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Effects of X-irradiation on the calcium channel of the mouse oocyte
Life Sciences, Vol. 60, No. 16, pp. 1377-1383, 1597 Copyright @ 1997 Elseier Science Inc. Printed in the USA. All rights rescind 0024-3205/97 $17.00 t .oo
ELSEVIER
PI1 SOO24-3205(97)00083-O
EFFECTS
OF X-IRRADIATION ON THE CALCIUM OF THE MOUSE OOCYIE
CHANNEL
Shigeru Yoshida Department of Physiology,
Nagasaki University School of Medicine, Nagasaki 852, Japan. (Received in final form January 17, 1997)
Effects of a single whole-body X-irradiation (0.2-100 Gy) were studied on Ca2+ channels in mature mouse oocytes using the two-electrode voltage-clamp method. No significant changes were observed in the electrophysiological properties of oocytes when the dose of X-rays was smaller than 10 Gy. However, the following changes, dependent on the dose of X-rays and on the time after irradiation, were observed with higher doses. 1) The resting potential and the input resistance of the oocyte were slightly reduced. 2) Inward caZ+ current was reduced in amplitude but the shape of the I-V (current-to-voltage) relations were preserved. 3) Both the activation and the inactivation processes of the W+ current became slower. These changes in the kinetics of the Ca2+ channel were observed even when no appreciable change was detected in the amplitude of the Cal+ current. 4) The steady-state inactivation curve of the Ca2+ current was shifted in a depolarizing direction and the slope of the curve became steeper. These data indicate that high doses of X-rays can affect Ca2+ channels in mouse oocytes and that the kinetics of the Ca2+ channel are more susceptible to irradiation than the amplitude of the Ca2+ current. Key Words: X-irradiation, calcium channel, mouse, oocyte
X-rays are widely used and are an indispensable tool in the diagnosis and therapy of diseases. A considerable amount of irradiation is sometimes necessary for the radiotherapy of malignant diseases. For example, 70 Gy X-irradiation has been used for treating lung cancer (1). During such treatment it is inevitable for some of the surrounding normal tissue to be exposed to high-doses of X-rays. Thus, effects of X-irradiation on animal cells, especially induced morphological changes, have been intensively studied. However, there are not many reports on the effect of X-rays on the dynamic functional properties of animal cells, especially not on the properties of ion channels, except for a few papers on Na+ channels (2-4). As for the effects of X-rays on Czi2+ channels, which are deeply involved in the regulation of the cell function by changing the intracellular concentration of free CL@+(5), there seems to be no available information. Hence the present study was designed to assess the effects of X-rays on the Ca2+ charmel of the mouse oocyte. This is a particularly suitable tissue to use because Ca2+ channels are the major type of ion channel present in mouse oocytes and no Na+ channels are present (6-9).
Present address: Department of Physiology,
Fukui Medical School, Fukui 910-11, Japan
1378
Vol. 60, No. 16, 1997
Effects of X-rays on Calcium Channels
USE GGCYIESZ Mature female mice (ICR strain, older than 8 weeks) were exposed, from the abdominal side, to a single whole-body X-irradiation of 0.2-100 Gy with the focal distance of 350 mm. X-rays were generated at 200 kV, 20 mA filtered by 0.5 mm copper and 0.5 mm aluminum, resulting in a I-IVL (half-value layer) of 0.6 mm copper. OF MQUSEOOCYTES: Oocytes were collected just before electrophysiological study; from the ampullary part of the oviduct of mice under ether anaesthesia; between 10 min and 50 h after X-irradiation. Mice were superovulated by intraperitoneal injections of PMSG (pregnant mare serum gonadotropin, Sankyo, Japan) and HCG (human chorionic gonadotropin, Sankyo) as described elsewhere (6). In order to facilitate the insertion of glass microelectrodes, collected oocytes were freed from surrounding follicular cells using 0.4 mgfml hyaluronidase (Type I-S, Sigma) for 25 min at room temperature (22-24”(Z). Hyaluronidase was dissolved in a solution consisting of: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgC&, 5 mM PIPES and 10 mM glucose (pH 7.4). EXPERIMENTAL Since mouse oocytes have voltage-dependent Caz+ channels but no Na+ channels (6-9), electrophysiological study was carried out using a solution containing high-Caz+ and no Na+ in order to evaluate the function of Ca2+ channels and Na+ was substituted by the membrane-impermeant cation TMA+ (tetramethylammonium+, Nakarai Chemicals, Japan). The composition of this experimental solution was 20 mM CaC12, 5 mM KCl, 1 mM MgC12, 113 mM ‘I&IA-Cl, 10 mM glucose, 5 r&I PIPES and 5 mg/ml BSA (bovine serum albumin, Sigma) (pH 7.4). ELECTROPHYSIOLOGY: Conventional two-electrode voltage-clamp recording was performed using a home-made voltage-clamp amplifier. The DC resistance of the glass microelectrodes was lo20 MS2 when filled with 3M-KCl. All experiments were carried out at room temperature (22-24’C).
B
A
7
C
I====-.
