1. Volume 137, Issue 3, Pages 955-964.e2 (September 2009)
Ca2+-Dependent K+ Efflux Regulates Deoxycholate-Induced Apoptosis of BHK-21 and Caco-2 Cells 
Andrea Gerbino, Marianna Ranieri, Stefania Lupo, Rosa Caroppo, Lucantonio Debellis, Isabella Maiellaro, Mariano F. Caratozzolo, Francesco Lopez, Matilde Colella 
Gastroenterology 
Volume 137, Issue 3, Pages e2 (September 2009)
DOI: /j.gastro
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2. Figure 1
Cell Vm measurements. (A) Representative trace recording of Vm of a BHK-21 cell. Arrow A indicates that the microelectrode is advanced from the perfusion bath into the cell. Arrow B indicates that the microelectrode is removed from the cell. High K+ Ringer's solution was used to assess the sensitivity of the microelectrode. (B and C) Frequency distribution of Vm values of (B) BHK-21 and (C) Caco-2 cells.
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3. Figure 2
Effects of DC on BHK-21 and Caco-2 Vm. DC induced a dose-dependent increase in (A and B) BHK-21 and (C and D) Caco-2 Vm. (B) BHK-21 Vm increased by −6.0 ± 0.7 mV (P < .05), −22.1 ± 1.1 mV (P < .001), and −25.0 ± 0.3 mV (P < .001) in response to 100, 250, and 400 μmol/L DC, respectively (n = 4). (D) Caco-2 Vm increased by −2.8 ± 0.1 mV, −5.9 ± 0.3 mV (P < .01), and −8.0 ± 0.7 mV (P < .05) in response to 100, 250, and 400 μmol/L DC, respectively (n = 4).
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4. Figure 3
Differential effects of Ca2+-elevating stimuli in BHK-21 cells. (A) Effect of Ca2+-mediated agonists on Vm. ATP and bradykinin (BK) caused a hyperpolarization similar to that elicited by DC. (B) Effect of store emptying on DC- and ATP/BK-induced membrane hyperpolarization. After prolonged treatment with CPA, the ATP/BK-induced hyperpolarization was suppressed while DC effect was unaltered. (C) Ionomycin induced a hyperpolarization comparable to the one elicited by DC. (D) Summary of kinetic parameters. Values ± SEM.
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5. Figure 4
DC-induced hyperpolarization is dependent on external Ca2+. (A) DC-induced hyperpolarization decreased by ∼80% after removal of Ca2+ and addition of EGTA. (B) ATP/bradykinin (BK)-induced hyperpolarization was reduced by ∼15% after Ca2+ removal. (C) Extracellular and intracellular Ca2+ buffering completely prevented membrane hyperpolarization.
Gastroenterology  , e2DOI: ( /j.gastro )
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6. Figure 5
Effect of Ca2+-dependent K+ channel blockers on cell membrane hyperpolarization induced by DC and ATP/bradykinin (BK). (A) The upper panel indicates that apamin (Ap) and charybdotoxin (Cbtx) suppressed DC-induced BHK-21 hyperpolarization. The bar graphs indicate the effect of Ap and Cbtx on DC- and agonist-induced membrane hyperpolarization. **P < .01 vs DC; •••P < .001 vs ATP/BK, paired data. (B) The DC-induced effect in Caco-2 cells was reduced from −6.9 ± 0.2 to −2.5 ± 0.9 mV (n = 4, P < .01, paired data). (C and D) Summary of data. In both cell types, the inhibitory effect of Ap and Cbtx applied together (BHK-21, n = 5; Caco-2, n = 7) was larger than when applied separately (BHK-21, n = 4 for Ap and n = 4 for Cbtx; Caco-2, n = 6 for Ap and n = 6 for Cbtx). ***P < .001 vs DC (BHK-21, n = 13; Caco-2, n = 19), °P < .05 vs DC + Ap + Cbtx, °°P < .01 vs DC + Ap + Cbtx.
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7. Figure 6
Measurement of extracellular K+ with ion-selective microelectrodes. (A) Representative trace recording in a cluster of BHK-21 cells. Top and bottom traces are recordings of reference and K+-sensitive barrel, respectively. The microelectrode was first calibrated by flushing the chamber with Ringer's solutions containing [K+] between 0.5 and 10.0 mmol/L. Afterward, the microelectrode tip was advanced into a cell and then into the extracellular space. (B) DC elicited an increase in [K+]e that was reduced (by 75%) after pretreatment with apamin (Ap) and charybdotoxin (Cbtx). (C and D) Summary of data.
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8. Figure 7
DC-induced apoptosis is strongly attenuated by pretreatment with apamin (Ap) and charybdotoxin (Cbtx). The effect of Ap and Cbtx on cell apoptosis induced in BHK-21 and Caco-2 cells exposed to DC (4 hours, 250 μmol/L and 400 μmol/L, respectively) was evaluated by (A and B) Hoechst staining of apoptotic nuclei (n = 9), (C and D) caspase-3/7 assay (n = 3), and (E and F) propidium iodide staining (n = 3). *P < .05, **P < .01, ***P < .01 vs control; •P < .05, ••P < .01, •••P < .01 vs control.
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9. Supplementary Figure 1
Effects of taurodeoxycholate (TDC) and taurochenodeoxycholate (TCDC) on BHK-21 and Caco-2 calcium signaling and membrane potential. Perfusion of cells with Ringer's solution containing either (A and B) TDC or (C and D) TCDC induced a dose-dependent increase in intracellular [Ca2+] as measured by Fura-2 in both cell lines. Increases in cytosolic calcium concentration were detected with concentrations of TDC and TCDC as low as 250 μmol/L for BHK-21 cells and 500 μmol/L for Caco-2 cells, respectively. At lower concentrations, both bile acids were ineffective. (E) BHK-21 Vm, expressed as ΔVm, hyperpolarized by −7.5 ± 1.7 mV (n = 4, P < .05) and −17.8 ± 1.7 mV (n = 4, P < .01) in response to 250 and 500 μmol/L TDC, respectively. Also, TCDC hyperpolarized the membrane potential by −8.5 ± 0.5 mV (n = 4, P < .05) and −13.8 ± 0.7 mV (n = 4, P < .05) in response to 250 and 500 μmol/L TCDC, respectively. (F) Caco-2 membrane potential hyperpolarized by −4.8 ± 0.5 mV (n = 4, P < .05) and −8.6 ± 0.8 mV (n = 4, P < .01) in response to 500 μmol/L and 1 mmol/L TDC, respectively. TCDC hyperpolarized the membrane potential by −3.1 ± 1.0 mV (n = 4, P < .05) and −7.7 ± 0.8 mV (n = 4, P < .01) in response to 500 μmol/L and 1 mmol/L TDC, respectively. °°P < .01 vs TDC 250 μmol/L; **P < .01 and ***P < .001 vs TCDC 250 μmol/L.
Gastroenterology  , e2DOI: ( /j.gastro )
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