sec. The current quickly recovered during CCCP washout (in ~30 sec). A second addition of CCCP again caused a loss of I_Ca in a reversible manner, but the recovery was partial, likely because of the absence of EGTA in the pipette that favors the rundown of Ca²⁺ channels (Fenwick et al., 1982). Original traces in the inset show that contrary to the cell dialyzed with 14 mM EGTA (B, trace a, inset), this cell exhibited clear current inactivation during the depolarizing pulse, before (A, trace a, inset) and during CCCP treatment (A, trace b, inset). The averaged initial I_Ca amplitude was 415 ± 68 pA (n = 8 cells). In the presence of CCCP, I_Ca was fully blocked with the much faster  of 16.7 ± 3.4 sec. From now on we will call this I_Ca loss inhibition, to distinguish this effect from the classic Ca²⁺-dependent current inactivation occurring during cell depolarization (Hagiwara and Byerly, 1981).

View larger version (25K): [in this window] [in a new window]   Figure 1.   CCCP provokes the inhibition of ICa in cells dialyzed with an EGTA-free intracellular solution, but not in the presence of EGTA. The two cells of A and B were voltage clamped at 80 mV and dialyzed with 14 mM EGTA (B) or with an intracellular solution without EGTA (A). To generate inward whole-cell Ca2+ channel currents (10 mM Ca2+ in the extracellular solution), cells were stimulated with 20 msec test depolarizing pulses to +10 mV at 15 sec intervals. Each black square shows the amplitude of peak ICa (ordinates) as a function of the time course shown in the abscissa. CCCP (2 µM) and Cd2+ (100 µM) were added with the extracellular solution (that continuously superfused the cells) during the time periods indicated by the horizontal black bars. Insets at the right in A and B show original current traces taken at the times shown by small letters. These are original records of two typical experiments, of eight for each protocol (see Results for averaged data).

The second cell (Fig. 1B) was dialyzed with an intracellular solution containing 14 mM EGTA to prevent surges of [Ca²⁺]_c during cell activation. The initial peak I_Ca was >600 pA, and the current in this cell was fairly stable, both before and during the application for 3 min of the mitochondrial protonophore CCCP (2 µM). Applied at the end of the recording period, Cd²⁺ (100 µM) fully and reversibly inhibited I_Ca. Original I_Ca traces showed no obvious inactivation (Fig. 1B, inset). Averaged results obtained in cells dialyzed with EGTA using this protocol showed an initial I_Ca amplitude of 608.5 ± 4 pA (n = 8 cells); in the presence of CCCP, peak I_Ca decreased by only 5.32 ± 2% (n = 8).

Similar experiments were performed using 10 mM Ba²⁺ as charge carrier. As shown previously, Ba²⁺ generated greater peak currents than Ca²⁺ (Albillos et al., 1994): although the initial I_Ca in 20 cells averaged 493 ± 48 pA, the initial I_Ba of another 17 cells amounted to 854 ± 94 pA. The cell of Figure 2A was dialyzed with an EGTA-free intracellular solution. The initial I_Ba of 1300 pA was reduced in CCCP to 900 pA in 1 min. Washout of CCCP allowed a partial gradual recovery of the current, which was fully blocked by Cd²⁺. Averaged results from nine cells gave an initial I_Ba amplitude of 783 ± 140 pA, which was reduced by 39 ± 4.9% in CCCP ( of 76.9 ± 13 sec). In the cell of Figure 2B that was dialyzed with 14 mM EGTA, the initial I_Ba amounted to 1200 pA. With 2 µM CCCP, the current measured along a 5 min period of cell superfusion remained unchanged. Cd²⁺ (100 µM) fully and reversibly blocked such current. In eight cells the initial I_Ba in the absence of CCCP was 933 ± 136 pA, and in its presence I_Ba decreased by only 5.3 ± 4%. Original current traces (insets at the right in A and B) show that I_Ba did not suffer inactivation during the depolarizing pulse, neither in the cell dialyzed with EGTA (B) nor in the cell dialyzed with an EGTA-free solution (A).