, --
--_
-6
_-
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I
100 mV
5*
50 ms
Fig. 1 Families of Ca*+ currents (lower traces) recorded from non-irradiated control (A) and X-irradiated (B-C) oocytes of the mouse under voltage-clamp conditions. Currents shown in B were recorded from an oocyte of a mouse which was exposed to a single whole-body irradiation of 50 Gy X-rays and then maintained for 28 h, and those in C from a mouse irradiated with 100 Gy and preserved for 6 h. Upper traces show the potential steps applied to the oocytes. The holding potential was -90 mV for all oocytes. The dashed lines indicate the zero-current level, and current and time calibrations are the same for all records.
G PGTWTIAI I AND QlE_INPUT RESISTANCE- The resting potential and the input resistance of non-irradiated control oocytes, measured in the experimental solution, were -40.5 f 5.0 mV and 167.8 + 33.1 MS2 (mean 2 SD., n = lo), respectively. These values were significantly reduced only in mouse oocytes which were exposed to excessive amount of X-rays and then maintained for longer than 2 days (p < 0.01, two-tailed Cochran-Cox’s r-test), which is in agreement with results reported by Schwarz and Fox for frog sciatic nerves (4). For example, the
Vol. 60,No. 16, 1997
Effects of X-rays on Calcium Channels
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present data show that the average resting potential and the input resistance of oocytes 4-8 h after exposure to 100 Gy were -36.8 k 4.6 mV and 143.0 2 28.5 MS2 (n = 6), not significantly different from those of control oocytes. However, the resting potential became significantly smaller (-32.2 2 3.4 mV, n = 5) by 2 days after 100 Gy exposure. The input resistance of oocytes was similarly affected by large-dose X-rays also in a time-dependent manner. For example, the mean input resistance of mouse oocytes 2 days after irradiation with 100 Gy was reduced to 85.2 2 19.2 MQ (n = 5).
164 6p ii
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0
10
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20
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30
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50
Time after X-irradiation
1
60 (h)
Fig. 2 Dose- and time-dependent effects of X-rays on the amplitude of the inward C.W+ current in mouse oocytes (holding potential = -90 mV). Each point represents the average value of the maximum amplitude of the inward Caz+ current, and attached bars indicate standard deviation. The open circle (0) shows non-irradiated control oocytes (n = lo), and the other symbols indicate oocytes which were exposed to X-rays of 10 Gy (II), 50 Gy (a), 75 Gy (I) and 100 Gy (A) (n = 5, for each dose of X-rays). Abscissa shows the time after exposure to a single whole-body X-irradiation. OF &RAYSCW CURBEPJE In order to assess the properties of Caz+ channels, pure Caz+ currents were measured in the experimental solution which contained 20 mM Ca*+ and no Na+ under voltage-clamp conditions (Fig. 1A). When the applied dose of X-rays was less than 10 Gy, no significant change was observed in current traces recorded from irradiated oocytes (not illustrated). At higher doses, Ca*+ currents were affected as displayed in Fig. lB-C, showing that the amplitude of the Ca*+ current was reduced by X-irradiation. Oocytes were exposed to 10,50,75 and 100 Gy and the effects of X-rays were examined at 0.5-2,4-8,25-31 and 45-51 h after irradiation (n = 4-6 at each intensity and time point). The time course of the averaged maximum amplitude of the Ca*+ current is displayed in Fig. 2 for different doses of X-rays. Since no significant change was observed in mouse oocytes irradiated with less than 10 Gy, such data are omitted from the graph. The control value of the maximum amplitude of the Ca*+ current obtained from non-irradiated oocytes was -4.95 2 0.51 nA (n = lo), and the value was decreased depending on dose of X-rays and time after irradiation (n = 5 for irradiated samples). Another prominent change was detected in the kinetics of the Ca*+ channel. As shown in Fig. 1, the time course of Ca*+ currents was slowed down in X-irradiated oocytes. The parameters “time-to-peak” and the “half-decay time” were measured for evaluating the activation and inactivation processes of the Ca*+ current. These values in non-irradiated control oocytes were 7.08 * 1.11 ms and 9.98 f 1.21 ms, respectively (n = 13) (Fig. 1A). In oocytes approximately 2 days after exposure to 100 Gy X-rays, the values were significantly prolonged to 13.68 f 4.71 ms and 28.00 2 9.60 ms @ < 0.001, n = 12). It is to be noted that the time course of the Ca*+ current was similarly slowed down even though no apparent change was observed in the amplitude of the current (Fig. 1B). For instance, the mean values of “time-to-peak” and the “half-decay time” were prolonged to 9.79 f 1.85 ms and 12.93 2 1.97 ms (n
Vol.60, No. 16, 1997
Effects of X-rayson CalciumChannels
1380
