1Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom;2Department of Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550; and 3Special Care Unit of Dentistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-0012, Japan
Submitted 3 October 2002 ; accepted in final form 28 August 2003
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ABSTRACT |
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endothelin receptors; urinary bladder
Two receptor subtypes for endothelin, ETA and ETB, have been described in mammlian tissues (see review in Refs. 1, 24). In the human bladder, Okamoto-Koizumi et al. (30) demonstrated the presence of both ETA and ETB receptors and showed that ETA receptors mediate the mechanical response (29, 30). However, the underlying mechanisms have not yet been examined even in terms of ionic channels in the plasma membrane.
Because the ET-1 produced in various cells within the urinary bladder presumably modulates the bladder tone (8, 34), it is intriguing to explore the mechanisms underlying ET-1-induced detrusor contractions. Furthermore, bladder ET receptor expression and activities are known to change under pathological conditions, such as urethral obstruction (20-22), diabetes (5, 26, 31), and ageing (35). Therefore, it is possible that ET receptors and/or their downstream intracellular mechanisms may become a target for pharmacological intervention in the treatment of urinary tract disorders and incontinence.
To address the receptor subtype and ion channels responsible for ET-1-induced urinary bladder contraction, and because the pig provides a good model for human micturition (17), we carried out whole cell patch-clamp experiments in isolated pig detrusor. We found that inward current oscillation is induced via ETA receptors and that Ca2+-activated Cl- channels are probably responsible for the oscillating current itself. Also, Ca2+ influx through nifedipine-insensitive cation channels seems to play an important role in pacemaking.
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METHODS |
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Membrane current recording. Whole cell membrane currents were recorded using a standard patch-clamp technique, as described previously (17, 33). A patch-clamp amplifier (EPC-7, List, Germany) was operated through a Macintosh computer equipped with an AD/DA converter (ITC-16, INSTRUTECH). The resistance of the patch pipette was 3-4 M, when a Cs+-rich pipette solution was used. After rupture of the cell membrane, the series resistance was normally less than 10 M
. Pig smooth muscle cells used in this investigation had a mean membrane capacitance of 62 ± 12 pF (n = 25). The capacitive surge was electrically compensated, and a cut-off frequency of 10 kHz was applied to reduce noise. Unless otherwise described, the cell membrane was clamped at -60 mV (the holding potential). All experiments were carried out at room temperature (20-24°C).
Solutions and chemicals. The "normal" extracellular solution had the following composition (in mM): 140 NaCl, 6 KCl, 2.4 CaCl2, 12 glucose, and 5 HEPES; pH was adjusted to 7.4 with Tris base. Isosmotic substitution was used for all changes in the ionic composition of the extracellular medium, including solutions used for cell isolation. In the Ca2+-free, Mg2+-free solution used for cell isolation, the osmotic pressure was also adjusted by modifying the NaCl concentration. The composition of the "normal" pipette solution was (in mM) 144 CsCl, 2 MgCl2, 0.025 CaCl2, 0.05 EGTA, 2 ATP, and 10 HEPES/Tris (pH 7.2). The free Ca2+ concentration in the pipette solution was estimated to be 106 nM using a commercial software EQCAL system (ChemCAD, Obernai, France). With the "normal" extracellular and pipette solutions, the equilibrium potential for Cl- ions calculated is -0.5 mV (at 20°C).
The following chemicals, drugs, and enzymes were used in the present study: ATP (disodium salt), N-methyl-D-glucamine (NMDG), nifedipine, niflumic acid, disodium 4,4'-diisothiocyanatostilbene-2,2'disulphonic acid (DIDS), ryanodine, caffeine, EGTA (free acid), trypsin inhibitor (type 1-S), collagenase (type H), papain, and carbamyl choline chloride (carbachol, CCh) from Sigma (St. Louis, MO); and ET-1, BQ-123, BQ-788, and sarafotoxin S6c from Calbiochem (San Diego, CA).