View larger version (26K): [in this window] [in a new window]   Figure 2.   CCCP affects little the amplitudes of whole-cell Ba2+ currents (IBa), independently of cell dialysis with EGTA or with an EGTA-free intracellular solution. The cell of B was dialyzed with 14 mM EGTA and that of A with a solution deprived of EGTA. Cells were superfused with 10 mM Ba2+ as charge carrier and stimulated with 20 msec test depolarizing pulses to +10 mV at 15 sec intervals to study the time course of their peak IBa. CCCP (2 µM) or Cd2+ (100 µM) was applied during the time periods shown by the horizontal black bars. Original traces at the right in A and B were taken at the times shown by small letters. Nine cells were tested for each protocol. Here we present the results obtained in one prototypical cell with each protocol. See Results for averaged results.

Time course of CCCP-induced inhibition of I_Ca and of its recovery, studied at high-frequency stimulation

Under physiological conditions in the intact adrenal gland, chromaffin cell Ca²⁺ channels are likely recruited at intervals of 1 sec, because splanchnic nerves fire action potentials at ~1 Hz. Hence, experiments were performed to study I_Ca decline during repetitive pulsing. Cells were dialyzed with an EGTA-free intracellular solution. In these experiments, control currents showed an initial decay of their amplitude during repetitive pulsing at 1 Hz; the peak I_Ca decayed slightly to reach a steady state with a  of 4.3 sec (n = 50 cells). Once I_Ca stabilized (in ~15 sec), CCCP (0.02-20 µM) was given for 45 sec (Fig. 3A). As shown in the Figure, the rate of I_Ca inhibition was dependent on the concentration of CCCP applied. Thus, at 0.02 µM, I_Ca peak was inhibited by only 6.3% at the end of the application period (45 sec). At 0.2 µM CCCP, I_Ca declined slowly ( of 21.1 sec). At 2 and 20 µM CCCP, I_Ca declined much faster ( of 6.5 sec at 2 µM and 3.6 sec at 20 µM CCCP). The highest concentrations used, 2 and 20 µM, inhibited I_Ca by 100%, with an average  of 8.6 ± 1.8 sec (n = 15 cells) and 6 ± 0.7 sec (n = 18 cells), respectively. Blockade induced by 0.2 µM CCCP amounted to 52.1 ± 8.9%, with a  of 25 ± 8.1 sec (n = 11 cells). No significant blockade of I_Ca was seen with 0.02 µM CCCP (6.3 ± 4.1% blockade; n = 6 cells). The one-factor ANOVA by Scheffe F test showed statistically significant differences between the low and high CCCP concentrations.

View larger version (23K): [in this window] [in a new window]   Figure 3.   ICa generated by depolarizing test pulses applied at high frequency: inhibition by CCCP and recovery after its washout. Cells were dialyzed with an EGTA-free intracellular solution and superfused with a 10 mM Ca2+-based extracellular solution. They were voltage clamped at 80 mV and 10 msec pulses to +10 mV applied at 1 sec intervals. A shows experiments performed with the concentrations of CCCP shown at the right. ICa was normalized as a fraction of the initial current peak. In B, ICa was elicited at 1 sec intervals by applying 20 msec depolarizing test pulses to +20 mV. Superfusion of CCCP (2 µM) provoked a progressive decline of peak ICa amplitude (data not shown). Currents were measured at the times shown in the abscissa after washout of CCCP. ICa that recovered after each washout time period was normalized as a fraction of the initial ICa before CCCP (ordinate). Data are means ± SEM of 18 cells;  for current recovery was 73.6 sec (n = 18 cells).

To study I_Ca recovery after blockade by CCCP, cells were superfused with 2 µM CCCP, which suppressed fully the current after 29 sec of pulsing. Then, CCCP was removed form the superfusion, and I_Ca was tested at different time intervals (5-240 sec) (Fig. 3B). At 5 sec there was no current recovery, but at 30 sec a sizable I_Ca was measured; after 2 min, most of the current was recovered. The current is expressed as a fraction of the initial I_Ca; the recovery curve could be well fitted to a single exponential, showing a  of 73.6 sec (n = 18 cells) (Fig. 3B).

