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J. Biol. Chem., Vol. 276, Issue 41, 37986-37992, October 12, 2001

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Rottlerin Is a Mitochondrial Uncoupler That Decreases Cellular ATP Levels and Indirectly Blocks Protein Kinase C Tyrosine Phosphorylation^*

Stephen P. Soltoff

From the Division of Signal Transduction, Harvard Institutes of Medicine, Boston, Massachusetts 02215

Received for publication, June 1, 2001, and in revised form, August 7, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein kinase C (PKC) is activated by stimuli that increase its tyrosine phosphorylation, including neurotransmitters that initiate fluid secretion in salivary gland (parotid) epithelial cells. Rottlerin, a compound reported to be a PKC-selective inhibitor, rapidly increased the rate of oxygen consumption (QO₂) of parotid acinar cells and PC12 cells. In parotid cells, this was distinct from the effects of the muscarinic receptor ligand carbachol, which promoted a sodium pump-dependent increase in respiration. Rottlerin increased the QO₂ of isolated rat liver mitochondria to a level similar to that produced when oxidative phosphorylation was initiated by ADP or when mitochondria were uncoupled by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The effects of rottlerin on mitochondrial QO₂ were neither mimicked nor blocked by the PKC inhibitor GF109203X. Rottlerin was not effective in blocking PKC activity in vitro. Exposure of freshly isolated parotid acinar cells to rottlerin and FCCP reduced cellular ATP levels and reduced stimuli-dependent increases in tyrosine phosphorylation of PKC. Neither rottlerin nor FCCP reduced stimuli-dependent PKC tyrosine phosphorylation in RPG1 cells (a salivary ductal line) or PC12 cells, consistent with their dependence on glycolysis rather than oxidative phosphorylation for energy-dependent processes. These results demonstrate that rottlerin directly uncouples mitochondrial respiration from oxidative phosphorylation. Previous studies using rottlerin should be evaluated cautiously.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Many receptor-initiated events involve the activation of members of the protein kinase C (PKC)¹ family of proteins, which are serine/threonine kinases (1-3). Although peptide-specific substrate motifs for different PKCs have been determined (4), a number of factors probably determine the biological substrates of these kinases, including the stimulus, enzyme location, and other conditions related to the cellular context. PKC family members are composed of three groups: the classical (cPKC) subtype (, I, II, and ), the novel (nPKCs) subtype (, , , and ), and the atypical (aPKCs) subtype ( and / ). The cPKCs and nPKCs are activated by the production of diacylglycerol or by phorbol esters. These agents bind to the two C2 domains found in the structure of these PKCs. The diversity of PKC family members and the expression of multiple PKC family members in most cells suggest that each PKC isoform may play a particular biological role in a cell type. Recent data utilizing a variety of techniques indicate that PKCs may play distinct and sometimes antagonist roles within a particular cell (1, 5).

Various chemical inhibitors of PKCs have been used to determine the biological roles of PKCs. Some of the inhibitors have various degrees of specificity for different PKC family members. Rottlerin, one of these inhibitors, has been reported to exhibit a specificity for PKC such that concentrations of rottlerin which are effective for PKC might not be expected to block a significant fraction of other PKC family members (6). Rottlerin has been used in an increasing number of studies that have suggested a role for PKC in a variety of biological events, including apoptosis (7, 8), cell differentiation (9), mitogen-activated protein kinase activation (10), and other cell processes (11-13).

We examined the effects of rottlerin on a variety of cells including freshly isolated rat parotid acinar cells, which initiate fluid and electrolyte secretion in the parotid salivary gland. Fluid secretion is mediated by the activation of muscarinic, substance P, and -adrenergic receptors, which are G protein-coupled receptors that are linked to phospholipase C. The phospholipase C-mediated production of diacylglycerol (DAG) results in the tyrosine phosphorylation of PKC, and this increases the enzymatic activity of this PKC family member in parotid acinar cells and PC12 cells (14). Many stimuli promote an increase in PKC tyrosine phosphorylation via an Src-related tyrosine kinase (13-17). Tyrosine phosphorylation plays a positive role in PKC enzyme activity in many cells stimulated by various stimuli (18-21). Initially we were interested in examining whether rottlerin affected various biological events that were downstream of receptor activation, and we were surprised to find that rottlerin itself produced an increase in the rate of O₂ consumption (QO₂) in multiple cells. Further studies were initiated to understand its mechanism of action on respiration. These studies indicated that rottlerin acts directly to uncouple mitochondria in a PKC-independent manner and facilitates a reduction in the levels of intracellular ATP. In some cells this may indirectly block PKC at rottlerin concentrations that are ineffective in blocking PKC activity in vitro. These results raise questions about the conclusions drawn from other studies that utilized rottlerin as a PKC-specific inhibitor.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- Carbamyl choline (carbachol) was purchased from Sigma. Ouabain was purchased from Sigma, Aldrich, and Calbiochem. Rottlerin was purchased from Calbiochem and from BioMol. GF109203X (3-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(indol-3-yl)-maleimide), Ro 31-8220 (3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide), PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), and nystatin (mycostatin) were purchased from Calbiochem. FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) was from Sigma. Anti-phosphotyrosine antibody was a generous gift of Dr. Thomas Roberts (Dana Farber, Boston, MA). Anti-PKC polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. Anti-PKC monoclonal antibody was purchased from Transduction Laboratories. Diacylglycerol (1,2-dioctanol-sn-glycerol) and L--phosphatidylserine were purchased from Avanti Polar Lipids, Inc. Essentially fatty acid-free bovine serum albumin (BSA) and fraction V BSA were purchased from Sigma. Protein A-Sepharose beads were bought from Amersham Pharmacia Biotech. Igepal was purchased from ICN Biomedicals, Inc. Dulbecco's modified Eagle's medium (DMEM) was purchased from Bio Whittaker. All other chemicals were reagent grade or better.

