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Intracellular protein changes during pentylenetetrazole induced bursting activity in snail neurons
Brain Research, 253 (1982) 271-279 Elsevier Biomedical Press
271
Intracellular Protein Changes During Pentylenetetrazole Induced Bursting Activity in Snail Neurons EIICHI SUGAYA, MINORU ONOZUKA, KENICHI KISHII, AIKO SUGAYA and TADASHI TSUDA
Department of Physiology, Kanagawa Dental College, 82, lnaoka-cho, Yokosuka and (A.S. and T.T.) Faculty of Pharmaceutical Sciences, Josai University, Keyakidai, Sakado, Saitama (Japan) (Accepted June 8th, 1982)
Key words: bursting activity - - seizure discharge - - calcium - - protein - - microdisk electrophoresis - - pentylenetetrazole - snail neuron - - cell membrane
The intracellular protein changes during pentylenetetrazole (PTZ)-induced bursting activity (BA) which is characteristic of seizure discharge were investigated using microdisk electrophoresis with 5 identified neurons. The identified neurons of the snail, Euhadra peliomphala, were used. The PTZ-sensitive neurons which manifest marked BA by application of PTZ were examined. PTZ induced in PTZ-sensitive neurons: (1) a prominent increase of 5-7 kdalton protein and (2) peak separation into 3 peaks of 10-15 kdalton protein. In the 5-7 and 10-15 kdalton protein, a marked increase in radioactivity of 4~Ca was observed after PTZ application. PTZ-non-sensitive neurons showed neither these protein changes nor 45Ca incorporation into these proteins. The above findings suggest that during PTZ-induced BA, intracellular protein changes occurred in relation to the intracellular calcium shift. INTRODUCTION In the cerebral cortex of the cat, the intracellular potential of cortical neurons manifests characteristic bursting activity (BA) when pentylenetetrazole (PTZ) is injected systematically 21. Exactly the same BA can be seen in the isolated PTZ-sensitive neurons of the Japanese land snail, Euhadra peliomphala, when PTZ is applied in a bathing solution 20. During BA in snail PTZ-sensitive neurons, the intracellular calcium was found to move toward the cell membrane, by examination using the intracellular calcium mapping technique with a computer controlled electron probe X-ray microanalyzer22; and the calcium stored in the cell organella is released with morphological change by PTZ treatmerit 2z. The combined study using the computer controlled electron probe X-ray microanalyzer and the ion shower milling machine with freeze dried single isolated PTZ-sensitive neurons of Euhadra revealed that the shifted calcium is probably attached to the inner surface of the cell membrane 24. Moreover, the binding state of calcium near the cell 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
membrane of PTZ treated PTZ-sensitive neurons is different from that of normal PTZ-sensitive neurons 25. If the intracellular released calcium is bound to the inner surface of the cell membrane, the substances with which the released calcium is bound are most likely the proteins related to the cell membrane. The present study was undertaken to clarify the substances to which intracellularly released calcium is bound using microdisk electrophoresis with 5 isolated identified cells. MATERIALS AND METHODS
Preparation of samples The PTZ-sensitive neurons in the RC-cluster of a suboesophageal ganglion of the Japanese land snail, Euhadra peliomphala, were used. The PTZsensitive neuron of Euhadra manifests a characteristic bursting activity with a sustained depolarization shift after 5-7 min of application of 5 × 10-2 M PTZ solution 2°. The PTZ-sensitive neurons (RC1-5) 2o were dissected under a binocular dissecting
272 microscope and a specially made dissecting apparatus which maintains a constant temperature in the surrounding snail Ringer solution. The identified cells were completely freed from surrounding tissue. Dissected single cells were picked up using a fine pipette and transferred to the cooled microwell of a 3034 microtest tissue culture plate (Falcon). The axon was cut and a cell body sample was prepared. Then the medium in the microwell was removed and replaced with 0.5 #1 of 0.1 M K2COa (pH 11.0) and homogenized with a glass rod. After mixing with 0.5 pl of 2'}.~i sodium dodecyl sulfate (SDS), samples were heated at 50 °C for 5 min. Then/3-mercaptoethanol was quickly introduced and mixed with 0.5 #1 of solution of a four-fold concentrated upper gel buffer, 30'}/~,sucrose and 1.0/~g//zl of dithiothreitol. According to the method of Gainer 7, we used the two reducing agents, fl-mercaptoethanol and dithiothreitol. This gave more stable results than those obtained when only /%mercaptoethanol was used. This is probably due to the relatively larger contact area between the internal surface of the glass capillary and acrylamide gel than with the normal disk electrophoresis tube.
Preparation of the capillary tube A Coming 4618 glass tube (inside diameter 0.4 mm) was used. The glass tube was dipped in a dichlorosulfate solution for 7 days ao.d washed with tap water for about 2-3 days. Then the tubes were washed with distilled water, at least 100 ml per tube, using a suction apparatus. The washed tube was dried and placed in a vacuum chamber for about 30 rain to force out gas and moisture.
Preparation of gel and buffer Preparation of the gel was performed principally according to the method of Gainer 7 and OsbornO 7. The compositions of the final gel and buffer solution were as follows. Lower gel (running gel) : 10 ~o acrylamid, 0.133 ~,, N,N'-methylenebisacrylamide, 0.0308 N HCI and 0.427 M Tris (pH 9.2). Upper gel (stacking gel): 3 % acrylamide, 0.2 ~o N,N'-methylenebisacrylamide, 0.0267 M H2SO4 and 0.0541 M Tris (pH 6.1). Upper gel buffer: 0.0267 M HzSO4 and 0.0541 M Tris (pH 6.1). Upper electrode buffer: 0.041 M Tris, 0.040 M boric acid and 0.1 ~ SDS (pH 8.6). Lower electrode buffer: 0.05 N HC1 and 0.062
M Tris (pH 7.5). The gels were polymerized with a final concentration of 0.15'Iii N,N,N',N'-tetramethylenediamine and 0.1"~i ammonium persulfate.
Preparation of capillary gel tube The degased lower gel solution was placed in a capillary tube by capillary force and pressed into a plasticine cushion. The upper part of the capillary was then filled with distilled water for polymerization. After polymerization of the lower gel, the superimposed water was removed by suction, and then the upper gel solution was added by a microsyringe with a 200 # m needle. Distilled water was superimposed and polymerization took place for 30-60 rain. After polymerization of the upper gel, the water was removed by suction and the upper gel buffer was added. The capillary was cut into an 80 mm length and the lower end was dipped in the lower electrode buffer for 2-3 h for stabilization.
