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Membrane Hyperpolarization Induced by Endoplasmic Reticulum Stress Facilitates Ca2+ Influx to Regulate Cell Cycle Progression in Brain Capillary Endothelial Cells
J Pharmacol Sci 125, 227 – 232 (2014)
Journal of Pharmacological Sciences © The Japanese Pharmacological Society
Short Communication
Membrane Hyperpolarization Induced by Endoplasmic Reticulum Stress Facilitates Ca2+ Influx to Regulate Cell Cycle Progression in Brain Capillary Endothelial Cells Hiroaki Kito1, Hisao Yamamura1, Yoshiaki Suzuki1, Susumu Ohya2, Kiyofumi Asai3, and Yuji Imaizumi1,* Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan 2 Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan 3 Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan 1
Received January 6, 2014; Accepted April 1, 2014
Abstract. Upregulation of the Kir2.1 channel during endoplasmic reticulum (ER) stress in t-BBEC117, an immortalized bovine brain endothelial cell line, caused a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) and a facilitation of cell death. Expressions of Ca2+ influx channels (TRPC, Orai1, STIM1) were unchanged by ER stress. The ER stress–induced [Ca2+]i increase was mainly attributed to the deeper resting membrane potential due to Kir2.1 upregulation. ER stress arrested at the G2/M phase and it was attenuated by an inhibitor of Kir2.1. These results indicate that Kir2.1 upregulation by ER stress facilitates cell death via regulation of cell cycle progression in t-BBEC117. Keywords: brain capillary endothelial cell, store operated calcium entry, endoplasmic reticulum stress
Brain capillary endothelial cell (BCEC) is the major component of the blood–brain barrier (BBB), which is structurally characterized by the intercellular tight junction and the surrounding astrocytes and pericytes (1). The proper balance between the proliferation and the cell death is essential for BBB integrity (2). In various types of cells, changes in intracellular Ca2+ concentration ([Ca2+]i) contribute to the regulation of cell functions, including cell proliferation and cell death (3, 4). It has been shown that membrane hyperpolarization due to the activation of inward rectifier K+ channel (Kir2.1) regulated [Ca2+]i to cause cell death in t-BBEC117 (5), which is an immortalized bovine brain endothelial cell line established by the transfection with SV40 large Tantigen-expressing vector (6). Endoplasmic reticulum (ER) is one of the major
organelles, which contribute to the regulation of [Ca2+]i. In addition, ER plays central roles in the folding of secreted proteins. Multiple stimuli and pathological conditions disturb ER homeostasis and result in ER stress (7) to induce damages and death of BCECs. Our previous studies indicated that the up-regulation of Kir2.1 was responsible for the elevated resting [Ca2+]i and cell death in ER stress–loaded t-BBEC117 (8). Membrane hyperpolarization via the activation of K+ channels is supposed to increase the electromotive force of Ca2+ entry (9). Store-operated Ca2+ entry (SOCE) via Ca2+ release-activated Ca2+ channel (CRAC) is required for sustained Ca2+ influx across the cell membrane (10). The resulting sustained [Ca2+]i elevation is essential for cell proliferation/death in BCECs (5, 8, 11). t-BBEC117 cells were cultured at 37°C, 5% CO2 in high-glucose (25 mM) Dulbecco’s modified Eagle’s medium (DMEM; Wako, Osaka) containing 10% fetal bovine serum, 100 U/ml penicillin (Wako), 100 mg/ml streptomycin (Meiji Seika, Tokyo), and 1.0 mg/ml G418
*Corresponding author. [email protected] Published online in J-STAGE on May 23, 2014 doi: 10.1254/jphs.14002SC
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Fig. 1. Relationship between membrane potential and [Ca2+]i in t-BBEC117. To obtain the relationship between membrane potential and [Ca2+]i, whole cell patch clamp techniques were applied to t-BBEC117, which was loaded with 50 mM fura-2 from the recording pipette. A: Change in [Ca2+]i and membrane potential. The holding potentials are shown at the top. The second hyperpolarization from −30 to −80 mV was performed in the presence of 10 mM La3+. B: Summarized data showing the relationship between membrane potential and [Ca2+]i. Data in the absence and presence of La3+ were obtained from 11 and 4 cells, respectively. **P < 0.01 vs. control. C: Store-operated Ca2+ entry (SOCE) and membrane potential. Ca2+ influx via SOCE was measured at holding potentials of −60 and 0 mV in t-BBEC117 under normal conditions. To induce SOCE, t-BBEC 117 were pretreated with 1 mM thapsigargin (TG), a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase inhibitor, for 30 min in Ca2+-free solution and the Ca2+ store was depleted. Then, the bath solution was changed from Ca2+-free solution to the standard solution containing 2.2 mM Ca2+. D: Transmitted and fluorescent images of t-BBEC117 loaded with fura-2 at the times indicated in C. Upper and lower panels represent fluorescent images at −60 and 0 mV, respectively. E: Summarized data showing the relationship between membrane potential and the SOCE activity under normal and tunicamycin-treated (TM) conditions. Data at −60 and 0 mV under normal conditons were obtained from 5 and 4 cells, respectively. **P < 0.01 vs. −60 mV. Data at −60 mV under tunicamycin-treated conditions were obtained from 7 cells.
