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Regulation of capacitative Ca2+ entry by tyrosine phosphorylation in the neural retina of chick embryo
Neuroscience Letters 272 (1999) 123±126
Regulation of capacitative Ca 21 entry by tyrosine phosphorylation in the neural retina of chick embryo Wen-Liang Zhou, Miho Sugioka, Yoko Sakaki 1, Masayuki Yamashita* Department of Physiology I, Nara Medical University, Shijo-cho 840, Kashihara 634-8521, Japan Received 31 May 1999; received in revised form 21 July 1999; accepted 21 July 1999
Abstract Capacitative Ca 21 entry occurs in the neural retina of chick embryo during neurogenesis. We studied the effects of blockers of capacitative Ca 21 entry (SK&F 96365, Zn 21, Ni 21), genistein, a tyrosine kinase inhibitor and vanadate, a protein-phosphotyrosine phosphatase inhibitor in the embryonic chick retina with fura-2 ¯uorescence measurements. After incubation of the retina in a Ca 21-free solution with or without an inhibitor of Ca 21 pumps of intracellular Ca 21 stores, re-introduction of extracellular Ca 21 caused capacitative Ca 21 entry which was inhibited by SK&F 96365 (10 mM), Zn 21 (1 mM), Ni 21 (5 mM) and genistein (100 mM). On the contrary, vanadate (1 mM) enhanced the Ca 21 entry. These results suggested that tyrosine phosphorylation was involved in the activation of capacitative Ca 21 entry. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Capacitative Ca 21 entry; Retina; SK&F96365; Genistein; Vanadate; Tyrosine phosphorylation; Chick embryo
Capacitative Ca 21 entry is originally thought to be induced by depletion of intracellular Ca 21 stores [5]. It can be induced by the application of inhibitors of Ca 21 pumps of the Ca 21 stores or Ca 21-mobilizing agonists in the absence of extracellular Ca 21 or simply by the incubation in a Ca 21-free medium [1]. We have shown that capacitative Ca 21 entry occurs in the neural retina of chick embryo during neurogenesis [7,10]. The capacitative Ca 21 entry is induced by the incubation of the retina in a Ca 21free medium and the application of agonists (extracellular ATP and acetylcholine) or thapsigargin with a Ca 21-free medium [6,7]. The present study was aimed at to characterize the pharmacological properties of the capacitative Ca 21 entry and to ®nd the regulatory mechanism for the activation of capacitative Ca 21 entry in the embryonic chick neural retina. We studied the effects of the blockers of capacitative Ca 21 entry, SK&F 96365 [3], Zn 21, Ni 21 and genistein, a tyrosine kinase inhibitor, and vanadate, a protein-phosphotyrosine phosphatase inhibitor [2]. The details of preparation and ¯uorescence measurements are described in the previous paper [7]. Brie¯y, the neural retina was isolated from an embryonic day 3 (E3) * Corresponding author. Tel.: 181-744-29-8827; fax: 181-74429-0306. E-mail address: [email protected] (M. Yamashita) 1 Deceased.
chick. The neural retina was loaded with fura-2 AM (10 mM, Dojin, Kumamoto, Japan) for 1 h at room temperature (,228C) in the normal bath solution (NBS) containing (mM): NaCl 137; KCl 5; CaCl2 2.5; MgCl2 1; HEPES 10; glucose 22; buffered to pH 7.3 by adding NaOH. The fura-2loaded retina was transferred to a recording chamber (volume, 0.2 ml). The recording chamber was continuously perfused with a bath solution at 2 ml/min. Bath solutions were changed from NBS to a test solution by multi-solenoid manifold valves (General Valve Corp., Fair®eld, NJ). A ¯uorescence measurement system OSP-3 with an inverted microscope (IMT-2, Olympus, Tokyo, Japan) was used. Fluorescence was excited at 340 nm (F340) and 380 nm (F380). When F340 increases and F380 decreases, an increase in the ratio of F340/F380 indicates a rise in [Ca 21]i. The ¯uorescence ratio is presented to describe relative changes in [Ca 21]i without conversion to absolute values of intracellular free-Ca 21 concentration. Mn 21 in¯ux was measured by quenching of fura-2 ¯uorescence excited at 360 nm. DBHQ (2,5-di-tert-butylhydroquinone, Biomol, Plymouth Meeting, PA), a reversible inhibitor of Ca 21 pumps of intracellular Ca 21 stores [4], and genistein (Wako, Osaka, Japan) were dissolved in DMSO at 25 mM for stock solutions. SK&F 96365 (Biomol) was dissolved in distilled water at 10 mM for a stock solution. Ca 21-free solutions were made by replacing Ca 21 with Na 1 and adding 1 mM EGTA. Recordings were performed at room temperature. Data are