= 7) at about 24 h after irradiation with 10 Gy @ c 0.01). With a dose lower than 10 Gy, no significant change was observed in the channel kinetics. I-V RET.m*‘CURRENT: Although the maximum amplitude of the Ca*+current was reduced by X-irradiation, the I-V (current-to-voltage) relations of the peak Ca*+current was not substantially affected as shown in Fig. 3. Both in control and irradiated oocytes (n = 4), the maximum value of the peak Ca*+current was attained around -15 mV, and the potential at which the Ca*+current became half maximum was around -30 mV (6). For further analysis of X-irradiated Cal+ channels, the steady-state inactivation process of the inward Ca*+current was examined by stepping from various conditioning pulses (duration = 10 s) to a constant test pulse of -15 mV around which the maximum current was obtained (see Fig. 3). Fig. 4 plots the normalized peak amplitude of the Ca*+current against the conditioning potential, i.e. the size of the current was divided by the largest observed current amplitude. Experimental plots were fitted by a curve calculated from the Boltzmann distribution relation: h, = (1 t exp[(V - V&J).* where h, is the normalized Ca*+current amplitude, V is the holding potential, Vt, is the potential at which h, becomes 0.5, and k is the slope parameter (10). The Vi, and the k values measured in control oocytes were -53.97 f 1.96 mV and 4.65 f 0.31 mV (n = 9), respectively. Again, no significant change was observed in the steady-state inactivation curve of the Ca*+current when the dose of X-rays was less than 10 Gy. However, the curve was shifted along the voltage axis to more positive values without appreciably changing its shape when the dose was elevated up to 50 Gy. For example, Vt, and k were -45.65 + 1.74 mV and 4.64 f 0.67 mV at 26-30 h after irradiation with 50 Gy (n = 5). Moreover, the slope of the inactivation curve became steeper when the dose was further increased. The Vt, and k values became -45.73 2 2.19 mV and 4.06 & 0.32 mV 25-31 h after exposure to 100 Gy (n = 6). -100
O-
-80
-60
-40
-20
0
20
40 V (mv)
-2 -
-4 -
-6 1 @A) 1 Fig. 3 Averaged I-V (current-to-voltage) relations of the peak Ca*+current recorded from control oocytes (O-O) and X-irradiated oocytes, 26-30 h after exposure to 50 Gy (O-O), and 25-31 h after exposure to 100 Gy (A-A). Vertical lines attached to each symbol represent the standard deviation of the mean (n = 5). Ordinate: Peak value of the inward Can+current. Abscissa: Membrane potential.
The aim of the present study was to evaluate the effects of a single whole-body X-irradiation on the dynamic properties of Ca*+channels which are important in the regulation of various types of cell functions (5), since to my knowledge no such reports have been made previously. The data
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1381
presented here would be helpful for estimating the alterations in cell functions in radiotherapy and accidental cases of human exposure to radiation. Such accidental cases usually occur as a single whole-body irradiation. A representative example is the explosion at the atomic power-station at Chernobyl in 1986. The workers were estimated to be exposed to OS-12 Gy radiation, and some of them survived longer than 30 days even exposed to 9-10 Gy (11). Atomic bomb explosions are another case of single whole-body irradiation of much higher dose. It was evaluated that the dose of irradiation at a distance of 100 m from the hypocenter of the atomic bomb explosion was as large as 150 Gy in Hiroshima and 310 Gy in Nagasaki (12).
hm 1.0
0.5
0 -90
-80
-70
-60
-50
-40
-30
ww
Fig. 4 Effects of X-rays on the steady-state inactivation process of Ca*+channels in mouse oocytes. Ca*+ currents were obtained by changing the membrane potential from various conditioning potentials to a test potential of -15 mV. Symbols represent non-irradiated control oocytes (0 -O), oocytes 28 h after exposure to 50 Gy X-rays (O- -O), and oocytes 25.5 h after exposure to 100 Gy (A-A). Curves were fitted according to the Boltzrnann distribution relation. In the present work, oocytes were obtained from mice which had been exposed to a single whole-body X-irradiation of 0.2-100 Gy. It is already known that irradiation on oocytes produces oocyte loss in the ovary (13) and premature menopause (14). The present data indicate that Ca*+ channels are considerably resistant to acute radiation exposure and that only high doses, greater than 10 Gy, of X-rays resulted in measurable effects on Ca*+ channels. The suppressive effect of irradiation on the amplitude of the Ca2+current was dependent not only on the dose of X-rays but also on the time after irradiation (Figs. 1 and 2). However, the kinetics of the Ca*+channel are more sensitive to X-rays than the current amplitude. Both the activation and inactivation processes were slowed down at lower doses than those required to reduce the amplitude (Fig. lB-C). Timedependent effects of X-irradiation on the CM+channel suggest that X-rays do not directly modify the structure of the Ca*+channel but may indirectly induce some chemical reactions which lead to the