Effects on I_Ca of the intracellular application of ruthenium red

Another means of blocking Ca²⁺ sequestration by mitochondria was the direct inhibition of the Ca²⁺ uniporter by RR (Montero et al., 2000). Because it is cell impermeant, this compound was given intracellularly by dialysis with the patch pipette. Figure 4A shows averaged results obtained in chromaffin cells dialyzed with an EGTA-free solution in the absence (control) or the presence of RR (4 µM). The  for the decay of I_Ca in control cells was 1110 sec (n = 10 cells), whereas that of cells dialyzed with ruthenium red was 228 sec (n = 11 cells). The inhibition of peak current after 5 min recording was 24.5 ± 4.5% in control cells and 55.5 ± 6.5% in cells dialyzed with ruthenium red (p < 0.01). When EGTA (14 mM) was present in the intracellular solution, either with or without ruthenium red, no current inhibition was observed after 5 min stimulation (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 4.   RR and oligomycin plus rotenone accelerated the inhibition of ICa with repetitive depolarizing pulsing. A shows averaged results (± SEM) obtained in 10 control cells (no RR) and in 11 cells dialyzed with RR. Cells were voltage clamped at 80 mV, dialyzed with an EGTA-free intracellular solution, and superfused with 10 mM Ca2+ as charge carrier. ICa was elicited by 20 msec depolarizing test pulses to +10 mV, applied at 15 sec intervals. B and C show the effects of the superfusion with a solution containing oligomycin (3 µM) plus rotenone (4 µM) on two different cells dialyzed with 14 mM EGTA (C) or with an intracellular solution without EGTA (B). The cells were stimulated with 20 msec test depolarizing pulses to +10 mV at 15 sec intervals using 10 mM Ca2+ as charge carrier. Each black square shows the amplitude of peak ICa as a function of the time course shown in the abscissa. Oligomycin plus rotenone was applied with the continued extracellular perfusion during the time periods indicated by the horizontal black bars.

Effects of oligomycin plus rotenone on I_Ca amplitude in cells dialyzed with or without EGTA

Ca²⁺ uptake into mitochondria can be also blocked by using a combination of oligomycin plus rotenone. Oligomycin blocks mitochondrial ATP production by direct inhibition of the ATP synthase, whereas rotenone is a specific inhibitor of the electron transport chain that blocks electron transfer and proton extrusion mechanisms, thereby decreasing mitochondrial membrane potential and ATP synthesis. Thus, the dissipation of the negative membrane potential of the mitochondria will prevent the Ca²⁺ uptake by the uniporter, leading Ca²⁺ to accumulate near the Ca²⁺ channels.

The cell shown in Figure 4B was dialyzed with an intracellular EGTA-free solution. Once I_Ca reached a stable value (~500 pA), superfusion of the cell with a mixture of oligomycin (3 µM) plus rotenone (4 µM) caused the loss of ~60% of the current in 3 min. After washout of the drugs, I_Ca recovered slowly and partially. In 11 cells, the superfusion of the cell with oligomycin plus rotenone induced a 67 ± 10% inhibition of I_Ca, with an averaged  of 102 ± 12 sec.

Figure 4C shows a similar experiment in one cell dialyzed with an intracellular solution containing 14 mM EGTA. In this cell, superfusion with the mixture of oligomycin plus rotenone caused a small inhibition of I_Ca, which recovered fully after washout of the drugs. Averaged results obtained in EGTA-dialyzed cells shows an inhibition of 17 ± 1% of I_Ca (n = 3 cells). This inhibition of I_Ca in cells dialyzed with EGTA-containing solutions seems to be related to oligomycin, as proven by the fact that superfusion of cells with only oligomycin induced a similar inhibition (19 ± 4%; n = 4 cells), whereas superfusion with rotenone alone had no effect on I_Ca in cells dialyzed with EGTA (data not shown).

CCCP increased the [Ca²⁺]_c in resting cells, whereas oligomycin plus rotenone did not

The differences observed between CCCP effects and those of oligomycin plus rotenone could be attributed to the ability of the former compound to release Ca²⁺ from mitochondria and other intracellular stores, in addition to its effect of preventing Ca²⁺ uptake into mitochondria. To test this possibility, we studied the effects of these compounds on the [Ca²⁺]_c levels in single fura-2-loaded bovine chromaffin cells. In the cell shown in Figure 5, superfusion of the cell with a solution containing oligomycin (1 µM) plus rotenone (4 µM) did not induce any significant change on [Ca²⁺]_c. However, superfusion of the cells with 2 µM CCCP during 90 sec induced a fast increase of [Ca²⁺]_c to approximately three to four times the basal levels (basal level amounted to 55 ± 1 nM; n = 10 cells). As shown in the Figure, effects of CCCP were reversible and reproducible after a second application of the compound. Similar results were obtained in the other eight cells, in which application of CCCP induced an increase of [Ca²⁺]_c levels to a peak of 187 ± 19 nM.