Electrophoresis The upper gel buffer above the upper gel was withdrawn by microsyringe and dense sample (about I /zl) was layered over the upper gel with a hand-made fine glass micropipette. The remaining space above the sample was filled with upper gel buffer and connected to the upper electrode buffer with polyethylene tubing. The bottom end of the gel tube was dipped into the lower electrode buffer. The electrode was a platinum wire with the upper and lower ends inserted in a plastic reservoir. The voltage source was DPI-506 (DIA Medical, Tokyo) type constant current supply apparatus specially made for this microelectrophoresis. The current of each gel was 45 ~A/tube for the upper gel and 30 #A/tube for the lower gel. The current was monitored by microammeters for each channel. The distance of electrophoresis was 30 mm from the point where the marker dye (bromophenol blue) reached the lower gel. The electrophoresis time was about 90 rain.
Staining, destaining and densitometry After the electrophoresis, the gel tube was removed from the polyethylene tube connecting it to the upper reservoir. The gel was pushed out using a piano wire with an internal diameter 90% of that of the capillary tube. The gels were stained with
273 Coomassie brilliant blue (0.25 ~) for about 12 h at 20 °C. Destaining was performed with a destaining solution (7 ~ acetic acid and 5 ~ methanol in water) for about 4-8 h. Densitometry was performed with a Joyse-Loeble 3CS microdensitometer at a 560 nm wave length.
Standard proteins As standard samples, albumin (67,000), ovalbumin (45,000) chymotrypsinogen (25,000), trypsin-inhibitor (21,500), ribonuclease (14,000), cytochrome (12,500), aprotinin (6,500) and insulin Kette B (3,400) were used. All standard samples were purchased from the Boehringer Co. Ltd.
Radioactive analysis The ganglia were incubated for 40 min at 0 °C in an ice cold snail Ringer solution containing 20/~Ci of 45Ca (New England Nuclear) and 2 mg/ml of glucose to obtain full intracellular uptake of 45Ca when the calcium pump was inhibited by low temperature. The preparatory experiment showed that 40 min was sufficient to saturate the intracellular 45Ca. Then, the ganglia were incubated in a normal and PTZ-Ringer solution containing the same quantity of 45Ca for 15 min at 22 °C. In the recovery experiment, a 60 min rinse with 45Ca containing normal Ringer was performed. After rinsing of the ganglion with isotonic phosphate buffer, the identified neurons were dissected and electrophoresed as described above. The identified cells were completely freed from surrounding tissue. The destained gels, containing radioactive calcium, were placed on a microscope slide and sectioned using a razor blade fragment into 1 mm long disks. Each disk was transferred to the scintillation vial. The disks were solubilized by the addition of 0.5 ml of a 30 ~ solution of H2Oz to the scintillation vials. The vials were loosely capped, and heated for 12-24 h at 50 °C. After the vials had cooled to room temperature, 7 ml of ACS II (Amersham, Lot 08908) counting mixture was added to each vial. The radioactivity was counted at 90 ~ efficiency for 14C in a LKB Packbeter 1215 liquid scintillation counter.
Preparation of subcellular fraction Cell membrane fraction. Desheathed ganglia of about 300 animals (each preparation in normal snail
Ringer or PTZ-containing snail Ringer) were rinsed in 0.32 M sucrose (pH 7.8 adjusted with NaHCOa), and homogenized in 1 ml of cold 0.32 M sucrose using a glass Teflon homogenizer (700 rpm, 10 strokes). The homogenate was centrifuged at 1,000 g for 10 min. The pellet was washed and centrifuged at 1,000 g for 10 min 3 times with 0.32 M sucrose and the suspension was homogenized using a glass Teflon homogenizer (2,000 rpm, 20 strokes). The homogenate was centrifuged at 1,000 g for 10 min to give a supernatant, S1, and a pellet, P1. The S1 was decanted and saved, whereas P1 was rehomogenized and centrifuged as described above, and supernatant $2 and pellet P2 were obtained. P2 was rehomogenized and centrifuged as described above and supernatant $3 was obtained. SI, $2 and $3 were pooled and centrifuged at 3,000 g for 60 rain. Supernatant, $4 and the loose layer on pellet P4 were centrifuged at 21,000 g for 30 min and supernatant $5 and pellet P5 were obtained. P5 was suspended with about 0.5 ml of 0.32 M sucrose and was layered on a discontinuous sucrose density gradient consisting of 1 ml each of 1.18, 1.16 and 1.13 density sucrose and centrifuged at 100,000 g for 120 min (3 × 5 ml swing-out head, Hitachi 65P). The fraction between 0.32 M and 1.13 density (0.99 M) sucrose was collected and suspended in 0.32 M sucrose (final concentration). The suspension was centrifuged at 30,000 g for 30 rain. The pellet was adjusted to 0.5-1.0 mg/ml protein content with 0.32 M sucrose solution and stored for assay at --80 °C. Lysosome fraction and microsome fraction. The process for preparing the lysosome fraction and microsome fraction from ganglia is shown below. All operations were carried out at 0-4 °C. The lysosome fraction, microsome fraction and plasma membrane fraction were examined by electron microscopy and they showed sufficient purity (Fig. 3). The biochemical characterization of the fractions (lysosome, microsome and plasma membrane) was performed according to the method of Marchbanks 15. Assay of the acid phosphatase activity according to King and Armstrong 13 and fl-glucuronidase activity according to Kato 12 for the lysosome fraction, RNA assay according to Schneider18 for the microsome fraction and Na +, K+-ATPase activity assay according to Gibbs et a1.11 for the plasma membrane fraction
274 homogenized I ml 0.32 M sucrose (pH 7.0) I withglass Teflon homogenizer 800-1,000rpm, 8 strokes, at 0 C 800 g, 20 min, at 4-C supernatant ] 10,000g, 15 rain, at 4 °C
I supernatant 105,000 × g, 60 rain, at 4 "C
residue ] 0.8 and 1.0 M sucrose gradient 55,000 g 15 rain, at 4 °C I
I I .....
