Intracellular transport of calcium from plasma membrane to mitochondria in adrenal H295R cells: implication for steroidogenesis

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Intracellular transport of calcium from plasma membrane to mitochondria in adrenal H295R cells: implication for steroidogenesis

LALEVEE, Nathalie, et al.


Angiotensin II and extracellular potassium stimulate aldosterone production in adrenal glomerulosa cells by mobilizing the calcium messenger system. This response requires calcium influx across the plasma membrane, followed by calcium uptake into the mitochondria. It has been proposed that calcium is transported to the mitochondria via the lumen of the endoplasmic reticulum, acting as a kind of intracellular calcium pipeline. This hypothesis has been tested in the present study by measuring intramitochondrial calcium variations in H295R cells with a new fluorescent calcium probe, ratiometric pericam. Calyculin A, a protein phosphatase inhibitor, induced the formation of a large cortical layer of actin filaments, removing the peripheral endoplasmic reticulum away from the plasma membrane and thereby physically uncoupling the calcium channels from the pipeline. The mitochondrial calcium response to potassium was markedly reduced after calyculin treatment, but that of AngII was unaffected. Under the same conditions, potassium-stimulated pregnenolone and aldosterone production was significantly reduced, whereas the [...]

LALEVEE, Nathalie, et al . Intracellular transport of calcium from plasma membrane to

mitochondria in adrenal H295R cells: implication for steroidogenesis. Endocrinology , 2003, vol.

144, no. 10, p. 4575-85

DOI : 10.1210/en.2003-0268 PMID : 12960050

Available at:

Disclaimer: layout of this document may differ from the published version.

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Intracellular Transport of Calcium from Plasma

Membrane to Mitochondria in Adrenal H295R Cells:

Implication for Steroidogenesis



Division of Endocrinology and Diabetology, Department of Internal Medicine (N.L., V.R., M.F.R.), Laboratory of Clinical Chemistry, Department of Pathology (M.F.R.), and Department of Physiology (S.A., N.D.), University Hospital, CH-1211 Geneva 14, Switzerland

Angiotensin II and extracellular potassium stimulate aldoste- rone production in adrenal glomerulosa cells by mobilizing the calcium messenger system. This response requires cal- cium influx across the plasma membrane, followed by calcium uptake into the mitochondria. It has been proposed that cal- cium is transported to the mitochondria via the lumen of the endoplasmic reticulum, acting as a kind of intracellular cal- cium pipeline. This hypothesis has been tested in the present study by measuring intramitochondrial calcium variations in H295R cells with a new fluorescent calcium probe, ratiometric pericam. Calyculin A, a protein phosphatase inhibitor, in- duced the formation of a large cortical layer of actin filaments, removing the peripheral endoplasmic reticulum away from the plasma membrane and thereby physically uncoupling the calcium channels from the pipeline. The mitochondrial cal- cium response to potassium was markedly reduced after ca-

lyculin treatment, but that of AngII was unaffected. Under the same conditions, potassium-stimulated pregnenolone and al- dosterone production was significantly reduced, whereas the steroidogenic response to AngII remained unchanged. The inhibitory action of calyculin A on the responses to potassium was not mediated by a modification of the calcium channel activity and was not accompanied by a reduction of the cy- tosolic calcium response. It therefore appears that, in H295R cells, the organization of the actin cytoskeleton at the cell periphery influences the steroidogenic action of potassium, but not the response to angiotensin II. The response to potas- sium is proposed to be dependent on the endoplasmic retic- ulum-mediated transfer of calcium entering through plasma membrane calcium channels to the mitochondria. (Endocri- nology144: 4575– 4585, 2003)


LDOSTERONE PRODUCTION by adrenal glomeru- losa cells in response to angiotensin II or extracellular potassium requires calcium influx, followed by calcium up- take into the mitochondria. Because both agents are known to depolarize the cells (1), in addition to enhance Ca2⫹influx (2, 3), voltage-operated Ca2⫹channels appear to be directly involved in the control of steroidogenesis. In glomerulosa cells, both low threshold (T-type) and high threshold (L-type) voltage-operated calcium channels have been characterized (4, 5). Recently, a clear dissociation between L- and T-type channel functions has been established, and Ca2⫹entering through each channel appears to have distinct roles and destinations (6). L-Type channels are major mediators of the large cytosolic free calcium concentration ([Ca2⫹]c) varia- tions observed in response to small, physiological increases in extracellular potassium concentrations, but selective in- hibition of these channels does not markedly affect steroi- dogenesis (6, 7). In contrast, despite of the fact that the cy- tosolic Ca2⫹increase resulting from T channel opening is barely detectable, the activity of T channels has been shown to be more closely related to aldosterone production (8, 9).

Although the angiotensin II- (AngII) and potassium-

induced control of aldosterone synthesis and the involve- ment of voltage-operated calcium channels in this process have been extensively documented, much less is known about the mechanism by which calcium entering through T channels is forwarded to mitochondria, the cellular site where it will control rate-limiting steps of steroidogenesis. To take into account the functional specificity of calcium chan- nels in glomerulosa cells and the poor diffusion of Ca2⫹

through the cytosol, we have proposed a model in which calcium would be transported from the T channels, embed- ded in the plasma membrane, to the mitochondria via the lumen of the endoplasmic reticulum (e.r.). The e.r. would therefore act like a kind of intracellular calcium pipeline, specifically connected to T channels (10). This hypothesis, however, implies 1) a nonrandom distribution of the calcium channels within the plasma membrane, and 2) the mainte- nance of a close physical relation between T channel-en- riched regions of the plasma membrane and specific portions of the e.r., able to rapidly accumulate local ambient Ca2. This second assumption has been tested in the present study using the human adrenocarcinoma aldosterone-producing cell line, H295R (11), by removing the e.r. away from the plasma membrane with calyculin A and therefore preventing the transfer of calcium from the channel into this putative pipeline.

