Effects of Secretagogues and Bile Acids on Mitochondrial Membrane Potential of Pancreatic Acinar Cells
2004; Elsevier BV; Volume: 279; Issue: 26 Linguagem: Inglês
10.1074/jbc.m311698200
ISSN1083-351X
AutoresSvetlana Voronina, Stephanie L. Barrow, Oleg V. Gerasimenko, Ole H. Petersen, Alexei V. Tepikin,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoIn this study, we investigated the effects of secretagogues and bile acids on the mitochondrial membrane potential of pancreatic acinar cells. We measured the mitochondrial membrane potential using the tetramethylrhodamine-based probes tetramethylrhodamine ethyl ester and tetramethylrhodamine methyl ester. At low levels of loading, these indicators appeared to have a low sensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and no response was observed to even high doses of cholecystokinin. When loaded at high concentrations, tetramethylrhodamine methyl ester and tetramethylrhodamine ethyl ester undergo quenching and can be dequenched by mitochondrial depolarization. We found the dequench mode to be 2 orders of magnitude more sensitive than the low concentration mode. Using the dequench mode, we resolved mitochondrial depolarizations produced by supramaximal and by physiological concentrations of cholecystokinin. Other calcium-releasing agonists, acetylcholine, JMV-180, and bombesin, also produced mitochondrial depolarization. Secretin, which employs the cAMP pathway, had no effect on the mitochondrial potential; dibutyryl cAMP was also ineffective. The cholecystokinin-induced mitochondrial depolarizations were abolished by buffering cytosolic calcium. A non-agonist-dependent calcium elevation induced by thapsigargin depolarized the mitochondria. These experiments suggest that a cytosolic calcium concentration rise is sufficient for mitochondrial depolarization and that the depolarizing effect of cholecystokinin is mediated by a cytosolic calcium rise. Bile acids are considered possible triggers of acute pancreatitis. The bile acids taurolithocholic acid 3-sulfate, taurodeoxycholic acid, and taurochenodeoxycholic acid, at low submillimolar concentrations, induced mitochondrial depolarization, resolved by the dequench mode. Our experiments demonstrate that physiological concentrations of secretagogues and pathologically relevant concentrations of bile acids trigger mitochondrial depolarization in pancreatic acinar cells. In this study, we investigated the effects of secretagogues and bile acids on the mitochondrial membrane potential of pancreatic acinar cells. We measured the mitochondrial membrane potential using the tetramethylrhodamine-based probes tetramethylrhodamine ethyl ester and tetramethylrhodamine methyl ester. At low levels of loading, these indicators appeared to have a low sensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and no response was observed to even high doses of cholecystokinin. When loaded at high concentrations, tetramethylrhodamine methyl ester and tetramethylrhodamine ethyl ester undergo quenching and can be dequenched by mitochondrial depolarization. We found the dequench mode to be 2 orders of magnitude more sensitive than the low concentration mode. Using the dequench mode, we resolved mitochondrial depolarizations produced by supramaximal and by physiological concentrations of cholecystokinin. Other calcium-releasing agonists, acetylcholine, JMV-180, and bombesin, also produced mitochondrial depolarization. Secretin, which employs the cAMP pathway, had no effect on the mitochondrial potential; dibutyryl cAMP was also ineffective. The cholecystokinin-induced mitochondrial depolarizations were abolished by buffering cytosolic calcium. A non-agonist-dependent calcium elevation induced by thapsigargin depolarized the mitochondria. These experiments suggest that a cytosolic calcium concentration rise is sufficient for mitochondrial depolarization and that the depolarizing effect of cholecystokinin is mediated by a cytosolic calcium rise. Bile acids are considered possible triggers of acute pancreatitis. The bile acids taurolithocholic acid 3-sulfate, taurodeoxycholic acid, and taurochenodeoxycholic