Cell Preparation and Solutions-- Parotid acinar cells were prepared from male Harlan Sprague-Dawley rats (Charles River Laboratories, Kingston, NY, or Taconic, Germantown, NY; 175-225 g) using previously established techniques (22, 23). Briefly, rat parotid glands were removed and treated with trypsin and collagenase to yield a suspension of single cells and small groups of cells. Cells were suspended at 1-2 mg of protein/ml in solution A, composed of the following: 116.4 mM NaCl, 5.4 mM KCl, 1 mM NaH₂PO₄, 25 mM NaHEPES, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 5 mM sodium butyrate, 5.6 mM glucose, pH 7.4. Cells were maintained on ice prior to use.

PC12 cells were cultured on 100-mm cell culture dishes and grown in DMEM containing 10% heat-inactivated calf serum, 5% heat-inactivated fetal calf serum, and 100 units/ml penicillin/100 µg/ml streptomycin. PC12 cells used for Western blotting were serum starved overnight in DMEM containing 0.1% serum. When used for QO₂ measurements, PC12 cells growing in cell culture dishes were washed in solution A, triturated off their dishes, and suspended in fresh solution A.

Rat parotid gland 1 cells (RPG1), which are an immortalized rat parotid ductal cell line (24), were cultured on 100-mm cell culture dishes and grown in a DMEM:Ham's F-12 medium (3:1, v/v) mixture supplemented with 1.8 × 10⁴ M adenine, 5 µg/ml insulin, 5 µg/ml transferrin, 2 × 10⁹ M triiodothyronine, 1.1 × 10⁶ M hydrocortisone, 1.64 × 10⁶ M epidermal growth factor, 5.5 × 10⁶ M epinephrine, and 10% fetal calf serum. RPG1 cells used for Western blotting were serum starved overnight in DMEM containing 0.1% serum.

Isolation of Rat Liver Mitochondria-- Rat livers from male Harlan Sprague-Dawley rats were homogenized in isolation medium containing 250 mM sucrose, 5 mM Tris, 1 mM EGTA, and 0.5% essentially fatty acid-free BSA, pH 7.4. The homogenate was subjected to centrifugation for 3 min at 1,075 × g. The supernatant was subjected to centrifugation at 11,750 × g for 10 min. The pellet was resuspended in isolation medium and sedimented again at 11,750 × g for 10 min. The pellet was resuspended in fresh isolation medium and maintained on ice prior to measuring the QO₂ (see below).

Oxygen Consumption Measurements-- Conditions were similar to those reported previously (22, 23). In brief, parotid acinar cells and PC12 cells suspended in solution A were prewarmed for 15 min at 37 °C prior to placing them in a stirred chamber maintained at 37 °C. A Clark-type O₂ electrode (model 125/05, Instech Laboratories) was used to record the disappearance of O₂ from the closed 400-µl chamber. The output from the O₂ electrode amplifier (Instech) was recorded using a Kipp & Zonen flatbed chart recorder. The oxygen tension was calculated by measuring the amount of room air oxygen that was dissolved in 150 mM NaCl at 37 °C. At the end of each measurement, a sample of the cell suspension was collected for protein analysis (25) so that each QO₂ could be normalized to the protein content of the cells. Fraction V BSA was used as the protein standard. The QO₂ value was calculated as the maximum linear QO₂ upon the addition of an agent to the chamber. The QO₂ values for parotid cells are the differences between the sustained O₂ consumption rates under particular conditions (control or ouabain) and the new maximal linear rates upon the addition of various agents (see figures).

For measurements of mitochondrial respiration, aliquots of mitochondria suspended in isolation medium were diluted ~1:9 with solution B, composed of the following: 120 mM KCl, 10 mM KH₂PO₄, 5 mM HEPES, 1 mM EGTA, pH 7.2. 10 mM pyruvate and 0.5 mM malate were added as substrates during the incubation of mitochondria at 37 °C for 4 min prior to closing the 1.85-ml chamber to commence the measurement of the QO₂. In some experiments the mitochondria were added to solution B that contained sufficient essentially fatty acid-free BSA for a final concentration of 5%. At the end of each QO₂ measurement, a sample of the mitochondrial suspension was collected for protein analysis.

