Male Sprague-Dawley rats weighing 180-200 g were obtained from the Beijing Laboratory Animal Research Center (Beijing, China). RPMI 1640 medium, penicillin, streptomycin, Fluo-4-acetoxymethyl (Fluo-4/AM), and poly-D-lysine were purchased from Invitrogen (United States). Collagenase IV, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), adenosine-triphosphate (ATP), CsOH, Taurocholic acid sodium salt, and SKF-96365 were obtained from Sigma (United States). Ethylene glycol tetraacetic acid (EGTA) was obtained from Solarbio (Beijing, China). Vasopressin, noradrenalin, thapsigargin (TG), and 2-aminoethoxydiphenyl borate (2-APB) were obtained from Merck KcaA (Darmstadt, Germany). Methanol and acetonitrile were of chromatographic grade; all other reagents were of reagent grade.

Rat hepatocytes were enzymatically isolated using a modification of the method originally reported by Seglen[23]. Briefly, rats were anesthetized with chloral hydrate (320 mg/kg body weight) and heparinized (1.5 U/g body weight) via intraperitoneal injection. A midline laparotomy was performed, and portal vein and the inferior vena cava were cannulated. The liver was initially perfused for 10 min at a constant flow rate of 25-30 mL/min with a modified oxygenated Ca²⁺-/Mg²⁺-free Hanks solution containing (in mmol/L) NaCl, 120; KCl, 5; Na₂HPO₄, 0.2; KH₂PO₄, 0.2; NaHCO₃, 25; EGTA, 0.5; and glucose, 5 (pH 7.4), followed by perfusion with Type IV collagenase (0.2 g/L) in RPMI 1640 medium for 10 min. The solution was gassed with 100% O₂ and warmed to 37  °C. After the perfusions, the three large cephalad lobes of the liver were excised and minced in a Ca²⁺-/Mg²⁺-free Hanks solution at 0  °C, before being filtered through a 74-μm nylon mesh and washed three times by centrifugation at 50 ×g for 2 min. The isolated hepatocytes (1 × 10⁵/mL; 85%-95% viability assessed by trypan blue exclusion) were incubated in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37  °C in a 95% air-5% CO₂ incubator for 1 h.

The rat model of HIRI was established according to our previously reported procedure[10]. Briefly, under anesthesia and heparinization (as above), a midline laparotomy was performed in each rat, and an atraumatic clip was used to interrupt the arterial and portal venous blood supply to the three cephalad lobes. After 20 min of hepatic ischemia, the clip was removed, and the liver was reperfused for a further 40 min. Sham-operated control animals were treated in an identical manner, with the omission of vascular occlusion.

Isolated hepatocytes were seeded in 35-mm glass-bottomed dishes (MatTek, Ashland, MA, United States), pretreated with poly-D-lysine (500 μg/mL in borate buffer) for 2 h, and loaded with 6.7 μmol/L fluo-4/AM (Figure 1A) in a recording solution containing (in mmol/L) NaCl, 116; KCl, 5.4; CaCl₂, 1.8; MgCl₂, 0.8; glucose, 5.05; and HEPES, 10 (pH 7.4) for 30 min at 37  °C, followed by three washes in PBS and a 15-min incubation to allow de-esterification of fluo-4/AM before imaging. A Lambda DG-4 high-speed wavelength switcher (Sutter Instruments, Novato, CA, United States) was used for fluo-4 excitation at 480 nm, and a cooled charge-coupled device (CCD) camera (CoolSnap FX, Roper Scientific, Princeton, NJ, United States) was used for image acquisition. Meta Fluor imaging software (Universal Imaging, Downingtown, PA, United States) was used for hardware control, image acquisition and image analysis. Typically, time-lapse recording of Ca²⁺ signals in hepatocytes was performed for a 2-min control period before and for a 10-min period after the application of the different agonists. The CCD camera was used with a sampling rate of one frame per 2 s, a typical exposure time of 50-350 ms, and a 4 × 4 binning. Quantitative measurements of changes of [Ca²⁺]_i were obtained by subtracting the average background intensity (measured in cell-free regions) from the average cellular fluo-4 fluorescence intensity values. Changes in [Ca²⁺]_i for each hepatocyte were then represented by the changes in relative fluo-4 fluorescence (∆F/F0), where F0 was the baseline intensity obtained from the 2-min control period.