(Wako). ER stress was loaded with tunicamycin as described previously (8). Although high glucose environments per se could induce ER stress (12), our previous study has demonstrated that the expression of CHOP, an ER stress marker, was significantly up-regulated under tunicamycin-treated conditions in t-BBEC117 under the cultivation in high-glucose DMEM medium. The fura-2 fluorescent was monitored using the ARGUS/HiSCA imaging system (Hamamatsu Photonics, Hamamatsu). Changes in membrane potential was also measured with a voltage-sensitive fluorescent dye, DiBAC4(3). Whole-cell patch clamp was performed in the same way as described previously (8). The pipette solution for
whole-cell patch clamp and the bath solution for the [Ca2+]i and membrane potential measurement were described previously (5, 8). To clarify the relationship between membrane potential and [Ca2+]i, [Ca2+]i was determined in t-BBEC117 loaded with 50 mM fura-2 from a recording pipette under the voltage clamp in the whole-cell patch configuration (Fig. 1A). The [Ca2+]i was reduced by depolarization from −30 to 0 mV and significantly increased by the hyperpolarization to −80 mV as described previously (11). The [Ca2+]i rise at −80 mV was significantly reduced by the application of 10 mM La3+ (n = 4, P < 0.01 vs. control) (Fig. 1B).
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Fig. 2. Expression analyses in the Ca2+ influx pathway of t-BBEC117 under tunicamycin-treated conditions. To obtain the relationship between membrane potential and [Ca2+]i in t-BBEC117 under tunicamycin-treated conditions, 50 mM fura-2 was applied from the recording pipette under whole cell voltage clamp. A: Changes in [Ca2+]i were measured during the voltage clamp protocol, which is shown at the top in the panel, in t-BBEC117 under normal and tunicamycin-treated conditions. B: Summarized data showing the relationship between membrane potential and [Ca2+]i. Data under normal conditions were replotted from Fig. 1B. Data in normal and tunicamycin-treated conditions were obtained from 11 and 5 cells, respectively. C: Quantitative analysis for mRNA expression of TRPC subfamily, Orai1, and STIM1. The mRNA levels are normalized to GAPDH. Data were collected from 4 dishes. Primer sets used in this study are shown as follows: bovine GAPDH (GenBank Accession No. BC102589, 138-249), TRPC1 (NM_174476, 1649-1770), TRPC3 (AB179743, 403-523), TRPC5 (XM_599990, 532-632), Orai1 (NM_001099002, 1037-1187) and STIM1 (NM_001035409, 895-1038).
Next, the influence of membrane potential on SOCE was examined at holding potentials of 0 mV and −60 mV (Fig. 1C). The peak of the [Ca2+]i rise at holding potential of −60 mV (n = 5) was significantly larger than 0 mV (n = 4, P < 0.01 vs. −60 mV), indicating that SOCE depends on transmembrane electromotive force for Ca2+. In addition, there was no significant difference in SOCE at −60 mV between normal and tunicamycin-treated conditions (Fig. 1E). In the next series of experiments, we examined the effects of tunicamycin-treatment on the relationship between membrane potential and Ca2+ influx in tBBEC117 under the voltage-clamp mode in the same manner as Fig. 1. In t-BBEC117 under ER stress, the Ca2+ entry depended upon the membrane potential as that in the control (Fig. 2A). The summarized data indicated that [Ca2+]i at −80 mV was significantly higher than at
−30 mV in t-BBEC117 regardless of tunicamycin treatment. There was, however, no significant difference in the [Ca2+]i at −80 mV between normal and tunicamycintreated conditions (Fig. 2B). Our previous study reported that mRNAs of TRPC1, TRPC3, and TRPC5 are expressed in t-BBEC117 (11). In the present study, the transcriptional expression analyses of TRPC, Orai1, and STIM1 in t-BBEC117 were performed under normal and tunicamycin-treated conditions. Orai1 and STIM1 comprise the major subunit composition responsible for CRAC and contribute to the regulation of Ca2+ signaling in a wide variety of cell types (13). Real-time PCR analyses show that no significant difference in mRNA expression of TRPC1, TRPC3, TRPC5, Orai1, and STIM1 was found between the control and tunicamycin-treated cells (Fig. 2C). However, a further study is required to draw a conclusion about