Immunoprecipitations and Western Blotting-- Parotid cells were exposed to various agents or vehicle (water or 0.1% dimethyl sulfoxide (Me₂SO)) and then were collected by sedimentation in a microcentrifuge (Brinkmann). The supernatant was removed, and cells were lysed in 1 ml of ice-cold lysis buffer (137 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EGTA, 1 mM EDTA, 10% (v/v) glycerol, and 1% v/v Igepal) containing the following phosphatase and protease inhibitors: 1 mM vanadate, 4.5 mM sodium pyrophosphate, 47.6 mM NaF, 9.26 mM -glycerophosphate, 0.5 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotenin, and 2 µg/ml AEBSF. The lysates were vortexed and then sedimented at 16,000 × g for 15 min at 4 °C. The cleared supernatants were transferred to fresh microfuge tubes and incubated with monoclonal PKC antibody (~0.5 µg/ml) for 3 h at 4 °C. The proteins were collected by exposure to 4 mg/ml protein A-Sepharose for an additional hour. At the end of this period, proteins were collected by sedimentation, and the immunoprecipitates were washed three times in phosphate-buffered saline and 1% Igepal. The pelleted proteins were diluted with 2× sample buffer, boiled for 4 min, and stored at 20 °C prior to electrophoresis. Samples were separated using SDS-polyacrylamide gel electrophoresis with an 8% separating gel and a 3% stacking gel as described previously (14). Immunoblots were sequentially probed overnight with 1 µg/ml monoclonal anti-Tyr(P) antibody and polyclonal anti-PKC antibody (~0.5 µg/ml). Proteins were visualized using a chemiluminescence system (PerkinElmer Life Sciences) and x-ray film (Kodak).

In experiments conducted using PC12 and RPG1 cells, the cells were serum-starved overnight in DMEM containing 0.1% serum. Inhibitors/uncouplers were added to this medium for 30-45 min followed by the addition of vehicle (0.1% Me₂SO) or 100 nM PMA for 5 min. Cells were washed twice with ice-cold phosphate-buffered saline solution (136.9 mM NaCl, 2.68 mM KCl, 1.47 mM KH₂PO₄, and 15.65 mM NaH₂PO₄, pH 7.4), lysed in 1 ml of lysis buffer, vortexed, and then sedimented at 16,000 × g for 15 min at 4 °C. PKC was immunoprecipitated and analyzed as was done for parotid acinar cells.

PKC Activity Assay-- PC12 cells growing on 100-mm dishes in normal serum-containing medium were lysed in 1 ml of ice-cold lysis buffer. After clearing the lysates by centrifugation for 15 min at 16,000 × g, the lysates were pooled and realiquoted into fresh tubes. Monoclonal anti-PKC antibody (~0.5 µg/ml) was added to each lysate for 2-3 h at 4 °C, and 4 mg/ml protein A-Sepharose was added for 1-2 h to collect PKC. The immunoprecipitates were washed twice in phosphate-buffered saline and 1% Igepal, once in 0.1 M Tris, pH 7.5, and 0.5 M LiCl, and twice in phosphate-buffered saline. All wash solutions were ice-cold. The beads were resuspended in assay buffer (5 mM MgCl₂, 0.5 mM EGTA, 10 µM PKC synthetic substrate peptide (AKRKRKGSFFYGG), 1 mM dithiothreitol, and 25 mM Tris-HCl, pH 7.5). As noted, in some experiments the assays were conducted in the presence of lipid cofactors (10 µM DAG plus 20 µg/ml phosphatidylserine (PS)) in the assay buffer. Immunoprecipitates were exposed to vehicle (0.02% Me₂SO) or various concentrations of rottlerin or GF109203X for 15 min at 30 °C prior to initiating the assay with the addition of ATP (50 µM ATP, 10 µCi [³²P]ATP (specific activity 10 µCi/µl)). The final volume of the assay mixture was 100 µl. Background activity was measured using protein A-Sepharose beads that were incubated with PC12 cell lysates without antibody. The samples were incubated for 30 min with intermittent mixing, and then duplicate 10-µl aliquots of each sample were spotted onto P-81 phosphocellulose paper. The P-81 papers were washed five or six times in 0.425% phosphoric acid, and the amount of ³²P was determined by liquid scintillation counting. The duplicate values from each immunoprecipitate were averaged and treated as one sample. Usually two or three immunoprecipitates were collected for each of the various conditions in each experiment. The samples were averaged and treated as the results from one experiment (n = 1). In each experiment, all values were normalized to the basal PKC activity in the presence of DAG and PS, which was 119,515 ± 13,618 cpm (n = 3 experiments). These assay conditions were similar to those used in a previous study in which the sequence of the substrate peptide, which is based on the pseudosubstrate region of PKC, was determined to be optimal for PKC (4). This assay also was used previously to analyze PKC activity in parotid and PC12 cells (14). The results from two or three experiments were averaged, and the S.E. was calculated (where n = 3).

ATP Assay-- Suspensions of parotid acinar cells were incubated at 37 °C for 10-20 min, after which they were exposed to 10 µM rottlerin or 10 µM FCCP for 2, 5, and 15 min at 37 °C. Control cells were treated with vehicle (0.1% Me₂SO). For each sample, cells were rapidly sedimented, and ATP was extracted from the cell pellets by lysing the cells in 6% perchloric acid. The supernatant was transferred to a fresh tube and neutralized using K₂CO₃, and the perchloric acid-precipitated proteins were saved for quantification via Lowry assay. The ATP content in the neutralized supernatant was measured spectrophotometrically (26) on the day of the experiment or after storing the sample at 20 °C. In each experiment, four samples of unstimulated (basal) cells were collected, and one or two samples of FCCP- and rottlerin-treated cells were collected at multiple time points. The results from two or three individual acinar cell preparations were averaged, and the S.E. was calculated (where n = 3).