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Figure 1 Agonist-induced Ca²⁺ oscillations are inhibited by blocking Ca²⁺ entry in freshly isolated rat hepatocytes. Traces show Ca²⁺ oscillations in freshly isolated rat hepatocytes, with the abscissa axis as time (min), the vertical axis as Fluo-4 fluorescence intensity (∆F/F0), which demonstrates the changes of [Ca²⁺]_i. The number of hepatocytes measured is indicated by “n”. A: The fluorescence image (left) and microscopic image (right) of freshly isolated hepatocytes loaded with fluo-4/acetoxymethyl. Ca²⁺ oscillations were initiated by 100 nmol/L noradrenaline in the presence (B, n = 27) or absence (C, n = 16) of extracellular Ca²⁺. Ca²⁺ oscillations induced by 0.5 nmol/L adenosine-triphosphate were inhibited by 20 μmol/L 2-APB in the presence (D, n = 14) or absence (E, n = 11) of extracellular Ca²⁺. 2-APB: 2-aminoethoxydiphenyl borate.

Measurements of net Ca²⁺ influx were performed using the non-invasive micro-test technique (NMT) system (BIO-IM, YoungerUnited States, Amherst, MA, United States) using our previously reported methods[24]. Briefly, isolated hepatocytes plated in a 35-mm dish (Figure 3A) were washed three times with a measuring solution containing (in mmol/L) NaCl, 136; KCl, 2.7; CaCl₂, 0.2; KH₂PO₄, 1.5; Na₂HPO₄, 8; and glucose, 5.05 (pH 7.4). The electrode was controlled to move with an excursion of 10 μm at a programmable frequency in the range of 0.3-0.5 Hz, chosen to minimize mixing of the bathing saline. To construct the microelectrodes, borosilicate micropipettes (2-4 μm aperture, XYPG120-2, Xuyue Sci. and Tech. Co., Ltd., Beijing, 100080, China) were silanized with tributylchlorosilane, and the tips were filled with calcium ionophore I-cocktail A (Sigma-Aldrich, St Louis, MO, United States). An Ag/AgCl wire electrode holder (XYEH01-1) was inserted in the back of the electrode to make electrical contact with the electrolyte solution. Only electrodes with Nernstian slopes between 25 and 29 mV/decade were used. Ca²⁺ fluxes were calculated by Fick’s law of diffusion: J₀ = -[D × (dC/dX)], where J₀ represents the net Ca²⁺ flux (in μmol per cm per s), D is the self-diffusion coefficient for Ca²⁺ (in cm²/s), dC is the difference value of Ca²⁺ concentrations between the two positions, and dX is the 10 μm excursion over which the electrode moved in our experiments. Data and image acquisition, preliminary processing, control of the three-dimensional electrode positioner, and stepper-motor-controlled fine focus of the microscope stage were performed with imFlux^® software.

Whole-cell patch-clamp recording was performed at room temperature (22-25  °C) using a computer-based patch-clamp amplifier (EPC-10, HEKA Electronics, Lambrecht/Pfalz, Germany) and PatchMaster software (HEKA Electronics, Lambrecht/Pfalz, Germany). The isolated hepatocytes were plated in 35-mm dishes and washed with a standard external solution containing (in mmol/L) NaCl, 140; CsCl, 4; MgCl₂, 2; CaCl₂, 10; glucose, 10; and HEPES, 10 (pH 7.4; adjusted with NaOH). In experiments investigating Ba²⁺ current in rat hepatocytes, the NaCl, MgCl₂ and CaCl₂ in the external solution were replaced with 30 mmol/L of NaCl and 100 mmol/L of BaCl₂. An automatic micropipette puller (Model P-97, Sutter Instruments, Novato, CA, United States) was used to pull the electrodes from the borosilicate glass. The pipette resistance was between 3 and 5 MΩ when filled with the pipette solution containing (in mmol/L) CsCl, 15; Cs glutamate, 135; EGTA, 10; and HEPES, 10 (pH 7.2; adjusted with CsOH). In all whole-cell patch-clamp recording experiments, recording started when the series resistance dropped to below 20 MΩ. After achieving the whole-cell configuration, voltage ramps of 50 ms duration, spanning a range from -100 mV to +100 mV, were immediately delivered from a holding potential of 0 mV every 2 s. Acquired currents were filtered at 2.9 kHz and sampled at 20 kHz. Capacitative currents were determined and compensated automatically by the EPC-10 amplifier. The voltages were corrected for a liquid-junction potential of 17 mV (estimated by JPCalc). The maximum SOC current (I_SOC) at -100 mV was applied for statistical analysis.