acid, at low submillimolar concentrations, induced mitochondrial depolarization, resolved by the dequench mode. Our experiments demonstrate that physiological concentrations of secretagogues and pathologically relevant concentrations of bile acids trigger mitochondrial depolarization in pancreatic acinar cells. Pancreatic acinar cells are polarized epithelial cells responsible for synthesis and secretion of digestive enzymes. The apical part of these cells contains a high density of secretory granules, whereas the basal region contains a well developed endoplasmic reticulum and the nucleus (1Bolender R.P. J. Cell Biol. 1974; 61: 269-287Crossref PubMed Scopus (206) Google Scholar, 2Ashby M.C. Camello-Almaraz C. Gerasimenko O.V. Petersen O.H. Tepikin A.V. J. Biol. Chem. 2003; 278: 20860-20864Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 3Gerasimenko O.V. 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Secretagogues (e.g. the circulating hormone CCK) utilize a calcium signaling cascade to trigger and regulate enzyme and fluid secretion in pancreatic acinar cells (reviewed in Refs. 9Ashby M.C. Tepikin A.V. Physiol. Rev. 2002; 82: 701-734Crossref PubMed Scopus (103) Google Scholar, 10Petersen O.H. J. Physiol. 1992; 448: 1-51Crossref PubMed Scopus (368) Google Scholar, 11Petersen O.H. Petersen C.C. Kasai H. Annu. Rev. Physiol. 1994; 56: 297-319Crossref PubMed Scopus (258) Google Scholar, 12Williams J.A. Annu. Rev. Physiol. 2001; 63: 77-97Crossref PubMed Scopus (194) Google Scholar). The secretagogue-induced calcium rise occurs primarily due to calcium release from internal stores. The stores are replenished by calcium influx through the basal portion of the plasma membrane and the tunneling action of the endoplasmic reticulum (13Lomax R.B. Camello C. Van Coppenolle F. Petersen O.H. Tepikin A.V. J. Biol. 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It has been hypothesized that such calcium toxicity is responsible for the damage to pancreatic tissue during acute pancreatitis (23Ward J.B. Petersen O.H. Jenkins S.A. Sutton R. Lancet. 1995; 346: 1016-1019Abstract PubMed Google Scholar). This hypothesis recently received considerable experimental support (20Kim J.Y. Kim K.H. Lee J.A. Namkung W. Sun A.Q. Ananthanarayanan M. Suchy F.J. Shin D.M. Muallem S. Lee M.G. Gastroenterology. 2002; 122: 1941-1953Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 21Kruger B. Albrecht E. Lerch M.M. Am. J. Pathol. 2000; 157: 43-50Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 22Raraty M. Ward J. Erdemli G. Vaillant C. Neoptolemos J.P. Sutton R. Petersen O.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13126-13131Crossref PubMed Scopus (283) Google Scholar). Mitochondria in pancreatic acinar cells accumulate calcium during cytosolic calcium responses (5Park M.K. Ashby M.C. Erdemli G. Petersen O.H. Tepikin A.V. 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In particular, the perigranular mitochondrial belt helps to restrain calcium signals to the apical region of the cell (5Park M.K. Ashby M.C. Erdemli G. Petersen O.H. Tepikin A.V. EMBO J. 2001; 20: 1863-1874Crossref PubMed Scopus (272) Google Scholar, 6Tinel H. Cancela J.M. Mogami H. Gerasimenko J.V. Gerasimenko O.V. Tepikin A.V. Petersen O.H. EMBO J. 1999; 18: 4999-5008Crossref PubMed Scopus (322) Google Scholar, 8Straub S.V. Giovannucci D.R. Yule D.I. J. Gen. Physiol. 2000; 116: 547-560Crossref PubMed Scopus (161) Google Scholar, 24Johnson P.R. Tepikin A.V. Erdemli G. Cell Calcium. 2002; 32: 59-69Crossref PubMed Scopus (25) Google Scholar). Important roles for calcium in stimulus-metabolism coupling (26Hajnoczky G. Robb-Gaspers L.D. Seitz M.B. Thomas A.P. Cell. 1995; 82: 415-424Abstract Full Text PDF PubMed Scopus (955) Google Scholar) (see also reviews in Refs. 27Duchen M.R. J. Physiol. 2000; 529: 57-68Crossref PubMed Scopus (948) Google Scholar, 28McCormack J.G. Halestrap A.P. Denton R.M. Physiol. Rev. 1990; 70: 391-425Crossref PubMed Scopus (1167) Google Scholar, 29Rizzuto R. Bernardi P. Pozzan T. J. Physiol. 2000; 529: 37-47Crossref PubMed Scopus (482) Google Scholar) and in mitochondria-mediated cell pathology (reviewed in Refs. 29Rizzuto R. Bernardi P. Pozzan T. J. Physiol. 