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A novel regulatory mechanism of the mitochondrial Ca²⁺ uniporter revealed by the p38 mitogen-activated protein kinase inhibitor SB202190 ¹

Instituto de Biología y Genética Molecular, Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Ramón y Cajal, 7, E-47005 Valladolid, Spain

²Correspondence: Instituto de Biología y Genética Molecular (IBGM), Departamento de Bioquímica y Biol. Mol. y Fisiología, Facultad de Medicina, Ramón y Cajal, 7, E-47005 Valladolid, Spain. E-mail: jalvarez{at}ibgm.uva.es

SPECIFIC AIMS

Ca²⁺ uptake by mitochondria has a large impact on overall cell Ca²⁺ homeostasis, allowing mitochondria to become important modulators of many different [Ca²⁺]_c-dependent processes, including apoptosis. However, little is known about the modulation of mitochondrial Ca²⁺ fluxes. The aim of this work was to explore regulation of the main mitochondrial Ca²⁺ import system, the Ca²⁺ uniporter.

PRINCIPAL FINDINGS

1. Size of the agonist-induced [Ca²⁺]_M peaks in HeLa cells
Measurements of [Ca²⁺]_M in HeLa cells using mitochondrially targeted aequorins with different Ca²⁺ affinities showed that histamine and carbachol induced rapid peaks of mitochondrial [Ca²⁺] ([Ca²⁺]_M) in HeLa cells, reaching values of 20–30 µM (Fig. 1 ). These are the mean [Ca²⁺]_M values of the entire mitochondrial population and there is probably great variability in the responses of individual mitochondria.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of SB202190 on the [Ca2+]M peaks induced by histamine and carbachol. In the upper panel, HeLa cells expressing mitmutAEQ (MM5 cell clone) reconstituted with wild type coelenterazine were stimulated with either 100 µM histamine or 500 µM carbachol. In experiments labeled ‘+SB’, 10 µM SB202190 was present during stimulation and for 5 min before the stimulus. In the lower panel, similar experiments were performed using MM5 cells reconstituted with coelenterazine n.

2. SB202190 strongly enhances agonist-induced [Ca²⁺]_M increase
The size of the agonist-induced mitochondrial [Ca²⁺] peak was dramatically increased (4-fold) when cells were preincubated briefly with 10 µM SB202190, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase (Fig. 1) . This effect was due to an fourfold increase in the rate of Ca²⁺ uptake by mitochondria.

3. SB202190 reduced agonist-induced cytosolic [Ca²⁺] ([Ca²⁺]_c) increase and did not modify Ca²⁺ release from the endoplasmic reticulum or Ca²⁺ entry from the extracellular medium
Preincubation with 10 µM SB202190 reduced the agonist-induced [Ca²⁺]_c increase and did not modify Ca²⁺ release from the endoplasmic reticulum as measured with targeted aequorin. Removal of extracellular Ca²⁺ did not modify the effects of SB202190 on the histamine-induced [Ca²⁺]_M peak. These findings clearly suggest that the primary effect of SB202190 is an increase in the rate of Ca²⁺ uptake by mitochondria.

4. SB202190 does not affect mitochondrial Ca²⁺ release
Using the specific inhibitor of mitochondrial Na⁺/Ca²⁺ exchange CGP37157, we show that inhibition of the main pathway for mitochondrial Ca²⁺ efflux produces an effect distinct and additive to that of SB202190.

5. The effects of SB202190 appear fast and are rapidly reversible
In intact HeLa cells, the effect of SB202190 on mitochondrial Ca²⁺ uptake was nearly maximum after 2 min of preincubation and was rapidly reversible within 2–5 min.

6. SB202190 reversibly activates mitochondrial Ca²⁺ uptake in permeabilized cells and only at physiological [Ca²⁺]_c
SB202190 produced a strong and reversible activation of mitochondrial Ca²⁺ uptake in permeabilized cells perfused with controlled amounts of Ca²⁺ (Fig. 2 ). The activation was much stronger (>10-fold) at low [Ca²⁺] (1–3 µM) and became smaller when [Ca²⁺] was increased above those levels. In fact, SB202190 produced no effect on mitochondrial Ca²⁺ uptake when the perfused [Ca²⁺] was 5 µM. The activated mitochondrial Ca²⁺ uptake was sensitive to ruthenium red, indicating it occurs through the Ca²⁺ uniporter.

View larger version (24K): [in this window] [in a new window]   Figure 2. Effect of SB202190 on mitochondrial Ca2+ uptake in permeabilized cells. MM5 cells expressing mitmutAEQ reconstituted with coelenterazine n were permeabilized. Buffers containing different [Ca2+] and/or 10 µM SB202190 were perfused. In the lower panels, traces obtained in the presence and in the absence of 10 µM SB202190 are superimposed for each [Ca2+]. In traces labeled +SB, the inhibitor was present for 2 min before and during perfusion of the [Ca2+] buffer.