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Fig. 3. Membrane hyperpolarization induced by SOCE in t-BBEC117 and the cell cycle arrest due to the Kir2.1 upregulation. A: The membrane potential changes induced by SOCE were monitored by 100 nM DiBAC4(3). At the end of each experiment, 140 mM K+ HEPES-buffered solution was applied and its fluorescence intensity was taken as F140K (1.0) to evaluate changes in intensity (F) as the ratio (F/F140K). The decrease in the ratio indicates membrane hyperpolarization. Time courses of typical changes in ratio of fluorescence under normal (black) and tunicamycin-treated conditions (gray). B: Summarized data showing changes in the ratio of fluorescence by the induction of SOCE at α in the time courses (A) (normal, n = 76; tunicamycin, n = 125; *P < 0.05 vs. normal). C: Summarized data on changes in the ratio of fluorescence by the addition of 100 mM Ba2+ were obtained as b-a in the time courses (A) (normal, n = 76; tunicamycin, n = 125; **P < 0.01 vs. normal). D: The effects of the treatment with tunicamycin (TM) in the absence and presence of Ba2+ on the cell cycle progression in t-BBEC117. Cell cycle progression was monitored by the detection of DNA content using flow cytometric analysis. t-BBEC117 were fixed and permeabilized, and then DNA was labeled with propidium iodide. (normal: G0/G1; 57.7%, S; 13.0%, G2/M; 29.3%, TM: G0/G1; 52.7%, S; 10.5%, G2/M; 36.8%, TM + Ba2+: G0/G1; 56.5%, S; 16.5%, G2/M; 27.0%). E, Summarized data showing the cell cycle distribution of t-BBEC117 under normal conditions (n = 6) and tunicamycin-treated conditions in the absence (n = 3) and presence (n = 5) of 100 mM Ba2+. *P < 0.05, **P < 0.01 vs. normal; ##P < 0.01 vs. tunicamycin.
whether the functional expression of Ca2+ entry channels per se is enhanced or not by ER stress in t-BBEC117. In Fig. 3A, the membrane potential changes in response to SOCE were monitored with 100 nM DiBAC4(3) in t-BBEC117. SOCE was induced by the addition of 2.2 mM Ca2+ to t-BBEC117, which were pretreated 1 mM TG in Ca2+-free solution. The addition of 2.2 mM Ca2+ decreased the relative fluorescent intensity (ratio of F/F140K), indicating that membrane hyperpolarization occurred in both the control and tunicamycin-treated cells examined. The decrease in F/F140K (a in Fig. 3A) in tunicamycin-treated cells was larger than that in the control. The summarized results indicated that the hyperpolarization in tunicamycin-treated cells was significantly larger than that in normal cells (n = 125 and 76,
respectively, P < 0.05 vs. normal) (Fig. 3B). The further application of 100 mM Ba2+ substantially increased F/F140K in tunicamycin-treated cells but not much in the control (Fig. 3A). We found two types of cell population according to the Ba2+-sensitivity of SOCE-induced hyperpolarization. Under the normal conditions, the dominant cell population showed that the SOCE-induced membrane hyperpolarization was Ba2+insensitive. On the other hand, under the tunicamycintreated conditions, the dominant cell population showed that the SOCE-induced membrane hyperpolarization was mostly recovered by Ba2+ application. In summary, Ba2+-sensitive hyperpolarization was observed in 14% of t-BBEC117 (11 of 76 cells) under normal conditions and in 54% of t-BBEC117 (68 of 125 cells) under tunic-
ER Stress & Membrane Potential in BCECs