Tension development. Fine strips (1.5-2 mm in length, 0.5-1 mm in diameter) were prepared as previously described (4, 10), transferred to a small chamber (0.2 ml in volume), and attached to a Dynamometer UFI transducer (Harvard Apparatus, Canterbury, UK) for isometric tension measurement at 35-37°C. The strips were strained by applying a 1-g (i.e., 10 mN) weight and allowed to equilibrate for 1 h before the start of the experiments. Drugs were administrated in the desired concentration in the superfusing solution (1.4 ml/min). The data were stored on DAT tapes with a digital audio tape deck through MacLab/8E system (ADInstrument, Chalgrove, UK) coupled with a Macintosh computer.
Statistics. Numerical data are expressed as means ± SD. The averaged amplitude of the inward current oscillation was calculated from the peaks of four to six currents when the amplitude of the oscillations had become stable. When differences between means were evaluated by ANOVA, a P value of <0.05 was taken as a statistically significant difference.
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RESULTS |
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Previous reports showed that two subtypes are recognized in smooth muscle, which are ETA and ETB (2, 14, 16). The effects of BQ-123 and BQ-788 were examined to determine which ET receptor subtype was responsible for the contractile response. As shown in Fig. 2A, in the presence of 1 µM BQ-123, a selective antagonist for ETA receptors, 10 nM ET-1 failed to induce a contraction. After washout of BQ-123 for 60 min, subsequent application of 10 nM ET-1 caused a tonic contraction [0.93 ± 0.02 (n = 6) vs. 1 µM CCh-induced contraction]. It is interesting to note that after the contraction was induced by ET-1, additional application of BQ-123 had little effect on the sustained contraction (Fig. 2B). On the other hand, BQ-788, an ETB receptor antagonist, affected neither resting tone nor ET-1-induced contraction even when preceding ET-1 application (Fig. 2C). Furthermore, we examined a potent ETB agonist, sarafotoxin S6c. Application of 10 nM sarafotoxin S6c induced a transient contraction of much smaller amplitude (0.14 ± 0.06 of 1 µM CCh-induced contraction, n = 6).
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Niflumic acid is known to block Ca2+-activated Cl- (ClCa) channels (12). As described later, the patch-clamp experiments in the present study suggested an important role of ClCa channels in the excitation-contraction (E-C) coupling of pig detrusor contraction via ETA receptors. We therefore examined the effect of niflumic acid on ET-1-induced contraction. As shown in Fig. 3A, after 10 nM ET-1-induced contraction had reached a plateau, niflumic acid was cumulatively applied. During application of 300 µM niflumic acid, ET-1-induced contraction was decreased to 0.16 ± 0.03 of the control (n = 5; Fig. 3B).
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Effects of ET-1 and ET antagonists on whole cell membrane current. With the use of the conventional whole cell patch-clamp technique, the effects of ET-1 were examined in isolated pig detrusor cells. The outward K+ currents were suppressed with Cs+ in the pipette, and intracellular Ca2+ was only weakly buffered (50 µM EGTA and 25 µM CaCl2 resulting in a free Ca2+ concentration of 106 nM). A holding potential of -60 mV was normally applied. In the majority of the cells, application of ET-1 (10 nM) induced a single transient inward current (334 ± 47 pA, n = 80) as shown in Fig. 4A. However, in 39% (n = 51) of cells responding to ET-1, the initial transient inward current was followed by oscillating inward currents. The initial single inward current was observed even in Ca2+-free solutions: the amplitude was 0.81 ± 0.04 of that seen in a normal extracellular solution (n = 4). On the other hand, the inward current oscillation was completely abolished by removal of extracellular Ca2+ (Fig. 4B). It may be noteworthy that ET-1 did not significantly affect the resting current level, whether a single transient inward current or inward current oscillation was induced.