Calyculin A is a serine/threonine phosphatase inhibitor that specifically blocks PP1 and PP2A enzymes and induces Abbreviations: AngII, Angiotensin II; [Ca2⫹]c, cytosolic free calcium

concentration; [Ca2⫹]m, mitochondrial calcium concentration; e.r., en- doplasmic reticulum; GFP, green fluorescent protein; mt, mitochondria- targeted; mtRFP, mitochondria-targeted red fluorescent protein; pCMV, cytomegalovirus promoter.



major cytoskeletal reorganization within the cell. Indeed, it has been shown in several cell types that after treatment with this drug, normal stress fibers disappear rapidly from the center of the cytoplasm, actin filaments are redistributed at the periphery, and actin becomes tightly condensed near the plasma membrane (12, 13). In addition, condensed actin bun- dles can be observed inside the cells, which may reflect the interaction of actin filaments with intermediate filaments (14). Such in situ modification of the actin cytoskeleton by steric obstruction prevents coupling between the e.r. and the plasma membrane Ca2⫹ channels, while not affecting the inositol 1,4,5-trisphosphate-mediated chemical coupling be- tween stimulated receptors and the release of stored Ca2⫹

(15). This approach has been recently used to demonstrate the requirement for a conformational interaction between intracellular Ca2⫹stores and the plasma membrane during activation of the Ca2⫹ capacitative influx (15). Moreover, calyculin action was reversible, and disassembly of the cor- tical actin layer resulting from washing out the drug per- mitted reestablishment of coupling and reactivation of Ca2⫹

entry. Thus, a physical actin barrier, while permitting trans- mission of the diffusible messenger inositol 1,4,5-trisphos- phate, prevents the close interaction between e.r. and the plasma membrane that is required to activate Ca2⫹entry.

Recently, a new fluorescent calcium probe, derived from circularly permuted green fluorescent proteins (GFP) and engineered to sense calcium, has been described for moni- toring calcium signaling within cell organelles (16). Indeed, like any protein, ratiometric pericam can be specifically tar- geted, for example, to the mitochondria, where its fluorescent spectral properties (excitation wavelength) change revers- ibly with intramitochondrial calcium fluctuations. Thanks to its high fluorescence yield, the signals elicited by ratiometric pericam can be easily detected and analyzed at the subcel- lular levels.

In the present study the consequence of calyculin A treat- ment on the mitochondrial calcium signal triggered by ex- tracellular potassium was assessed in H295R cells by record- ing intramitochondrial calcium variations with ratiometric pericam. Here we show that the formation of a large actin layer at the periphery of the cell significantly reduces the mitochondrial Ca2⫹ signaling and the activation of steroi- dogenesis in response to potassium, but only minimally af- fects the response to AngII. Moreover, this treatment does not modify channel activation elicited by cell depolarization or the rise of cytosolic calcium concentration induced by potassium, suggesting that only the transfer of Ca2⫹from the channel to the mitochondria is affected by calyculin A.

Materials and Methods Materials

ATP, BSA, tetraethylammonium-Cl, and HEPES were purchased from Sigma-Aldrich Corp. (St. Louis, MO), and AngII was obtained from Bachem AG (Bubendorf, Switzerland). [3H]Pregnenolone was obtained from NEN Life Science Products (Geneva, Switzerland), and antipreg- nenolone antiserum was purchased from Biogenesis Ltd. (Poole, UK).

Calyculin A was purchased from Alexis Biochemicals (Lausen, Swit- zerland), and fura-2/AM, MitoTracker Red, and Cs4-1,2-bis-(2-amino- phenoxy)ethane-N,N,N,N-tetraacetic acid were obtained from Molec- ular Probes (Eugene, OR). WIN 19758 was purchased from Farillon (London, UK). Plasmids (pcDNA3) containing DNA coding for the

mitochondrial ratiometric pericam or the mitochondria-targeted red fluorescent protein (mtRFP) were provided by Dr. Tullio Pozzan (Uni- versity of Padova, Padova, Italy).

Cell culture

H295R cells were obtained from Dr. W. E. Rainey (University of Texas, Dallas, TX). Cells were grown in DMEM/Ham’s F-12 (1:1) me- dium with 15 mmHEPES, supplemented with 120 IU/ml penicillin, 120

␮g/ml streptomycin, 6 IU/ml nystatin (Life Technologies, Inc., Gaith- ersburg, MD), 0.5␮g/ml fungizone (Squibb AG, Zurich, Switzerland), 40␮g/ml garamycin (ESSEX Chemie AG, Lucerne, Switzerland), 1% ITS Plus (insulin, transferrin, selenium, BD Biosciences, Bedford, MA), and 2% Ultroser SF (BioSepra SA, Villeneuve la Garenne, France). Cells were routinely grown in 75-cm2flasks in a humidified atmosphere at 37 C (95% air/5% CO2). The medium was changed every 3 d.

Before experiments, cells were detached with 0.25% trypsin-EDTA and plated into petri dishes or on glass coverslips. A few days after trypsinization, the medium was replaced by DMEM/Ham’s F-12 with- out insulin/transferrin/selenium and Ultroser during the 24 h preced- ing the start of the various treatments.