View larger version (15K): [in this window] [in a new window]   Figure 5.   Changes in the [Ca2+]c in fura-2-loaded cells induced by CCCP, oligomycin, and rotenone. Typical records of changes in the [Ca2+]c in one fura-2-loaded chromaffin cell are shown. The cell was superfused for 90 sec with CCCP (2 µM) or a mixture of oligomycin (1 µM) plus rotenone (4 µM), as shown by the horizontal bars. At the end of the experiment, after the washout of CCCP, a K+ pulse (70 mM isotonic K+, 2.5 mM Ca2+, 5 sec) was applied as shown by the arrow to assess the viability of the cell. The [Ca2+]c is expressed in nanomolar (ordinate). Similar results were obtained in the other eight cells (see Results for averaged results).

Different effects of CCCP on the various subtypes of Ca²⁺ channels in chromaffin cells

We have studied the effects of the superfusion with CCCP (2 µM) on L-, N-, and P/Q-type Ca²⁺ channels in bovine chromaffin cells. In these experiments, cells were dialyzed with an intracellular EGTA-free solution. To isolate P/Q-type Ca²⁺ channels, cells were superfused with a combination of nisoldipine (an L-type Ca²⁺ channel blocker; 3 µM) plus -agatoxin GVIA (an N-type Ca²⁺ channel blocker; 1 µM). To isolate N-type Ca²⁺ channels, cells were superfused with a combination of nisoldipine plus -agatoxin IVA (a P/Q-type Ca²⁺ channel blocker; 2 µM). To isolate L-type Ca²⁺ channels, cells were superfused with -conotoxin GVIA (1 µM) plus -agatoxin IVA (2 µM).

Figure 6A shows a typical experiment in a cell treated with nisoldipine plus -conotoxin GVIA. Once the current stabilized, we studied the effects of CCCP on the remaining current, mostly P/Q Ca²⁺ channel current, by superfusing the cell with 2 µM CCCP. This treatment led to a prompt decrease of I_Ca, which slowly and partially recovered after washout of CCCP. Averaged results showed that under these conditions, I_Ca was inhibited by 86.4 ± 3% (n = 10) in the first 10 sec of superfusion with CCCP (Fig. 6E). Inhibition of I_Ca was almost complete and developed with a  of 7.2 ± 1 sec (n = 10 cells) (Fig. 6D,F). When cells were treated with a combination of -conotoxin GVIA plus -agatoxin IVA to isolate the L-type Ca²⁺ channel current (Fig. 6B), superfusion of the cells with 2 µM CCCP induced a slower blockade of the remaining L-type Ca²⁺ channel current, with a  of 19.1 ± 2 sec (n = 10 cells) (Fig. 6D,F). Only 54 ± 8% (n = 10 cells) of the I_Ca was blocked during the first depolarizing pulse in the presence of CCCP (Fig. 6E). Finally, in cells treated with -agatoxin IVA plus nisoldipine to isolate N-type channel currents, superfusion of the cells with 2 µM CCCP induced a fast ( of 11.4 ± 1 sec; n = 14 cells) (Fig. 6C,F) and almost complete blockade of I_Ca of 80.5 ± 4% in the first depolarizing pulse (n = 14 cells) (Fig. 6E).

