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1 Supporting Information Gerasimenko et al..73/pnas.39 SI Materials and Methods Reagents used in this study include Fluo-4/Fura- (Invitrogen), thapsigargin (albiochem), collagenase (Worthington), palmitoleic acid ethyl ester (POAEE) (ayman hemical), and from GSK (,6-difluoro-N-(-(4-hydroxy--(trifluoromethyl)benzyl)-H-pyrazol-3-yl)benzamide), compound 36 from patent WO/89 A (). All other chemicals were purchased from Sigma. Pancreatic acinar cells were isolated from mice as described previously (). The extracellular solutioncontained:4mmnal,4.7mmkl,mmhepes (KOH), mm Mgl,mMglucose,pH7.,andal ( mm as described in the text). hanges in the cytoplasmic a + concentration ([a + ] i ) were monitored by Fluo-4 or Fura- fluorescence measurements and necrosis assessed by propidium iodide (PI) uptake (3). We assessed protease activation with the generic fluorescent substrate bis-l-aspartic acid amide rhodamine (D-R) and trypsinogen activation with the trypsin fluorescent substrate Rhodamine, bis-(z-l-isoleucyl-l-prolyl-l-arginine amide), dihydrochloride (ZiPAR) (4, 5). Fluorescence signals were plotted as F/F (F is the initial level of fluorescence). The polynomial curve fit method was used to estimate I 5. Statistical significance and P values were calculated using t test or one-way ANOVA. Data presented as mean ± SEM. In Na + -free solution, Na + was replaced by N- methyl-d-glucamine (NMDG + ). During standard patch clamp whole-cell recordings, the pipette solution contained mm Kl, mm Mgl, mm MgATP, 5 mm Hepes, mm APTA and mm al. Patch pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus). The pipettes had a resistance of 3 5 megaohms when filled with an intracellular solution (containing mm Kl). Whole cell currents were sampled at KHz using an EPS-8 amplifier and Pulse software (HEKA) or Multilamp7 amplifier and plamp software (Molecular Devices). In the standard protocol the membrane voltage was clamped at 5 mv. For investigations of current voltage relationships voltage ramps were applied from to + 4 mv (the slope was 4 mv/s). hanges in [a + ] in the intracellular stores were assessed by fluorescence measurements in cells loaded with Fluo-5N AM (Invitrogen) simultaneously with measurements of whole cell currents. The protocol for primary hepatocyte isolation was as described in ref. 6. All animal work was carried out in accordance with the UK Animals (Scientific Procedures) Act (986) and approved by the Ethical Review ommittee of ardiff University. riefly, the liver was perfused with buffer I without a + : 4 mm Nal; 4.7 mm Kl; mm Hepes; mm D-glucose; μm EGTA; (ph 7.4); the rate of perfusion was 5 ml/min at 37. Then the liver was perfused with buffer I in the presence of.3 mm al and collagenase I (Sigma) for min at 37. Dissociated hepatocytes were centrifuged at 5 g for min and transferred into buffer I containing mm Mgl and.3 mm al, ph 7.4. AR4J cells (EA, 9368) were maintained in RPMI 64 as described previously (3, 7). For intracellular calcium imaging isolated hepatocytes were loaded with fluo-4 in AM form at room temperature for 45 min.. Derler I, et al. (3) The action of selective RA channel blockers is affected by the Orai pore geometry. ell alcium 53(): Gerasimenko JV, et al. () Menadione-induced apoptosis: Roles of cytosolic a( + ) elevations and the mitochondrial permeability transition pore. J ell Sci 5(Pt 3): Ferdek PE, et al. () A novel role for cl- in regulation of cellular calcium extrusion. urr iol (3): Raraty M, et al. () alcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells. Proc Natl Acad Sci USA 97(4): Gerasimenko JV, et al. (9) Pancreatic protease activation by alcohol metabolite depends on a + release via acid store IP 3 receptors. Proc Natl Acad Sci USA 6(6): Li W-, Ralphs KL, Tosh D () Isolation and culture of adult mouse hepatocytes. Methods Mol iol 633: Gallacher DV, et al. (99) Substance P and bombesin elevate cytosolic a + by different molecular mechanisms in a rat pancreatic acinar cell line. J Physiol 46:93 7. Gerasimenko et al. of7