Endoplasmic reticulum, mitochondria, and actin filament fluorescence labeling

The e.r. was labeled by transfecting the H295R cells with a cytomeg- alovirus promoter (pCMV) plasmid coding for an e.r.-targeted GFP (pCMV/myc/ER/GFP, Invitrogen, San Diego, CA). H295R cells (200,000) were plated on 25-mm glass coverslips, and at about 60%

confluence, they were incubated for 6 h at 37 C with the plasmid (340 ng/ml) mixed with Effecten and reagents (Qiagen, Basel, Switzerland), as indicated by the manufacturer. After transfection, cells were washed and left in culture medium supplemented with serum for 2–3 additional d before being employed for confocal microscopic analysis. In some preparations, cells were cotransfected with pCMV/myc/ER/GFP (260 ng/ml) and a pcDNA3 plasmid (80 ng/ml) containing an mtRFP. To visualize actin filaments in e.r.-labeled cells, transfected cells were grown on glass coverslips for 3 d before being fixed with 2% parafor- maldehyde (Polysciences, Inc., Warrington, PA) in PBS (Life Technol- ogies, Inc.) for 10 min at room temperature and washed three times in the same buffer with 8% NaCl for 10 min. Cells were then permeabilized in 0.5% Triton X-100 (Fluka Chemie AG, Buchs, Switzerland) in PBS for 10 min, washed three times in PBS with 1% BSA, and incubated for 10 min in this solution. Coverslips were subsequently inverted on a drop of 10␮g/ml rhodamine (TRITC)-phalloidin (Sigma-Aldrich Corp.) in PBS in the dark for 1 h at room temperature. Finally, coverslips were washed three times in PBS, and observation was begun after 20 min.

High resolution confocal microscopy

High resolution image acquisition was performed on a spinning disk confocal microscope. The incident beam from a 2.5-W Kr/Ar water- cooled ion laser in multiline mode (Innova 70C Spectrum, Coherent, Santa Clara, CA) was directed on an Acousto-Optical Tunable Filter controlled for wavelength selection and intensity modulation by a four- channel multifrequency driver (AOTF from Visitech International, Sun- derland, UK). The AOTF output was coupled by a single-mode fiber optic (Oz Optics, Ontario, Canada) to a Yokogawa spinning disk con- focal scan-head (QLC100 from Visitech International, Sunderland, UK) mounted on an inverted microscope (Axiovert 200M, Carl Zeiss AG, Feldbach, Switzerland). Fluorescence images were collected by a100 1.4 NA Plan-Apochromat oil immersion objective (Carl Zeiss AG) and captured with a cooled, 12-bit TE/CCD interlined Coolsnap HQ Pho- tometrics camera (Ropper Scientific, Trenton, NJ). To record simulta- neously mitochondria-targeted (mt) Pericam and MitoTracker Red flu- orescence, both markers were excited at 488 nm, and emissions were separated with a 565 DCXR dichroic filter (Chroma Technology Corp., Brattleboro, VT) mounted on a Dual-View Micro-Imager (Optical In- sights, LLC, Santa Fe, NM), which permits simultaneous acquisition of green and red emissions (D525/50m and D615/60m emission filters from Chroma Technology Corp.) on each half of the CCD image sensor.

Wavelength selection with the AOTF, camera control, and all motorized functions of the microscope were controlled for image acquisition by


Metamorph/Metafluor 5.0r6 software (Universal Imaging, West Ches- ter, PA).

For optimal representation, Nipkow disk confocal fluorescence image stacks were deconvoluted after acquisition by treatment on a Dell Pre- cision 530MT workstation with the use of Huygens Essential 2.4.1 soft- ware (Scientific Volume Imaging BV, Hilversum, The Netherlands).

Shadow projections for three-dimensional rendering were constructed using Imaris 2.7 software on a Silicon Graphics Octane workstation (Bitplane AG, Zurich, Switzerland).

Mitochondrial calcium measurements

Mitochondrial calcium fluctuations were recorded using mt ratio- metric pericam probes (16). Plasmid (pcDNA3) containing DNA coding for ratiometric pericam fused in-frame on the 5extremity to the first 36 nucleotides of the mitochondrial cytochrome C oxidase subunit IV (Cox IV) was introduced in H295R cells by transient transfection with Ef- fecten, as described above for e.r. labeling. After transfection (plasmid at 340 ng/ml for 9 –16 h), cells were washed and left in culture medium supplemented with serum for 2–3 additional d before being employed in the experiments. Specific targeting of mt Pericam within the mito- chondria was demonstrated in some cells by costaining mt Pericam- expressing H295R cells with MitoTracker Red, CMXRos (Molecular Probes Europe BV, Leiden, The Netherlands). This cell-permeant, spe- cific mitochondrial marker was introduced simply by incubating the cells in PBS medium for 2 min at room temperature in the presence of the dye (500 nm).

Variations in mitochondrial calcium concentrations were recorded by imaging pericam fluorescence on an Axiovert S100TV microscope, using a⫻100 (or⫻40), 1.3NA oil immersion objective (Carl Zeiss AG). Exci- tation was alternatively performed at 48010 and 41010 nm with the use of a DeltaRam monochromator (Photon Technology Interna- tional, Inc., Monmouth Junction, NJ) through a 505 DCXR dichroic mirror (Chroma Technology Corp.) and emission filtered at 535 nm (535DF25, Omega Optical, Brattleboro, VT). Image acquisition (1 image every 10 –15 sec at each excitation wavelength), and analysis was per- formed with the Metamorph/Metafluor 4.1.2 software. Fluorescence ratio (fluorescence intensity at 480 nm minus background/fluorescence intensity at 410 nm minus background) was calculated pixel by pixel and averaged within each manually delimited entire cell. Changes in fluo- rescence ratio (reflecting mitochondrial calcium variations) were then expressed as a function of time.

Electrophysiological recordings

Patch-clamp recordings were performed in the whole cell configu- ration using an Axopatch 1D patch-clamp amplifier (Axon Instruments, Inc., Foster City, CA). Membrane currents, elicited by cell depolarization as indicated in the figure legends, were filtered at 1–2 kHz and sampled at 5 kHz using a TL-1–125 interface (Axon Instruments). The bath so- lution contained 117 mmtetraethylammonium chloride, 20 mmBaCl2, 0.5 mmMgCl2, and 37 mmsucrose and was buffered to pH 7.5 with 10 mmHEPES (CsOH). The patch pipette (3– 6 MOhm; Clark 150T, Read- ing, UK) were filled with 85 mm CsCl, 10 mmtetraethylammonium chloride, 6 mmMgCl2, 5 mmMg-ATP, 0.04 mmNa-GTP, 0.9 mmCaCl2, and 11 mmCs4-1,2-bis-(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid; pH was adjusted to 7.2 with 20 mmHEPES (CsOH). Cells were relatively homogenous in size, with a membrane capacitance of 8.19 1.86 pF (meansd; n29). The leak current was automatically sub- tracted by a P/4 protocol (pClamp V.6, Axon Instruments, Inc.).