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In Fig. 5, the effects of ET agonists and antagonists were examined on detrusor cells showing a single transient inward current upon ET-1 application. Ten nanomolar ET-1 failed to induce any membrane current in the presence of 1 µM BQ-123. However, after washout of BQ-123 for 30 min, reapplication of 10 nM ET-1 elicited a single transient inward current in the same cell (Fig. 5A). On the other hand, 1 µM BQ-788 had no effect on ET-1-induced transient inward current (Fig. 5B). The mean amplitude of the inward current induced by 10 nM ET-1 in the presence of 1 µM BQ-788 was 280.8 ± 79.8 pA (n = 9) [not significantly different (P < 0.05) from the control]. Furthermore, S6c (10 nM) failed to induce any inward current, whereas subsequent application of ET-1 (10 nM) elicited a single transient inward current in the same cell (Fig. 5C).
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Receptor subtypes were also investigated in detrusor cells, showing inward current oscillation upon ET-1 application. Figure 6A shows that a 1-min application of 10 nM ET-1 caused an inward current oscillation that lasts for a while after removal of ET-1. After washout for 30 min, application of BQ-123 (1 µM) itself had little effect on the membrane current; however, additional application of ET-1 (10 nM) failed to induce inward current oscillation. Furthermore, the fact that this cell remained capable of producing an inward current oscillation upon subsequent application of 10 µM CCh (in the presence of BQ-123) indicated that the underlying intracellular mechanisms were still preserved during this long patch-clamp measurement. It should also be noted that BQ-123 had no effect on the inward current oscillation induced by a preceding ET-1 application (Fig. 6B). The relative amplitude and frequency of the inward current oscillation induced by ET-1 (10 nM) were 0.96 ± 0.01 and 1.07 ± 0.15 (vs. control), when 1 µM BQ-123 was additionally applied (n = 4). This feature of BQ-123 inhibition (requirement of preapplication) was in good agreement with its action on the ET-1-induced contaction (Fig. 2, A and B). On the other hand, BQ-788 (1 µM) had no significant effect on either resting membrane current or ET-1-induced oscillating inward currents (amplitude: 164 ± 77 pA, frequency: 0.035 ± 0.014 Hz, n = 5; Fig. 6C). Furthermore, sarafotoxin S6c (10 nM) did not induce an inward current oscillation in detrusor cells that show oscillatory responses to ET-1 (n = 5; Fig. 6D). Taken together, it is suggested that whether transient or persistent, electrical activities seen during and after ET-1 applications are caused via ETA receptors.
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General features of ET-1-induced inward current oscillation. From the essential similarities on the BQ-123 inhibitory effects and persistence after washout of ET-1, inward current oscillation was considered to be the underlying mechanism in the sustained contraction induced by ET application. We thus examined the properties of inward current oscillation in terms of the ET concentration and membrane potential.
The effects of cumulative application of ET-1 on oscillating inward currents were examined at 1, 10, and 100 nM. The frequency of the inward current oscillation was comparable between 1 and 10 nM ET-1 (0.040 ± 0.007 Hz at 1 nM and 0.045 ± 0.004 Hz at 10 nM), whereas it was significantly increased by increasing the ET-1 concentration to 100 nM (P < 0.05, 0.070 ± 0.007 Hz). On the other hand, no statistically significant change was observed in the mean amplitude of the oscillating inward current among the three ET-1 concentrations: 196 ± 31 pA at 1 nM; 301 ± 110 pA at 10 nM; and 305 ± 47 pA at 100 nM (n = 4-7).
Figure 7 shows the voltage dependency of an inward current oscillation induced by 10 nM ET-1. As shown in Fig. 7, the frequency of the inward current oscillation was clearly increased at more negative membrane potentials. In contrast, the amplitude was normally maximal at -60 mV. In the detrusor cell used in Fig. 7Aa, ramp pulses (from -120 to +40 mV for 1,000 ms) were also applied in the absence of ET-1 as a control () and during an oscillating inward current in the presence of ET-1 (
). Figure 7Ab shows the instantaneous current-voltage (I-V) relationship of the resting membrane current and oscillating current. The subtraction of the I-V relationship
from
yielded an ohmic I-V relationship over the range between +40 and -120 mV with the reversal potential (Erev) of -0.82 ± 0.93 mV (n = 6).