High-performance liquid chromatography (HPLC) analysis was performed using LC-10A apparatus (Shimizu, Japan) with an Agilent Extend C18 column (416 × 150 mm, 5 μm). To create a fine chromatographic separation of taurocholate, a 60:20 (v/v) mixture of mobile phases A (methanol and acetonitrile 1:1) and B (5 mmol/L KH₂PO₄; pH 3.0) was employed. A constant flow rate of 0.8 mL/min was used for the quantitative determination of the standard solutions and test samples, with a column temperature of 30  °C, sample size of 10 μL, and detection wavelength of 210 nm. Freshly isolated hepatocytes were plated at 5 × 10⁵/mL in 35-mm dishes at 37  °C in a 95% air-5% CO₂ incubator and treated with 100 μmol/L 2-APB, 100 μmol/L La³⁺ or 10 μmol/L SKF-96365 for 12 h. The culture solution was collected and centrifuged at 500 rpm for 3 min. The supernatant was stored frozen at -20  °C. Supernatant (0.5 mL) was mixed intensively with 0.5 mL of absolute ethyl alcohol, incubated in a 60  °C water bath for 3 min, then centrifuged at 3000 rpm for 10 min. Supernatant (0.3 mL) was run through Sep-PAK columns, washed with 10 mL of pure water, and eluted with 3 mL of methanol. This eluent was placed in a water bath (70  °C), blown dry with nitrogen, and mixed with 3 mL of mobile phase.

The method of Mosmann[25] for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used with modifications for functional studies on cell survival/injury. Freshly isolated rat hepatocytes in 1640 medium were plated at 5 × 10⁴ cells per well in 96-well microtiter plates. After 3 h, various SOC antagonists were added, or 0.025% (v/v) dimethylsulfoxide (DMSO) vehicle was used as control. Cells were cultured after drug exposure for 12 h at 37  °C in a 95% air-5% CO₂ incubator, which is sufficient time for evidence of drug-induced cell death, increased cell survival or dampened cell injury to become apparent, as quantified by the generation of the formazan product from the MTT substrate. The number of hepatocytes per microtiter well was proportional to the absorbance of the solubilized formazan. After addition of MTT (5 mg/mL, 20 μL) and incubation for 4 h, the medium was discarded. MTT formazan crystals were then resolubilized with 150 μL DMSO per well and mixed on a microshaker for 10 min. The plate was then read immediately on a scanning multiwell spectrophotometer (Termo Multiscan MK3, Finland) at 490 nm.

IGOR Pro 5.01 software (Wavemetrics, Portland, OR, United States) was used to conduct an analysis of whole-cell patch-clamp recording data. All current traces were corrected for leak currents. Mageflux (http://www.xuyue.net/mageflux) was used to process the data of the NMT. All results are expressed as mean ± SD. Statistical significance of differences between test samples and controls was determined using the Student’s t-test. P values less than 0.05 were considered statistically significant differences.

Using calcium imaging, we investigated the relationship between Ca²⁺ entry and Ca²⁺ oscillations in hepatocytes under physiological conditions, with Ca²⁺ oscillations induced by noradrenaline in freshly isolated hepatocytes (Figure 1B). We found that Ca²⁺ oscillations were dependent on Ca²⁺ entry through the plasma membrane (Figure 1C). Ca²⁺ oscillations induced by ATP (activator of phospholipase C) were inhibited by 2-APB[26] (an inhibitor of SOCs, Figure 1D), which inhibited entry of extracellular Ca²⁺ but not release of Ca²⁺ from ER (Figure 1E).

TG, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺ pump (SERCA)[27], was used to passively eliminate hepatocyte ER calcium stores, evoking two peaks (one for the release of Ca²⁺ from the ER and the other for the subsequent Ca²⁺ entry through the plasma membrane; Figure 2A). Ca²⁺ oscillations in freshly isolated rat hepatocytes could be inhibited by 2-APB (Figures 1D and E). Moreover, Ca²⁺ oscillations could also be inhibited by the SOC inhibitor, SKF-96365[28,29] (Figure 2B), the phospholipase C (PLC) inhibitor, U73122[27], and the phospholipase A2 inhibitor, tetrandrine[30,31] (Figures 2C and D).