modification of the channel, as has been proposed for the Na+ channel (4). The present work suggests that X-ray exposure may ultimately lead to a modification in the structure of the Ca*+ channel since the observed changes in the steady-state inactivation curve (Fig. 4) which could indicate a modification of amino acid side chains in the channel protein (4, 15). It should be noted that the oocytes used in the present experiments were in a state of recently resumed meiotic division and do not necessarily reflect the physiology of other types of cells which are in an arrested or mitotically dividing state. Studies on Na+ channels show that they are more resistant to X-rays than Ca*+ channels. Sodium currents were reduced by X-rays at doses in excess of 80 Gy in frog sciatic nerves (4) and by gamma radiation beyond a threshold dose of 200-300 Gy in neuroblastoma (N18) cells (3). Also, Gaffey (2) reported that as much as 1,600-2,000 Gy of X-rays were necessary to attenuate the
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Effects of X-rayson CalciumChannels
Vol.60, No. 16, NT7
amplitude of compound action potentials in frog sciatic nerves. The duration of the compound action potential, in contrast, was prolonged when the dose was beyond 100-200 Gy (2). This finding that the kinetics of the Na+ channel were more susceptible to irradiation than its amplitude is compatible with the present data for the Caz+channel. The reason for Caz+ channels being more susceptible to X-rays than Na+ channels is unknown, but the reported size of the Caz+ channel (m.w. 447,000) (16) is larger than that of the Na+ channel (m.w. 290,000) (17). This size difference may account for some of the difference in the sensitivity of Ca2+ and Na+ channels to X-rays assuming that the possibility of capturing incoming irradiation increases with channel size. Furthermore, it is said that undifferentiated cells including mammalian oocytes are highly susceptible to radiation. For example, chromosome anomalies can be induced in mouse oocytes by X-rays less than 1 Gy (18-20). It is also known that Ca2+ channels are abundant in undifferentiated cells and the number of Ca2+ channels decreases during development whereas that of Na+ channels increases with differentiation of cells (21). Considering that the present data indicate that X-rays may indirectly induce chemical reactions leading to the modification of Ca2+channels, it is suggested that undifferentiated cells are more prone to develop such intracellular chemical reactions than differentiated cells. Another possibility is that there would be an increased entry of Caz+ into irradiated oocytes: the toxic effects of high-level intracellular Car+ have been reported in various types of cells (22). The present work has shown that the prolongation of the channel kinetics appears earlier than the reduction of the current amplitude. This change in the channel kinetics would lead to the entry Car+ via Ca2+channels: compare the area under the curves illustrated in Fig. 1A and B. Thus, the time-dependent late effect of X-rays, i.e. reduction in the current amplitude (Fig. 2), might be ascribed to the toxic effect of Cal+ which would gradually build up inside the cell. Moreover, it is known that Ca2+ plays an important role in fertilization of oocytes (23-24). A series of repetitive Ca2+ transients triggered by the sperm at fertilization causes completion of meiosis and a block to polyspermy. This increase in the intracellular Ca2+ concentration is supported by an entry of Car+ from extracellular space. It is therefore speculated that X-rays affect fertilization by decreasing the amplitude of the Caz+ current and by changing the kinetics of the Ca2+channel.
I thank Prof. H. Okumura for the use of his facility to irradiate mice with X-rays; Drs. S. Macmillan and A. J. Pennington for their comments on the manuscript; Mr. M. Yogata and Mrs. N. Momosaki for preparing the figures. The work was supported by the Grant-in-Aids for Scientific Research from the Ministry of Education, Science and Culture of Japan.
1. 2. :* 5: 6. :* 9: 10. :;: 13. 14.
G.S. SIBLEY, A.J. MUNDT, C. SHAPIRO, R. JACOBS, G. CHEN, R. WEICHSELBAUM and S. VIJAYAKUMAR, Int. J. Radiat. Oncol. Biol. Phys. 3Z lOOl1007 (1995). C.T. GAFFEY, Radiat. Res. &311-325 (1971). J.E. FRESCHI and A. MORAN, B&him. Biophys. Acta 85831-37 (1986). W. SCHWARZ and J.M. FOX, Experientia 35 1200-1201(1979). R.W. TSIEN and R.Y. TSIEN, Ann. Rev. Cell Biol. 4715-760 (1990). H. OKAMOTO, K. TAKAHASHI and N. YAMASHITA, J. Physiol. (Land.) 2h2.465-495 f.‘z&ES J Physiol (Lond ) 3%. 573-588 (1987) S. YOSHIDA, J. Physiol. (L&d.) 33$!631-642 (1983). S . YOSHIDA, Dev. Biol. lBl200-206 (1985). N. YAMASI-IITA,J. Physiol. (Lond.) 329263-280 (1982). A.K. GUSKGVA and A.E. BARANOV, Hematology Rev: 39-21(1989). . Of
-search_ R.L. DOBSON and J.S. FELTON, Am. J. Ind. Med. 4175-190 (1983). M.D. DAMEWOODand L.B. GROCHOW, Fertil. Steril. 45443-459 (1986).
.
.
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17.
18. 19. 20. ;:: 23. 24.