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Effects of ionic substitution and channel blockers. To elucidate the ionic nature of the ET-1-induced oscillating inward currents, effects of ionic substitution and channel blockers were examined. As shown in Fig. 8A, 100 nM nifedipine, which would cause complete suppression of voltage-sensitive (L-type) Ca2+ current in detrusor smooth muscle (27), had little effect on inward current oscillation (226 ± 92 pA, 0.036 ± 0.016 Hz, n = 6). This result suggests that although Ca2+ influx from the extracellular space is essential for the maintenance of inward current oscillation, voltage-sensitive Ca2+ channels are not required.
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Because the Erev was close to 0 mV, the ion channel candidates for the ET-1-induced oscillating inward currents under the conditions used are nonselective cation channels and Cl- channels. In the experiment shown in Fig. 8B, the extracellular Na+ was replaced with a large cation, NMDG, which does not permeate through nonselective cation channels (e.g., Ref. 38). Application of 10 nM ET-1 still induced inward current oscillation, and the subsequent removal of extracellular Ca2+ completely abolished it. On the other hand, 100 µM niflumic acid, a blocker for ClCa channels (12), significantly and reversibly suppressed ET-1-evoked inward current oscillation in normal solution (Fig. 8C). Furthermore, DIDS (500 µM), another ClCa channel blocker, also abolished it (Fig. 8D). These results suggest that Cl- is the charge carrier for the oscillating inward current.
It has been reported that ET-1 activates L-type voltage-sensitive Ca2+ channels in guinea pig portal vein (15). In detrusor smooth muscle, the same L-type voltage-sensitive Ca2+ channels are well known to play an important role in E-C coupling. Thus we examined effects of ET-1 on voltage-sensitive Ca2+ channel current. Application of ET-1 up to 100 nM, however, had little effect on the I-V relationship (peak current amplitude vs. step voltage) of voltage-sensitive Ca2+ channel current (2.5 mM extracellular Ca2+ were used as a charge carrier). The intracellular Ca2+ was buffered with 4 mM EGTA (n = 4, data not shown).
Involvement of intracellular Ca2+ stores. Possible involvement of Ca2+ release from the intracellular Ca2+ stores in the oscillating inward current was examined. In Fig. 9A, 10 mM caffeine was applied to a pig detrusor cell showing oscillating inward currents in the presence of 10 nM ET-1. A transient inward current was evoked just after application of caffeine, and oscillating inward currents ceased during the exposure. Ryanodine (30 µM) also abolished the oscillating inward currents induced by ET-1 (Fig. 9B). The results agree well with the hypothesis that periodic Ca2+ release underlies oscillating inward currents.
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We further examined whether G proteins are involved in ET-1-induced inward currents. As shown in Fig. 9C, when 1 mM GDPS was contained in the patch pipette, ET-1 was unable to induce any inward current (even a transient one). Applications of caffeine (10 mM) or ATP (100 µM), however, readily induced transient inward currents, as seen in detrusor cells dialyzed with "normal" intracellular solution (n = 5). (The inward currents induced by the former and latter drugs were presumably through ClCa channels and nonselective cation channels.) Furthermore, step pulse depolarizations readily evoked voltage-sensitive inward currents (data not shown).
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DISCUSSION |
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ET-1-induced contractions and oscillating inward currents share a number of features. First, the ET-1-induced contraction was persistent, and the tension only slowly decreased during washout of ET-1. This persistent contraction is also observed in other smooth muscles (19, 37). Similarly, as shown in Fig. 6A, inward current oscillations in isolated detrusor cells last for some while after washout of ET-1. The similar persistence seen in contraction and electrical activity may be explained by the following two hypotheses: 1) a very high affinity of ET-1 to its receptor in detrusor cells and/or a very low dissociation rate and 2) ET-1-induced persistent intracellular signal(s) that can promote mechanisms underlying inward current oscillation and tonic contraction.