I
i
residue (microsome fraction)
residue (lysosome fraction)
showed considerably higher values than those of the other fractions and the starting homogenate (Table I). RESULTS Microdisk electrophoresis pattern changes of P T Z sensitive neurons during BA Fig. 1A is the densitometry pattern of the microdisk electrophoresis pattern of normal PTZ-sensitive neurons and that of P T Z treated PTZ-sensitive neurons. The prominent differences between these two disk patterns were: (1) the single peak of approximately 10-15 kdalton peak in the normal state was divided into 3 peaks when a PTZ-sensitive neuron was incubated for 15 min in P T Z (5 × 10 -2 M); and (2) the flat part of approximately 5-7 kdalton in the normal state became prominent in PTZ incubated PTZ-sensitive neurons. The part with a high molecular weight of more than 30 kdalton was almost constant with some fluctuation. Although the possibility of differences in the higher molecular weight part between PTZ-treated and normal PTZ-sensitive cell exists, the present investi-
intermediate layer
gatlon was focused only on the above-mentioned marked changes. Although the total protein of the samples could not be measured because of the small quantity, the samples used were always almost equal in size to the 5 identified neurons. Judging from the comparison of band width of the standard protein and that of neurons, the total protein of the 5 measured neurons was approximately 30-50 ng. The neurons rinsed for 60 min after P T Z treatment showed almost the same disk pattern as normal neurons (Fig. 1A, lower most curve). The PTZ-non-sensitive neurons (RC-6, LO-1 and RO-1) never manifest BA by P T Z even with prolonged and concentrated application 2°. The disk patterns of normal and P T Z treated PTZ-non-sensitive neurons are shown in Fig. lB. Unlike PTZsensitive neurons, the PTZ-treated PTZ-non-sensitive neurons never changed their disk pattern. Microdisk electrophoresis of fractionated parts To obtain some clue about which subcellular components are responsible for forming the 10-15 and 5-7 kdalton peaks, microdisk electrophoresis of the fractionated parts obtained from clusters which
TABLE 1 Biochemical characteristics of fractions
Whole homogenate Lysosome fraction Microsome fraction Plasma membrane fraction
Na+-K+-ATPase (l~moles/h/mgprotein)
RNA (lzg/mgprotein)
.4cid phosphatase (l~g/h/mgprotein)
tl-gluculonidase (f*g/h/mgprotein)
8.16 1.11 4.98 14.75
5.16 3.87 13.59 4.08
79.1 124.8 48.2 5.3
1.13 4.00 0.22 0.82
275
electrophoresis pattern of a lysosome fraction (100 mg total protein both in normal and PTZ-treated samples). This clearly demonstrates that the 10-15 kdalton peak is prominent in normal, and in PTZ-
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Fig. 1. Microdisk electrophoresis patterns of normal and PTZ-treated neurons. A : PTZ-sensitive neuron. B: PTZ-nonsensitive neuron. Thick line, normal neuron; dotted line, neuron after 15 rain incubation with PTZ; dot-dash line, recovery by 60 min rinsing with normal snail Ringer.
contain a great majority of the PTZ-sensitive neurons was performed. In the Euhadra suboesophageal ganglion, PTZ-sensitive and PTZ-non-sensitive neurons are found. The number of the PTZ-non-sensitive neurons is quite limited (RO-1, LO-1, RC-6, LC-1 and LC-2 only). Most of the other cells, especially neurons in the cluster situated in the dorsal part of the ganglion, are highly PTZ-sensitive (RC-1-5) or moderately PTZ-sensitive (RC-7-13 and most of neurons not named in the other clusters). Therefore, if the dorsal clusters are collected and neurochemical experiments are performed, the results represent the characteristics of the PTZ-sensitive neurons. The desheathed cluster forms, like a brindle resembling balloons, with axons as the strings. The neurons were collected by cutting axons. Thus contamination of non-neuronal elements was limited. Fig. 2A shows a microdisk
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Fig. 2. Microdisk electrophoresis patterns of fractionated neurons. A: lysosome fraction. B: microsome fraction. C" plasma membrane fraction. Thick line, normal state; dotted line, after PTZ incubation. Arrows represent the parts changed by PTZ.
276
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Fig. 4. 45Ca Incorporation into microgels of PTZ-sensitive neurons. Electrophoresis of 100 identified neurons. Thick line and solid circles, normal neurons; dotted line and open circles, after PTZ incubation. Fig. 3. Electron mlcrographs of lysosome fraction (A), microsome fraction (B) and plasma membrane fi-action (C). Bars: 0.5 ,, 11.
treated samples (Fig. 2A, arrow) ; but there is no 5-7 kdalton peak in both normal and PTZ-treated samples. Fig. 2B shows a microdisk electrophoresis pattern of the lnicrosome fraction (100 mg total protein, both in normal and PTZ treated samples). In the micros•me fraction, the 10-15 kdalton peak is very prominent (Fig. 2B, arrow) but there is no 5-7 kdalton w-ak. Fig. 2C shows a microdisk electrophoresis pattern of the plasma membrane fraction (100 mg total protein both in normal and PTZtreated samples). Unlike the micros•me and lysosome fractions, the 5-7 kdalton peak forms a marked elevation in the PTZ-treated sample in the case of the plasma membrane fraction (Fig. 2C, arrow). The 5-7 kdalton peak never appeared in the lysosome and micros•me fraction.s: and the 10-15 kdalton peak never appeared in the plasma membrane fraction. These 3 microdisk electrophoresis patterns suggest that the 5-7 kdalton peak mainly originates from the plasma membrane and the 10-15 kdalton peak mainly originates from the micros•me and lysosome.
Changes of 45Ca incorporation into the intracellular proteins during BA To clarify the relationship between intracellular protein and calcium behavior, 45Ca incorporation into the molecular weight distribution of proteins in the cell was examined. After incubation in normal
snail Ringer containing 20 #Ci of 45Ca for 40 min at 0 '-~C as described in Methods, the PTZ-sensitive neurons were incubated in PTZ snail Ringer containing the same quantity of 45Ca for 15 min at 22 °C. Twenty neurons were used for one gel. The gels were sectioned into 1 m m disks and the radioactivity was measured with a liquid scintillation counter (Fig. 4). Each point in Fig. 4 represents the total dpm/gel slice of 5 gels. The PTZ-treated samples showed a prominent peak of radioactivity of 45Ca at 10-15 and 5-7 kdalton proteins. Judging from the disk patterns of the subcellular fraction., the peak at 5-7 kdaltons suggests an increase of 45Ca in.corporation into the plasma membrane. This result is consistent with the results obtained by computer controlled electron probe X-ray microanalysis 22 as well as that with an ion shower etching of the freeze dried cell membrane z4.