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M. RACK, S. HU, N. RUBLY and C. WASCHOW, Pfltigers Archiv 4Qa403-408 (1984). T. TANABE, H. TAKESHIMA, A. MIKAMI, V. FLOCKERZI, H. TAKAHASHI, K. KANAGAWA, M. KOJIMA, H. MATSUO, T. HIROSE and S. NUMA, Nature 328 313318 (1987). M. NODA, S. SHIMIZU, T. TANABE, T. TAKAI, T. KAYANO, T. IKEDA, H. TAKAHASHI, H. NAKAYAMA, Y.KANAOKA, N. MINAMINO, K. KANAGAWA, H. MATSUO, M.A. RAFIERY, T. HIROSE, S. INAYAMA, H. HAYASHIDA, T. MIYATA and S. NUMA, Nature ?t12 121-127 (1984). C.S. GRIFFIN, C. TEASE and G. FISHER, Mutat. Res. 2X137-142 (1990). I. HANSMANN, J. JENDERNY and H.D.PROBECK, Hum. Genet. f&190-192 (1982). C. TEASE and G. FISHER, Mutat. Res. m211-215 (1986). S. YOSHIDA, Cell. Mol. Neurobiol. 14227-244 (1994). S. ORRENIUS, D.J. McCONKEY and P. NICOTERA, Adv. Exp. Med. Biol. 28.3419-425 (1991). M. WHITAKER and R. PATEL, Development l.Q&525-542 (1990) S. MIYAZAKI, Jpn. J. Physiol. 43 409-434 (1993)
ELSEVIER
PI1 SOO24-3205(97)00083-O
EFFECTS
OF X-IRRADIATION ON THE CALCIUM OF THE MOUSE OOCYIE
CHANNEL
Shigeru Yoshida Department of Physiology,
Nagasaki University School of Medicine, Nagasaki 852, Japan. (Received in final form January 17, 1997)
Effects of a single whole-body X-irradiation (0.2-100 Gy) were studied on Ca2+ channels in mature mouse oocytes using the two-electrode voltage-clamp method. No significant changes were observed in the electrophysiological properties of oocytes when the dose of X-rays was smaller than 10 Gy. However, the following changes, dependent on the dose of X-rays and on the time after irradiation, were observed with higher doses. 1) The resting potential and the input resistance of the oocyte were slightly reduced. 2) Inward caZ+ current was reduced in amplitude but the shape of the I-V (current-to-voltage) relations were preserved. 3) Both the activation and the inactivation processes of the W+ current became slower. These changes in the kinetics of the Ca2+ channel were observed even when no appreciable change was detected in the amplitude of the Cal+ current. 4) The steady-state inactivation curve of the Ca2+ current was shifted in a depolarizing direction and the slope of the curve became steeper. These data indicate that high doses of X-rays can affect Ca2+ channels in mouse oocytes and that the kinetics of the Ca2+ channel are more susceptible to irradiation than the amplitude of the Ca2+ current. Key Words: X-irradiation, calcium channel, mouse, oocyte
X-rays are widely used and are an indispensable tool in the diagnosis and therapy of diseases. A considerable amount of irradiation is sometimes necessary for the radiotherapy of malignant diseases. For example, 70 Gy X-irradiation has been used for treating lung cancer (1). During such treatment it is inevitable for some of the surrounding normal tissue to be exposed to high-doses of X-rays. Thus, effects of X-irradiation on animal cells, especially induced morphological changes, have been intensively studied. However, there are not many reports on the effect of X-rays on the dynamic functional properties of animal cells, especially not on the properties of ion channels, except for a few papers on Na+ channels (2-4). As for the effects of X-rays on Czi2+ channels, which are deeply involved in the regulation of the cell function by changing the intracellular concentration of free CL@+(5), there seems to be no available information. Hence the present study was designed to assess the effects of X-rays on the Ca2+ charmel of the mouse oocyte. This is a particularly suitable tissue to use because Ca2+ channels are the major type of ion channel present in mouse oocytes and no Na+ channels are present (6-9).
Present address: Department of Physiology,
Fukui Medical School, Fukui 910-11, Japan
1378
Vol. 60, No. 16, 1997
Effects of X-rays on Calcium Channels
USE GGCYIESZ Mature female mice (ICR strain, older than 8 weeks) were exposed, from the abdominal side, to a single whole-body X-irradiation of 0.2-100 Gy with the focal distance of 350 mm. X-rays were generated at 200 kV, 20 mA filtered by 0.5 mm copper and 0.5 mm aluminum, resulting in a I-IVL (half-value layer) of 0.6 mm copper. OF MQUSEOOCYTES: Oocytes were collected just before electrophysiological study; from the ampullary part of the oviduct of mice under ether anaesthesia; between 10 min and 50 h after X-irradiation. Mice were superovulated by intraperitoneal injections of PMSG (pregnant mare serum gonadotropin, Sankyo, Japan) and HCG (human chorionic gonadotropin, Sankyo) as described elsewhere (6). In order to facilitate the insertion of glass microelectrodes, collected oocytes were freed from surrounding follicular cells using 0.4 mgfml hyaluronidase (Type I-S, Sigma) for 25 min at room temperature (22-24”(Z). Hyaluronidase was dissolved in a solution consisting of: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgC&, 5 mM PIPES and 10 mM glucose (pH 7.4). EXPERIMENTAL Since mouse oocytes have voltage-dependent Caz+ channels but no Na+ channels (6-9), electrophysiological study was carried out using a solution containing high-Caz+ and no Na+ in order to evaluate the function of Ca2+ channels and Na+ was substituted by the membrane-impermeant cation TMA+ (tetramethylammonium+, Nakarai Chemicals, Japan). The composition of this experimental solution was 20 mM CaC12, 5 mM KCl, 1 mM MgC12, 113 mM ‘I&IA-Cl, 10 mM glucose, 5 r&I PIPES and 5 mg/ml BSA (bovine serum albumin, Sigma) (pH 7.4). ELECTROPHYSIOLOGY: Conventional two-electrode voltage-clamp recording was performed using a home-made voltage-clamp amplifier. The DC resistance of the glass microelectrodes was lo20 MS2 when filled with 3M-KCl. All experiments were carried out at room temperature (22-24’C).
B
A
7
C
I====-.
, --
--_
-6
_-
i-
pis=yF
I
100 mV
5*
50 ms
Fig. 1 Families of Ca*+ currents (lower traces) recorded from non-irradiated control (A) and X-irradiated (B-C) oocytes of the mouse under voltage-clamp conditions. Currents shown in B were recorded from an oocyte of a mouse which was exposed to a single whole-body irradiation of 50 Gy X-rays and then maintained for 28 h, and those in C from a mouse irradiated with 100 Gy and preserved for 6 h. Upper traces show the potential steps applied to the oocytes. The holding potential was -90 mV for all oocytes. The dashed lines indicate the zero-current level, and current and time calibrations are the same for all records.