Cytosolic calcium measurements

For cytosolic calcium determinations, H295R cells were trypsinized and maintained in solution (6106cells/ml) in a Krebs medium (con- taining 136 mmNaCl, 1.8 mmKCl, 1.2 mmKH2PO4, 1.2 mmMgSO4, 5 mmNaHCO3, 1.2 mmCaCl2, and 5.5 mm d-glucose, buffered to pH 7.4 with 20 mmHEPES) supplemented with 1% BSA. Cells were incubated for 45 min at 37 C in the presence of 2␮mfura-2/AM. After washing the excess dye, cells were incubated for 20 min at 37 C in Krebs medium before being used for determination of the fura-2 fluorescence (alter- native excitation at 340 and 380 nm and emission at 500 nm) in a CAF-110 fluorometer (Jasco, Hachioji City, Japan). The fluorescence signal was

digitized (DaQSys 2.0, Sicmu, University of Geneva), and cytosolic cal- cium was calibrated using the ratio values of emitted fluorescence (340/

380 nm) as described by Grynkiewiczet al.(17).

Determination of steroid production

Pregnenolone and aldosterone productions were determined by di- rect RIA of the steroids in the cell medium. Cells were incubated in a Krebs medium containing either 3 mmKCl (basal production) and 12 mmKCl or 3 mmKCl and 10⫺7mAngII. For this purpose, H295R cells were cultured for 3 d in 24-well plates. The day of the experiment, 10 nmcalyculin A or vehicle (control cells) was added in wells for 3.5 h. At the time of stimulation, cells were washed with a Krebs buffer and incubated for 1.5 h in the same buffer, supplemented or not with 9 mm KCl (12 mmfinal concentration) and 10⫺7mAngII, and with calyculin A or vehicle. For pregnenolone measurement, WIN 19758 (5␮m) was present in wells throughout the stimulation period to prevent the con- version of pregnenolone to progesterone. At the end of the incubation, the ambient medium was removed for pregnenolone and aldosterone assays, as previously described (18, 19). Aldosterone RIA was performed using a commercially available kit (Diagnostic Systems Laboratories, Inc., Webster, TX). Adherent stimulated cells were washed and detached in NaOH (0.5m) to determine protein content by the Coomassie Blue method (Bio-Rad Laboratories, Inc., Munich, Germany).


Results are expressed as the meansemunless stated otherwise. The statistical significance of the changes induced by treatments was ana- lyzed by unpairedttests.


Effect of calyculin A on H295R cell morphology and actin distribution

Calyculin A was used to create a physical barrier of actin between the cell plasma membrane and the mitochondria to disturb the intracellular calcium pathway putatively respon- sible for steroid synthesis. We first assessed, by fluorescence imaging, whether calyculin A effectively acts on actin fila- ment distribution in H295R cells as previously described in other cell types. For this purpose, the e.r. of H295R cells was first labeled in green by transfecting the cells with a pCMV plasmid coding for an e.r.-targeted GFP, and actin micro- filaments were then stained in red, after fixing the cells, with rhodamine-phalloidin (Fig. 1, A–F). Confocal Nipkow mi- croscopy analysis revealed that under control conditions, actin filaments are evenly dispersed throughout the cytosol between the e.r. network (Fig. 1A), and most importantly, that the peripheral e.r. freely reaches the edge of the cell, where it apparently can make physical contact in some places with the plasma membrane (Fig. 1B). In contrast, after treat- ment with calyculin A (10 nm, for 3.5 h), a significant reor- ganization of the actin microfilaments toward the periphery of the cell was observed (Fig. 1C). Indeed, a redistribution of actin staining into a tight cortical layer, subjacent to the plasma membrane, was visible and prevented the e.r. from interacting with the plasma membrane by physically exclud- ing the organelle from the vicinity of the membrane (Fig. 1D).

After treatment, some cells became more rounded and some- times plasma membrane surface blebs were apparent (Fig.

1E). These budding structures were also delimited by a ring of filamentous actin and contained a portion of the e.r. This effect, associated with the presence of a thicker actin layer (Fig. 1F) and a propensity of the cells to detach from petri dishes, was attributed to the disappearance of stress fibers


and was observed in an increased number of cells upon longer treatments or when higher concentrations of calyculin were used. However, the effect of the drug was reversed within 1 h after washing out calyculin A (not shown).

The presence of actin-labeled cells that are negative for e.r.-GFP in the various images (Fig. 1, A, C, and E) reflects the 100% efficiency of actin labeling after cell fixation com- pared with the modest e.r. labeling by transfection in 35– 45%

of the cells.

We also tested the possible effect of calyculin A on the

interaction between the e.r. and the mitochondria after co- transfecting the cells with the e.r.-GFP and an mtRFP. The distribution of mitochondria, which appears as red oblong structures by confocal microscopy (Fig. 1G), among the e.r.

network suggests the presence in control H295R cells of multiple sites of interaction between these organelles, as we have previously observed at the level of the electron micros- copy resolution in bovine adrenal glomerulosa cells (20). The effect of calyculin A was determined by analyzing the dis- tribution of the e.r. (Fig. 1, H and I) and the mitochondria FIG. 1. Effect of calyculin A on H295R cell morphology

and filamentous actin distribution. A–F, Endoplasmic reticulum- and actin microfilament-labeled H295R cells. H295R cells expressing e.r.-targeted GFP (green staining) were incubated for 3.5 h with vehicle (A) or 10 nMcalyculin A (C and E) before being fixed and stained with rhodamine-phalloidin (redstaining). Images were then acquired on a confocal microscope, as described in Materials and Methods.Redcells are labeled with phal- loidin, but do not express e.r.-GFP. White rectangles indicate the limits of the corresponding magnified im- ages, shown in the right panels(B, D, and F). G–K, Endoplasmic reticulum- and mitochondria-labeled cells.