Another characteristic feature shared in contraction and electrical activity is the effects of antagonists. Prior application of BQ-123, an ETA receptor antagonist, completely suppressed both contraction and inward current oscillation induced by ET-1, whereas BQ-788, an ETB receptor antagonist, had no effect on either (Figs. 2 and 6). Furthermore, sarafotoxin S6c, an ETB receptor agonist, produced only a very small contraction and no inward current (Figs. 2 and 6). These results suggest that inward current oscillation induced via ETA receptors is the primary electrical activity underlying ET-1-induced contraction. When application of BQ-123 was preceded by ET-1, neither ET-1-induced contraction nor inward current oscillation was suppressed. This order-dependent inhibitory effect of BQ-123 could be explained by the hypotheses described in the previous paragraph.
ET-1 increased the frequency of inward current oscillation in a dose-dependent manner. It can be deduced that at high concentrations of ET-1, summation of oscillating inward currents results in a large tonic contraction of detrusor muscle. Gap junction channels, although less present than in other smooth muscle tissues (3), may also make an important contribution in the summation of electrical activity in the detrusor, i.e., sustained depolarization. Nifedipine, a selective blocker for L-type voltage-sensitive Ca2+ channels, had little effect on ET-1-induced inward current oscillation (Fig. 8A) but largely suppressed ET-1-induced tonic contraction (data not shown). We also observed these paradoxical responses to nifedipine in porcine coronary artery in which the high sensitivity to a dihydropyridine Ca2+ channel antagonist of the ET-1-induced contraction represents a tissue-specific rather than agonistspecific property (18). Although the tissue specificity was taken into consideration, these facts suggest that L-type Ca2+ channels play a central role in E-C coupling in detrusor muscle but that this Ca2+ pathway is not always required to maintain inward current oscillation.
In vascular smooth muscle, it has been shown that voltage-sensitive L-type Ca2+ channels are directly facilitated through ET-1 receptor stimulation even under voltage-clamp conditions (11, 15). However, we noted that ET-1 had no effect on voltage-sensitive Ca2+ channel current in detrusor cells (data not shown), supporting the conclusion that the major action of ET-1 is to produce oscillating inward currents, which depolarize the cell membrane and subsequently activate voltage-sensitive Ca2+ channels. Further evidence was obtained using a K+-rich solution in the patch pipette. When the cell membrane was clamped at -60 or -40 mV, only oscillating inward currents were observed after application of ET-1. A periodic outward component that accompanied the oscillating inward current was produced by depolarizing the membrane to -20 mV (data not shown).
In the majority of detrusor cells, application of ET-1 promptly evoked a single transient inward current (Fig. 4A), but about one-third of the cells showed persistent inward current oscillation even after the removal of ET-1 (Fig. 6A). The single inward current was observed even in Ca2+-free solutions, but on the other hand, the oscillatory inward currents were terminated by removal of extracellular Ca2+ (Fig. 4). These results suggest that distinct mechanisms are operated in the single transient inward current and persistent inward current oscillation, although the same ETA receptors are responsible for both. Because inward current oscillation seems to underlie ET-1-induced tonic contractions, we focused in the present study on the ionic nature of this electrical activity.
The Erev of the ET-1-induced oscillating inward current was very close to 0 mV (Fig. 7Ab). Nonselective cation channels and Cl- channels are candidates for carrying this current under the conditions used in the present study. As shown in Fig. 8B, ET-1 still induced oscillating inward currents, even when extracellular Na+ was replaced with a large cation, NMDG, which does not permeate through nonselective cation channels (e.g., Ref. 38). On the other hand, niflumic acid, a Ca2+-activated Cl- channel blocker, significantly suppressed inward current oscillation (Fig. 8C). Taken together, these results suggest that Ca2+-activated Cl- channels are the major conductance for the oscillating inward currents induced by ET-1 and that these Cl- channels are activated presumably by periodic increases in the intracellular Ca2+ concentration ([Ca2+]i).