2000; 529: 37-47Crossref PubMed Scopus (482) Google Scholar, 30Duchen M.R. Cell Calcium. 2000; 28: 339-348Crossref PubMed Scopus (274) Google Scholar, 31Hajnoczky G. Davies E. Madesh M. Biochem. Biophys. Res. Commun. 2003; 304: 445-454Crossref PubMed Scopus (393) Google Scholar) have been documented for a number of cell types. We have recently characterized the correlation between cytosolic calcium, mitochondrial calcium, and mitochondrial NADH production in pancreatic acinar cells (32Voronina S. Sukhomlin T. Johnson P.R. Erdemli G. Petersen O.H. Tepikin A. J. Physiol. 2002; 539: 41-52Crossref PubMed Scopus (95) Google Scholar). Specifically, we found that even relatively short (a few seconds) apical calcium signals are able to produce a considerable increase of mitochondrial NADH. It is important to note that, depending on the status of the mitochondria, both positive and negative changes of NADH concentrations could be induced by calcium signals (32Voronina S. Sukhomlin T. Johnson P.R. Erdemli G. Petersen O.H. Tepikin A. J. Physiol. 2002; 539: 41-52Crossref PubMed Scopus (95) Google Scholar). Both positive and negative NADH changes were also recorded in whole cell patch clamp experiments when the patch pipette solution contained 2 mm of ATP, suggesting that these changes are not due to fluctuations of ATP concentration but, most probably, due to a direct effect of calcium on the mitochondria. The mitochondrial membrane potential (ΔΨm) is a very important parameter, controlling different aspects of mitochondrial metabolism and ultimately ATP production. Our previous attempts to resolve changes of mitochondrial membrane potential induced by calcium-releasing secretagogues were unsuccessful (22Raraty M. Ward J. Erdemli G. Vaillant C. Neoptolemos J.P. Sutton R. Petersen O.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13126-13131Crossref PubMed Scopus (283) Google Scholar). These attempts were undertaken using loading of the cells with a low concentration of a mitochondrial probe (tetramethylrhodamine methyl ester (TMRM)). In the current study, we decided to employ both the conventional low concentration and a high concentration dequench mode for evaluation of the mitochondrial membrane potential. The two modes of measurements are based on different principles. The low concentration mode relies on the Nernst distribution of the positively charged, yet membrane-permeable, fluorescent indicator between cytosol and mitochondria. Depolarization of mitochondria should result in redistribution of the indicator from the mitochondria to the cytosol, causing a decrease of mitochondrial fluorescence and increase of cytosolic fluorescence. The dequench mode requires high concentrations of indicators (tetramethylrhodamine ethyl ester (TMRE) or TMRM in our experiments). When loaded at high concentrations, these probes further concentrate in the negatively charged mitochondria and, as a result of such dense packaging, become quenched. Depolarization of the mitochondria results in release of the indicator from mitochondria to the cytosol, dequench of the indicator, and therefore an overall increase of cell fluorescence (for a detailed review of the techniques of ΔΨm measurements and techniques for studies of other mitochondrial functions, see Ref. 33Duchen M.R. Surin A. Jacobson J. Methods Enzymol. 2003; 361: 353-389Crossref PubMed Scopus (193) Google Scholar). It is important to note that complete dequench of an indicator does not necessarily mean complete dissipation of ΔΨm. Mitochondria could still be substantially hyperpolarized when dequench response reaches its maximum (33Duchen M.R. Surin A. Jacobson J. Methods Enzymol. 