amycin-treated conditions. The changes in fluorescent ratio (DF/F140K) after the application of 100 mM Ba2+ measured in Fig. 3A were summarized as b-a in Fig. 3C. There was a significant difference between the tunica mycin-treated cells and the control cells. (n = 125 and 76, respectively). We have reported previously that ATP stimulation increased [Ca2+]i and triggered membrane hyperpolarization in most of t-BBEC117 under control conditions (5). The ATP-induced membrane hyperpolarization was mostly blocked by apamin, a small conductance Ca2+activated K+ (SK2)-channel blocker. The Ba2+-insensitive component of membrane hyperpolarization induced by SOCE was also blocked by 100 nM UCL1684 (normal: 0.66 ± 0.014 tunicamycin: 0.70 ± 0.019, n = 23 and 33 respectively, P = 0.17) (data not shown) and is supposed to be due to SK2 channel activation as has been suggested in the previous reports (5, 8). The membrane hyperpolarization caused an increase of Kir2.1 conductance in t-BBEC117. The excessive membrane hyper polarization mediated by SK2 and Kir2.1 channels induced further Ca2+ entry (5, 8). These results indicated that Ca2+ influx in ER stress– loaded cells sequentially activated SK2 and Kir2.1 channels, and the subsequent membrane hyperpolarization induced further Ca2+ entry. Since it has been demonstrated that upregulation of Kir2.1 by ER stress facilitated cell death in t-BBEC117 (5, 8), effects of ER stress on cell cycle progression was examined in this study. The staining of t-BBEC117 with propidium iodide was performed by flow cytometry. The summarized data showed that tunicamycin treatment resulted in cell cycle arrest at the G2/M phase: normal: G0/G1, 58.1% ± 0.32%; S, 14.1% ± 0.75%; G2/M, 27.8% ± 1.0%, n = 6 and tunicamycin: G0/G1, 53.0% ± 0.21%; S, 10.4% ± 0.15%; G2/M, 36.6% ± 0.13%, n = 3; Fig. 3: D and E). Moreover, the application of Ba2+ attenuated the G2/M cell cycle arrest in tunicamycintreated cells (tunicamycin + Ba2+: G0/G1, 55.2% ± 0.63%; S, 15.4% ± 0.33%; G2/M, 29.4% ± 0.93%, n = 5). These results suggest that the facilitation of cell death in tBBEC117 due to the upregulation of Kir2.1 is involved in the arrest of the G2/M phase during cell cycle progression. The treatment with tunicamycin produced an approximately 10% change in cell cycle phase in tBBEC117. Although this change was modest, it appears to be sufficient for the regulation of cell proliferation and/or cell death. It has been shown that the knockdown of Orai1 suppresses cell proliferation and arrests cells at the G2/M phase in human umbilical vein endothelial cells (HUVECs) (14). Orai1 knockdown induced a small increase in the fraction of cell population in the G2/M phase only by approximately 7% but significantly sup-
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pressed cell growth and reduced the cell number to less than 50%. Another report has also shown that a small change in the cell population in the G0/G1 phase induced substantial cell death (15). In conclusion, ER stress induced upregulation of Kir2.1, which contributes to the deep resting membrane potential in t-BBEC117, whereas it had little effect on the pathway of Ca2+ influx. These results strongly suggest that the Ca2+ influx is regulated mainly by membrane potential changes in t-BBEC117 regardless of the tunica mycin-treatment. Subsequently, the Kir2.1 upregulation under tunicamycin-treated conditions arrested the cell cycle at the G2/M phase in t-BBEC117. It is conceivable that the membrane hyperpolarization via the upregulation of Kir2.1 induces sustained [Ca2+]i rise under ER stress conditions and regulates ER stress–induced cell death in t-BBEC117 via the regulation of cell cycle progression. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (20056027 and 23136512, to Y.I.) from the Ministry of Education, Culture, Sports, Science and Technology; a Grant-inAid for Scientific Research (B) (23390020, to Y.I.) from the Japan Society for the Promotion of Science; and a Grant-in-Aid for JSPS Fellows (25-10244, to H.K.) from the Japan Society for the Promotion of Science.
Conflicts of Interest The authors indicated no potential conflicts of interest in this study.