View larger version (33K): [in this window] [in a new window]   Figure 6.   CCCP caused a faster inhibition of ICa through non-L, as compared with L-type Ca2+ channels. Cells were voltage clamped at 80 mV and stimulated with 20 msec test depolarizing pulses to +10 mV at 15 sec intervals using 10 mM Ca2+ as charge carrier and dialyzed without EGTA. A shows the time course of peak ICa recorded in one cell superfused with nisoldipine (3 µM) and -conotoxin GVIA (GVIA; 1 µM) to block L- and N-type Ca2+ channels, as well as the blocking effects of CCCP (2 µM) of the remaining current. B shows a similar experiment in which N- and P/Q-type Ca2+ channels were blocked by superfusing the cell with GVIA and -agatoxin IVA (AGA; 2 µM); after this, CCCP was applied at 2 µM. C shows an experiment in which L- and P/Q-type Ca2+ channels were blocked by superfusing the cell with nisoldipine and -agatoxin IVA; after this, CCCP was applied at 2 µM. D shows the normalized averaged time course of CCCP effects under these conditions. Blockade of N- and P/Q-type Ca2+ channels developed faster, with a  of 9.2 sec (n = 14) and 8.2 sec (n = 10 cells), respectively, than that of L-type Ca2+ channels ( = 20.6 sec; n = 10 cells) as shown in F. E shows averaged data of the blockade of the first depolarizing pulse induced by CCCP under each experimental condition. The data are means ± SEM of 10-14 cells. *p < 0.05, **p < 0.001.

Effects of CCCP on Na⁺ channel currents and nicotinic receptor currents

It was of interest to test the specificity of the effects of CCCP on Ca²⁺ channel currents; thus, its effects on Na⁺ channel currents and nicotinic receptor currents were also studied. Figure 7A shows the time course of peak I_Na in a chromaffin cell dialyzed with an EGTA-free solution. Test pulses to 10 mV produced initial I_Na of near 1200 pA amplitude. Addition of 2 µM CCCP for 2 min did not affect significantly the amplitude of the current; neither the kinetics of activation nor the kinetics of inactivation of the current was affected, as seen in the original I_Na traces shown in the inset. The  for inactivation of I_Na before CCCP was 1 msec, and after 90 sec superfusion with CCCP the  amounted to 0.998 msec (inset). Averaged results obtained with this protocol show a peak I_Na amplitude of 851 ± 119 pA (10 cells) and 800 ± 113 pA (10 cells), respectively, before and during CCCP superfusion (6.1% of current inhibition); the  values for I_Na inactivation were 0.997 ± 0.001 and 0.998 ± 0.001 msec, respectively.

View larger version (16K): [in this window] [in a new window]   Figure 7.   Inward currents through voltage-dependent Na+ channels (INa) and through nicotinic receptor channels (IACh) were not affected by CCCP. A shows the time course of peak INa elicited by 16 msec depolarizing test pulses to 10 mV, applied at 15 sec intervals to a cell voltage clamped at 80 mV. The cell was superfused with an extracellular solution containing 137 mM Na+ and 0 mM Ca2+ and dialyzed with an intracellular EGTA-free solution. CCCP was superfused as shown by the black horizontal bar. Insets show original traces taken immediately before (control), at 90 sec of superfusion with CCCP, and 2 min after washout of CCCP. Similar results were obtained in the other nine cells; averaged values of INa for all cells are given in Results. The stimulation protocol for recording of IACh is shown at the top of B. The cell was voltage clamped at 80 mV, and then stimulation was applied at 120 sec intervals as follows. First, a 1 sec depolarizing pulse to +20 mV was applied, and then, after 300 msec, an acetylcholine (ACh) pulse of 500 msec duration was given. Typical current traces obtained before (control) and at the 90 sec of superfusion with 2 µM CCCP are shown.

The protocol used to explore sequentially the effects of CCCP on Ca²⁺ channels and nicotinic receptor channel currents is shown at the top of Figure 7B. The control trace shows first the I_Ca generated by a long (1 sec) depolarizing pulse to +20 mV, followed by the current induced by 500 msec application of 100 µM ACh (I_ACh). I_Ca (372 pA) inactivated with a  of 120.2 msec. I_ACh had a greater amplitude (1160 pA) and desensitized with a  of 98.5 msec. In the presence of 2 µM CCCP (120 sec superfusion), peak I_Ca was greatly inhibited (20.5 pA) but I_ACh was unaffected (1049 pA), showing a  for its desensitization of 92.3 msec. In 17 cells, I_Ca (in 10 mM Ca²⁺) amounted to 284 ± 36 pA and was reduced to 63 ± 88 pA in the presence of CCCP (75% current inhibition). However, I_ACh was 1091 ± 98 and 972 ± 95 pA, before and after superfusion of the cells with 2 µM CCCP (~11% of current loss).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The central finding of this study was the inhibition by CCCP of the amplitude of peak I_Ca generated by repeated depolarizing pulses of voltage-clamped chromaffin cells. This effect seemed to be quite selective for Ca²⁺ channel currents. In fact, other inward currents through voltage-dependent Na⁺ channels or nicotinic receptor channels were scarcely affected by CCCP.