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Figure 2 Store-operated calcium channel blockers inhibit Ca²⁺ oscillations of freshly isolated hepatocytes. A: Ca²⁺ stores were depleted with 4 μmol/L thapsigargin (TG), causing two peaks, the first being Ca²⁺ released from endoplasmic reticulum (ER), and the second peak being Ca²⁺ entry through the plasma membrane (n = 16); B: 10 μmol/L SKF-96365 inhibited Ca²⁺ oscillations (n = 12); C: 25 μmol/L U73122 inhibited Ca²⁺ oscillations (n = 10); D: 100 μmol/L tetrandrine inhibited Ca²⁺ oscillations (n = 10).

Using NMT, maximum net Ca²⁺ flux values of 1163 ± 279 nmol cm^-2 s^-1 were recorded in freshly isolated hepatocytes (n = 10; Figure 3B). Nifedipine (an inhibitor of VDCCs; 10 μmoL/L) had no effect on net Ca²⁺ fluxes (1108 ± 298 nmol cm^-2 s^-1) (n = 15; Figure 3C). The SOC blockers, SKF-96365, La³⁺ and 2-APB (all at 100 μmol/L), reduced net Ca²⁺ flux to 738 ± 195, 452 ± 136, and 84 ± 37 nmol cm^-2 s^-1, respectively (Figures 3D, E and F). These effects of SOC blockers on net Ca²⁺ fluxes were statistically significant (Figure 3G).

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Figure 3 Ca²⁺ flux in freshly isolated hepatocytes. A: A screen-printed picture of a cell measured by NMT; B-F: Net Ca²⁺ fluxes of a freshly isolated rat hepatocyte (B) and repeated in the presence of 10 μmol/L nifedipine (C), 100 μmol/L SKF-96365 (D), 100 μmol/L La³⁺ (E), and 100 μmol/L 2-APB (F); G: Bar graph of the maximum net Ca²⁺ fluxes in five groups. ^aP < 0.01, control group vs SKF-96365, La³⁺ or 2-APB group. 2-APB: 2-aminoethoxydiphenyl borate.

IP₃ (10 μmol/L) and EGTA (10 mmol/L) induced currents in hepatocytes (Figures 4A and C), with a reverse potential of approximately +40 mV. The current-voltage relationship revealed that the currents were inwardly rectifying (Figures 4A, B and C). To further characterize the recorded currents, we substituted Ca²⁺ for Ba²⁺ in the external solution. The Ba²⁺ current (79 ± 7 pA) was significantly greater than the Ca²⁺ current (32 ± 1 pA, t = 5.683, P = 0.002), but much more transient, dropping to a lower level after less than 2 min (Figures 4D and E). The SOC inhibitor, 2-APB (100 mmol/L), inhibited the current induced by 10 mmol/L EGTA (Figure 4F). An inhibitor of non-selective cation channels, A9C (10 μmol/L), did not influence current magnitude (Figure 4G).

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Figure 4 Store-operated calcium currents in freshly isolated hepatocytes. A: I-V curves of I_SOC induced by 10 μmol/L IP₃ and 10 mmol/L ethylene glycol tetraacetic acid (EGTA) in hepatocytes, recorded with the voltage ramps from -100 mV to +100 mV, showing the beginning of whole-cell patch-clamp recording (red) and reaching peak current (green) (n = 14); B: Time course of I_SOC development, taken at holding potentials of -100 mV and +100 mV for inward and outward currents, respectively; C: I-V curves of I_SOC obtained by subtraction of baseline from peak current (the red and green sections of the trace, respectively, as described in A), at the time marked “▲” in B; D: Currents induced by 10 mmol/L EGTA following replacement of Ca²⁺ with Ba²⁺ in the external solution (n = 6); E: I-V curves at the three time points indicated by the arrows in panel D: 1, SOC was fully activated by 10 mmol/L EGTA in the pipette solution, 2, 3, The amplitude of I_SOC at -100 mV as a function of time when the external solution was replaced by a solution containing 100 mmol/L Ba²⁺ and no Ca²⁺ or Mg²⁺ for the period indicated in D (n = 6); F: Currents stimulated by 10 mmol/L EGTA. The green trace shows the instantaneous current density-voltage relationship in the range of -100 mV to +100 mV obtained in response to a voltage ramp at the time when inward current was fully developed. The black trace was obtained in response to the presence of 100 μmol/L 2-APB (n = 6); G: Effect of 10 μmol/L A9C on current induced by 10 mmol/L EGTA. The green trace shows the instantaneous current density-voltage relationship in the range of -100 mV to 100 mV obtained in response to a voltage ramp at the time when inward current is fully developed. The black trace was obtained in the presence of 10 μmol/L A9C (n = 5).