References 1 Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci. 2006;7: 41–53. 2 Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev. 2005;57:173–185. 3 Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol. 2000;1: 11–21. 4 Lang F, Foller M, Lang KS, Lang PA, Ritter M, Gulbins E, et al. Ion channels in cell proliferation and apoptotic cell death. J Membr Biol. 2005;205:147–157. 5 Yamazaki D, Kito H, Yamamoto S, Ohya S, Yamamura H, Asai K, et al. Contribution of Kir2 potassium channels to ATP-induced cell death in brain capillary endothelial cells and reconstructed HEK293 cell model. Am J Physiol Cell Physiol. 2011;300: C75–C86. 6 Sobue K, Yamamoto N, Yoneda K, Hodgson ME, Yamashiro K, Tsuruoka N, et al. Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors. Neurosci Res. 1999;35:155–164. 7 Kim I, Xu W, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev
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Drug Discov. 2008;7:1013–1030. 8 Kito H, Yamazaki D, Ohya S, Yamamura H, Asai K, Imaizumi Y. Up-regulation of Kir2.1 by ER stress facilitates cell death of brain capillary endothelial cells. Biochem Biophys Res Commun. 2011;411:293–298. 9 Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev. 2001;81:1415–1459. 10 Lewis RS. Calcium signaling mechanisms in T lymphocytes. Annu Rev Immunol. 2001;19:497–521. 11 Yamazaki D, Aoyama M, Ohya S, Muraki K, Asai K, Imaizumi Y. Novel functions of small conductance Ca2+-activated K+ channel in enhanced cell proliferation by ATP in brain endo thelial cells. J Biol Chem. 2006;281:38430–38439. 12 Takahashi S, Izawa Y, Suzuki N. Astroglial pentose phosphate
pathway rates in response to high-glucose environments. ASN Neuro. 2012;4:art:e00078. 13 Muik M, Schindl R, Fahrner M, Romanin C. Ca2+ release-activated Ca2+ (CRAC) current, structure, and function. Cell Mol Life Sci. 2012;69:4163–4176. 14 Abdullaev IF, Bisaillon JM, Potier M, Gonzalez JC, Motiani RK, Trebak M. Stim1 and Orai1 mediate CRAC currents and storeoperated calcium entry important for endothelial cell proliferation. Circ Res. 2008;103:1289–1299. 15 Duan J, Yu Y, Li Y, Yu Y, Li Y, Zhou X, et al. Toxic effect of silica nanoparticles on endothelial cells through DNA damage response via Chk1-dependent G2/M checkpoint. PLoS One. 2013;8:e62087.
Journal of Pharmacological Sciences © The Japanese Pharmacological Society
Short Communication
Membrane Hyperpolarization Induced by Endoplasmic Reticulum Stress Facilitates Ca2+ Influx to Regulate Cell Cycle Progression in Brain Capillary Endothelial Cells Hiroaki Kito1, Hisao Yamamura1, Yoshiaki Suzuki1, Susumu Ohya2, Kiyofumi Asai3, and Yuji Imaizumi1,* Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan 2 Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan 3 Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan 1
Received January 6, 2014; Accepted April 1, 2014
Abstract. Upregulation of the Kir2.1 channel during endoplasmic reticulum (ER) stress in t-BBEC117, an immortalized bovine brain endothelial cell line, caused a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) and a facilitation of cell death. Expressions of Ca2+ influx channels (TRPC, Orai1, STIM1) were unchanged by ER stress. The ER stress–induced [Ca2+]i increase was mainly attributed to the deeper resting membrane potential due to Kir2.1 upregulation. ER stress arrested at the G2/M phase and it was attenuated by an inhibitor of Kir2.1. These results indicate that Kir2.1 upregulation by ER stress facilitates cell death via regulation of cell cycle progression in t-BBEC117. Keywords: brain capillary endothelial cell, store operated calcium entry, endoplasmic reticulum stress
Brain capillary endothelial cell (BCEC) is the major component of the blood–brain barrier (BBB), which is structurally characterized by the intercellular tight junction and the surrounding astrocytes and pericytes (1). The proper balance between the proliferation and the cell death is essential for BBB integrity (2). In various types of cells, changes in intracellular Ca2+ concentration ([Ca2+]i) contribute to the regulation of cell functions, including cell proliferation and cell death (3, 4). It has been shown that membrane hyperpolarization due to the activation of inward rectifier K+ channel (Kir2.1) regulated [Ca2+]i to cause cell death in t-BBEC117 (5), which is an immortalized bovine brain endothelial cell line established by the transfection with SV40 large Tantigen-expressing vector (6). Endoplasmic reticulum (ER) is one of the major