G PGTWTIAI I AND QlE_INPUT RESISTANCE- The resting potential and the input resistance of non-irradiated control oocytes, measured in the experimental solution, were -40.5 f 5.0 mV and 167.8 + 33.1 MS2 (mean 2 SD., n = lo), respectively. These values were significantly reduced only in mouse oocytes which were exposed to excessive amount of X-rays and then maintained for longer than 2 days (p < 0.01, two-tailed Cochran-Cox’s r-test), which is in agreement with results reported by Schwarz and Fox for frog sciatic nerves (4). For example, the
Vol. 60,No. 16, 1997
Effects of X-rays on Calcium Channels
1379
present data show that the average resting potential and the input resistance of oocytes 4-8 h after exposure to 100 Gy were -36.8 k 4.6 mV and 143.0 2 28.5 MS2 (n = 6), not significantly different from those of control oocytes. However, the resting potential became significantly smaller (-32.2 2 3.4 mV, n = 5) by 2 days after 100 Gy exposure. The input resistance of oocytes was similarly affected by large-dose X-rays also in a time-dependent manner. For example, the mean input resistance of mouse oocytes 2 days after irradiation with 100 Gy was reduced to 85.2 2 19.2 MQ (n = 5).
164 6p ii
I
4-1 2-
0'
)
I
0
10
I
20
I
30
I
40
I
50
Time after X-irradiation
1
60 (h)
Fig. 2 Dose- and time-dependent effects of X-rays on the amplitude of the inward C.W+ current in mouse oocytes (holding potential = -90 mV). Each point represents the average value of the maximum amplitude of the inward Caz+ current, and attached bars indicate standard deviation. The open circle (0) shows non-irradiated control oocytes (n = lo), and the other symbols indicate oocytes which were exposed to X-rays of 10 Gy (II), 50 Gy (a), 75 Gy (I) and 100 Gy (A) (n = 5, for each dose of X-rays). Abscissa shows the time after exposure to a single whole-body X-irradiation. OF &RAYSCW CURBEPJE In order to assess the properties of Caz+ channels, pure Caz+ currents were measured in the experimental solution which contained 20 mM Ca*+ and no Na+ under voltage-clamp conditions (Fig. 1A). When the applied dose of X-rays was less than 10 Gy, no significant change was observed in current traces recorded from irradiated oocytes (not illustrated). At higher doses, Ca*+ currents were affected as displayed in Fig. lB-C, showing that the amplitude of the Ca*+ current was reduced by X-irradiation. Oocytes were exposed to 10,50,75 and 100 Gy and the effects of X-rays were examined at 0.5-2,4-8,25-31 and 45-51 h after irradiation (n = 4-6 at each intensity and time point). The time course of the averaged maximum amplitude of the Ca*+ current is displayed in Fig. 2 for different doses of X-rays. Since no significant change was observed in mouse oocytes irradiated with less than 10 Gy, such data are omitted from the graph. The control value of the maximum amplitude of the Ca*+ current obtained from non-irradiated oocytes was -4.95 2 0.51 nA (n = lo), and the value was decreased depending on dose of X-rays and time after irradiation (n = 5 for irradiated samples). Another prominent change was detected in the kinetics of the Ca*+ channel. As shown in Fig. 1, the time course of Ca*+ currents was slowed down in X-irradiated oocytes. The parameters “time-to-peak” and the “half-decay time” were measured for evaluating the activation and inactivation processes of the Ca*+ current. These values in non-irradiated control oocytes were 7.08 * 1.11 ms and 9.98 f 1.21 ms, respectively (n = 13) (Fig. 1A). In oocytes approximately 2 days after exposure to 100 Gy X-rays, the values were significantly prolonged to 13.68 f 4.71 ms and 28.00 2 9.60 ms @ < 0.001, n = 12). It is to be noted that the time course of the Ca*+ current was similarly slowed down even though no apparent change was observed in the amplitude of the current (Fig. 1B). For instance, the mean values of “time-to-peak” and the “half-decay time” were prolonged to 9.79 f 1.85 ms and 12.93 2 1.97 ms (n
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= 7) at about 24 h after irradiation with 10 Gy @ c 0.01). With a dose lower than 10 Gy, no significant change was observed in the channel kinetics. I-V RET.m*‘CURRENT: Although the maximum amplitude of the Ca*+current was reduced by X-irradiation, the I-V (current-to-voltage) relations of the peak Ca*+current was not substantially affected as shown in Fig. 3. Both in control and irradiated oocytes (n = 4), the maximum value of the peak Ca*+current was attained around -15 mV, and the potential at which the Ca*+current became half maximum was around -30 mV (6). For further analysis of X-irradiated Cal+ channels, the steady-state inactivation process of the inward Ca*+current was examined by stepping from various conditioning pulses (duration = 10 s) to a constant test pulse of -15 mV around which the maximum current was obtained (see Fig. 3). Fig. 4 plots the normalized peak amplitude of the Ca*+current against the conditioning potential, i.e. the size of the current was divided by the largest observed current amplitude. Experimental plots were fitted by a curve calculated from the Boltzmann distribution relation: h, = (1 t exp[(V - V&J).