Data-- For QO₂ measurements, the mean ± S.E. of n number of replicates from a single preparation of cells or mitochondria are shown in the figures. The numbers of different preparations are as indicated in the figure legends. Western blots are representative of at least three different experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Rottlerin Increases the QO₂ of Intact Parotid Acinar Cells-- 10 µM rottlerin produced a large increase in the QO₂ of intact parotid acinar cells (Fig. 1A). 10 µM carbachol, a ligand that activates the M3 muscarinic receptor in these cells, also increased the QO₂ of intact parotid cells. The changes in the QO₂ were rapid for both agents, although the time to reach the maximal linear peak QO₂ occurred somewhat more rapidly in response to carbachol compared with rottlerin. Initially, the reason for the response to rottlerin was unclear, but it was presumed to involve PKC and/or other PKC family members. The QO₂ response to carbachol was caused by the activation of the sodium pump and was secondary to increases in intracellular Na⁺ and decreases in intracellular K⁺ which occur upon the activation of Na⁺ entry pathways and K⁺ channels in carbachol-treated parotid acinar cells. In many epithelial cells, the sodium pump is tightly and functionally coupled to mitochondrial respiration because of the regulation of oxidative phosphorylation by the ADP (and perhaps inorganic phosphate) generated from ATP hydrolysis by the sodium pump (Na,K-ATPase) (27). In these cells, inhibition of the sodium pump with ouabain will block the sodium pump component of oxidative phosphorylation (22, 28). The addition of ouabain to unstimulated parotid cells reduced the basal QO₂ (Fig. 1B). Although the response to carbachol was substantially blocked by pretreatment of the parotid acinar cells with ouabain, the increase in QO₂ by exposure of the cells to rottlerin was equally effective in the absence or presence of ouabain (Fig. 1, B and D). This characteristic of the effects of rottlerin on parotid cell QO₂ suggested that rottlerin might be acting as a mitochondrial uncoupler, analogous to the equivalent effects of the mitochondrial uncoupler FCCP on the QO₂ of isolated renal epithelial cells in the presence and absence of ouabain (29). In fact, FCCP also stimulated the QO₂ of parotid acinar cells independent of the presence of ouabain (Fig. 1).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of rottlerin and various compounds on the QO2 of rat parotid acinar cells in suspension. Panels A-C, reductions in the amount of O2 present in a closed system as a function of time. The numbers in parentheses are the QO2 (in nmol of O2/mg of protein/min) that were calculated from the maximal linear portion of the disappearance of O2 for each condition. The following concentrations of agents were added: 10 µM carbachol, 10 µM rottlerin, 10 µM FCCP, 4 mM ouabain, and 10 µM GF109203X. Carbachol, rottlerin, and FCCP were added in the absence (panel A) or presence (panel B) of ouabain. In panel C, the closed chamber was opened temporarily (dotted line) so that the O2 level could be increased to extend the time of measurement prior to anoxia. Panel D, calculations of increases (QO2) in the basal QO2 level (± ouabain) in cells exposed to carbachol, rottlerin, GF109203X, and FCCP. The data are the mean ± S.E. of replicates (numbers at the bottom of the bar) from one parotid acinar cell preparation. In this preparation, the control basal QO2 was 14.4 ± 0.3 (n = 28) nmol of O2/mg/min, and the addition of ouabain to unstimulated cells reduced the basal QO2 by 4.0 ± 0.4 (n = 11) nmol of O2/mg/min. The traces in panels A-C and the values in panel D are representative of two other experiments.

Rottlerin also increased the QO₂ in cells that first were exposed sequentially to carbachol, which increases the sodium pump activity, and ouabain, which blocks the activated sodium pump (Fig. 1C). Rottlerin also increased the QO₂ when cells were treated sequentially with nystatin and ouabain (not shown). Nystatin, a cationophore, produces a rapid increase in the QO₂ of parotid acinar cells in a ouabain-sensitive manner because of its effects on increasing Na⁺ and decreasing K⁺ within the cell (22, 30). These results indicated that the effects of rottlerin were not mediated by alterations in the concentrations of intracellular Na⁺ and K⁺.

In contrast to the stimulatory effects of rottlerin on the QO₂, 10 µM GF109203X, a PKC inhibitor effective on a broad spectrum of PKC isoforms, did not increase the QO₂ (Fig. 1D). In addition, rottlerin stimulated the QO₂ in the presence of GF109203X (Fig. 1D). These results indicated that the effects of rottlerin were not caused by inhibiting or stimulating PKC activity.