Hepatocellular Ca²⁺ overload is thought to play a crucial role in HIRI. To explore the effects of SOCs on the pathogenesis of HIRI, SOC currents induced by 10 mmol/L EGTA in hepatocytes were recorded in the HIRI and control groups (Figures 5A, B and C). The results showed that the SOC currents were significantly increased (from 31.6 ± 2.7 pA in controls to 57.0 ± 7.5 pA in HIRI hepatocytes; t = 2.682, P = 0.036; Figure 5D). In HIRI hepatocytes, the fully developed inward currents were rapidly reduced by 100 μmol/L La³⁺ (Figures 5E and F).

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Figure 5 Store-operated calcium channel participates in hepatocellular Ca²⁺ overload in HIRI. A: I-V curves of the I_SOC induced by 10 mmol/L ethylene glycol tetraacetic acid (EGTA) in HIRI hepatocytes, recorded with voltage ramps from -100 mV to +100 mV. Red trace shows the beginning of whole-cell patch-clamp recording, the green trace shows the peak current (n = 12); B: The time course for development of I_SOC in HIRI hepatocytes (n = 12); C: I-V curves of I_SOC obtained by subtraction of baseline from peak current (the red and green sections of the trace, respectively, as described in A) at the time marked “▲” in B; D: Bar graph of the mean current density of maximal I_SOC recorded at a holding potential of -100 mV in HIRI hepatocytes (n = 12) and controls (n = 24), ^bP < 0.01; E: I_SOC was recorded in HIRI hepatocytes before (green trace) or after (black trace) the application of 100 μmol/L La³⁺ (n = 7); F: The time course of I_SOC development in the presence of 100 μmol/L La³⁺ in HIRI hepatocytes (n = 7). HIRI: Hepatic ischemia-reperfusion injury.

To explore the influence of SOC blockers on cell survival, we performed an MTT assay. No significant differences (P > 0.05) in cell survival rate (compared with normal hepatocytes) were observed after exposure to the DMSO vehicle or either of the two SOC inhibitors (Figure 6A). In addition, there was a similar survival rate in HIRI hepatocytes compared with hepatocytes pretreated with 0.025% (v/v) DMSO or 100 μmol/L La³⁺. However, the average optical absorbance value in hepatocytes pretreated with 100 μmol/L 2-APB (0.79 ± 0.05) was higher than in untreated HIRI hepatocytes (0.69 ± 0.04), P = 0.027, t = 4.047 (Figure 6B). Compared with normal hepatocytes, HIRI hepatocytes incurred cell damage (P = 0.029, t = 2.882; Figure 6C).

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Figure 6 Effects of store-operated calcium channel blockers on cell survival or injuries of normal and hepatic ischemia-reperfusion injuried hepatocytes. A: MTT assay of normal hepatocytes alone or co-cultivated with 0.025% (v/v) DMSO, 100 μmol/L 2-APB, or 100 μmol/L La³⁺ (n = 4), the cell survival rate is not significantly different (P > 0.05); B: MTT assay of HIRI hepatocytes alone or cocultivated with 0.025% (v/v) DMSO, 100 μmol/L 2-APB or 100 μmol/L La³⁺ (n = 4), the average optical absorbance value in hepatocytes pretreated with 100 μmol/L 2-APB (0.79 ± 0.05) was higher than in untreated hepatocytes (0.69 ± 0.04), ^aP = 0.027; C: Compared with normal hepatocytes, HIRI hepatocytes incurred cell damage (n = 4), ^aP = 0.029. HIRI: Hepatic ischemia-reperfusion injury; 2-APB: 2-aminoethoxydiphenyl borate; DMSO: Dimethylsulfoxide; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