Effect of extracellular calcium concentration on the 45Ca incorporation into proteins during BA To obtain some clue concerning where the calcium which binds to the protein comes from, the 45Ca radioactivity of a disk of 5-7 kdalton protein was measured in a calcium free and four-fold calcium extracellular medium. When extracellular calcium was freed, 45Ca incorporation into this protein decreased, although the peak remained (Fig. 5C). In the four-fold extracellular calcium concentration, the incorporation increased slightly compared with that in normal Ringer, but not in proportion to the extracellular calcium concentration (Fig. 5B).
277 - e - Normal
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incorporation with calcium-free medium. Thick line and solid circles, normal state; dotted line and open circles, after PTZ incubation.
Time course of the 45Ca incorporation To elucidate the role of intracellular calcium movement, the time course of the 45Ca incorporation into proteins during BA was examined. The BA occurred 5-7 men after PTZ application electrophysiologically. The 45Ca incorporation into proteins increased within 5 men (Fig. 6); in particular, the 80-90 % saturation of 45Ca incorporation into 5-7 kdalton protein occurred far earlier than BA manifestation (Fig. 6, upper part). These findings suggest that the increase of 45Ca incorporation is probably the origin and not the result of BA. DISCUSSION Intracellular protein was markedly changed during PTZ-induced BA compared with the normal state in the identified PTZ-sensitive neurons• This change showed recovery after 60 men rinsing with normal Ringer. The PTZ-non-sensitive neurons ma-
278 nifested neither BA nor intracellular protein changes. In the examination using an intracellular calcium mapping technique, the intrace[lu[ar calcium distribution showed a clear difference between the BA state and the normal state in PTZ-sensitive neurons, and PTZ-non-sensitive neurons never showed these intracellular calcium changes 16,~'~. During BA the intracellularly shifted calcium was shown to gather at the inner surface of the cell membrane by an examination using an ion shower milling machine and calcium measurement using a flameless atomic analyzer '~'1. Moreover, the binding state change of calcium occurred at the same membrane site '5. These experimental facts prompted us to seek the substances to which the shifted calcium binds. The present experiment reveals that the substances are most likely the 5-7 and 10-15 kdalton proteins. The detailed characteristics of these proteins, however, are difficult to determine at this stage because of the insufficient qua~.tity of the samples. The high molecular weight part of more than 30 kdalton of the protein showed an almost unchanged pattern with some fluctuation in PTZ-treated and normal PTZ-sensitive neurons. The significance of the higher molecular part in the manifestation of BA, however, is still obscure and requires further investigation. Gainer and his colleagues performed an extensive study on the protein synthesis of a single identified neuron of Otala and Aplysia 8,u. They concluded that the 5 and 12.5 kdalton proteins are specific to spontaneously bursting cells, cell 10 in Otala and R I 5 in Aplysia. Thereafter, in neurosecretory cells of Aplysia, conversion from higher molecular weight protein to low molecular weight protein or peptide was demonstrated1-a,14,19; for example, from 29 to 6 and 3 kdaltons protein 14, and from 18 to 3 kdaltons protein a. Strumwasser and Wilson demonstrated a temporal processing of 12 to 6-9 kdalton protein in the R 15 neuron of Aplysia using a doublelabel technique 19, All of these processes from higher molecular weight protein to lower molecular weight protein required at least 2 h. In our present experiment, 15 min incubation in PTZ resulted in the appearance of the 5-10 kdalton protein and the change in the 10-15 kdalton protein accompanied by calcium incorporation. Since 15 min incubation in PTZ is not sufficient time to show the synthesis of
new proteins, it would be more natural to imagine a shift from one cell compartment to another. The calcium binding to these two proteins prominently increased after PTZ treatment. The fact that the specific calcium binding to PTZ-treated samples after rather drastic treatment such as that with flmercaptoethanol, dithiothreitol and SDS suggests that calcium binding to these two proteins might not be labile. Burgess et al. observed calcium binding to calmodulin in the presence of SDS 4. The calcium binding of our protein is probably of a similar nature. Since a marked release of stored calcium from lysosomes with clear morphological changes has been observed previously 2a and 10-15 kdalton protein most likely originated from lysosomes and microsomes in the present experiment using fractionated materials, we assume that the calcium release from lysosomes will be accompanied by 10-15 kdalton protein. The previous experiment showed calcium gathering at the inner surface of the cell membrane after PTZ treatment 22 with a calcium binding state change 2s. The substance to which the shifted calcium binds is most likely 5-7 kdalton protein. In any case, PTZ-induced BA required calcium incorporation to 10-15 kdalton protein which is related to lysosome and also to 5-7 kdalton protein which is related to plasma membrane. The relationship between 10-15 and 5-7 kdalton proteins, however, is a subject of further investigation. The calcium shift to the 5-7 kdalton protein occurred earlier than the complete electrophysiological manifestation of BA. Even in calcium free medium, the 45Ca intracellularly incorporated in advance shifted to the 5 7 kdalton protein and 10-15 kdalton protein. The study by computer controlled electron probe X-ray microanalysis showed that the shifted calcium was mainly of intracellular origin z2. Electrophysiological observation of PTZ induced BA with cobalt chloride-containing medium to eliminate the calcium influx also showed that the manifestation of BA did not essentially require calcium influx (tmpublished observation). These experimental facts lead us to conclude that the shifted calcium is mainly of intracellular origin, and occurs prior to BA manifestation. In the examination using a computer-controlled electron probe Xray microanalyzer, the calcium binding state after PTZ treatment of PTZ-sensitive neurons was found
279 to be different f r o m the n o r m a l state 25. In the e x a m i n a t i o n using the freeze fracture technique, the i n t r a m e m b r a n e o u s particles were f o u n d to be c h a n g e d b y P T Z t r e a t m e n t 26. The p r o m i n e n t increase o f 45Ca r a d i o a c t i v i t y b y P T Z t r e a t m e n t in 5-7 k d a l t o n p r o t e i n suggests that the shifted intracellular calcium binds with the intracellular cell memb r a n e related protein. Calcium and calcium-dependent regulatory proteins are i m p o r t a n t for the regulation o f various aspects o f cell functions 5,6. B A is a p a t h o l o g i c a l change which is characteristic o f seizure discharge.