Because it is an uncoupler of oxidative phosphorylation, CCCP could cause ATP depletion and a deficit of energy supply to Ca²⁺-ATPases and hence inhibition of I_Ca. However, the intracellular pipette solution contained 5 mM ATP that surely served to fuel intracellular as well as plasmalemmal Ca²⁺-ATPases. Another possibility is that CCCP causes direct pharmacological inhibition of Ca²⁺ channels, as reported by Stapleton et al. (1994) and Park et al. (1996). This is also unlikely because a compound causing direct blockade of Ca²⁺ channels will do so regardless of the use of Ca²⁺ or Ba²⁺ as charge carrier, or regardless of the presence or absence of EGTA in the intracellular solution. This is the case, for instance, for the neuroprotectant lubeluzole, which blocks L- and N- as well as P/Q-type Ca²⁺ channel currents regardless of the use of Ca²⁺ or Ba²⁺ as charge carrier (Hernández-Guijo et al., 1997). This was not the case for CCCP, which caused the quick inhibition of I_Ca (Fig. 1A) but only a mild inhibition of I_Ba (Fig. 2A). In addition, the inhibition of I_Ca was fully prevented by intracellular EGTA.

Rather, we attribute the effects of CCCP on I_Ca to its well known effects on mitochondria. CCCP collapses the proton gradient and the electrical potential, causing mitochondrial depolarization. This has two immediate consequences: blockade of Ca²⁺ uptake by the mitochondrial uniporter and the release of any stored mitochondrial Ca²⁺. This will cause an increase of the bulk [Ca²⁺]_c (Fig. 5) that will reach tenths of micromolar at subplasmalemmal sites, after cell depolarization, as we recently demonstrated using mitochondrially targeted aequorin and electroporated bovine chromaffin cells (Montero et al., 2000). We believe that this high local [Ca²⁺]_c is responsible for the inhibition of I_Ca. Such inhibition is gradual during repeated application of depolarizing pulses at 1 Hz (Fig. 3). This was likely caused by the progressive elevation of [Ca²⁺]_c near the Ca²⁺ channels, because Ca²⁺ enters the cell through those channels during each pulse, and Ca²⁺ cannot be buffered by CCCP-poisoned mitochondria. A subpopulation of mitochondria is capable of taking up vast amounts of Ca²⁺, which reach mitochondrial [Ca²⁺] near the millimolar. These mitochondria can sense up to 50 µM [Ca²⁺]_c, and hence they must be located close to the plasmalemma, where such large [Ca²⁺]_c gradients are possible (Montero et al., 2000).

A second mechanism relates to the release of mitochondrial Ca²⁺ as a consequence of the dissipation of the proton gradient. If CCCP is applied when mitochondria is still full of Ca²⁺, then CCCP will induce a prompt release of mitochondrial Ca²⁺ that generates the increase in local [Ca²⁺]_c near the internal mouth of the Ca²⁺ channel (Fig. 5). This increase will promote the Ca²⁺-induced inhibition of Ca²⁺. This second mechanism can also explain the differences observed between CCCP and the other agents used to prevent Ca²⁺ uptake into mitochondria, i.e., slower and partial I_Ca decay after ruthenium red dialysis or extracellular superfusion with oligomycin plus rotenone. As shown in Figure 5, CCCP favors Ca²⁺ release from mitochondria and other intracellular store in fura-2-loaded chromaffin cells, whereas oligomycin plus rotenone did not.