2 µm nm Ah µm 5 nm Ah µm 5 pm K-8. Ratio s. Ratio s D nm Ah E nm Ah F. Ratio G Pre-incubation: M. Ratio 5 s 5 s 4 3 M Ah control 7975A pre-treated min H Area under the curve, Fluo-4 (a.u.) 4 3. Ratio Ah s Ah * Ah Ah -3s 3-6s I µm TG µm TG + µm J K mm a + mm a +.3 P <... Ratio s. Ratio s. trl Fig. S. does not affect [a + ] i spike generation induced by low concentrations of Ah or K. (A ) Acute application of μm does not interrupt a + oscillations induced by low (quasiphysiological) concentrations of Ah [ nm (A) and 5 nm ()] or a low physiological concentration of K [5 pm ()]. (D and E) Preincubation with μm does not reduce a + oscillations induced by a low concentration ( nm) of Ah (E, n = 9) compared with control (D, n = 3). (F) omparison of the areas under [a + ] i changes induced by nm Ah in the presence or absence of μm. Data represent mean values ± SEM. P >. (, nonsignificant difference). Experiments were performed in standard buffer containing mm al.(g) GSK- 7975A inhibits the sustained phase of the [a + ] i elevation induced by a high concentration of Ah. Average traces of [a + ] i changes in response to μm Ah in control cells (blue trace, n = 4) and in cells preincubated with μm for min (red trace, n = ). ars represent ±SEM. (H) Quantitative analysis of experiments in G. Averaging [a + ] i elevations induced by Ah recorded during the first 3 s ( 3 s) shows similar values (area under the curve, ± 8.5 and 335. ± 8.4 au, P =.97). However, very different values were obtained during the last 3 s (3 6 s) in cells pretreated with ( μm) (red bars) and control cells (blue bars) (48.3 ± 5. and 8.7 ± 6.3 au, *P < 6 ). (I K) Average traces of [a + ] i rise (together with SEM) due to storeoperated a + influx in the presence (J, n = 38) or absence (I, n = 36) of μm. (K) omparison of the amplitudes of the increases in [a + ] i in response to admission of a + to the external solution (from I and J) in the presence and absence of μm, P <.. Gerasimenko et al. of7
3 . -AP M Ra o, Fura % necrotic cells, (PI) p=.5 ntrl -AP 3 4 -AP Fig. S. locker of RA channels -AP induces rise in [a + ] i and necrosis in pancreatic acinar cells, which cannot be inhibited by. (A) Averaged traces (together with error bars) of rise in [a + ] i induced by μm -AP (n = 4). () Preincubation of pancreatic acinar cells with μm -AP induces substantial necrosis (35 ± %, P <.5, n = 4 series, with >8 cells in each). does not induce significant necrosis by itself compared with control (P >.) and does not protect against the necrosis induced by -AP (P >.8). Gerasimenko et al. 3of7
4 3 P >.8 Area under the curve, au 35 3, POAEE, mm a POAEE + % necrotic cells, (PI) control mm a 3 POAEE 4 5 mm a POAEE POAEE Fig. S3. Elevating the external a + concentration from mm to mm counteracts the protective effect of against the POAEE-induced [a + ] i rise and necrosis. (A)omparisonofthe areas under the curve of the POAEE ( μm)-induced [a + ] i responses with the normal mm external [a + ](n =, typical example shown in Fig. 4A) and the responses induced by μmpoaeeinthepresenceofmmexternala + and ( μm) (cells pretreated with GSK- 7975A for min, n = 6). No significant difference was found (P >.8). () POAEE(μM)-induced necrosis ( % necrotic cells) was dramatically reduced in cells treated with μm for min (. ±.8%, P <.3, compared with POAEE treatment alone). However, if after treatment with ( μm, min) POAEE ( μm) was applied in the presence of mm al in the external medium, the level of necrosis was not significantly different (P >.4) from POAEE-induced necrosis at an external a + concentration of mm and without (6 ±.3%). In a control series of experiments (no POAEE treatment, mm al ), the level of necrosis was 8.3 ±.5%. The level of necrosis in the presence of mm al increased to.7 ±.3% (P <., compared with control). n = 3 for all series of experiments with number of tested cells in each group >35. Gerasimenko et al. 4of7