2003; 361: 353-389Crossref PubMed Scopus (193) Google Scholar). The first aim of our study was to characterize the relative sensitivities of the low concentration and the dequench mode of evaluation of changes in ΔΨm. Thereafter, we used the more sensitive of the two methods to identify and investigate ΔΨm changes, induced by physiological and supramaximal doses of CCK and other calcium-releasing agonists. We also compared the effects of CCK with that of the calcium-releasing agonist JMV-180, which cannot induce a sustained toxic calcium elevation (34Stark H.A. Sharp C.M. Sutliff V.E. Martinez J. Jensen R.T. Gardner J.D. Biochim. Biophys. Acta. 1989; 1010: 145-150Crossref PubMed Scopus (72) Google Scholar, 35Thorn P. Petersen O.H. J. Biol. Chem. 1993; 268: 23219-23221Abstract Full Text PDF PubMed Google Scholar) and with the secretagogue secretin, employing cAMP rather than Ca2+ as a second messenger (36Robberecht P. Conlon T.P. Gardner J.D. J. Biol. Chem. 1976; 251: 4635-4639Abstract Full Text PDF PubMed Google Scholar, 36Robberecht P. Conlon T.P. Gardner J.D. J. Biol. Chem. 1976; 251: 4635-4639Abstract Full Text PDF PubMed Google Scholar, 37Deschodt-Lanckman M. Robberecht P. De Neef P. Labrie F. Christophe J. Gastroenterology. 1975; 68: 318-325Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 38Gardner J.D. Conlon T.P. Adams T.D. Gastroenterology. 1976; 70: 29-35Abstract Full Text PDF PubMed Scopus (66) Google Scholar). The final aim of this investigation was to elucidate the effect of putative activators of acute pancreatitis, namely bile acids, on ΔΨm. Cell Preparation—Pancreata were obtained from adult mice (CD1) killed by cervical dislocation in accordance with the Animal (Scientific Procedure) Act of 1986. Pancreatic acinar cells were prepared by injecting 1 ml of 200 units ml-1 collagenase CLSPA (Worthington) and digesting for 16–17 min at 37 °C with permanent agitation. After digestion, the pancreas was agitated manually to release single cells or small clusters in solution. Cells were washed three times by centrifugation in standard "extracellular" solution. All experiments were performed at room temperature (23–25 °C), and cells were used within 3–4 h after isolation. During experiments, the cells were placed on a glass coverslip coated with poly-l-lysine (0.01%), which was attached to an open perfusion chamber. Solutions—The standard extracellular solution, used for cell preparation and for perfusion of cells during experiments, contained 140 mm NaCl, 4.7 mm KCl, 1.13 mm MgCl2;1mm CaCl2;10mm d-glucose; 10 mm Hepes (adjusted to pH 7.2 by NaOH). Solutions with the required concentrations of CCK, ACh, secretin, dibutyryl cyclic AMP, bombesin, and bile acids were prepared by dissolving these compounds in the extracellular solution. CCCP was initially prepared as a 10 mm stock solution in ethanol and then diluted in the extracellular solution before beginning experiments; the concentration of ethanol did not exceed 0.1%. JMV-180 was initially prepared as a 200 μm stock solution in Me2SO and then diluted in the extracellular solution; the concentration of Me2SO did not exceeded 0.1%. During experiments, solutions were exchanged by perfusion using a gravity-fed system. The intracellular solution (solution for loading into patch pipettes) contained 130 mm KCl, 10 mm NaCl, 1.5 mm MgCl2, 2 mm ATP, 0.1 mm EGTA, and 10 mm HEPES (adjusted to pH 7.2 by KOH). 10 mm BAPTA and 2 mm CaCl2 were added to the intracellular solution to obtain an intrapipette solution with highly buffered calcium. Loading of Fluorescent Indicators and Optical Imaging of Cells—For the low concentration mode of measurement, cells were loaded with 50–100 nm TMRM (or TMRE) for 20–25 min at 37 °C. For the dequench mode of measurement, cells were loaded with 10–20 μm TMRM (or TMRE) for 20–25 min at 37 °C. TMRM and TMRE were initially prepared as 10 mm stock solutions in Me2SO and diluted to the required concentration before loading into the cells. In the majority of experiments, fluorescence imaging of cells was conducted using a Leica SP2 confocal microscope with a × 63 water immersion objective. Fluorescence was excited by a 543-nm laser line, and emission was collected above 560 nm. In some experiments, a Zeiss confocal microscope LSM 510 was used with similar excitation/emission conditions. During processing of fluorescence data, generated in experiments with low concentrations of TMRM/TMRE, the signals from mitochondria and cytosol were analyzed separately (e.g. see Fig. 1B). The regions of interest were chosen