These results, which were obtained with freshly isolated rat parotid acinar cells, suggested that rottlerin directly or indirectly uncoupled mitochondrial respiration from oxidative phosphorylation. Like the mitochondrial uncoupler FCCP, rottlerin was effective in the presence of ouabain and under conditions in which the normally low concentrations of intracellular Na⁺ and high concentrations of K⁺ were reversed. Freshly isolated rat parotid acinar cells rely primarily on oxidative metabolism to supply energy for the sodium pump and other ATP-dependent processes. Therefore, we tested the effects of rottlerin on the respiration of PC12 cells, which rely heavily on glycolysis (31-33) for their energetic needs. Rottlerin also produced a rapid increase in the QO₂ of PC12 cells (Fig. 2). The basal QO₂ of PC12 cells (19.8 ± 2.0 nmol of O₂/mg/min, n = 3) increased by 12.1 ± 0.3 (n = 3) nmol of O₂/mg/min in the acute presence of 10 µM rottlerin. Thus, rottlerin promoted respiratory increases in cells that rely heavily on glycolysis for ATP production as well as in cells that rely primarily on oxidative metabolism.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Rottlerin promotes an increase in the QO2 of PC12 cells. The reduction in the amount of O2 present in a closed system as a function of time is shown. The addition of 10 µM rottlerin produced a rapid increase in the QO2 until the closed chamber was depleted of O2. The numbers in parentheses are the QO2 values (in nmol of O2/mg of protein/min) that were calculated from the maximal linear portion of the consumption of O2 under each condition.

Rottlerin Increases the QO₂ of Isolated Rat Liver Mitochondria-- To determine if rottlerin had effects directly on mitochondria, the QO₂ of isolated rat liver mitochondria was measured. Rottlerin produced a large and rapid increase in the QO₂ of the isolated mitochondria (Fig. 3A). Similar increases were produced by FCCP and ADP (Fig. 3, A and B). The ADP-stimulated QO₂, in which O₂ consumption is coupled to the production of ATP, was classified by Chance and Williams (34) as the state 3 rate of mitochondrial O₂ consumption. The QO₂ of ADP-treated mitochondria returned to a slower rate upon the conversion of ADP to ATP (Fig. 3A). However, the enhanced QO₂ produced by rottlerin or FCCP continued until the O₂ levels in the chamber were depleted. The effectiveness of rottlerin in stimulating the QO₂ of both intact cells and isolated mitochondria indicated that rottlerin promoted the uncoupling of mitochondrial respiration from the production of ATP. In contrast to the effects of 10 µM rottlerin, 10 µM GF109203X did not alter the mitochondrial QO₂ and also did not block the effects of rottlerin (Fig. 3, A and B). These results suggest that the effects of rottlerin on respiration were not mediated by blocking (or stimulating) PKC activity.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Rottlerin stimulates the QO2 of isolated rat liver mitochondria in a PKC-independent manner. The effects of various agents on the QO2 of isolated rat liver mitochondria in suspension are shown. Panel A, representative recordings of the decrease of O2 in a closed system are shown as a function of time. At the arrows, the following additions were made: 0.25 mM ADP, 10 µM rottlerin, 10 µM GF109203X, and 10 µM FCCP. The numbers in parentheses are the QO2 values (in nmol of O2/mg of protein/min) that were calculated from the maximal linear portion of the disappearance of O2. Panel B, QO2 values of unstimulated (basal) mitochondria and mitochondria exposed to various agents as shown in panel A. The PKC inhibitor GF109203X did not produce significant alterations in the basal mitochondrial QO2 and did not block the stimulatory effects of rottlerin on the QO2. The data are the mean QO2 values ± S.E. of replicates (numbers at the bottom of the bars) from one mitochondrial preparation. In this preparation, ADP increased the QO2 to 4.7 ± 0.2 (n = 7) times the basal rate, suggesting that the mitochondrial membranes were intact and that mitochondrial O2 consumption was coupled to ATP production. Similar effects were observed in one other preparation of rat liver mitochondria. Panel C, concentration dependence of rottlerin and FCCP on mitochondrial QO2. Mitochondria were exposed to 0.25 mM ADP or various concentrations (0.1-10 µM) of rottlerin and FCCP in the presence of 0.05% BSA (BSA) or 5% BSA (+BSA). The presence of 5% BSA lowered the effectiveness of 1 µM but not 10 µM FCCP and rottlerin. The data are the mean QO2 values ± S.E. of replicates (numbers at bottom of the bars) from one mitochondrial preparation.

The dependence of the mitochondrial respiration on the concentration of rottlerin was compared with that of FCCP (Fig. 3C). At 1 and 10 µM, both compounds stimulated the largest rates, which were similar to the ADP-stimulated state 3 rate of respiration; but FCCP was more effective than rottlerin at 0.1 µM. High (5%) concentrations of BSA in the mitochondrial solution did not alter the effectiveness of 10 µM FCCP and rottlerin, but the responses to 1 µM FCCP were reduced more than responses to 1 µM rottlerin. BSA did not reduce the response to ADP, consistent with BSA acting by binding to both uncoupling agents rather than by compromising the ability of the mitochondria to respond with an increase in QO₂. These results demonstrate that the potency of the uncouplers is affected by high levels of proteins, a consideration to bear in mind when cells are in serum-containing medium. BSA has been shown to lower the potency of many compounds, including mitochondrial respiratory chain inhibitors added to intact cells (35).