Endoplasmic reticulum-targeted GFP and mtRFP (red) were coexpressed in the same cells by cotransfection (G).

The distribution of the e.r. (H and I) and that of the mitochondria (J and K) are shown in the same cell before and 15 min after the addition of 100 nMcalyculin A.

Rounding of the cell was used as an indicator of calyculin action. L and M, Mitochondrial expression of the mt ratiometric Pericam. Cells were transfected with the plasmid coding for the mt ratiometric Pericam (green staining in M), as explained inMaterials and Methods.

After expression of the protein within the mitochondria, the same cells were incubated for 2 min in the presence of 500 nMMitoTracker Red (redstaining in L). Confocal images were acquired simultaneously at both wave- lengths with a dichroic mirror separating the emission beam. Mitochondria of cells labeled with MitoTracker, but not Pericam, are visible at thebottomof the image.

Scale bars, 5m.


(Fig. 1, J and K) in the same cell, before and after the addition of the drug. Despite a clear rounding of the cell induced by calyculin, no obvious redistribution of the organelles was observed, suggesting that the interaction between the e.r. and the mitochondria should not be affected by the treatment.

Inhibition by calyculin A of the potassium-induced mitochondrial calcium response

We then assessed the effect of calyculin A treatment on mitochondrial calcium in H295R cells by recording the fluc- tuations in the calcium concentration within the mitochon- drial matrix with the fluorescent probe, ratiometric pericam.

Three days after cell transfection with DNA coding for the mitochondrial pericam, the fluorescence emitted by this probe was punctuated and essentially perinuclear in most of the cells, limited to regions of the cytoplasm known to be rich in mitochondria (Fig. 1M). As expected, the nucleus was devoid of fluorescence. Nevertheless, the specificity of peri- cam targeting within the right organelles was confirmed by comparing, in the same cells, its distribution with that of MitoTracker Red, a recognized mitochondrial marker (Fig.

1L). Images of ratiometric pericam and of MitoTracker flu- orescence distribution obtained by confocal microscopy were almost identical, confirming that the pericam expressed in H295R cells under our experimental conditions was correctly addressed to the mitochondria. The cell-permeant Mito-

Tracker Red labeled mitochondria in all cells, whereas only 35– 45% cells were transfected with Pericam, which explains the presence of a red signal in some cells negative for pericam.

Typical mitochondrial responses to stimulation by potas- sium and AngII in control (untreated) and calyculin A-treated H295R cells are shown in Fig. 2A. The stimulation of control cells by 12 mmKCl led to a biphasic response, with an immediate transient increase in the mitochondrial calcium concentration ([Ca2⫹]m), followed by a more sustained phase. This response was markedly reduced in cells treated for 3.5 h with 10 nmcalyculin A (lower trace). In contrast, the [Ca2⫹]mresponse to AngII (100 nm), which was also biphasic, was only minimally affected by calyculin A treatment. After AngII stimulation, 1␮mcarbonyl cyanidem-chlorophenyl- hydrazone, a proton ionophore, was added in the medium to abolish the mitochondrial Hgradient and therefore the driving force for Ca2⫹uptake into the organelles. This inhi- bition led to a rapid decrease in the fluorescence ratio, con- firming the mitochondrial localization of pericam. Finally, ionomycin (2␮m), a calcium ionophore, was used to elicit a maximal calcium increase within the mitochondria, which was used to normalize the previous responses.

The amplitude of the response to each agonist, expressed as a percentage of the response to ionomycin, was highly variable from one cell to the other, with a coefficient of

FIG. 2. Inhibition by calyculin A of the KCl-induced mi- tochondrial calcium response in H295R cells. H295R cells were transiently transfected with a plasmid coding for the mt ratiometric Pericam, as indicated in detail inMaterials and Methods, and maintained for 3 d in culture. Before recording, cells were incubated for 3– 4 h with vehicle (Con- trol) or 10 nM calyculin A (Caly A). A, Typical traces of [Ca2⫹]m measurements obtained in control or calyculin A-treated H295R cells that were sequentially exposed to 12 mMKCl, 100 nMAngII, 10Mcarbonyl cyanidem-chloro- phenylhydrazone, and 2Mionomycin. B, KCl- and AngII- induced mitochondrial calcium responses in each cell (mea- sured at the peak) were normalized to the maximal calcium responses induced by ionomycin in the same cell, and re- sults were averaged for control and calyculin A-treated cells (meanSEM; n15–21 cells from four independent experiments). ***,P0.001.


variation greater than 75%. Some control cells (20%) re- sponded better to AngII than to KCl, whereas others (30%) were more sensitive to potassium. Nevertheless, as shown in Fig. 2B, calyculin A pretreatment induced a significant de- crease in the [Ca2⫹]mresponse to KCl in H295R cells. On the average, the response was reduced by 77% compared with that of control cells. In contrast, the response to AngII in the same cells was much less affected (17%) by calyculin A, a decrease that was not statistically significant.

To determine whether the inhibition of the mitochondrial signal induced by calyculin A was due to the formation of the cortical actin layer or to the reduction of the microfilament concentration in the center of the cells, we performed addi- tional experiments in which cells (n⫽11) were pretreated for 1.5 h with a cocktail of cytoskeleton inhibitors composed of 10␮mcytochalasin B, 20␮mcolchicine, 10␮mdemecolcine, and 10␮m nocodazole. The effect of this treatment, which normally does not affect the interaction between the e.r. and the plasma membrane (15), could be easily visualized by a change in cell morphology (not shown) and was accompa- nied by a reduction of the mitochondrial calcium response to both potassium and AngII, by approximately 70% and 35%, respectively. These data suggest that the integrity of the cytoskeleton at the proximity of the mitochondria could also be required to maintain the e.r. closely connected to these organelles and to allow the transfer of calcium from the pipeline into the mitochondria.