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 59 1- 1
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W.-L. Zhou et al. / Neuroscience Letters 272 (1999) 123±126
presented as mean ^ SD and the number of retinae tested (n). When an E3 chick retina was bathed in a Ca 21-free solution (CFS) for 10 min, a small transient increase and a subsequent decrease were observed in F340/F380 (Fig. 1A, upper trace). Changing bath solutions from CFS to NBS evoked a rapid rise in F340/F380 in all the retinae tested (n 68, Fig. 1A, lower trace). The averaged peak amplitude of the rapid rise in F340/F380 as measured from foot to top was 0:53 ^ 0:15 (n 9, Fig. 1C). Capacitative Ca 21 entry was more intensely induced by the addition of DBHQ (50 mM) to CFS (Fig. 1B). The averaged peak amplitude of the rapid rise in F340/F380 was 1:31 ^ 0:22 (n 9, Fig. 1C). The effects of SK&F 96365, Zn 21 and Ni 21 were examined on both of the Ca 21 rises induced by DBHQ with CFS
and by CFS alone. The Ca 21 rise by CFS alone was decreased to 39:7^2:2%, 6:0^1:0% and 6:5 ^ 0:8% of the control responses by SK&F 96365 (10 mM), Zn 21 (1 mM) and Ni 21 (5 mM), respectively, (n 4, Fig. 1D, open columns). The Ca 21 rise by DBHQ (50 mM) with CFS was decreased to 51:5^5:2%, 12:3 ^ 2:5% and 10:0^1:1% of the control responses by SK&F 96365, Zn 21 and Ni 21, respectively, (n 4, Fig. 1D, closed columns). The effect of genistein was also examined on both of the Ca 21 rises induced by DBHQ with CFS and by CFS alone. The Ca 21 rise by CFS alone was markedly inhibited by genistein (100 mM, Fig. 2A(b)) and the averaged peak amplitude was reduced to 28:3^1:5% (n 4) of the control response (Fig. 2C, open columns). Surprisingly, after wash out of genistein, the recovery response was increased to 276:7^26:9% of the control response (Fig. 2A(c), Fig.
Fig. 1. (A) Ca 21 ¯uorescence responses to the application of a Ca 21-free solution in a neural retina of an E3 chick embryo. The Ca 21-free solution was applied for the period indicated by the white bar (10 min). NBS: normal bath solution containing 2.5 mM Ca 21. Upper and lower traces were continuously recorded. (B) Ca 21 ¯uorescence responses to the application of DBHQ with a Ca 21-free solution. DBHQ (50 mM) was applied for the period indicated by the thinner bar. (C) The mean amplitudes of the rise in F340/F380 (DF340/F380) as measured from foot to top when the bath solutions were changed from a Ca 21-free solution to NBS without DBHQ (left column, n 9) and with DBHQ (right column, n 9). Error bars indicate SD. (D) The inhibitory effects of SK&F 96365 (10 mM), Zn 21 (1 mM) and Ni 21 (5 mM) on the rise in F340/F380 when the bath solutions were changed from a Ca 21-free solution to NBS without DBHQ (open column, n 4) and with DBHQ (closed column, n 4). The mean percentages of relative changes in F340/F380 are indicated: (*) P , 0:05; (***) P , 0:001).
W.-L. Zhou et al. / Neuroscience Letters 272 (1999) 123±126
125
Fig. 2. The inhibitory effects of genistein (100 mM) on the rise in F340/F380 when the bath solutions were changed from a Ca 21-free solution (applied for 10 min) to NBS without DBHQ (A) and with DBHQ (B). (C) The mean percentages of relative changes in F340/F380 when the bath solutions were changed from a Ca 21-free solution to NBS without DBHQ (open column, n 4) and with DBHQ (closed column, n 4). (*) P , 0:05.