organelles, which contribute to the regulation of [Ca2+]i. In addition, ER plays central roles in the folding of secreted proteins. Multiple stimuli and pathological conditions disturb ER homeostasis and result in ER stress (7) to induce damages and death of BCECs. Our previous studies indicated that the up-regulation of Kir2.1 was responsible for the elevated resting [Ca2+]i and cell death in ER stress–loaded t-BBEC117 (8). Membrane hyperpolarization via the activation of K+ channels is supposed to increase the electromotive force of Ca2+ entry (9). Store-operated Ca2+ entry (SOCE) via Ca2+ release-activated Ca2+ channel (CRAC) is required for sustained Ca2+ influx across the cell membrane (10). The resulting sustained [Ca2+]i elevation is essential for cell proliferation/death in BCECs (5, 8, 11). t-BBEC117 cells were cultured at 37°C, 5% CO2 in high-glucose (25 mM) Dulbecco’s modified Eagle’s medium (DMEM; Wako, Osaka) containing 10% fetal bovine serum, 100 U/ml penicillin (Wako), 100 mg/ml streptomycin (Meiji Seika, Tokyo), and 1.0 mg/ml G418
*Corresponding author. [email protected] Published online in J-STAGE on May 23, 2014 doi: 10.1254/jphs.14002SC
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Fig. 1. Relationship between membrane potential and [Ca2+]i in t-BBEC117. To obtain the relationship between membrane potential and [Ca2+]i, whole cell patch clamp techniques were applied to t-BBEC117, which was loaded with 50 mM fura-2 from the recording pipette. A: Change in [Ca2+]i and membrane potential. The holding potentials are shown at the top. The second hyperpolarization from −30 to −80 mV was performed in the presence of 10 mM La3+. B: Summarized data showing the relationship between membrane potential and [Ca2+]i. Data in the absence and presence of La3+ were obtained from 11 and 4 cells, respectively. **P < 0.01 vs. control. C: Store-operated Ca2+ entry (SOCE) and membrane potential. Ca2+ influx via SOCE was measured at holding potentials of −60 and 0 mV in t-BBEC117 under normal conditions. To induce SOCE, t-BBEC 117 were pretreated with 1 mM thapsigargin (TG), a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase inhibitor, for 30 min in Ca2+-free solution and the Ca2+ store was depleted. Then, the bath solution was changed from Ca2+-free solution to the standard solution containing 2.2 mM Ca2+. D: Transmitted and fluorescent images of t-BBEC117 loaded with fura-2 at the times indicated in C. Upper and lower panels represent fluorescent images at −60 and 0 mV, respectively. E: Summarized data showing the relationship between membrane potential and the SOCE activity under normal and tunicamycin-treated (TM) conditions. Data at −60 and 0 mV under normal conditons were obtained from 5 and 4 cells, respectively. **P < 0.01 vs. −60 mV. Data at −60 mV under tunicamycin-treated conditions were obtained from 7 cells.
(Wako). ER stress was loaded with tunicamycin as described previously (8). Although high glucose environments per se could induce ER stress (12), our previous study has demonstrated that the expression of CHOP, an ER stress marker, was significantly up-regulated under tunicamycin-treated conditions in t-BBEC117 under the cultivation in high-glucose DMEM medium. The fura-2 fluorescent was monitored using the ARGUS/HiSCA imaging system (Hamamatsu Photonics, Hamamatsu). Changes in membrane potential was also measured with a voltage-sensitive fluorescent dye, DiBAC4(3). Whole-cell patch clamp was performed in the same way as described previously (8). The pipette solution for
whole-cell patch clamp and the bath solution for the [Ca2+]i and membrane potential measurement were described previously (5, 8). To clarify the relationship between membrane potential and [Ca2+]i, [Ca2+]i was determined in t-BBEC117 loaded with 50 mM fura-2 from a recording pipette under the voltage clamp in the whole-cell patch configuration (Fig. 1A). The [Ca2+]i was reduced by depolarization from −30 to 0 mV and significantly increased by the hyperpolarization to −80 mV as described previously (11). The [Ca2+]i rise at −80 mV was significantly reduced by the application of 10 mM La3+ (n = 4, P < 0.01 vs. control) (Fig. 1B).
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Fig. 2. Expression analyses in the Ca2+ influx pathway of t-BBEC117 under tunicamycin-treated conditions. To obtain the relationship between membrane potential and [Ca2+]i in t-BBEC117 under tunicamycin-treated conditions, 50 mM fura-2 was applied from the recording pipette under whole cell voltage clamp. A: Changes in [Ca2+]i were measured during the voltage clamp protocol, which is shown at the top in the panel, in t-BBEC117 under normal and tunicamycin-treated conditions. B: Summarized data showing the relationship between membrane potential and [Ca2+]i. Data under normal conditions were replotted from Fig. 1B. Data in normal and tunicamycin-treated conditions were obtained from 11 and 5 cells, respectively. C: Quantitative analysis for mRNA expression of TRPC subfamily, Orai1, and STIM1. The mRNA levels are normalized to GAPDH. Data were collected from 4 dishes. Primer sets used in this study are shown as follows: bovine GAPDH (GenBank Accession No. BC102589, 138-249), TRPC1 (NM_174476, 1649-1770), TRPC3 (AB179743, 403-523), TRPC5 (XM_599990, 532-632), Orai1 (NM_001099002, 1037-1187) and STIM1 (NM_001035409, 895-1038).