A b n o r m a l b e h a v i o r o f the intracellular calcium a n d t h a t o f the related proteins might induce seizure discharge. ACKNOWLEDGEMENTS The a u t h o r s are grateful to Prof. S. H i r a n o , Prof. K. U e m u r a a n d Dr. A. A s o for advice a n d criticism d u r i n g the course o f this experiment. This research was s u p p o r t e d in p a r t by a grant f r o m the J a p a n e s e M i n i s t r y o f Education, Science a n d Culture, a n d by a g r a n t f r o m the Prof. Genichi K a t o M e m o r i a l F u n d for P h y s i o l o g y a n d Medicine.
REFERENCES 1 Arch, S., Smock, T. and Earley, P., Precursor and product processing in the bag cell neurons of Aplysia californica, J. gen. PhysioL, 68 (1976) 211-225. 2 Asward, D. W., Biosynthesis and processing of presumed neurosecretory proteins in single identified neurons of Aplysia cali]brnica, J. Neurobiol., 9 (1978) 267-284. 3 Berry, R. W. and Schwartz, A. W., Axonal transport and axonal processing of low molecular weight proteins from the abdominal ganglion of Aplysia, Brain Research, 129 (1977) 75-90. 4 Burgess, W. H., Jemiolo, D. K. and Kertsinger, R. H., Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate, Biochim. biophys. Acta, 623 (1980) 257-270. 5 Cheng, W. Y., Calmodulin plays a pivotal role in cellular regulation, Science, 207 (1979) 19-27. 6 Klee, C. B., Crouch, T. H. and Richman, P. G., Calmodulin, Ann. Rev. Biochem., 49 (1980) 489-515. 7 Gainer, H., Micro disc electrophoresis in sodium dodecyl sulfate: an application to the study of protein synthesis in individual, identified neurons, Anal Biochem., 44 (1971) 589-605. 8 Gainer, H., Patterns of protein synthesis in individual, identified molluscan neurons, Brain Research, 39 (1972) 369-385. 9 Gainer, H., Effects of experimentally induced diapause on the electrophysiology and protein synthesis patterns of identified molluscan neurons, Brain Research, 39 (1972) 387-402. 10 Gainer, H., Electrophysiological behavior of an endogenously active neurosecretory cell, Brain Research, 39 (1972) 403--418. 11 Gibbs, R., Roddy, P. M. and Titus, E., Preparation, assay and properties of an Na+,K+-requiring adenosine triphosphate from beef brain, J. biol. Chem., 240 (1965) 2181-2187. 12 Kato, K., Synthesis of p-nitrophenyl-fl-o-glucopyrosiduronic acid and its utilization as a substrate for the assay of fl-glucuronidase activity, Chem. pharm. Bull., 8 (1960) 239-242. 13 King, E. J. and Armstrong, A. R., A convenient method for determining serum and bile phosphatase activity, Canad. reed. Ass. J., 31 (1934) 367-381. 14 Lob. Y. P.. Sarne, Y., Daniels, M. P. and Gainer, H.,
15
16
17 18
19 20
21
22 23 24 25
26
Subcellular fractionation studies related to the processing of neurosecretory proteins in Aplysia neurons, J. Neurochem., 29 (1977) 135-139. Marchbanks, R. M., Isolation and study of synaptic vesicles. In N. Marks and R. Rodnight (Eds.), Research Method in Neurochemistry, VoL 2, Plenum Press, New York, 1974, pp. 79-98. Onozuka, M., Sugaya, E., Sugaya, A. and Usami, M., Intracellular calcium distribution map and chemical shift of neurons examined by computer controlled electron probe X-ray microanalyzer, Proc. int. Congr. Electron Microscopy (Toronto) VoL H, (1978) 126-127. Osborne, N. N., Microchemical Analysis of Nervous Tissue, Pergamon Press, New York, 1974, pp. 153-215. Schneider, W. C., Determination of nucleic acid in tissue by pentose analysis. In S. P. Colowick and N. O. Kaplan (Eds.), Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, pp. 680-684. Strumwasser, F. and Wilson, D. L., Patterns of proteins synthesized in the R15 neuron ofAplysia, J. gen. Physiol., 67 (1976) 691-702. Sugaya, A., Sugaya, E. and Tsujitani, M., Pentylenetrazole-induced intracellular potential changes of the neuron of the Japanese land snail Euhadra peliomphala, Jap. J. PhysioL, 23 (1973) 261-274. Sugaya, E., Goldring, S. and O'Leary, J. L., Intracellular potentials associated with direct cortical response and seizure discharge in cat, Electroenceph. clin. NeurophysioL, 17 (1964) 661-669. Sugaya, E. and Onozuka, M., Intracellular calcium: its movement during pentylenetetrazole-induced bursting activity, Science, 200 (1978) 797-799. Sugaya, E. and Onozuka, M., Intracellular calcium: its release from granules during bursting activity in snail neurons, Science, 202 (1978) 1195-1197. Sugaya, E. and Onozuka, M., Ion shower milling: its application to cell membrane removal, Science, 202 (1978) 1197-1198. Sugaya, E., Onozuka, M., Furuichi, H. and Sugaya, A., Cellular calcium binding state change during pentylenetetrazole induced bursting activity in snail neurons, Experientia, 37 (1981) 1080-1081. Sugaya, E., Onozuka, M., Furuichi, H. and Sugaya, A., lntramembraneous particle change during pentylenetetrazole induced bursting activity in snail neurons, Experientia, 37 (1981) 1081-1082.