Lack of effect of calyculin A on the activation of voltage- operated calcium channels

To exclude the possibility that the action of calyculin A on the [Ca2⫹]mresponse to KCl was simply due to impairment of channel activation upon cell depolarization, we analyzed channel activity in control and treated H295R cells with the patch-clamp technique, in the whole cell configuration. The presence of both low threshold, T-type, and high threshold, L-type, calcium channels in these cells has been previously demonstrated (21). To discriminate between T- and L-type currents, we used distinct electrophysiological properties of the corresponding channels, namely the slower deactivation of T channels and the slower inactivation of L channels (21).

Thus, for determining T-type channel activity, tail current was elicited by repolarizing the cell at ⫺65 mV after a 20- msec depolarization at⫹20 mV from a HP of⫺90 mV (Fig.

3A). The slowly deactivating Ca2⫹current evoked upon cell repolarization was exclusively due to T channel activity be- cause of the very rapid L channel deactivation at this voltage (t1/2⫽ ⬃0.2 msec). Tail current was fitted to a single expo- nential function, and the value of T current present at the time of membrane repolarization was then determined by extrapolation.

To specifically determine L-type current amplitude, we depolarized the cell from a holding potential (HP) of⫺90 mV to 0 mV and maintained this potential for 600 msec (Fig. 3B).

The amplitude of the inward current was measured 500 msec after the depolarization, when T-type channels were almost completely inactivated.

The density of T-tail currents (i.e.the amplitude of the current divided by the cell capacitance) was 22.5⫾3.4 pA/pF

(mean⫾sem; n⫽13), and that of L-type currents was 9.0⫾ 1.3 pA/pF (n⫽9) in control cells. We did not observe any significant modification in T-type (24.4⫾3.4 pA/pF; n⫽11) or L-type (6.1 ⫾0.9 pA/pF; n ⫽9) current densities after treatment of the cells for 3.5 h with 10 nm calyculin A (Fig. 3C).

Because T-type channels have been directly involved in the stimulation of steroidogenesis by potassium in adrenal glo- merulosa and H295R cells, we also verified that calyculin A did not modify other electrophysiological properties of this channel. The dependence of T channel opening on the mem- brane potential was assessed by establishing activation and steady state inactivation curves as a function of voltage, as previously described in detail (22). As shown in Fig. 3D, the half-potential of channel activation was similar in normal (⫺31.3 ⫾ 0.9 mV; n ⫽ 12) and calyculin A-treated cells (⫺28.5⫾1.15 mV; n ⫽ 10). Similarly, calyculin A did not affect the half-potential of channel steady state inactivation, which was ⫺47.4 ⫾ 1.2 mV in control cells (n ⫽ 6) and

⫺44.7⫾1.1 in treated cells (n⫽8). As a consequence, the size of the permissive window of voltage, in which activation and inactivation curves overlap and which allows a sustained influx of calcium through T channels, was not reduced after calyculin A treatment.

Lack of effect of calyculin A on the cytosolic calcium responses induced by KCl or AngII

We also carried out experiments to assess whether the cytosolic calcium ([Ca2⫹]c) rise induced by KCl and AngII in H295R cells was affected by calyculin A treatment. For this purpose, fura-2-loaded cells were stimulated, after 3.5-h pre- treatment in the presence or absence of 10 nmcalyculin A, consecutively with 12 mm KCl, 100 nm AngII, and 2 ␮m ionomycin. As shown in Fig. 4A, KCl induced a small, but relatively sustained, [Ca2⫹]c response in both control and calyculin A-treated cells. The amplitude of this response to KCl, which has been shown to be essentially due to activation of L-type channels in adrenal glomerulosa cells (6), was not significantly different in the cell groups (Fig. 4B). Compared with the response induced in the same cells by the calcium ionophore ionomycin, the [Ca2⫹]cresponse elicited by KCl was 9⫾2% (n⫽11) in control cells and 7⫾1% (n⫽12) in calyculin A-treated cells.

The response to AngII was clearly biphasic and involved Ca2⫹ release from intracellular stores as well as voltage- dependent and -independent calcium entry pathways (19).

Once again, calyculin A treatment had no significant effect on the size of the peak responses, which, on the average, were 32⫾4% (n⫽11) and 27⫾3% (n⫽12) of the response to ionomycin in control and calyculin A-treated cells, respectively.

Inhibition of KCl- and AngII-stimulated steroidogenesis by calyculin A

Finally, we investigated the ability of calyculin A to inhibit the steroidogenic function of H295R cells. For this purpose, we carried out experiments to evaluate the production of two steroids: pregnenolone, which is the first metabolite of cho- lesterol produced in the steroidogenic cascade, and aldoste-


rone (Fig. 5). Basal steroid production was not inhibited by 3.5-h pretreatment with 10 nmcalyculin A. In contrast, ca- lyculin A significantly decreased both pregnenolone (Fig.