2C, open columns). The Ca 21 rise by DBHQ with CFS was also decreased by genistein (Fig. 2B(b)) to 32:3^2:1% (n 4, Fig. 2C, closed columns) of the control response without any rebound after wash out of genistein (Fig. 2B(c)). On the contrary to the inhibitory effect of genistein, vanadate (1 mM) enhanced the CFS-induced Ca 21 rise by about 4 times (Fig. 3). We found that both of the Ca 21 rises induced by DBHQ with CFS and by CFS alone were inhibited by SK&F 96365, Zn 21 and Ni 21 to almost similar extent. These facts could indicate that the same Ca 21 entry pathway was involved in
both of the Ca 21 rises. However, the Ca 21 rise by CFS alone was not likely to be evoked by depletion of intracellular Ca 21 stores, since Ca 21 release from intracellular Ca 21 stores is induced even 10 min after the incubation of the retina in CFS [8,11]. We further found that quenching of fura-2 ¯uorescence by Mn 21 in¯ux occurred immediately after decreasing [Ca 21]0 and this quenching was blocked by SK&F 96365 (data not shown), suggesting that the depletion of Ca 21 stores is not absolutely necessary for the induction of capacitative Ca 21 entry. Genistein inhibited both of the Ca 21 rises induced by
Fig. 3. The enhancement by vanadate (sodium orthovanadate, Na3VO4, 1 mM) of the rise in F340/F380 when the bath solutions were changed from a Ca 21-free solution (applied for 10 min) to NBS (A). (B) The mean percentages of relative changes in F340/F380 when the bath solutions were changed from a Ca 21-free solution to NBS (n 4). (*) P , 0:05.
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W.-L. Zhou et al. / Neuroscience Letters 272 (1999) 123±126
DBHQ with CFS and by CFS alone to similar extent. These facts suggested that tyrosine kinases are involved in the activation of capacitative Ca 21 entry. To our surprise, a much larger Ca 21 rise than the control was evoked by CFS alone after wash out of genistein and this Ca 21 rise showed almost the same size as that induced by DBHQ with CFS (cf. Fig. 2A(c),B(a)). This could be explained by rebound activity of tyrosine kinases. The inhibitory effect of genistein on capacitative Ca 21 entry has been also shown in rat glioma C6 cells [9]. On the contrary, the proteinphosphotyrosine phosphatase inhibitor, vanadate clearly enhanced the CFS-induced Ca 21 entry. These results could suggest that tyrosine phosphorylation is involved in the activation of capacitative Ca 21 entry. [1] Berridge, M.J., Capacitative calcium entry. Biochem. J., 312 (1995) 1±11. [2] Gordon, J.A., Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol., 201 (1991) 477± 482. [3] Merritt, J.E., Armstrong, W.P. and Benham, C.D., SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem. J., 271 (1990) 515±522.
[4] Moore, G.A., McConkey, D.J., Kass, G.E.N., O'Brien, P.J. and Orrenius, S., 2,5-Di(tert-butyl)-1,4-benzohydroquinone ± a novel inhibitor of liver microsomal Ca 21 sequestration. FEBS Lett., 224 (1987) 331±336. [5] Putney Jr, J.W. and St. J. Bird, G., The inositol phosphatecalcium signaling system in non-excitable cells. Endocr. Rev., 14 (1993) 610±631. [6] Sakaki, Y., Fukuda, Y. and Yamashita, M., Muscarinic and purinergic Ca 21 mobilizations in the neural retina of early embryonic chick. Int. J. Dev. Neurosci., 14 (1996) 691±699. [7] Sakaki, Y., Sugioka, M., Fukuda, Y. and Yamashita, M., Capacitative Ca 21 in¯ux in the neural retina of chick embryo. J. Neurobiol., 32 (1997) 62±68. [8] Sugioka, M., Fukuda, Y. and Yamashita, M., Ca 21 responses to ATP via purinoceptors in the early embryonic chick retina. J. Physiol. (Lond.), 493 (1996) 855±863. [9] Takemura, H., Sakano, S., Kaneko, M. and Ohshika, H., Inhibitory effects of tyrosine kinase inhibitors on capacitative Ca 21 entry in rat glioma C6 cells. Life Sci., 62 (1998) 271± 276. [10] Yamashita, M. and Sugioka, M., Calcium mobilization systems during neurogenesis. News Physiol. Sci., 13 (1998) 75±79. [11] Zhou, W.L., Sugioka, M. and Yamashita, M., Lysophosphatidic acid-induced Ca 21 mobilization in the neural retina of chick embryo. J. Neurobiol., (1999) in press.