271
Intracellular Protein Changes During Pentylenetetrazole Induced Bursting Activity in Snail Neurons EIICHI SUGAYA, MINORU ONOZUKA, KENICHI KISHII, AIKO SUGAYA and TADASHI TSUDA
Department of Physiology, Kanagawa Dental College, 82, lnaoka-cho, Yokosuka and (A.S. and T.T.) Faculty of Pharmaceutical Sciences, Josai University, Keyakidai, Sakado, Saitama (Japan) (Accepted June 8th, 1982)
Key words: bursting activity - - seizure discharge - - calcium - - protein - - microdisk electrophoresis - - pentylenetetrazole - snail neuron - - cell membrane
The intracellular protein changes during pentylenetetrazole (PTZ)-induced bursting activity (BA) which is characteristic of seizure discharge were investigated using microdisk electrophoresis with 5 identified neurons. The identified neurons of the snail, Euhadra peliomphala, were used. The PTZ-sensitive neurons which manifest marked BA by application of PTZ were examined. PTZ induced in PTZ-sensitive neurons: (1) a prominent increase of 5-7 kdalton protein and (2) peak separation into 3 peaks of 10-15 kdalton protein. In the 5-7 and 10-15 kdalton protein, a marked increase in radioactivity of 4~Ca was observed after PTZ application. PTZ-non-sensitive neurons showed neither these protein changes nor 45Ca incorporation into these proteins. The above findings suggest that during PTZ-induced BA, intracellular protein changes occurred in relation to the intracellular calcium shift. INTRODUCTION In the cerebral cortex of the cat, the intracellular potential of cortical neurons manifests characteristic bursting activity (BA) when pentylenetetrazole (PTZ) is injected systematically 21. Exactly the same BA can be seen in the isolated PTZ-sensitive neurons of the Japanese land snail, Euhadra peliomphala, when PTZ is applied in a bathing solution 20. During BA in snail PTZ-sensitive neurons, the intracellular calcium was found to move toward the cell membrane, by examination using the intracellular calcium mapping technique with a computer controlled electron probe X-ray microanalyzer22; and the calcium stored in the cell organella is released with morphological change by PTZ treatmerit 2z. The combined study using the computer controlled electron probe X-ray microanalyzer and the ion shower milling machine with freeze dried single isolated PTZ-sensitive neurons of Euhadra revealed that the shifted calcium is probably attached to the inner surface of the cell membrane 24. Moreover, the binding state of calcium near the cell 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
membrane of PTZ treated PTZ-sensitive neurons is different from that of normal PTZ-sensitive neurons 25. If the intracellular released calcium is bound to the inner surface of the cell membrane, the substances with which the released calcium is bound are most likely the proteins related to the cell membrane. The present study was undertaken to clarify the substances to which intracellularly released calcium is bound using microdisk electrophoresis with 5 isolated identified cells. MATERIALS AND METHODS
Preparation of samples The PTZ-sensitive neurons in the RC-cluster of a suboesophageal ganglion of the Japanese land snail, Euhadra peliomphala, were used. The PTZsensitive neuron of Euhadra manifests a characteristic bursting activity with a sustained depolarization shift after 5-7 min of application of 5 × 10-2 M PTZ solution 2°. The PTZ-sensitive neurons (RC1-5) 2o were dissected under a binocular dissecting
272 microscope and a specially made dissecting apparatus which maintains a constant temperature in the surrounding snail Ringer solution. The identified cells were completely freed from surrounding tissue. Dissected single cells were picked up using a fine pipette and transferred to the cooled microwell of a 3034 microtest tissue culture plate (Falcon). The axon was cut and a cell body sample was prepared. Then the medium in the microwell was removed and replaced with 0.5 #1 of 0.1 M K2COa (pH 11.0) and homogenized with a glass rod. After mixing with 0.5 pl of 2'}.~i sodium dodecyl sulfate (SDS), samples were heated at 50 °C for 5 min. Then/3-mercaptoethanol was quickly introduced and mixed with 0.5 #1 of solution of a four-fold concentrated upper gel buffer, 30'}/~,sucrose and 1.0/~g//zl of dithiothreitol. According to the method of Gainer 7, we used the two reducing agents, fl-mercaptoethanol and dithiothreitol. This gave more stable results than those obtained when only /%mercaptoethanol was used. This is probably due to the relatively larger contact area between the internal surface of the glass capillary and acrylamide gel than with the normal disk electrophoresis tube.
Preparation of the capillary tube A Coming 4618 glass tube (inside diameter 0.4 mm) was used. The glass tube was dipped in a dichlorosulfate solution for 7 days ao.d washed with tap water for about 2-3 days. Then the tubes were washed with distilled water, at least 100 ml per tube, using a suction apparatus. The washed tube was dried and placed in a vacuum chamber for about 30 rain to force out gas and moisture.
Preparation of gel and buffer Preparation of the gel was performed principally according to the method of Gainer 7 and OsbornO 7. The compositions of the final gel and buffer solution were as follows. Lower gel (running gel) : 10 ~o acrylamid, 0.133 ~,, N,N'-methylenebisacrylamide, 0.0308 N HCI and 0.427 M Tris (pH 9.2). Upper gel (stacking gel): 3 % acrylamide, 0.2 ~o N,N'-methylenebisacrylamide, 0.0267 M H2SO4 and 0.0541 M Tris (pH 6.1). Upper gel buffer: 0.0267 M HzSO4 and 0.0541 M Tris (pH 6.1). Upper electrode buffer: 0.041 M Tris, 0.040 M boric acid and 0.1 ~ SDS (pH 8.6). Lower electrode buffer: 0.05 N HC1 and 0.062
M Tris (pH 7.5). The gels were polymerized with a final concentration of 0.15'Iii N,N,N',N'-tetramethylenediamine and 0.1"~i ammonium persulfate.
Preparation of capillary gel tube The degased lower gel solution was placed in a capillary tube by capillary force and pressed into a plasticine cushion. The upper part of the capillary was then filled with distilled water for polymerization. After polymerization of the lower gel, the superimposed water was removed by suction, and then the upper gel solution was added by a microsyringe with a 200 # m needle. Distilled water was superimposed and polymerization took place for 30-60 rain. After polymerization of the upper gel, the water was removed by suction and the upper gel buffer was added. The capillary was cut into an 80 mm length and the lower end was dipped in the lower electrode buffer for 2-3 h for stabilization.
Electrophoresis The upper gel buffer above the upper gel was withdrawn by microsyringe and dense sample (about I /zl) was layered over the upper gel with a hand-made fine glass micropipette. The remaining space above the sample was filled with upper gel buffer and connected to the upper electrode buffer with polyethylene tubing. The bottom end of the gel tube was dipped into the lower electrode buffer. The electrode was a platinum wire with the upper and lower ends inserted in a plastic reservoir. The voltage source was DPI-506 (DIA Medical, Tokyo) type constant current supply apparatus specially made for this microelectrophoresis. The current of each gel was 45 ~A/tube for the upper gel and 30 #A/tube for the lower gel. The current was monitored by microammeters for each channel. The distance of electrophoresis was 30 mm from the point where the marker dye (bromophenol blue) reached the lower gel. The electrophoresis time was about 90 rain.
Staining, destaining and densitometry After the electrophoresis, the gel tube was removed from the polyethylene tube connecting it to the upper reservoir. The gel was pushed out using a piano wire with an internal diameter 90% of that of the capillary tube. The gels were stained with
273 Coomassie brilliant blue (0.25 ~) for about 12 h at 20 °C. Destaining was performed with a destaining solution (7 ~ acetic acid and 5 ~ methanol in water) for about 4-8 h. Densitometry was performed with a Joyse-Loeble 3CS microdensitometer at a 560 nm wave length.