That the dialysis of the cells with 14 mM EGTA completely prevented the current inhibition by CCCP (Fig. 1B) strongly supports the hypothesis that the sequestration of Ca²⁺ by mitochondria plays a major role in maintaining functional Ca²⁺ channels during repeated stimulation of chromaffin cells. This was corroborated also by three additional experimental findings. First, ruthenium red, which blocks the Ca²⁺ uniporter thus preventing mitochondrial Ca²⁺ uptake, also caused the gradual inhibition of I_Ca during repeated depolarization pulses (Fig. 4A). Second, combined oligomycin plus rotenone, which also collapses the mitochondrial potential, also caused gradual I_Ca inhibition (Fig. 4B), which was again prevented by 14 mM intracellular EGTA (Fig. 4C). Third, when Ba²⁺ was used as charge carrier instead of Ca²⁺, CCCP caused little inhibition of I_Ba. This might be explained by the fact that Ba²⁺ is a poor substrate for the Ca²⁺ transport systems (Schilling et al., 1989; Wagner-Mann et al., 1992). In addition, Ba²⁺ has also been described as inducing the inactivation of Ca²⁺ channels, although with an affinity for the inactivation site on the Ca²⁺ channels 100 times slower than that of Ca²⁺ (Ferreira et al., 1997). The partial inhibition of I_Ba induced by CCCP could be also attributed to Ba²⁺-induced release of Ca²⁺ from intracellular stores or binding sites (von Rüden et al., 1993). It is plausible that this Ca²⁺ may be taken up by mitochondria in control conditions (Montero et al., 2000). However, treatment of the cells with CCCP will preclude this mitochondrial Ca²⁺ removal, and this Ca²⁺ might thus contribute to the partial blockade of Ca²⁺ channel currents seen when Ba²⁺ is used as charge carrier.

We believe that the full inhibition of I_Ca by CCCP, indicating that L- and N- as well as P/Q-type Ca²⁺ channels were affected, is a most interesting finding (Fig. 6). Modulation by Ca²⁺ of L-type channels is well illustrated; however, little evidence is available for N and P/Q channels. The Ca²⁺-dependent inactivation of Ca²⁺ channels seems to be more sensitive to Ca²⁺ in the case of L-type Ca²⁺ channels, as compared with non-L-type Ca²⁺ channels (Plant, 1988; Kasai and Neher, 1992). However, we find here that N and P/Q channels were inhibited faster than L channels after repeated application of depolarizing pulses to CCCP-treated cells (Fig. 6D). This raises the question of whether the classic Ca²⁺-dependent inactivation of Ca²⁺ channel currents (Hagiwara and Byerly, 1981) and the Ca²⁺-dependent inhibition of such currents (this work) are different manifestations of the same underlying mechanism of modulation of the channels by Ca²⁺ ions.

In any case, the idea that clearly emerges from this study is that as on other cell types (Bassani et al., 1992; Rizzuto et al., 1992; Friel and Tsien, 1994; Drummond and Fay, 1996; Park et al., 1996; Greenwood et al., 1997), mitochondria have an important role in shaping the [Ca²⁺]_c transients generated under our experimental conditions (i.e., brief depolarizing pulses applied at 0.1-1 Hz to dialyzed cells). It might be that under more physiological stimulation conditions of chromaffin cells (i.e., by short-cut repeated depolarizing pulses), mitochondria could sense the high [Ca²⁺]_c that can be reached only underneath the plasma membrane (Montero et al., 2000). Hence, we believe that by limiting the extent and duration of such large local [Ca²⁺]_c transients, mitochondria strategically located near the plasma membrane play the important function of maintaining the L, N, and P/Q Ca²⁺ channels ready to be recruited under stressful conditions that cause repetitive stimulation of chromaffin cells, by endogenously released acetylcholine in the intact adrenal gland.

	FOOTNOTES

Received Dec. 11, 2000; revised Jan. 6, 2001; accepted Jan. 7, 2001.

This work was supported in part by grants from DGICYT (Dirección General de Investigación Científica y Técnica; No. PB99-0004 and No. PB99-0005), CAM (Comunidad Autónoma de Madrid; No. 08.5/0002/98 and No. 08.5/0007/98), Programa de Grupos Estratégicos CAM/UAM, and Fundación Teófilo Hernando, Spain. We thank Ricardo de Pascual for the preparation of cell cultures. J.M.H.G. and A.R.N. are fellows of CAM, Madrid, Spain. We also thank Drs. Javier García-Sancho and Javier Alvarez for their suggestions and criticisms.

Correspondence should be addressed to Luis Gandía, Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, 28029 Madrid Spain. E-mail: luis.gandia{at}uam.es.

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