5A) and aldosterone (Fig. 5B) responses to 12 mmKCl by 57%

and 39%, respectively (the drug effect on the response was calculated after subtracting basal values). In the case of stim- ulation with AngII (100 nm), pregnenolone formation was only slightly inhibited (by 24%) by calyculin A, whereas aldosterone was not affected by the drug. To exclude the possibility that calyculin A, by increasing the phosphoryla- tion level of the cell, directly affects the activity of the various

steroidogenic enzymes (what would be, however, in appar- ent contradiction with the lack of effect of the drug on the aldosterone response to AngII), we also tested the effect of calyculin on the aldosterone production supported by an analog of cholesterol. Indeed, 25-hydroxycholesterol freely enters the mitochondria and can therefore be converted into steroids independently from agonist stimulation and cal- cium mobilization (23). We found in this independent series of experiments that the aldosterone produced upon stimu- lation with KCl was reduced to 59.5⫾13.5% (n⫽3) of control values after treatment with calyculin, but the conversion of FIG. 3. Lack of effect of calyculin A treatment on volt-

age-gated calcium currents in H295R cells. Cells plated on glass coverslips were treated for 3.5 h with 10 nM

calyculin A or vehicle immediately before being used for the patch-clamp experiments in the whole cell configu- ration. When appropriate, calyculin A was maintained in the recording solution. A, Example of a slowly deac- tivating tail current elicited upon repolarization of a control cell to65 mV after a 20-msec period of channel activation at20 mV from a HP of90 mV. The am- plitude of the maximal T-type current was determined by extrapolating the tail current fitted to a single ex- ponential. B, Inward current activated, in the same cell as in A, by a step depolarization from90 to 0 mV and maintained for 600 msec. The amplitude of the L-type current was measured at 500 msec after depolarization.

C, The amplitude of T- and L-type currents was deter- mined in control and treated cells using the voltage protocols indicated in A and B and then normalized for the cell capacitance (meanSEM; n9 –13 cells). D, The voltage-dependent activation and steady state inactiva- tion properties of T channels were examined in the same cells with the patch-clamp technique as previously de- scribed (22). Slowly deactivating currents were recorded at65 mV after 20-msec activation at various poten- tials or after steady state inactivation for 10 sec at var- ious potentials, followed by a 20-msec stimulation at

20 mV. The amplitude of the maximal tail current occurring upon repolarization to 65 mV was deter- mined and plotted as a function of test potential to be fitted to Boltzmann’s equation. The parameters of the equation were then determined for each cell, and the currents were normalized to the maximal current (I0) before being averaged. Mean activation (EandF) and steady state inactivation (andf) curves were estab- lished for control and calyculin A-treated cells (mean

SEM; n6 –12 cells).


10␮m25-hydroxycholesterol into aldosterone in calyculin- treated cells was 105.3⫾17.7% (n⫽3) of that measured in the respective control cells. Aldosterone production from 25-hydroxycholesterol-supplemented cells was quantita- tively similar to that elicited by KCl in untreated cells. Thus, this result demonstrates that the ability of the steroidogenic enzymes to convert cholesterol into aldosterone remains in- tact in H295R cells after treatment with calyculin A.


In this study we show that restructuring actin cytoskeletal organization at the periphery of H295R cells with calyculin A leads to an impairment of calcium signal transduction from the plasma membrane to the mitochondria that normally occurs upon cell stimulation with extracellular potassium and therefore reduces the steroidogenic response to this ag- onist. This inhibitory action of calyculin A was observed without any change in calcium channel activity or modifi- cation of the size of the cytosolic calcium response.

This approach had been previously used by others to dem- onstrate, in smooth muscle (15), platelet (24), and lympho- cyte (25) cell lines, thatin situredistribution of filamentous actin into a tight cortical layer subjacent to the plasma mem- brane displaces cortical e.r. and prevents coupling between the e.r. and the plasma membrane Ca2⫹channels while not affecting inositol 1,4,5-trisphosphate-mediated store release.

The parallel and reversible inhibition of the capacitative in- flux observed in these studies strongly suggested that a phys- ical interaction with a portion of the e.r. was mandatory for activating store-operated calcium channels, and a model of regulation of the capacitative influx, based on vesicle traf- ficking and docking on plasma membrane, has been pro- posed (15).

A similar mechanism is believed to occur in aldosterone- producing cells, where a specific interaction between plasma membrane T-type calcium channels and the e.r., acting in these cells as a sort of intracellular calcium pipeline to convey the cation to the mitochondria (10), would be required to maintain the functional specificity attributed to the various calcium channels (6, 7). Indeed, the fact that selective acti- vation of L channels leads to a large increase in [Ca2⫹]c, but to a poor steroidogenic response, strongly suggests that rate- limiting steps controlled by calcium are not located within the cytosol. This assumption is reinforced by the observation that stimulation of steroid production in response to in- FIG. 4. Lack of effect of calyculin A on the potassium- and AngII-

induced cytosolic calcium responses in H295R cells. Cells were pre- treated for 3.5 h with 10 nMcalyculin A or vehicle before being loaded with fura-2, as described inMaterials and Methods, and sequentially exposed to 12 mMKCl, 100 nMAngII, and 2Mionomycin. A, Rep- resentative traces of [Ca2]cresponses obtained in control and caly- culin-treated cells. B, Mean KCl- and AngII-induced cytosolic calcium responses determined in control and calyculin-treated cells. The mean responses to ionomycin (Iono) were 46884 and to 46265 nMin control and treated cells, respectively, and were not significantly different. Results are the meanSEMfrom 11–12 traces recorded

within three independent experiments. FIG. 5. Inhibition by calyculin A of the KCl- and AngII-induced ste- roidogenesis in H295R cells. H295R cells were pretreated for 3.5 h with 10 nMcalyculin A or vehicle before being incubated for 1.5 h, as indicated in Materials and Methods, in the presence of 3 mM KCl (basal), 12 mMKCl, or 3 mMKCl plus 100 nMAngII. For pregnenolone determination, an inhibitor of the conversion to progesterone was added in the medium. Pregnenolone (A) and aldosterone (B) produc- tion in the medium was assessed by RIA, and results were normalized to the cell protein. Data are the meanSEMfrom three independent experiments performed in triplicate. *,P0.05; **,P0.01.


creased ambient calcium in permeabilized glomerulosa cells is sensitive to ruthenium red, an efficient blocker of mito- chondrial calcium uptake (26), as well as by the demonstra- tion of activation of mitochondrial cholesterol transport by increasing cytosolic calcium (23, 27). In addition, important modification of steroidogenesis follows pharmacological modulation of T-type channel activity, which results in barely detectable changes in [Ca2⫹]c, (8, 9, 21, 28). It was therefore proposed that calcium entering the cell through T channels (the steroidogenic calcium) must be conveyed to the mitochondria (the steroidogenic organelles) without cross- ing the cytosol, in which the diffusion of the cation is known to be only limited to microdomains (Fig. 6A). Microscopic morphological analysis of adrenal glomerulosa cells revealed the presence of a continuous e.r. network in contact with both regions of the plasma membrane and many mitochondria (20), suggesting that the e.r. could be responsible for this intracellular calcium transport, and a specific docking pro- cess of the e.r. close to T channels has been proposed as an important factor allowing the cell to discriminate between calcium flowing through T and non-T channels.