5 Ratio, Fura-.5.5 M Tg M Ah ontrol pre-treated hour Ratio, Fura ontrol ns Ratio, Fura- D Ah µm p =.4 control a + a + control no a + Ratio, Fura-.3. p <.. control a + a + control no a + Fig. S4. does not significantly affect the amount of a + that can be released from the ER in resting pancreatic acinar cells and only has a relatively minor effect on a + store reloading. (A) ells were treated with μm for h (red averaged trace (together with SEM), n = ) and compared with control [blue averaged trace (together with SEM), n = 4], then external a + was removed (nominally a + -free solution) and cells were stimulated with μm Ah together with μm thapsigargin for maximal response. () omparison of the maximal amplitudes of the rises in [a + ] i shown in A. ontrol (n = 4) and (n = ) were not significantly different (P >.5). () ells were stimulated with a high concentration of Ah ( μm) for min and then, after a long period of recovery, were stimulated for a second time with the same Ah concentration. The traces show the averaged second responses to Ah (together with SEM) in experiments carried out under three different conditions: standard external a + concentration ( mm) without any blocker (blue trace, n = 8), in the presence of μm and mm external a + (red trace, n = ) and nominally a + -free external solution (green trace, n = ). (D) omparison of the amplitudes of the second responses to Ah in the traces shown in. Averaged amplitude of the responses in control mm external a + was.7 ±.5, n = 8. Averaged amplitude of the responses in the presence of was slightly, but significantly lower (. ±., n =, P =.4) than in control. The averaged amplitude of the response in the absence of external a + was substantially lower (.89 ±.8, n =, P <.) than in the presence of and a +. Gerasimenko et al. 5of7
6 .5 TG 5 mm a TG 5 mm a % p=.3 M Tg 5 mm a M Tg M ' 5 mm a Fig. S5. Standard store-operated a + entry protocol (as in Fig. ), but this time carried out in freshly isolated hepatocytes. only has a relatively small inhibitory effect on store-operated a + entry. (A) ontrol representative trace of [a + ] i changes evoked by thapsigargin ( μm) in the absence of external a + and then following introduction of the external solution containing 5 mm a +.() Same protocol as in A, but this time in presence of μm GSK- 7975A. Store-operated a + entry has been reduced, but only to a limited degree. () omparison of the responses induced by a + entry in control (95.6 ± 5 au, n = ) and in the presence of μm (56. ± 8.4 au, n = ). has caused a relatively minor reduction in the amplitude of the a + entry response (P <.3). Gerasimenko et al. 6of7
7 .6 M PA 5 mm a M PA + M 5 mm a P >. ontrol Fig. S6. does not affect store-operated a + entry in rat pancreatic tumor cell line (AR4J). (A) ontrol averaged traces (together with error bars) of changes in [a + ] i induced in AR4J cells first by exposure to μm cyclopiazonic acid (PA) in the absence of external a + and thereafter by introducing an external solution containing 5 mm a + (n = 3). () Same protocol as in A, but this time in the presence of μm (n = 7). The rise in [a + ] i following admission of the external solution containing 5 mm a + is very similar to that observed in the absence of. () omparison of the amplitudes of the rise in [a + ] i induced by store-operated a + entry in control (A) and in the presence of μm (). There is no significant difference (P >.). Gerasimenko et al. 7of7
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