Next, the influence of membrane potential on SOCE was examined at holding potentials of 0 mV and −60 mV (Fig. 1C). The peak of the [Ca2+]i rise at holding potential of −60 mV (n = 5) was significantly larger than 0 mV (n = 4, P < 0.01 vs. −60 mV), indicating that SOCE depends on transmembrane electromotive force for Ca2+. In addition, there was no significant difference in SOCE at −60 mV between normal and tunicamycin-treated conditions (Fig. 1E). In the next series of experiments, we examined the effects of tunicamycin-treatment on the relationship between membrane potential and Ca2+ influx in tBBEC117 under the voltage-clamp mode in the same manner as Fig. 1. In t-BBEC117 under ER stress, the Ca2+ entry depended upon the membrane potential as that in the control (Fig. 2A). The summarized data indicated that [Ca2+]i at −80 mV was significantly higher than at
−30 mV in t-BBEC117 regardless of tunicamycin treatment. There was, however, no significant difference in the [Ca2+]i at −80 mV between normal and tunicamycintreated conditions (Fig. 2B). Our previous study reported that mRNAs of TRPC1, TRPC3, and TRPC5 are expressed in t-BBEC117 (11). In the present study, the transcriptional expression analyses of TRPC, Orai1, and STIM1 in t-BBEC117 were performed under normal and tunicamycin-treated conditions. Orai1 and STIM1 comprise the major subunit composition responsible for CRAC and contribute to the regulation of Ca2+ signaling in a wide variety of cell types (13). Real-time PCR analyses show that no significant difference in mRNA expression of TRPC1, TRPC3, TRPC5, Orai1, and STIM1 was found between the control and tunicamycin-treated cells (Fig. 2C). However, a further study is required to draw a conclusion about
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Fig. 3. Membrane hyperpolarization induced by SOCE in t-BBEC117 and the cell cycle arrest due to the Kir2.1 upregulation. A: The membrane potential changes induced by SOCE were monitored by 100 nM DiBAC4(3). At the end of each experiment, 140 mM K+ HEPES-buffered solution was applied and its fluorescence intensity was taken as F140K (1.0) to evaluate changes in intensity (F) as the ratio (F/F140K). The decrease in the ratio indicates membrane hyperpolarization. Time courses of typical changes in ratio of fluorescence under normal (black) and tunicamycin-treated conditions (gray). B: Summarized data showing changes in the ratio of fluorescence by the induction of SOCE at α in the time courses (A) (normal, n = 76; tunicamycin, n = 125; *P < 0.05 vs. normal). C: Summarized data on changes in the ratio of fluorescence by the addition of 100 mM Ba2+ were obtained as b-a in the time courses (A) (normal, n = 76; tunicamycin, n = 125; **P < 0.01 vs. normal). D: The effects of the treatment with tunicamycin (TM) in the absence and presence of Ba2+ on the cell cycle progression in t-BBEC117. Cell cycle progression was monitored by the detection of DNA content using flow cytometric analysis. t-BBEC117 were fixed and permeabilized, and then DNA was labeled with propidium iodide. (normal: G0/G1; 57.7%, S; 13.0%, G2/M; 29.3%, TM: G0/G1; 52.7%, S; 10.5%, G2/M; 36.8%, TM + Ba2+: G0/G1; 56.5%, S; 16.5%, G2/M; 27.0%). E, Summarized data showing the cell cycle distribution of t-BBEC117 under normal conditions (n = 6) and tunicamycin-treated conditions in the absence (n = 3) and presence (n = 5) of 100 mM Ba2+. *P < 0.05, **P < 0.01 vs. normal; ##P < 0.01 vs. tunicamycin.