We propose that upon treatment of the cells with calyculin A, removing the e.r. away from the plasma membrane by condensation of cortical actin results in a failure to fill up the pipeline and therefore to supply mitochondria with calcium.

This model is fully supported by our observation that the [Ca2⫹]m response to potassium is significantly reduced by calyculin A as well as the production of steroids, particularly that of an early intermediate such as pregnenolone, which is highly sensitive to calcium variations (23, 29).

The mitochondrial response to AngII was much less af- fected by calyculin A. In fact, we analyzed the peak response to the hormone, reflecting essentially the release phase of the signal. This lack of effect of calyculin A confirmed the ob- servation previously made in other cell types that the inositol 1,4,5-trisphosphate response is insensitive to this drug (15, 25) and that calcium stores are not altered, nor is the ability of the mitochondria to accumulate calcium. The plateau phase of the response to AngII was not taken into account because of the complexity of the factors contributing to its maintenance. Indeed, the plateau level is not only the inte- gration of the activation of multiple voltage-dependent and

FIG. 6. Hypothetical model of intracellular cal- cium transport from T channels to mitochondria.

A, Control cells: docking of a portion of the e.r.

network in regions of the plasma membrane spe- cifically enriched in T channels facilitates the transport of calcium entering the cell through these channels to the mitochondria, where the cation stimulates early and limiting steps of ste- roidogenesis. Other channels, not linked to the pipeline, are less efficient for stimulating steroid production. B, Calyculin A treatment: after reor- ganization of the cortical actin filaments induced by calyculin A, this physical barrier uncouples T channels from the pipeline, decreasing the trans- fer of calcium to the mitochondria, and therefore steroidogenesis. Calyculin treatment is not ex- pected to modify channel activity or the cytosolic response.


-independent influx pathways, but is also modulated by the activity of the calcium extrusion mechanisms (30). AngII has been shown to modulate most of these factors (31), in many cases through the control of protein kinases and phospha- tases (22, 32, 33), that could be a prioridirectly affected by calyculin A treatment. Moreover, steroid production upon AngII challenge is not really expected to faithfully reflect Ca2⫹transport into the mitochondria because Ca2⫹-depen- dent and -independent mechanisms have been clearly shown to coexist in the control of steroidogenesis by the hormone (23, 32, 34). However, AngII has been used in this study essentially to demonstrate that calyculin A by itself does not impair calcium release from the stores, calcium uptake into the mitochondria, or the steroidogenic capacity of the cells.

In contrast, the response elicited by extracellular potas- sium is much more straightforward, involving opening of plasma membrane calcium channels upon cell depolariza- tion, transfer of a part of this calcium into the mitochondria, and stimulation of cholesterol conversion into pregnenolone and steroid intermediates. Any interference with one of these steps is generally detected on steroid output. In fact, we found an excellent correlation between the decrease in the mitochondrial calcium response after calyculin treatment and pregnenolone formation. The smaller effect of calyculin on aldosterone production could be due to the presence of other rate-limiting steps (different from those controlled by calcium) for late steroidogenic reactions, possibly explaining the low ratio of aldosterone on pregnenolone production in H295R cells.

The identity of the plasma membrane channel connected to the pipeline has not been definitely demonstrated, but T channels are clearly among the best candidates. In prelimi- nary experiments we found, as expected, that specific inhib- itors of L channels have no effect on the mitochondrial re- sponse, which is markedly reduced by nickel. Additional experiments are currently performed with the same exper- imental approach to determine the involvement of the var- ious T channel isoforms in the mitochondrial calcium re- sponse to potassium.

The inhibition of the response to potassium induced by calyculin A is proposed to be due to the increased distance, sterically maintained by the enlarged actin layer, between the e.r. and the plasma membrane and not to a toxic effect of the drug on the channel activity, as demonstrated by electro- physiological recording of the currents elicited upon cell depolarization, or recording of the cytosolic calcium re- sponse to extracellular potassium. This is in contrast with the many pharmacological tools previously tested in our labo- ratory and reported to affect KCl-induced steroid production in glomerulosa cells through inhibition of calcium channels, such as dihydropyridines (6, 19), tetrandrine (8), mibefradil (9), diacylglycerol or phorbol esters (6, 22, 35), peripheral- type benzodiazepines (28), and zonisamide (6).

In summary, we have shown in this study that the orga- nization of the actin cytoskeleton, which is probably in- volved in the formation of physical contacts between specific regions of the e.r. with the plasma membrane or the mito- chondria, influences the calcium signal transduction elicited by extracellular potassium in H295R cells and leads to the activation of steroidogenesis. Although not proving it, these

observations support our model of intracellular transport of the steroidogenic calcium from the plasma membrane to the mitochondria through a physical pipeline formed by the e.r.

network. Additional work is now necessary to identify the molecular determinants responsible for maintaining this pipeline in place and for regulating its activity.


We are grateful to Dr. Pozzan for providing us with the plasmids coding for the mt ratio pericam and the mtRFP.

Received February 28, 2003. Accepted June 30, 2003.

Address all correspondence and requests for reprints to: Dr. Michel F. Rossier, Division of Endocrinology and Diabetology, University Hos- pital, 24 rue Micheli du Crest, CH-1211 Geneva 14, Switzerland. E-mail:

This work was supported by Swiss National Science Foundation Grant 32-58948.99.


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