Rottlerin Reduces the Intracellular ATP Content-- The preceding data indicated that rottlerin alters oxidative metabolism, which epithelial and other cells rely upon for the production of ATP. To determine the degree to which intracellular ATP levels were affected, the ATP content of parotid acinar cells was measured in cells exposed to rottlerin and FCCP for various periods of time (Fig. 4). 10 µM FCCP reduced the level of ATP to about 10% of control values within 2 min. Rottlerin also reduced the intracellular ATP content, but the reduction occurred more slowly than that produced by exposure to FCCP. After cells were exposed to 10 µM rottlerin, about half of the total cell ATP was reduced within 2 min, and about 80% was reduced within 15 min. Presumably, the ATP levels are reduced by the consumption of ATP for various energetic demands of the cells when FCCP and rottlerin uncouple mitochondrial ATP synthesis.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Rottlerin and FCCP rapidly reduce cellular ATP levels in parotid acinar cells. Suspensions of cells were treated with 10 µM rottlerin or 10 µM FCCP for 2-15 min, and the cellular ATP content was measured and compared with control cells. Exposure of cells to FCCP produced a more rapid reduction in ATP levels than did rottlerin. The results were normalized to the control value of ATP in each of three experiments and are presented as the mean ± S.E. (where n = 3). The ATP level in control cells was 7.6 ± 0.9 (n = 3) nmol of ATP/mg of protein.

Rottlerin Reduces the Phosphorylation of PKC on Tyrosine Residues-- The reduction of intracellular ATP could block a variety of ATP-dependent processes, including kinase-mediated cell signaling events. One such event is the tyrosine phosphorylation of PKC, which is mediated by Src-related tyrosine kinases in various cells (see the Introduction), including parotid and PC12 cells. Therefore, the effect of rottlerin on the tyrosine phosphorylation of PKC was investigated. Carbachol- and PMA-promoted increases in PKC tyrosine phosphorylation in parotid acinar cells were decreased in cells exposed to rottlerin (Fig. 5A). In contrast, the PKC inhibitor GF109203X did not reduce the tyrosine phosphorylation of PKC in these cells (not shown), but exposure of the cells to FCCP blocked this phosphorylation. Exposure of the parotid acinar cells to PP2, an inhibitor of Src and Src-related tyrosine kinases (36), also reduced the carbachol- and PMA-mediated increases in PKC tyrosine phosphorylation. Because PKC in parotid acinar (14) and other cells (see the Introduction) is activated by increases in its phosphorylation on tyrosine residues, the activation of PKC will be blocked in cells exposed to rottlerin and FCCP.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Effects of rottlerin, FCCP, and other agents on the tyrosine phosphorylation of PKC in various cells. Cells were treated with vehicle (0.1% Me2SO), 10 µM rottlerin, 10 µM FCCP, 10 µM GF109203X, and 10 µM PP2 prior to stimuli. The arrows indicate the location of the tyrosine-phosphorylated form of PKC. Panel A, rat parotid acinar cells were exposed to Me2SO (), rottlerin, FCCP, and PP2 for 20 min followed by exposure to vehicle (0.1% water) for 1 min, 10 µM carbachol for 1 min, or 100 nM PMA for 5 min. Cells were lysed, and PKC was immunoprecipitated (IP), subjected to SDS-polyacrylamide gel electrophoresis, and sequentially immunoblotted (IB) using P-Tyr and PKC antibodies. Panel B, RPG1 cells were exposed to Me2SO (), rottlerin, FCCP, and GF109203X for 30 min followed by exposure to vehicle (0.1% Me2SO) or 100 nM PMA for 5 min. Cells were then lysed and treated as in panel A. Panel C, PC12 cells were exposed to Me2SO (), rottlerin, FCCP, GF109203X, and PP2 for 30 min and then exposed to vehicle (0.1% Me2SO) or 100 nM PMA for 5 min. Cells were lysed and treated as in panel A. Each blot in panels A, B, and C is representative of at least three experiments.

The effects of rottlerin and FCCP on the tyrosine phosphorylation of PKC also were examined in two cell lines: RPG1 cells, an immortalized rat parotid ductal cell line, and PC12 cells. Neither rottlerin nor FCCP significantly reduced the PMA-promoted increase in PKC tyrosine phosphorylation in these two cell types under the conditions of these experiments, although inhibition of Src-related kinases with PP2 blocked this phosphorylation event² (Fig. 5, B and C). GF109203X (Fig. 5, B and C) and Ro 31-8220 (not shown), another PKC inhibitor, also did not block stimuli-dependent increases in PKC tyrosine phosphorylation in these cells. The effectiveness of rottlerin on parotid acinar cells (Fig. 5A) and the lack of effect of rottlerin in these two cell lines (Fig. 5, B and C) is probably the result of differences in metabolism between freshly isolated cells and cultured cell lines (see "Discussion").