whether the functional expression of Ca2+ entry channels per se is enhanced or not by ER stress in t-BBEC117. In Fig. 3A, the membrane potential changes in response to SOCE were monitored with 100 nM DiBAC4(3) in t-BBEC117. SOCE was induced by the addition of 2.2 mM Ca2+ to t-BBEC117, which were pretreated 1 mM TG in Ca2+-free solution. The addition of 2.2 mM Ca2+ decreased the relative fluorescent intensity (ratio of F/F140K), indicating that membrane hyperpolarization occurred in both the control and tunicamycin-treated cells examined. The decrease in F/F140K (a in Fig. 3A) in tunicamycin-treated cells was larger than that in the control. The summarized results indicated that the hyperpolarization in tunicamycin-treated cells was significantly larger than that in normal cells (n = 125 and 76,
respectively, P < 0.05 vs. normal) (Fig. 3B). The further application of 100 mM Ba2+ substantially increased F/F140K in tunicamycin-treated cells but not much in the control (Fig. 3A). We found two types of cell population according to the Ba2+-sensitivity of SOCE-induced hyperpolarization. Under the normal conditions, the dominant cell population showed that the SOCE-induced membrane hyperpolarization was Ba2+insensitive. On the other hand, under the tunicamycintreated conditions, the dominant cell population showed that the SOCE-induced membrane hyperpolarization was mostly recovered by Ba2+ application. In summary, Ba2+-sensitive hyperpolarization was observed in 14% of t-BBEC117 (11 of 76 cells) under normal conditions and in 54% of t-BBEC117 (68 of 125 cells) under tunic-
ER Stress & Membrane Potential in BCECs
amycin-treated conditions. The changes in fluorescent ratio (DF/F140K) after the application of 100 mM Ba2+ measured in Fig. 3A were summarized as b-a in Fig. 3C. There was a significant difference between the tunica mycin-treated cells and the control cells. (n = 125 and 76, respectively). We have reported previously that ATP stimulation increased [Ca2+]i and triggered membrane hyperpolarization in most of t-BBEC117 under control conditions (5). The ATP-induced membrane hyperpolarization was mostly blocked by apamin, a small conductance Ca2+activated K+ (SK2)-channel blocker. The Ba2+-insensitive component of membrane hyperpolarization induced by SOCE was also blocked by 100 nM UCL1684 (normal: 0.66 ± 0.014 tunicamycin: 0.70 ± 0.019, n = 23 and 33 respectively, P = 0.17) (data not shown) and is supposed to be due to SK2 channel activation as has been suggested in the previous reports (5, 8). The membrane hyperpolarization caused an increase of Kir2.1 conductance in t-BBEC117. The excessive membrane hyper polarization mediated by SK2 and Kir2.1 channels induced further Ca2+ entry (5, 8). These results indicated that Ca2+ influx in ER stress– loaded cells sequentially activated SK2 and Kir2.1 channels, and the subsequent membrane hyperpolarization induced further Ca2+ entry. Since it has been demonstrated that upregulation of Kir2.1 by ER stress facilitated cell death in t-BBEC117 (5, 8), effects of ER stress on cell cycle progression was examined in this study. The staining of t-BBEC117 with propidium iodide was performed by flow cytometry. The summarized data showed that tunicamycin treatment resulted in cell cycle arrest at the G2/M phase: normal: G0/G1, 58.1% ± 0.32%; S, 14.1% ± 0.75%; G2/M, 27.8% ± 1.0%, n = 6 and tunicamycin: G0/G1, 53.0% ± 0.21%; S, 10.4% ± 0.15%; G2/M, 36.6% ± 0.13%, n = 3; Fig. 3: D and E). Moreover, the application of Ba2+ attenuated the G2/M cell cycle arrest in tunicamycintreated cells (tunicamycin + Ba2+: G0/G1, 55.2% ± 0.63%; S, 15.4% ± 0.33%; G2/M, 29.4% ± 0.93%, n = 5). These results suggest that the facilitation of cell death in tBBEC117 due to the upregulation of Kir2.1 is involved in the arrest of the G2/M phase during cell cycle progression. The treatment with tunicamycin produced an approximately 10% change in cell cycle phase in tBBEC117. Although this change was modest, it appears to be sufficient for the regulation of cell proliferation and/or cell death. It has been shown that the knockdown of Orai1 suppresses cell proliferation and arrests cells at the G2/M phase in human umbilical vein endothelial cells (HUVECs) (14). Orai1 knockdown induced a small increase in the fraction of cell population in the G2/M phase only by approximately 7% but significantly sup-
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pressed cell growth and reduced the cell number to less than 50%. Another report has also shown that a small change in the cell population in the G0/G1 phase induced substantial cell death (15). In conclusion, ER stress induced upregulation of Kir2.1, which contributes to the deep resting membrane potential in t-BBEC117, whereas it had little effect on the pathway of Ca2+ influx. These results strongly suggest that the Ca2+ influx is regulated mainly by membrane potential changes in t-BBEC117 regardless of the tunica mycin-treatment. Subsequently, the Kir2.1 upregulation under tunicamycin-treated conditions arrested the cell cycle at the G2/M phase in t-BBEC117. It is conceivable that the membrane hyperpolarization via the upregulation of Kir2.1 induces sustained [Ca2+]i rise under ER stress conditions and regulates ER stress–induced cell death in t-BBEC117 via the regulation of cell cycle progression. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (20056027 and 23136512, to Y.I.) from the Ministry of Education, Culture, Sports, Science and Technology; a Grant-inAid for Scientific Research (B) (23390020, to Y.I.) from the Japan Society for the Promotion of Science; and a Grant-in-Aid for JSPS Fellows (25-10244, to H.K.) from the Japan Society for the Promotion of Science.
Conflicts of Interest The authors indicated no potential conflicts of interest in this study.
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