To explore the influence of SOCs on the secretory function of hepatocytes, taurocholate secretion by hepatocytes was investigated using HPLC analysis. Taurocholate concentration in the culture supernatant of normal hepatocytes was 38.58 ± 7.35 μg/mL (n = 6), notably higher than in hepatocytes treated with 100 μmol/L 2-APB (28.85 ± 8.18 μg/mL; P < 0.05, n = 6), 100 μmol/L La³⁺ (27.76 ± 9.86 μg/mL; P < 0.05, n = 6) or in HIRI hepatocytes (19.9 ± 3.8 μg/mL; P < 0.05, n = 6) (Figure 7). Interestingly, 100 μmol/L2-APB, 100 μmol/L La³⁺ and 10 μmol/L SKF-96365 could reverse the taurocholate level to 27.42 ± 4.74, 26.58 ± 6.67 and 25.52 ± 7.30 μg/mL, respectively, in HIRI hepatocytes, P < 0.05, n = 6.

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Figure 7 Taurocholate measurements in hepatic ischemia-reperfusion injuried hepatocytes. Bar graph of taurocholate secreted by hepatocytes is distinctly higher in the supernatant of cultured normal hepatocytes (38.58 ± 7.35 μg/mL, n = 6) than for cells after exposure to 100 μmol/L 2-APB (28.85 ± 8.18 μg/mL, ^aP < 0.05, n = 6), 100 μmol/L La³⁺ (27.76 ± 9.86 μg/mL, ^aP < 0.05, n = 6) or HIRI hepatocytes (19.92 ± 3.75 μg/mL, ^aP < 0.05, n = 6). Whereas 100 μmol/L 2-APB, 100 μmol/L La³⁺ and 10 μmol/L SKF-96365 could respectively reverse the taurocholate level to 27.42 ± 4.74, 26.58 ± 6.67 and 25.52 ± 7.30 μg/mL in HIRI hepatocytes, ^aP < 0.05, n = 6. HIRI: Hepatic ischemia-reperfusion injury; 2-APB: 2-aminoethoxydiphenyl borate.

Hepatic ischemia-reperfusion injury (HIRI) can occur in the liver in a wide variety of clinical and operative situations. The pathogenesis of HIRI is multifactorial, such as in hepatocellular Ca²⁺ overload. Variation of [Ca²⁺]_i has been shown to play a significant role. [Ca²⁺]_i may be increased by releasing Ca²⁺ from the endoplasmic reticulum (ER) or the sarcoplasmic reticulum (SR), or by stimulating Ca²⁺ entry from the extracellular space through calcium channels. The concept of a “store-operated calcium current” was proposed in 1986, and store-operated calcium channels (SOCs) may play an important role in HIRI.

SOCs have been verified as Ca²⁺ channels in that the amount of Ca²⁺ in the stores controls the extent of Ca²⁺ influx in non-excitable cells. However, the role of SOCs in rat hepatocytes has not been elucidated. In this study, the authors have further confirmed the important role of SOCs in rat hepatocytes and the pivotal role of SOCs in HIRI. SOC blockers assisted the recovery of secretory function in HIRI hepatocytes.

This is the first study that has used multiple experimental techniques to investigate the important role of SOCs in rat hepatocytes from various perspectives, particularly with regard to pathogenesis of HIRI on the cellular electrophysiological level and on the clinical research level.

Cytoplasmic Ca²⁺ overload could result in injury of liver cells. By understanding the role of SOCs in HIRI, this research will contribute to clarifying the exact mechanism of hepatocellular Ca²⁺ overload. This study might indicate a novel class of effective drugs targeted at Ca²⁺ channels in hepatocytes for the prevention or therapy of HIRI.

SOCs are a family of Ca²⁺-permeable ion channels expressed by most cells. The signal for the activation of SOCs is a decrease in the [Ca²⁺]_i in the ER Ca²⁺ store, and this is believed to be an essential and ubiquitous component of Ca²⁺-signaling pathways. Stimulation of a diverse range of plasma membrane receptors converges on and activates phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C and results in the generation of diacylglycerol and IP₃, which induces the subsequent activation of Ca²⁺ release from the ER via IP₃ receptors and Ca²⁺ influx across the plasma membrane. STIM1, identified as the ER Ca²⁺ sensor, and Orai1, as a pore-forming subunit of SOCs, have both been shown to be necessary for SOC function.

This is a well conducted study with a clear objective and the data reflects the quality of the work by these investigators. Significance of the data from the study complies with the background objectives. All sections including Materials and Methods, Results, Discussion and References conform well to the style of the journal.