around perigranular mitochondria (the largest group of mitochondria in the cell) and in the cytoplasm devoid of mitochondria. As expected from the low concentration mode of measurements, the depolarization of mitochondria resulted in a decrease in mitochondrial fluorescence and an increase of fluorescence in the mitochondria-free parts of the cell. The fluorescence changes were similar in basal and apical nonmitochondrial regions; however, it was usually more convenient to analyze basal regions. For analyses of the dequench mode experiments, regions of interest included whole cells. We have chosen this form of analysis, because both the mitochondrial region and the cytosol responded to depolarization of mitochondria with an increase of fluorescence. The increase of fluorescence in the mitochondrial region is probably due to the fact that the fluorescence increases in regions of the cytosol located between mitochondria in the mitochondrial belt more than compensate for the decrease of fluorescence in mitochondria. One potential artifact in employing charged membrane-permeant indicators for measurements of ΔΨm is that the fluorescence can be influenced by translocation of the indicators across the plasma membrane. Because of the presence of calcium-dependent ionic channels in the plasma membrane, the pancreatic acinar cells are able to generate rapid changes of membrane potential during calcium responses (10Petersen O.H. J. Physiol. 1992; 448: 1-51Crossref PubMed Scopus (368) Google Scholar). To avoid a possible artifact due to the redistribution of indicators across the plasma membrane, we removed the indicators from the extracellular solution prior to experiments. A few control experiments conducted in the continuous presence of TMRM in the extracellular solution are mentioned under "Effects of Physiological and Supramaximal Doses of CCK, Measured in Dequench Mode." The fluorescence was usually corrected for bleaching, using the initial parts of the curves. Intracellular calcium was measured with fluo-4 on the Zeiss confocal microscope. In these experiments, 3 μm fluo-4, AM (membrane permeable form of the indicator; AM stands for acetoxy-methyl ester) were loaded into cells for 20–25 min at room temperature. Fluo-4 was excited by a 488-nm laser line, and emission was collected through a 505–550-nm band pass filter. Patch Clamp Recording—The electrophysiological recordings of calcium-dependent Cl- currents were used as a measure of changes in cytosolic calcium. Whole cell current recordings were made using an EPC-8 amplifier and PULSE software (HEKA). The holding potential was -30 mV. The pipette resistance usually was 2–3 megaohms. Chemicals—TMRM, TMRE, and fluo-4, AM were purchased from Molecular Probes, Inc. (Eugene, OR). Bombesin, dibutyryl cyclic AMP, taurolithocholic acid 3-sulfate (TLC-S), taurodeoxycholic acid (TDC), taurochenodeoxycholic acid (TCDC), taurocholic acid (TC), and other chemicals were purchased from Sigma and were of the highest grade available. JMV-180 was purchased from Research Plus, Inc. (Bayonne, NJ). Fig. 1 shows results of experiments designed to reveal the difference in sensitivity of the two modes of measurements of the mitochondrial membrane potential. Fig. 1A represents the dequench mode of measurements, whereas Fig. 1B illustrates results obtained with low concentration loading of TMRM. Application of the protonophore CCCP (Fig. 1A) resulted in increases of fluorescence in cells loaded with a high concentration (10 μm) of TMRM (n = 16). The threshold CCCP concentration, able to produce a response, was between 50 and 500 pm. No responses were found at 5 pm CCCP (n = 6; not shown). The majority of cells did not respond to 50 pm CCCP (11 of 16 cells, with small responses in five cells), whereas all cells (n = 16) responded to 500 pm CCCP. The subsequent addition of 5 and 50 nm CCCP resulted in further increases of fluorescence in all cells (Fig. 1A). During CCCP-induced responses, the fluorescence increased in all regions of the cells (not shown). These results are consistent with the dequench mode of measurements. Single application of high doses of CCCP (500 nm or higher) resulted in a fast and large increase of fluorescence followed by a slower decline, due to loss of the indicator from the cells (see examples in Figs. 2, 3, 4, 5, 6, 7). Such additions of the protonophore were used at the end of the experiments to reveal the presence of dequenched indicator in the mitochondria. In separate experiments, we determined the amplitude of the fluorescence rise induced by application of a very high concentration of CCCP (10 μm). In these experiments, the fluorescence increased by 82 ± 8% (n = 31). Here, and below, the values represent averaged changes of fluorescence divided by the fluorescence value taken just before the addition of the relevant substance and multiplied by 100% ± S.E. of such measurements.Fig. 3Calcium dependence of CCK-induced mitochondrial depolarization and mitochondrial depolarization triggered by thapsigargin. Simultaneous recordings of mitochondrial membrane potential and cytosolic calcium responses induced by 2 nm CCK (A and B). Whole cell current recordings of calcium-dependent chloride currents were used to monitor cytosolic calcium changes. The holding potential was -30 mV. The upper traces for both A and B show recordings of calcium-dependent current, and the lower traces show changes of TMRM fluorescence (in both panels, an arrow links a fluorescence trace with an appropriate axis). A, calcium-dependent Cl- current (upper trace) and TMRM fluorescence (lower trace) in a patch-clamped cell with low calcium buffering (0.1 mm EGTA in the patch pipette solution). B, inhibition of calcium current and mitochondrial depolarization by high calcium buffering (10 mm of BAPTA, 2 mm CaCl2 in the patch pipette). C, changes of TMRM fluorescence in intact (nonpatched) pancreatic acinar cell induced by 2 μm thapsigargin (Tg).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Depolarization of mitochondrial membrane potential induced by the calcium-releasing agonists bombesin and JMV-180. Evaluation of changes in mitochondrial membrane potential was performed on cells loaded with 10 μm TMRM. A, sequential application of 50 pm, 5 nm bombesin, and 10 μm CCCP stimulated measurable elevation of TMRM fluorescence. An inset shows the effect of 50 pm bombesin on expanded scale. B, stimulation of mitochondrial depolarization by 20 nm JMV-180 and 10 μm CCK. The subsequent application of 10 mm CCCP produced further depolarization. Expanded time and fluorescence scales are used in the inset to highlight the effect of JMV-180 and CCK.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5The lack of changes of mitochondrial membrane potential on application of dibutyryl cyclic AMP (dcAMP) and secretin.A, an example of response from the cell loaded with 10 μm TMRM on subsequent application of 0.1 mm dibutyryl cyclic AMP, 2 nm CCK, and 10 μm CCCP. B, the lack of changes in fluorescence on application of 30 pm, 1 nm, and 100 nm of secretin, followed by a strong increase of TMRM fluorescence upon the addition of CCCP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Depolarization of mitochondrial membrane potential induced by TLC-S.A, changes of fluorescence produced by sequential application of 10 and 25 μm TLC-S and 10 μm CCCP in pancreatic acinar cells loaded with 10 μm TMRM (dequench mode). Expanded time and fluorescence scales are used in the inset to highlight the effect of 10 μm TLC-S. B, normalized fluorescence changes induced by 100 and 200 μm TLC-S, followed by 10 μm CCCP. C, Ca2+ chelator BAPTA suppressed responses to a low (25 μm) but not to a high (100 μm) concentration of TLC-S (dequench mode). D, the lack of changes in fluorescence upon application of 400 μm TLC-S to acinar cells, loaded with 100 nm TMRM (low concentration mode).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Changes of mitochondrial membrane potential induced by TC, TDC, and TCDC. Evaluation of changes in mitochondrial membrane potential were performed using the dequench mode of TMRM measurement. CCCP was applied at the end of each of the illustrated experiments to verify the presence of the dequen
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