PKC Enzyme Activity-- The concentration dependence of rottlerin on PKC activity in vitro was examined and compared with the effects of GF109203X. As in previous studies (14), the basal PKC activity measured in the presence of DAG and PS was much higher than in the absence of these lipid cofactors (Fig. 6). In the presence of the cofactors, 1 and 10 µM rottlerin had almost no effect ( 10% reduction) on PKC activity, and 30 µM rottlerin blocked only 24.6% (n = 2) of the PKC activity. In contrast, rottlerin produced a concentration-dependent increase in PKC activity in the absence of the cofactors; in fact, 10 µM rottlerin increased the activity to 6.0 ± 1.2 (n = 3) times the basal activity. GF109203X effectively blocked PKC activity at low micromolar concentrations in both the presence and absence of DAG and PS. Thus, rottlerin did not inhibit PKC enzyme activity in vitro at a concentration (10 µM) at which it had various biological effects on intact cells.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Concentration-dependent effects of rottlerin and GF109203X on PKC activity. The activity of PKC immunoprecipitated from PC12 cells was measured in the presence (+) and absence () of DAG and PS and in various concentrations of rottlerin and GF109203X. The results of two or three separate experiments are shown. In each experiment, the PKC activities were normalized to the activity of PKC in the presence of DAG and PS with no inhibitors present. Results are presented as the mean ± S.E. (n = 3). Although GF109203X produced a concentration-dependent decrease in PKC activity in the presence and absence of lipid cofactors, rottlerin had little effect in the presence of cofactors and produced a stimulatory effect in the absence of the cofactors.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Rottlerin has been used extensively as a PKC-specific inhibitor. In many studies of a variety of cells, the alterations of biological events by rottlerin have been interpreted as a positive indication for the involvement of PKC in these events. The results presented here indicate that these conclusions are open to question for the following reasons: 1) rottlerin was ineffective in blocking PKC activity in vitro; 2) rottlerin uncoupled isolated mitochondria and mitochondria in intact cells; and 3) rottlerin promoted the rapid decrease of ATP to low levels in intact cells. Rottlerin also blocked the tyrosine phosphorylation of PKC, and this would be expected to inhibit the activation of PKC, although this effect may be coincidental with the reduction in ATP rather than the result of any selectivity of rottlerin for PKC (see below).

In the initial study of rottlerin as a PKC inhibitor, it was reported to exert selectivity for PKC among other PKC isoforms when examined for its ability to block the phosphorylation of protamine in vitro (6). This suggested that rottlerin might be a useful pharmacological inhibitor and perhaps could be used as a template for designing inhibitors of specific PKC isoforms. Subsequently, rottlerin has been widely used as a PKC-selective inhibitor in many different cellular systems, and its use has increased dramatically during the last several years. In some studies, the effects of rottlerin have been compared simultaneously with other experimental approaches, but many studies have relied on the classification of rottlerin as a PKC-specific inhibitor for interpreting its effects at low micromolar concentrations. However, rottlerin did not block a significant amount of PKC enzyme activity in vitro in the presence of lipid cofactors (Fig. 6), which also was reported recently by other investigators (37), and it increased PKC activity in the absence of lipids. There are multiple implications to these findings. First, because 10 µM rottlerin did not display a significant inhibitory activity toward PKC, this belies its classification as a PKC inhibitor. In addition, the results of the in vitro assay performed in the absence of lipid cofactors suggest that rottlerin might increase PKC activity in intact cells under some conditions. In either condition, the effects of rottlerin on intact cells would not be caused by the direct inhibition of PKC. The in vitro substrate phosphorylation assays shown in Fig. 6 were performed using a peptide containing the optimum sequence for a PKC substrate (4), whereas the in vitro assay cited in the original report (6) was conducted using protamine, a more generic substrate. It is not known whether this alone accounts for the drastic differences between the findings of the original study and the present one.

The uncoupling actions of rottlerin on isolated mitochondria and intact cells were neither mimicked nor blocked by the PKC inhibitor GF109203X, suggesting that rottlerin did not act indirectly via inhibiting PKC or by altering GF109203X-sensitive PKC family members. Because rottlerin was effective in uncoupling isolated mitochondria, this suggests that rottlerin did not uncouple mitochondria in intact cells via a cytosolic mediator. 10 µM rottlerin was as effective as 10 µM FCCP in stimulating the QO₂ of isolated mitochondria (Fig. 3), but it was less effective than FCCP on intact parotid cells, at least when added acutely to cells (Fig. 1). This difference in effectiveness may indicate that rottlerin was less permeable to intact parotid cells than was FCCP. Differences between FCCP and rottlerin in altering intracellular ATP levels (Fig. 4) are consistent with differences between rottlerin and FCCP in uncoupling the mitochondria of intact parotid acinar cells.

PKC translocates to various locations and compartments within cells, including the mitochondria (8, 38, 39). PKC has been reported to play multiple roles in apoptosis involving separate and distinct roles upstream and downstream of mitochondria (1, 7, 40), including altering the mitochondrial membrane potential in a PMA-dependent manner in intact cells (39) and contributing to the loss of mitochondrial membrane potential by apoptosis-inducing agents (41). Thus, without knowledge of the present findings, it might seem appropriate that rottlerin, a putative PKC inhibitor, could have PKC-dependent effects on mitochondrial O₂ consumption. However, the lack of effect of GF109203X on the QO₂ of cells and isolated mitochondria and the inability of GF109203X to block the effects of rottlerin on the increases in the QO₂ (Figs. 1 and 3) indicate that the uncoupling action of rottlerin is not mediated indirectly via PKC.