7. Other structurally related p38 MAP kinase inhibitors produced little or no effect on mitochondrial Ca²⁺ uptake
SB202190 increased mitochondrial Ca²⁺ uptake even at concentrations as low as 1 µM, although the maximum effect was obtained only at 10–20 µM. However, several structurally related p38 MAP kinase inhibitors produced little (SB203580) or no effect (PD169316 and SB220025) at higher concentrations (30 µM).

CONCLUSIONS AND SIGNIFICANCE

We show in this paper a novel regulatory mechanism of mitochondrial Ca²⁺ uptake that may have physiological relevance for fine-tuning of mitochondrial and cytosolic [Ca²⁺] after cell stimulation. Our data show that the mitochondrial Ca²⁺ uniporter can increase its activity at constant [Ca²⁺]_c by more than one order of magnitude, rapidly and reversibly, just by adding or removing the p38 MAP kinase inhibitor SB202190. This effect occurs only at low [Ca²⁺]_c (1–4 µM), precisely those more important from a physiological point of view. Thus, when this mechanism is activated, the increased mitochondrial Ca²⁺ uptake may damp [Ca²⁺]_c changes, inhibiting [Ca²⁺]_c-dependent processes. In intact HeLa cells, the increased activity of the Ca²⁺ uniporter induced by SB202190 led to a large increase in the mitochondrial Ca²⁺ peaks and a concomitant decrease in cytosolic [Ca²⁺] peaks. Therefore, the increase in mitochondrial Ca²⁺ uptake was able to modulate [Ca²⁺]_c. To fully appreciate this effect, it has to be realized that the [Ca²⁺]_M and [Ca²⁺]_c peaks have a similar time course, so that most of the Ca²⁺ accumulated in mitochondria is being released back into the cytosol before the end of the [Ca²⁺]_c peak. Moreover, local [Ca²⁺]_c changes in locations close to mitochondria are expected to be reduced by a much greater extent.

SB202190 belongs to a family of pyridinyl imidazole compounds that have been shown to be very potent and specific inhibitors of p38 and p38ß at the concentrations used. Therefore, the new regulation of the Ca²⁺ uniporter described here could be controlled by this pathway. However, the lack of effect of several structurally related p38 MAP kinase inhibitors (SB203580, PD169316, SB220025) suggests that a different but closely related protein kinase may be involved. On the other hand, the striking difference between the effects of SB202190 and these compounds, which are similar structurally, indicates that SB202190 binds to its target with high specificity.

In conclusion, our results suggest that an SB202190-sensitive protein kinase keeps a tonic inhibition of the mitochondrial Ca²⁺ uniporter in HeLa cells, which is rapidly and reversibly relieved by this compound (Fig. 3 ). That the effect of SB202190 develops fast in permeabilized cells and is rapidly reversible provides insight regarding the mechanism of the effect. Apart from excluding participation of soluble cytosolic factors, these data suggest that all the elements responsible for the regulation are tightly attached to mitochondria. Further work is necessary to identify the precise mechanism of this regulation and how can it be modulated under physiological conditions. Regarding the kinetic mechanism of the modulation of the Ca²⁺ uniporter, the observed shift in the [Ca²⁺]_c activation curve suggests that the protein kinase activity may be reducing the [Ca²⁺]_c affinity of the uniporter.

View larger version (37K): [in this window] [in a new window]   Figure 3. Schematic model of the regulation of the Ca2+ uniporter. The protein kinase sensitive to SB202190 and the associated phosphatase are both tentatively depicted as attached to the inner mitochondrial membrane. On phosphorylation of the Ca2+ uniporter, its Ca2+ affinity decreases and remains closed (left). In the presence of SB202190 (right), dephosphorylation of the uniporter increases its Ca2+ affinity, facilitating opening at relatively low cytosolic [Ca2+].

Our finding opens a broad new field of interactions among plasma membrane receptors and mitochondrial Ca²⁺ uptake. There is considerable evidence in different cell types that mitochondrial Ca²⁺ uptake modulates [Ca²⁺]_c transients. Therefore, mitochondrial Ca²⁺ uptake may modulate many [Ca²⁺]_c-dependent processes such as secretion, neurotransmission, or cell contraction. In chromaffin cells, for example, we and others have shown previously that catecholamine secretion is strongly enhanced when mitochondrial Ca²⁺ uptake is abolished. The question then was how this mitochondrial Ca²⁺ uptake could be modulated to have physiological significance. The new regulatory pathway described in this paper may be the link required to allow mitochondria to perform a flexible regulation of [Ca²⁺]_c-dependent phenomena. We can now predict, for example, that stimulation of this protein kinase pathway in neuronal cells should lead to inhibition of mitochondrial [Ca²⁺] uptake, local increase in [Ca²⁺]_c, and potentiation of secretion. Agonists acting through this pathway may therefore prime the secretory response by blocking mitochondrial Ca²⁺ buffering. On the other hand, several inhibitors of p38 MAP kinase are being used now in a series of preclinical and clinical studies as possible oral therapeutic agents to treat several inflammatory and cardiovascular diseases. The new effect we show here of SB202190 on mitochondrial Ca²⁺ uptake may be highly relevant when interpreting some of the clinical effects of these drugs.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0553fje; to cite this article, use FASEB J. (October 4, 2002) 10.1096/fj.02-0553fje

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