Regulation of capacitative Ca 21 entry by tyrosine phosphorylation in the neural retina of chick embryo Wen-Liang Zhou, Miho Sugioka, Yoko Sakaki 1, Masayuki Yamashita* Department of Physiology I, Nara Medical University, Shijo-cho 840, Kashihara 634-8521, Japan Received 31 May 1999; received in revised form 21 July 1999; accepted 21 July 1999
Abstract Capacitative Ca 21 entry occurs in the neural retina of chick embryo during neurogenesis. We studied the effects of blockers of capacitative Ca 21 entry (SK&F 96365, Zn 21, Ni 21), genistein, a tyrosine kinase inhibitor and vanadate, a protein-phosphotyrosine phosphatase inhibitor in the embryonic chick retina with fura-2 ¯uorescence measurements. After incubation of the retina in a Ca 21-free solution with or without an inhibitor of Ca 21 pumps of intracellular Ca 21 stores, re-introduction of extracellular Ca 21 caused capacitative Ca 21 entry which was inhibited by SK&F 96365 (10 mM), Zn 21 (1 mM), Ni 21 (5 mM) and genistein (100 mM). On the contrary, vanadate (1 mM) enhanced the Ca 21 entry. These results suggested that tyrosine phosphorylation was involved in the activation of capacitative Ca 21 entry. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Capacitative Ca 21 entry; Retina; SK&F96365; Genistein; Vanadate; Tyrosine phosphorylation; Chick embryo
Capacitative Ca 21 entry is originally thought to be induced by depletion of intracellular Ca 21 stores [5]. It can be induced by the application of inhibitors of Ca 21 pumps of the Ca 21 stores or Ca 21-mobilizing agonists in the absence of extracellular Ca 21 or simply by the incubation in a Ca 21-free medium [1]. We have shown that capacitative Ca 21 entry occurs in the neural retina of chick embryo during neurogenesis [7,10]. The capacitative Ca 21 entry is induced by the incubation of the retina in a Ca 21free medium and the application of agonists (extracellular ATP and acetylcholine) or thapsigargin with a Ca 21-free medium [6,7]. The present study was aimed at to characterize the pharmacological properties of the capacitative Ca 21 entry and to ®nd the regulatory mechanism for the activation of capacitative Ca 21 entry in the embryonic chick neural retina. We studied the effects of the blockers of capacitative Ca 21 entry, SK&F 96365 [3], Zn 21, Ni 21 and genistein, a tyrosine kinase inhibitor, and vanadate, a protein-phosphotyrosine phosphatase inhibitor [2]. The details of preparation and ¯uorescence measurements are described in the previous paper [7]. Brie¯y, the neural retina was isolated from an embryonic day 3 (E3) * Corresponding author. Tel.: 181-744-29-8827; fax: 181-74429-0306. E-mail address: [email protected] (M. Yamashita) 1 Deceased.
chick. The neural retina was loaded with fura-2 AM (10 mM, Dojin, Kumamoto, Japan) for 1 h at room temperature (,228C) in the normal bath solution (NBS) containing (mM): NaCl 137; KCl 5; CaCl2 2.5; MgCl2 1; HEPES 10; glucose 22; buffered to pH 7.3 by adding NaOH. The fura-2loaded retina was transferred to a recording chamber (volume, 0.2 ml). The recording chamber was continuously perfused with a bath solution at 2 ml/min. Bath solutions were changed from NBS to a test solution by multi-solenoid manifold valves (General Valve Corp., Fair®eld, NJ). A ¯uorescence measurement system OSP-3 with an inverted microscope (IMT-2, Olympus, Tokyo, Japan) was used. Fluorescence was excited at 340 nm (F340) and 380 nm (F380). When F340 increases and F380 decreases, an increase in the ratio of F340/F380 indicates a rise in [Ca 21]i. The ¯uorescence ratio is presented to describe relative changes in [Ca 21]i without conversion to absolute values of intracellular free-Ca 21 concentration. Mn 21 in¯ux was measured by quenching of fura-2 ¯uorescence excited at 360 nm. DBHQ (2,5-di-tert-butylhydroquinone, Biomol, Plymouth Meeting, PA), a reversible inhibitor of Ca 21 pumps of intracellular Ca 21 stores [4], and genistein (Wako, Osaka, Japan) were dissolved in DMSO at 25 mM for stock solutions. SK&F 96365 (Biomol) was dissolved in distilled water at 10 mM for a stock solution. Ca 21-free solutions were made by replacing Ca 21 with Na 1 and adding 1 mM EGTA. Recordings were performed at room temperature. Data are