* where h, is the normalized Ca*+current amplitude, V is the holding potential, Vt, is the potential at which h, becomes 0.5, and k is the slope parameter (10). The Vi, and the k values measured in control oocytes were -53.97 f 1.96 mV and 4.65 f 0.31 mV (n = 9), respectively. Again, no significant change was observed in the steady-state inactivation curve of the Ca*+current when the dose of X-rays was less than 10 Gy. However, the curve was shifted along the voltage axis to more positive values without appreciably changing its shape when the dose was elevated up to 50 Gy. For example, Vt, and k were -45.65 + 1.74 mV and 4.64 f 0.67 mV at 26-30 h after irradiation with 50 Gy (n = 5). Moreover, the slope of the inactivation curve became steeper when the dose was further increased. The Vt, and k values became -45.73 2 2.19 mV and 4.06 & 0.32 mV 25-31 h after exposure to 100 Gy (n = 6). -100
O-
-80
-60
-40
-20
0
20
40 V (mv)
-2 -
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-6 1 @A) 1 Fig. 3 Averaged I-V (current-to-voltage) relations of the peak Ca*+current recorded from control oocytes (O-O) and X-irradiated oocytes, 26-30 h after exposure to 50 Gy (O-O), and 25-31 h after exposure to 100 Gy (A-A). Vertical lines attached to each symbol represent the standard deviation of the mean (n = 5). Ordinate: Peak value of the inward Can+current. Abscissa: Membrane potential.
The aim of the present study was to evaluate the effects of a single whole-body X-irradiation on the dynamic properties of Ca*+channels which are important in the regulation of various types of cell functions (5), since to my knowledge no such reports have been made previously. The data
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presented here would be helpful for estimating the alterations in cell functions in radiotherapy and accidental cases of human exposure to radiation. Such accidental cases usually occur as a single whole-body irradiation. A representative example is the explosion at the atomic power-station at Chernobyl in 1986. The workers were estimated to be exposed to OS-12 Gy radiation, and some of them survived longer than 30 days even exposed to 9-10 Gy (11). Atomic bomb explosions are another case of single whole-body irradiation of much higher dose. It was evaluated that the dose of irradiation at a distance of 100 m from the hypocenter of the atomic bomb explosion was as large as 150 Gy in Hiroshima and 310 Gy in Nagasaki (12).
hm 1.0
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0 -90
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Fig. 4 Effects of X-rays on the steady-state inactivation process of Ca*+channels in mouse oocytes. Ca*+ currents were obtained by changing the membrane potential from various conditioning potentials to a test potential of -15 mV. Symbols represent non-irradiated control oocytes (0 -O), oocytes 28 h after exposure to 50 Gy X-rays (O- -O), and oocytes 25.5 h after exposure to 100 Gy (A-A). Curves were fitted according to the Boltzrnann distribution relation. In the present work, oocytes were obtained from mice which had been exposed to a single whole-body X-irradiation of 0.2-100 Gy. It is already known that irradiation on oocytes produces oocyte loss in the ovary (13) and premature menopause (14). The present data indicate that Ca*+ channels are considerably resistant to acute radiation exposure and that only high doses, greater than 10 Gy, of X-rays resulted in measurable effects on Ca*+ channels. The suppressive effect of irradiation on the amplitude of the Ca2+current was dependent not only on the dose of X-rays but also on the time after irradiation (Figs. 1 and 2). However, the kinetics of the Ca*+channel are more sensitive to X-rays than the current amplitude. Both the activation and inactivation processes were slowed down at lower doses than those required to reduce the amplitude (Fig. lB-C). Timedependent effects of X-irradiation on the CM+channel suggest that X-rays do not directly modify the structure of the Ca*+channel but may indirectly induce some chemical reactions which lead to the modification of the channel, as has been proposed for the Na+ channel (4). The present work suggests that X-ray exposure may ultimately lead to a modification in the structure of the Ca*+ channel since the observed changes in the steady-state inactivation curve (Fig. 4) which could indicate a modification of amino acid side chains in the channel protein (4, 15). It should be noted that the oocytes used in the present experiments were in a state of recently resumed meiotic division and do not necessarily reflect the physiology of other types of cells which are in an arrested or mitotically dividing state. Studies on Na+ channels show that they are more resistant to X-rays than Ca*+ channels. Sodium currents were reduced by X-rays at doses in excess of 80 Gy in frog sciatic nerves (4) and by gamma radiation beyond a threshold dose of 200-300 Gy in neuroblastoma (N18) cells (3). Also, Gaffey (2) reported that as much as 1,600-2,000 Gy of X-rays were necessary to attenuate the