Standard proteins As standard samples, albumin (67,000), ovalbumin (45,000) chymotrypsinogen (25,000), trypsin-inhibitor (21,500), ribonuclease (14,000), cytochrome (12,500), aprotinin (6,500) and insulin Kette B (3,400) were used. All standard samples were purchased from the Boehringer Co. Ltd.
Radioactive analysis The ganglia were incubated for 40 min at 0 °C in an ice cold snail Ringer solution containing 20/~Ci of 45Ca (New England Nuclear) and 2 mg/ml of glucose to obtain full intracellular uptake of 45Ca when the calcium pump was inhibited by low temperature. The preparatory experiment showed that 40 min was sufficient to saturate the intracellular 45Ca. Then, the ganglia were incubated in a normal and PTZ-Ringer solution containing the same quantity of 45Ca for 15 min at 22 °C. In the recovery experiment, a 60 min rinse with 45Ca containing normal Ringer was performed. After rinsing of the ganglion with isotonic phosphate buffer, the identified neurons were dissected and electrophoresed as described above. The identified cells were completely freed from surrounding tissue. The destained gels, containing radioactive calcium, were placed on a microscope slide and sectioned using a razor blade fragment into 1 mm long disks. Each disk was transferred to the scintillation vial. The disks were solubilized by the addition of 0.5 ml of a 30 ~ solution of H2Oz to the scintillation vials. The vials were loosely capped, and heated for 12-24 h at 50 °C. After the vials had cooled to room temperature, 7 ml of ACS II (Amersham, Lot 08908) counting mixture was added to each vial. The radioactivity was counted at 90 ~ efficiency for 14C in a LKB Packbeter 1215 liquid scintillation counter.
Preparation of subcellular fraction Cell membrane fraction. Desheathed ganglia of about 300 animals (each preparation in normal snail
Ringer or PTZ-containing snail Ringer) were rinsed in 0.32 M sucrose (pH 7.8 adjusted with NaHCOa), and homogenized in 1 ml of cold 0.32 M sucrose using a glass Teflon homogenizer (700 rpm, 10 strokes). The homogenate was centrifuged at 1,000 g for 10 min. The pellet was washed and centrifuged at 1,000 g for 10 min 3 times with 0.32 M sucrose and the suspension was homogenized using a glass Teflon homogenizer (2,000 rpm, 20 strokes). The homogenate was centrifuged at 1,000 g for 10 min to give a supernatant, S1, and a pellet, P1. The S1 was decanted and saved, whereas P1 was rehomogenized and centrifuged as described above, and supernatant $2 and pellet P2 were obtained. P2 was rehomogenized and centrifuged as described above and supernatant $3 was obtained. SI, $2 and $3 were pooled and centrifuged at 3,000 g for 60 rain. Supernatant, $4 and the loose layer on pellet P4 were centrifuged at 21,000 g for 30 min and supernatant $5 and pellet P5 were obtained. P5 was suspended with about 0.5 ml of 0.32 M sucrose and was layered on a discontinuous sucrose density gradient consisting of 1 ml each of 1.18, 1.16 and 1.13 density sucrose and centrifuged at 100,000 g for 120 min (3 × 5 ml swing-out head, Hitachi 65P). The fraction between 0.32 M and 1.13 density (0.99 M) sucrose was collected and suspended in 0.32 M sucrose (final concentration). The suspension was centrifuged at 30,000 g for 30 rain. The pellet was adjusted to 0.5-1.0 mg/ml protein content with 0.32 M sucrose solution and stored for assay at --80 °C. Lysosome fraction and microsome fraction. The process for preparing the lysosome fraction and microsome fraction from ganglia is shown below. All operations were carried out at 0-4 °C. The lysosome fraction, microsome fraction and plasma membrane fraction were examined by electron microscopy and they showed sufficient purity (Fig. 3). The biochemical characterization of the fractions (lysosome, microsome and plasma membrane) was performed according to the method of Marchbanks 15. Assay of the acid phosphatase activity according to King and Armstrong 13 and fl-glucuronidase activity according to Kato 12 for the lysosome fraction, RNA assay according to Schneider18 for the microsome fraction and Na +, K+-ATPase activity assay according to Gibbs et a1.11 for the plasma membrane fraction
274 homogenized I ml 0.32 M sucrose (pH 7.0) I withglass Teflon homogenizer 800-1,000rpm, 8 strokes, at 0 C 800 g, 20 min, at 4-C supernatant ] 10,000g, 15 rain, at 4 °C
I supernatant 105,000 × g, 60 rain, at 4 "C
residue ] 0.8 and 1.0 M sucrose gradient 55,000 g 15 rain, at 4 °C I
I I .....
I
i
residue (microsome fraction)
residue (lysosome fraction)
showed considerably higher values than those of the other fractions and the starting homogenate (Table I). RESULTS Microdisk electrophoresis pattern changes of P T Z sensitive neurons during BA Fig. 1A is the densitometry pattern of the microdisk electrophoresis pattern of normal PTZ-sensitive neurons and that of P T Z treated PTZ-sensitive neurons. The prominent differences between these two disk patterns were: (1) the single peak of approximately 10-15 kdalton peak in the normal state was divided into 3 peaks when a PTZ-sensitive neuron was incubated for 15 min in P T Z (5 × 10 -2 M); and (2) the flat part of approximately 5-7 kdalton in the normal state became prominent in PTZ incubated PTZ-sensitive neurons. The part with a high molecular weight of more than 30 kdalton was almost constant with some fluctuation. Although the possibility of differences in the higher molecular weight part between PTZ-treated and normal PTZ-sensitive cell exists, the present investi-
intermediate layer
gatlon was focused only on the above-mentioned marked changes. Although the total protein of the samples could not be measured because of the small quantity, the samples used were always almost equal in size to the 5 identified neurons. Judging from the comparison of band width of the standard protein and that of neurons, the total protein of the 5 measured neurons was approximately 30-50 ng. The neurons rinsed for 60 min after P T Z treatment showed almost the same disk pattern as normal neurons (Fig. 1A, lower most curve). The PTZ-non-sensitive neurons (RC-6, LO-1 and RO-1) never manifest BA by P T Z even with prolonged and concentrated application 2°. The disk patterns of normal and P T Z treated PTZ-non-sensitive neurons are shown in Fig. lB. Unlike PTZsensitive neurons, the PTZ-treated PTZ-non-sensitive neurons never changed their disk pattern. Microdisk electrophoresis of fractionated parts To obtain some clue about which subcellular components are responsible for forming the 10-15 and 5-7 kdalton peaks, microdisk electrophoresis of the fractionated parts obtained from clusters which
TABLE 1 Biochemical characteristics of fractions
Whole homogenate Lysosome fraction Microsome fraction Plasma membrane fraction
Na+-K+-ATPase (l~moles/h/mgprotein)
RNA (lzg/mgprotein)
.4cid phosphatase (l~g/h/mgprotein)
tl-gluculonidase (f*g/h/mgprotein)
8.16 1.11 4.98 14.75
5.16 3.87 13.59 4.08
79.1 124.8 48.2 5.3
1.13 4.00 0.22 0.82
275
electrophoresis pattern of a lysosome fraction (100 mg total protein both in normal and PTZ-treated samples). This clearly demonstrates that the 10-15 kdalton peak is prominent in normal, and in PTZ-
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Fig. 1. Microdisk electrophoresis patterns of normal and PTZ-treated neurons. A : PTZ-sensitive neuron. B: PTZ-nonsensitive neuron. Thick line, normal neuron; dotted line, neuron after 15 rain incubation with PTZ; dot-dash line, recovery by 60 min rinsing with normal snail Ringer.