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 59 1- 1
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W.-L. Zhou et al. / Neuroscience Letters 272 (1999) 123±126
presented as mean ^ SD and the number of retinae tested (n). When an E3 chick retina was bathed in a Ca 21-free solution (CFS) for 10 min, a small transient increase and a subsequent decrease were observed in F340/F380 (Fig. 1A, upper trace). Changing bath solutions from CFS to NBS evoked a rapid rise in F340/F380 in all the retinae tested (n 68, Fig. 1A, lower trace). The averaged peak amplitude of the rapid rise in F340/F380 as measured from foot to top was 0:53 ^ 0:15 (n 9, Fig. 1C). Capacitative Ca 21 entry was more intensely induced by the addition of DBHQ (50 mM) to CFS (Fig. 1B). The averaged peak amplitude of the rapid rise in F340/F380 was 1:31 ^ 0:22 (n 9, Fig. 1C). The effects of SK&F 96365, Zn 21 and Ni 21 were examined on both of the Ca 21 rises induced by DBHQ with CFS
and by CFS alone. The Ca 21 rise by CFS alone was decreased to 39:7^2:2%, 6:0^1:0% and 6:5 ^ 0:8% of the control responses by SK&F 96365 (10 mM), Zn 21 (1 mM) and Ni 21 (5 mM), respectively, (n 4, Fig. 1D, open columns). The Ca 21 rise by DBHQ (50 mM) with CFS was decreased to 51:5^5:2%, 12:3 ^ 2:5% and 10:0^1:1% of the control responses by SK&F 96365, Zn 21 and Ni 21, respectively, (n 4, Fig. 1D, closed columns). The effect of genistein was also examined on both of the Ca 21 rises induced by DBHQ with CFS and by CFS alone. The Ca 21 rise by CFS alone was markedly inhibited by genistein (100 mM, Fig. 2A(b)) and the averaged peak amplitude was reduced to 28:3^1:5% (n 4) of the control response (Fig. 2C, open columns). Surprisingly, after wash out of genistein, the recovery response was increased to 276:7^26:9% of the control response (Fig. 2A(c), Fig.
Fig. 1. (A) Ca 21 ¯uorescence responses to the application of a Ca 21-free solution in a neural retina of an E3 chick embryo. The Ca 21-free solution was applied for the period indicated by the white bar (10 min). NBS: normal bath solution containing 2.5 mM Ca 21. Upper and lower traces were continuously recorded. (B) Ca 21 ¯uorescence responses to the application of DBHQ with a Ca 21-free solution. DBHQ (50 mM) was applied for the period indicated by the thinner bar. (C) The mean amplitudes of the rise in F340/F380 (DF340/F380) as measured from foot to top when the bath solutions were changed from a Ca 21-free solution to NBS without DBHQ (left column, n 9) and with DBHQ (right column, n 9). Error bars indicate SD. (D) The inhibitory effects of SK&F 96365 (10 mM), Zn 21 (1 mM) and Ni 21 (5 mM) on the rise in F340/F380 when the bath solutions were changed from a Ca 21-free solution to NBS without DBHQ (open column, n 4) and with DBHQ (closed column, n 4). The mean percentages of relative changes in F340/F380 are indicated: (*) P , 0:05; (***) P , 0:001).
W.-L. Zhou et al. / Neuroscience Letters 272 (1999) 123±126
125
Fig. 2. The inhibitory effects of genistein (100 mM) on the rise in F340/F380 when the bath solutions were changed from a Ca 21-free solution (applied for 10 min) to NBS without DBHQ (A) and with DBHQ (B). (C) The mean percentages of relative changes in F340/F380 when the bath solutions were changed from a Ca 21-free solution to NBS without DBHQ (open column, n 4) and with DBHQ (closed column, n 4). (*) P , 0:05.