Because rottlerin uncouples mitochondria, it is not surprising that it greatly decreased the levels of ATP in freshly isolated parotid acinar cells. Freshly isolated epithelial cells, excised tissue, and cells in vivo rely heavily on oxidative phosphorylation for energy production, especially for the ATP necessary to support active ion transport (42), although some epithelial cellular functions may depend on glycolysis (43). FCCP decreased the ATP level in freshly isolated renal epithelial cells to about 10% of the control levels within 1-2 min (29), similar to the effects of FCCP on parotid acinar cells. One would predict that such large decreases could compromise ATP-dependent events such as the Src kinase-dependent tyrosine phosphorylation of PKC. This phosphorylation produces an increase in PKC enzyme activity in both parotid acinar cells and PC12 cells (14). These results suggest that by reducing the level of ATP and blocking the tyrosine phosphorylation of PKC, rottlerin and FCCP could block the biological effects of PKC. In this way, rottlerin could block PKC via a mechanism unrecognized by its users. However, this would be coincidental to nonselective effects of rottlerin on other kinases and enzymes that rely on ATP, which also could be responsible for some of the biological effects of rottlerin. Moreover, rottlerin also increased PKC activity in vitro, suggesting that the mechanism(s) of its biological effects on PKC in intact cells might be very complicated to predict or to interpret. Furthermore, in another study that found that rottlerin did not inhibit PKC in vitro, rottlerin potently inhibited a number of other kinases in vitro (37); and in the initial study of rottlerin, it also inhibited calmodulin-dependent kinase III in vitro (6). Therefore, it may be unlikely that most of the biological effects of rottlerin reported in the literature are the results of compromising the phosphorylation of PKC on tyrosine residues. Of interest, a long term (6-12 h) exposure of human U-937 myeloid leukemia cells to 10 µM rottlerin produced a release of cytochrome c and an increase in apoptosis (8). It is tempting to speculate that the uncoupling of mitochondria played a role in promoting these effects.

Cultured cells and cell lines, including PC12 cells, rely on a combination of glycolysis and oxidative metabolism for ATP production (31, 42, 44). The lack of a detectable effect of rottlerin and FCCP (30-45 min) on the tyrosine phosphorylation of PKC in PC12 and RPG1 cells (Fig. 5) suggests that mitochondrial uncouplers do not affect the ATP levels in these cells to the same degree or with the same time course as cells that rely more exclusively on oxidative phosphorylation for ATP production. In fact, ATP levels in PC12 cells were reduced by only ~60% in cells exposed to a high concentration (30 µM) of FCCP for 2 h (45) and were reduced by only ~70% after a 3-h exposure to inhibitors of both glycolysis and oxidative phosphorylation (46).

The activation of muscarinic and other receptors on parotid acinar cells initiates fluid secretion because of the activation of Ca²⁺ -sensitive Cl and K⁺ channels and the entry of extracellular Na⁺ via several ion exchange and cotransport systems (47). Subsequent to intracellular ionic changes, the sodium pump activity is increased, resulting in a ouabain-sensitive increase in the QO₂ (22). Ouabain lowered the basal QO₂ of parotid acinar cells by about 25-30% because of its inhibition of the constitutive sodium pump activity in the presence of submaximal concentrations of sodium (Fig. 3). In retrospect it was not surprising that the effects of FCCP and rottlerin on intact cells were not blocked by ouabain, which would not be expected to inhibit mitochondrial uncouplers. In contrast, it was somewhat surprising that ouabain did not fully prevent carbachol from producing an initial increase in QO₂ (Fig. 1D). However, under these conditions the increase in QO₂ by carbachol in the presence of ouabain was extremely transient (Fig. 1B, dotted line) and returned to the lower rate within about 1 min, unlike the sustained carbachol-initiated increase that occurred in the absence of ouabain (Fig. 1A) or the rottlerin-initiated increase that occurs in the presence of ouabain (Fig. 1B). Presumably, the large influx of Na⁺ and efflux of K⁺ which occur upon exposure to carbachol (22, 23) were sufficient to activate the sodium pump partially and transiently within the first 2 min of ouabain exposure. An alternative explanation may be that additional sodium pumps are recruited to the membrane by activation of the muscarinic M3 receptors.

In conclusion, these studies reinforce the interrelationship between the mitochondria and ATP-dependent events in cells that have a large dependence on oxidative metabolism. Rottlerin is a mitochondrial uncoupler, and this may be the mechanism of action for some of its effects on a variety of cells, including those that rely on oxidative metabolism as well as those that also depend on glycolysis for energy. These findings suggest that the use of rottlerin as an inhibitor of PKCs and PKC should be curtailed in all cells, regardless of the type of metabolism upon which they rely for energy production.

	ACKNOWLEDGEMENTS

I thank Dr. Mary Chamberlin (Ohio University) for conversations about mitochondrial respiration and Dr. Cyril Benes (Beth Israel Deaconess Medical Center) for helpful discussions.

	FOOTNOTES

^* This work was supported by Grant DE-10877 from the NIDCR, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article is dedicated to the late Dr. Lazaro J. Mandel. Laz's friendship and scientific expertise in epithelia are greatly missed.

To whom correspondence should be addressed: Division of Signal Transduction, Harvard Institutes of Medicine, Room 1025, Beth Israel Deaconess Medical Center, Dept. of Medicine, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-0949; Fax: 617-667-0957; E-mail: ssoltoff@caregroup.harvard.edu.

Published, JBC Papers in Press, August 9 2001, DOI 10.1074/jbc.M105073200

² S. P. Soltoff, unpublished results.

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; QO₂, rate of oxygen consumption; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; RPG1 cells, rat parotid gland 1 cells; Me₂SO, dimethyl sulfoxide; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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