With respect to the contractile response, niflumic acid suppressed the ET-1-induced contraction with IC50 between 100 and 300 µM (Fig. 3). It may be noteworthy that phasic contractions appeared when niflumic acid significantly reduced the amplitude of the ET-1-induced tonic contraction. This might be evidence for the summation of phasic activities (contraction and/or electrical oscillations) induced via ETA receptors. Compared with patch-clamp experiments, a slightly higher concentration was required to inhibit the contractile response to ET-1. This is presumably due to higher spontaneous activity at a higher temperature (35-37°C) used for tension recordings and/or due to the low permeability (efficacy) of niflumic acid in tissue level experiments. Also, it has been reported that niflumic acid activates K+ currents (25). This effect might be involved in the inhibitory action of niflumic acid on the ET-1-induced contraction. In addition, it is important to consider the contribution of K+ channels on the detrusor excitability (36), although this was not the subject of the present study.
The mechanism(s) of the periodic [Ca2+]i increases seems to require Ca2+ influx from the extracellular space (Figs. 4B and 8B) through nifedipine-insensitive Ca2+-permeable channels (Fig. 8A). The fact that the frequency of the ET-1-induced inward current oscillation was increased by increasing the negativity of the membrane potential over the range -20 to -100 mV supports the involvement of a Ca2+ permeability other than voltage-sensitive Ca2+ channels. On the other hand, the amplitude of the currents decreased despite the increase in frequency. We hypothesize that the periodic [Ca2+]i increase that underlies ET-1-induced inward current oscillation is mainly due to Ca2+ release from the sarcoplasmic reticulum. The inhibitory effects of caffeine and ryanodine on the inward current oscillation (Fig. 9, A and B) support this hypothesis. If the release is triggered by Ca2+ influx through the nifedipine-insensitive Ca2+ permeability, the increase in frequency of the oscillating inward current at -100 mV could be due to amplification of the driving force for Ca2+ entry, and the decrease in size might reflect the insufficiency of refilling Ca2+ in the intracellular Ca2+ stores due to shorter intervals.
The inclusion of GDPS in the patch pipette completely abolished ET-1-induced electrical activities (both single and oscillatory inward currents; Fig. 9C), implying that factors (or mechanisms) causing periodic changes in [Ca2+]i are downstream of GTP binding protein(s) activated via ETA receptors (6, 28). Further experiments are necessary to elucidate details of intracellular signals: for example, whether G protein-related signals primarily activate periodic Ca2+ release from the intracellular Ca2+ stores or enhance the nifedipine-insensitive Ca2+ permeability in the plasma membrane consequently activating it.
What is the role of ET-1 and its receptors in urinary bladder function? It seems likely that ET-1 may be released from some tissues in the bladder wall during micturition and may play some facilitatory role in enhancing the responses to acetylcholine released from the parasympathetic nerves. The sustained nature of the oscillating inward currents induced by ET-1 may contribute to the maintenance of the pressure rise, preventing the buildup of residual urine. It is further speculated that changes in the amount of released ET-1 might alter the duration of sustained activation of individual smooth muscle cells and consequently alter the threshold for activation of afferent nerves and initiation of the next micturition. Ageing (35) and some popular diseases, such as diabetes mellitus (5, 26, 31), are known to alter ET receptor expression and bladder activity.
In conclusion, we demonstrated that ET-1 induces inward current oscillation via ETA receptors in a considerable number of detrusor cells. The inward current oscillation is associated with tonic and persistent contraction in the smooth muscle. The results with ionic substitution and channel blockers suggested that the oscillating inward currents flow through Ca2+-activated Cl- channels that are periodically activated. Ca2+ influx through nifedipine-insensitive Ca2+ permeability plays an important role in pacing the oscillation.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by a Medical Research Council project grant of UK. S. Nakayama was supported by grants-in-aid for Scientific Research from Ministry of Education, Science and Culture of Japan.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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