2C, open columns). The Ca 21 rise by DBHQ with CFS was also decreased by genistein (Fig. 2B(b)) to 32:3^2:1% (n 4, Fig. 2C, closed columns) of the control response without any rebound after wash out of genistein (Fig. 2B(c)). On the contrary to the inhibitory effect of genistein, vanadate (1 mM) enhanced the CFS-induced Ca 21 rise by about 4 times (Fig. 3). We found that both of the Ca 21 rises induced by DBHQ with CFS and by CFS alone were inhibited by SK&F 96365, Zn 21 and Ni 21 to almost similar extent. These facts could indicate that the same Ca 21 entry pathway was involved in
both of the Ca 21 rises. However, the Ca 21 rise by CFS alone was not likely to be evoked by depletion of intracellular Ca 21 stores, since Ca 21 release from intracellular Ca 21 stores is induced even 10 min after the incubation of the retina in CFS [8,11]. We further found that quenching of fura-2 ¯uorescence by Mn 21 in¯ux occurred immediately after decreasing [Ca 21]0 and this quenching was blocked by SK&F 96365 (data not shown), suggesting that the depletion of Ca 21 stores is not absolutely necessary for the induction of capacitative Ca 21 entry. Genistein inhibited both of the Ca 21 rises induced by
Fig. 3. The enhancement by vanadate (sodium orthovanadate, Na3VO4, 1 mM) of the rise in F340/F380 when the bath solutions were changed from a Ca 21-free solution (applied for 10 min) to NBS (A). (B) The mean percentages of relative changes in F340/F380 when the bath solutions were changed from a Ca 21-free solution to NBS (n 4). (*) P , 0:05.
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W.-L. Zhou et al. / Neuroscience Letters 272 (1999) 123±126
DBHQ with CFS and by CFS alone to similar extent. These facts suggested that tyrosine kinases are involved in the activation of capacitative Ca 21 entry. To our surprise, a much larger Ca 21 rise than the control was evoked by CFS alone after wash out of genistein and this Ca 21 rise showed almost the same size as that induced by DBHQ with CFS (cf. Fig. 2A(c),B(a)). This could be explained by rebound activity of tyrosine kinases. The inhibitory effect of genistein on capacitative Ca 21 entry has been also shown in rat glioma C6 cells [9]. On the contrary, the proteinphosphotyrosine phosphatase inhibitor, vanadate clearly enhanced the CFS-induced Ca 21 entry. These results could suggest that tyrosine phosphorylation is involved in the activation of capacitative Ca 21 entry. [1] Berridge, M.J., Capacitative calcium entry. Biochem. J., 312 (1995) 1±11. [2] Gordon, J.A., Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol., 201 (1991) 477± 482. [3] Merritt, J.E., Armstrong, W.P. and Benham, C.D., SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem. J., 271 (1990) 515±522.
[4] Moore, G.A., McConkey, D.J., Kass, G.E.N., O'Brien, P.J. and Orrenius, S., 2,5-Di(tert-butyl)-1,4-benzohydroquinone ± a novel inhibitor of liver microsomal Ca 21 sequestration. FEBS Lett., 224 (1987) 331±336. [5] Putney Jr, J.W. and St. J. Bird, G., The inositol phosphatecalcium signaling system in non-excitable cells. Endocr. Rev., 14 (1993) 610±631. [6] Sakaki, Y., Fukuda, Y. and Yamashita, M., Muscarinic and purinergic Ca 21 mobilizations in the neural retina of early embryonic chick. Int. J. Dev. Neurosci., 14 (1996) 691±699. [7] Sakaki, Y., Sugioka, M., Fukuda, Y. and Yamashita, M., Capacitative Ca 21 in¯ux in the neural retina of chick embryo. J. Neurobiol., 32 (1997) 62±68. [8] Sugioka, M., Fukuda, Y. and Yamashita, M., Ca 21 responses to ATP via purinoceptors in the early embryonic chick retina. J. Physiol. (Lond.), 493 (1996) 855±863. [9] Takemura, H., Sakano, S., Kaneko, M. and Ohshika, H., Inhibitory effects of tyrosine kinase inhibitors on capacitative Ca 21 entry in rat glioma C6 cells. Life Sci., 62 (1998) 271± 276. [10] Yamashita, M. and Sugioka, M., Calcium mobilization systems during neurogenesis. News Physiol. Sci., 13 (1998) 75±79. [11] Zhou, W.L., Sugioka, M. and Yamashita, M., Lysophosphatidic acid-induced Ca 21 mobilization in the neural retina of chick embryo. J. Neurobiol., (1999) in press.