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Volume 130, Issue 3, Pages 781-793 (March 2006)
Fatty Acid Ethyl Esters Cause Pancreatic Calcium Toxicity via Inositol Trisphosphate Receptors and Loss of ATP Synthesis David N. Criddle, John Murphy, Gregorio Fistetto, Stephanie Barrow, Alexei V. Tepikin, John P. Neoptolemos, Robert Sutton, Ole H. Petersen Gastroenterology Volume 130, Issue 3, Pages (March 2006) DOI: /j.gastro Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 1 (A) Typical effects of palmitoleic acid ethyl ester (100 μmol/L POAEE) applied internally to the acinar cell via patch pipette (whole-cell recording configuration) on calcium-activated chloride current and [Ca2+]C. Inset shows isolated acinar cell doublet with patch on recorded cell. (B) Absence of effect of ethanol (850 mmol/L) alone applied internally on calcium-activated chloride current and [Ca2+]C. (C) Representative recording showing the comparative effects of intracellular and extracellular application of palmitoleic acid ethyl ester (100 μmol/L) on [Ca2+]C, to produce transient and sustained elevations, respectively (n = 8). The Ca2+-sensitive Fluo 4 fluorescence is given as the fluorescence ratio (F/F0), where F is the fluorescence that changes with time, increasing with increasing [Ca2+]C, and F0 is the initial (prestimulation) basal fluorescence. Inset shows isolated acinar cell doublet from which recordings were taken, with patch on upper cell. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 2 (A) Typical inhibitory effects of 20 mmol/L caffeine on the oscillatory [Ca2+]C transients induced by palmitoleic acid ethyl ester (100 μmol/L POAEE; applied via patch pipette) in a doublet of acinar cells (patched and adjacent cell traces shown). Palmitoleic acid ethyl ester responses were inhibited by caffeine in 11 of 11 cells. Ca2+-sensitive Fluo 4 fluorescence is given as the fluorescence ratio (F/F0), as in Figure 1. (B) Increases in [Ca2+]C (measured with Fura Red) induced by ethanol (850 mmol/L) or palmitoleic acid ethyl ester (100 μmol/L POAEE with 850 mmol/L ethanol), applied extracellularly, are not inhibited by 20 mmol/L caffeine. Data are shown as mean ± SEM expressed as percentage changes from the control response before addition of caffeine. Numbers of cells tested are in parentheses on the control bars. (C) Absence of caffeine effect on extracellular palmitoleic acid (POA)-induced [Ca2+]C elevation, measured with Fura Red. Quasiphysiologic (25 nmol/L) acetylcholine (ACh) stimulation evokes characteristic oscillatory increases of [Ca2+]C that are prevented by 20 mmol/L caffeine through inositol trisphosphate receptor blockade. Continuous caffeine administration does not prevent 50 μmol/L palmitoleic acid from inducing typical [Ca2+]C elevation (n = 7). The Ca2+-sensitive fluorescence of Fura Red is given as the fluorescence ratio F0/F because Fura Red fluorescence falls with increasing [Ca2+]C. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 3 (A) Typical effect of 200 μmol/L bis-(4-nitrophenyl) phosphate (BNPP), a fatty acid ethyl ester hydrolase inhibitor, to inhibit the sustained elevation of [Ca2+]C (measured with Fluo 4) induced by externally perfused palmitoleic acid ethyl ester (100 μmol/L POAEE; 6 of 8 cells). (B) Typical light-transmitted and propidium iodide fluorescence images of isolated acinar cells after 1-hour incubation with (i) 100 μmol/L palmitoleic acid ethyl ester alone, showing morphologic disruption and propidium iodide staining of the nucleus, and (ii) 200 μmol/L bis-(4-nitrophenyl) phosphate and 100 μmol/L palmitoleic acid ethyl ester together. (iii) Propidium iodide fluorescence intensity following staining of cells after 1-hour coexposure to 100 μmol/L palmitoleic acid ethyl ester and 200 μmol/L bis-(4-nitrophenyl) phosphate, compared with cells exposed to 100 μmol/L palmitoleic acid ethyl ester alone (n = 6), showing significant protective effects of bis-(4-nitrophenyl) phosphate. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 4 Representative traces showing the effects of (A) palmitoleic acid ethyl ester (100 μmol/L POAEE, with 850 mmol/L ethanol) and (B) palmitoleic acid (50 μmol/L POA) on [Ca2+]ER (blue; Ca2+-sensitive Mg Fluo-4 fluorescence ratio [F/F0]) and NADH autofluorescence ratio (red; F/F0) measured simultaneously with external calcium present in the physiologic bathing solution (n = 5–7). Acetylcholine (10 μmol/L ACh) was present briefly prior to palmitoleic acid in some experiments. NB, The sharp decrease in Mg-Fluo-4 fluorescence seen at the end of the trace in (A) at approximately 1100 seconds was due to cell lysis and subsequent loss of the indicator from the cell. Light-transmitted and Mg Fluo-4 fluorescence images are also included to show the deleterious effects of prolonged exposure to palmitoleic acid ethyl ester (100 μmol/L) and palmitoleic acid (50 μmol/L) on doublets of acinar cells. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 5 Representative traces showing the effects of (A) 100 μmol/L palmitoleic acid ethyl ester (POAEE in the presence of 850 mmol/L ethanol, n = 10) and (B) palmitoleic acid (50 μmol/L POA, n = 15) on [Ca2+]ER (blue; Mg Fluo-4 fluorescence ratio F/F0) and NADH autofluorescence ratio (red; F/F0) measured simultaneously in Mg Fluo-4-loaded acinar cells under calcium-free (1 mmol/L EGTA) conditions. (C) Typical images from a triplet of isolated acinar cells showing the time-dependent reduction of [Ca2+]ER by 50 μmol/L palmitoleic acid under calcium-free conditions (1 mmol/L EGTA), indicated by a decrease of Mg Fluo-4 fluorescence predominantly in the basolateral part of the cells, which is dominated by the endoplasmic reticulum. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 6 (A) Typical effect of palmitoleic acid (100 μmol/L POA) on Mg Green fluorescence demonstrating increase of cytosolic free magnesium ion concentration [Mg2+]C; magnesium ions bind with much higher affinity to ATP than ADP, so, when the cellular ATP concentration is reduced, for example by preventing mitochondrial ATP production, magnesium ions are released into the cytosol. Subsequent addition of the protonophore CCCP, which depolarizes the inner mitochondrial membrane, caused no further change in [Mg2+]C, suggesting complete ATP depletion. (B) Control experiment showing the effect of CCCP alone, indicative of comparable ATP depletion to that induced by palmitoleic acid. Mg Green fluorescence is expressed as the fluorescence ratio F/F0. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 7 Effects of palmitoleic acid (POA) on the mitochondrial membrane potential (green; TMRM fluorescence ratio F/F0) and [Ca2+]C (blue; Fluo 4 fluorescence ratio F/F0) or NADH autofluorescence ratio (red; F/F0) measured simultaneously in dual-loaded cells. (A) Characteristic effect of palmitoleic acid (50 μmol/L POA) showing mitochondrial membrane depolarization with a global, sustained elevation of [Ca2+]C (9 of 9 cells). (B) Light-transmitted, NADH autofluorescent, TMRM fluorescent, and superimposed NADH and TMRM fluorescent images with a plot of typical changes demonstrating effects of palmitoleic acid (100 μmol/L POA). Note the precise perigranular correspondence of the TMRM and NADH signals used to detect changes in mitochondrial membrane potential and energetics. Following BAPTA pretreatment, there is no change in mitochondrial membrane potential, but NADH fluorescence still falls, although not as completely as in Figures 4 and 5. (C) Effects of low-dose palmitoleic acid (POA 10 μmol/L), showing well-maintained mitochondrial membrane potential during development of a modest, global, sustained elevation of [Ca2+]C (cf scale for Fluo 4 in (A) and rises in [Ca2+]C). The protonophore CCCP (10 μmol/L) was added after palmitoleic acid to induce complete mitochondrial depolarization (n = 16). Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 8 (A) Representative traces showing the oscillatory elevations of [Ca2+]C in patched (whole-cell recording configuration with direct access from pipette interior to cytosol) cells (ATP in internal pipette solution) induced by extracellular palmitoleic acid ethyl ester (100 μmol/L POAEE; 10 of 11 cells), compared with the predominantly sustained response observed in nonpatched cells (6 of 9 cells showed only a sustained plateau increase in [Ca2+]C, whereas the 3 remaining cells demonstrated oscillatory signals superimposed on this plateau elevation). (B) Typical recordings showing the lack of effect of palmitoleic acid (100 μmol/L POA) on [Ca2+]C in patched acinar cells with pipette delivery of ATP (8 out of 11 cells), compared with nonpatched cells, which showed a characteristic sustained elevation of [Ca2+]C (29 of 29 cells). [Ca2+]C is measured by the Fluo 4 fluorescence ratio F/F0. Inset shows recorded cells with patch pipette applied to 1 cell of isolated doublet and separate isolated cell (“nonpatched”) some distance away. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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