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amplitude of compound action potentials in frog sciatic nerves. The duration of the compound action potential, in contrast, was prolonged when the dose was beyond 100-200 Gy (2). This finding that the kinetics of the Na+ channel were more susceptible to irradiation than its amplitude is compatible with the present data for the Caz+channel. The reason for Caz+ channels being more susceptible to X-rays than Na+ channels is unknown, but the reported size of the Caz+ channel (m.w. 447,000) (16) is larger than that of the Na+ channel (m.w. 290,000) (17). This size difference may account for some of the difference in the sensitivity of Ca2+ and Na+ channels to X-rays assuming that the possibility of capturing incoming irradiation increases with channel size. Furthermore, it is said that undifferentiated cells including mammalian oocytes are highly susceptible to radiation. For example, chromosome anomalies can be induced in mouse oocytes by X-rays less than 1 Gy (18-20). It is also known that Ca2+ channels are abundant in undifferentiated cells and the number of Ca2+ channels decreases during development whereas that of Na+ channels increases with differentiation of cells (21). Considering that the present data indicate that X-rays may indirectly induce chemical reactions leading to the modification of Ca2+channels, it is suggested that undifferentiated cells are more prone to develop such intracellular chemical reactions than differentiated cells. Another possibility is that there would be an increased entry of Caz+ into irradiated oocytes: the toxic effects of high-level intracellular Car+ have been reported in various types of cells (22). The present work has shown that the prolongation of the channel kinetics appears earlier than the reduction of the current amplitude. This change in the channel kinetics would lead to the entry Car+ via Ca2+channels: compare the area under the curves illustrated in Fig. 1A and B. Thus, the time-dependent late effect of X-rays, i.e. reduction in the current amplitude (Fig. 2), might be ascribed to the toxic effect of Cal+ which would gradually build up inside the cell. Moreover, it is known that Ca2+ plays an important role in fertilization of oocytes (23-24). A series of repetitive Ca2+ transients triggered by the sperm at fertilization causes completion of meiosis and a block to polyspermy. This increase in the intracellular Ca2+ concentration is supported by an entry of Car+ from extracellular space. It is therefore speculated that X-rays affect fertilization by decreasing the amplitude of the Caz+ current and by changing the kinetics of the Ca2+channel.
I thank Prof. H. Okumura for the use of his facility to irradiate mice with X-rays; Drs. S. Macmillan and A. J. Pennington for their comments on the manuscript; Mr. M. Yogata and Mrs. N. Momosaki for preparing the figures. The work was supported by the Grant-in-Aids for Scientific Research from the Ministry of Education, Science and Culture of Japan.
1. 2. :* 5: 6. :* 9: 10. :;: 13. 14.
G.S. SIBLEY, A.J. MUNDT, C. SHAPIRO, R. JACOBS, G. CHEN, R. WEICHSELBAUM and S. VIJAYAKUMAR, Int. J. Radiat. Oncol. Biol. Phys. 3Z lOOl1007 (1995). C.T. GAFFEY, Radiat. Res. &311-325 (1971). J.E. FRESCHI and A. MORAN, B&him. Biophys. Acta 85831-37 (1986). W. SCHWARZ and J.M. FOX, Experientia 35 1200-1201(1979). R.W. TSIEN and R.Y. TSIEN, Ann. Rev. Cell Biol. 4715-760 (1990). H. OKAMOTO, K. TAKAHASHI and N. YAMASHITA, J. Physiol. (Land.) 2h2.465-495 f.‘z&ES J Physiol (Lond ) 3%. 573-588 (1987) S. YOSHIDA, J. Physiol. (L&d.) 33$!631-642 (1983). S . YOSHIDA, Dev. Biol. lBl200-206 (1985). N. YAMASI-IITA,J. Physiol. (Lond.) 329263-280 (1982). A.K. GUSKGVA and A.E. BARANOV, Hematology Rev: 39-21(1989). . Of
-search_ R.L. DOBSON and J.S. FELTON, Am. J. Ind. Med. 4175-190 (1983). M.D. DAMEWOODand L.B. GROCHOW, Fertil. Steril. 45443-459 (1986).
.
.
Vol. 60, No. 16, 1997
15. 16.
17.
18. 19. 20. ;:: 23. 24.
Effects of X-rays on Calcium Channels
1383
M. RACK, S. HU, N. RUBLY and C. WASCHOW, Pfltigers Archiv 4Qa403-408 (1984). T. TANABE, H. TAKESHIMA, A. MIKAMI, V. FLOCKERZI, H. TAKAHASHI, K. KANAGAWA, M. KOJIMA, H. MATSUO, T. HIROSE and S. NUMA, Nature 328 313318 (1987). M. NODA, S. SHIMIZU, T. TANABE, T. TAKAI, T. KAYANO, T. IKEDA, H. TAKAHASHI, H. NAKAYAMA, Y.KANAOKA, N. MINAMINO, K. KANAGAWA, H. MATSUO, M.A. RAFIERY, T. HIROSE, S. INAYAMA, H. HAYASHIDA, T. MIYATA and S. NUMA, Nature ?t12 121-127 (1984). C.S. GRIFFIN, C. TEASE and G. FISHER, Mutat. Res. 2X137-142 (1990). I. HANSMANN, J. JENDERNY and H.D.PROBECK, Hum. Genet. f&190-192 (1982). C. TEASE and G. FISHER, Mutat. Res. m211-215 (1986). S. YOSHIDA, Cell. Mol. Neurobiol. 14227-244 (1994). S. ORRENIUS, D.J. McCONKEY and P. NICOTERA, Adv. Exp. Med. Biol. 28.3419-425 (1991). M. WHITAKER and R. PATEL, Development l.Q&525-542 (1990) S. MIYAZAKI, Jpn. J. Physiol. 43 409-434 (1993)