contain a great majority of the PTZ-sensitive neurons was performed. In the Euhadra suboesophageal ganglion, PTZ-sensitive and PTZ-non-sensitive neurons are found. The number of the PTZ-non-sensitive neurons is quite limited (RO-1, LO-1, RC-6, LC-1 and LC-2 only). Most of the other cells, especially neurons in the cluster situated in the dorsal part of the ganglion, are highly PTZ-sensitive (RC-1-5) or moderately PTZ-sensitive (RC-7-13 and most of neurons not named in the other clusters). Therefore, if the dorsal clusters are collected and neurochemical experiments are performed, the results represent the characteristics of the PTZ-sensitive neurons. The desheathed cluster forms, like a brindle resembling balloons, with axons as the strings. The neurons were collected by cutting axons. Thus contamination of non-neuronal elements was limited. Fig. 2A shows a microdisk
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276
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Fig. 4. 45Ca Incorporation into microgels of PTZ-sensitive neurons. Electrophoresis of 100 identified neurons. Thick line and solid circles, normal neurons; dotted line and open circles, after PTZ incubation. Fig. 3. Electron mlcrographs of lysosome fraction (A), microsome fraction (B) and plasma membrane fi-action (C). Bars: 0.5 ,, 11.
treated samples (Fig. 2A, arrow) ; but there is no 5-7 kdalton peak in both normal and PTZ-treated samples. Fig. 2B shows a microdisk electrophoresis pattern of the lnicrosome fraction (100 mg total protein, both in normal and PTZ treated samples). In the micros•me fraction, the 10-15 kdalton peak is very prominent (Fig. 2B, arrow) but there is no 5-7 kdalton w-ak. Fig. 2C shows a microdisk electrophoresis pattern of the plasma membrane fraction (100 mg total protein both in normal and PTZtreated samples). Unlike the micros•me and lysosome fractions, the 5-7 kdalton peak forms a marked elevation in the PTZ-treated sample in the case of the plasma membrane fraction (Fig. 2C, arrow). The 5-7 kdalton peak never appeared in the lysosome and micros•me fraction.s: and the 10-15 kdalton peak never appeared in the plasma membrane fraction. These 3 microdisk electrophoresis patterns suggest that the 5-7 kdalton peak mainly originates from the plasma membrane and the 10-15 kdalton peak mainly originates from the micros•me and lysosome.
Changes of 45Ca incorporation into the intracellular proteins during BA To clarify the relationship between intracellular protein and calcium behavior, 45Ca incorporation into the molecular weight distribution of proteins in the cell was examined. After incubation in normal
snail Ringer containing 20 #Ci of 45Ca for 40 min at 0 '-~C as described in Methods, the PTZ-sensitive neurons were incubated in PTZ snail Ringer containing the same quantity of 45Ca for 15 min at 22 °C. Twenty neurons were used for one gel. The gels were sectioned into 1 m m disks and the radioactivity was measured with a liquid scintillation counter (Fig. 4). Each point in Fig. 4 represents the total dpm/gel slice of 5 gels. The PTZ-treated samples showed a prominent peak of radioactivity of 45Ca at 10-15 and 5-7 kdalton proteins. Judging from the disk patterns of the subcellular fraction., the peak at 5-7 kdaltons suggests an increase of 45Ca in.corporation into the plasma membrane. This result is consistent with the results obtained by computer controlled electron probe X-ray microanalysis 22 as well as that with an ion shower etching of the freeze dried cell membrane z4.
Effect of extracellular calcium concentration on the 45Ca incorporation into proteins during BA To obtain some clue concerning where the calcium which binds to the protein comes from, the 45Ca radioactivity of a disk of 5-7 kdalton protein was measured in a calcium free and four-fold calcium extracellular medium. When extracellular calcium was freed, 45Ca incorporation into this protein decreased, although the peak remained (Fig. 5C). In the four-fold extracellular calcium concentration, the incorporation increased slightly compared with that in normal Ringer, but not in proportion to the extracellular calcium concentration (Fig. 5B).
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incorporation with calcium-free medium. Thick line and solid circles, normal state; dotted line and open circles, after PTZ incubation.
Time course of the 45Ca incorporation To elucidate the role of intracellular calcium movement, the time course of the 45Ca incorporation into proteins during BA was examined. The BA occurred 5-7 men after PTZ application electrophysiologically. The 45Ca incorporation into proteins increased within 5 men (Fig. 6); in particular, the 80-90 % saturation of 45Ca incorporation into 5-7 kdalton protein occurred far earlier than BA manifestation (Fig. 6, upper part). These findings suggest that the increase of 45Ca incorporation is probably the origin and not the result of BA. DISCUSSION Intracellular protein was markedly changed during PTZ-induced BA compared with the normal state in the identified PTZ-sensitive neurons• This change showed recovery after 60 men rinsing with normal Ringer. The PTZ-non-sensitive neurons ma-
278 nifested neither BA nor intracellular protein changes. In the examination using an intracellular calcium mapping technique, the intrace[lu[ar calcium distribution showed a clear difference between the BA state and the normal state in PTZ-sensitive neurons, and PTZ-non-sensitive neurons never showed these intracellular calcium changes 16,~'~. During BA the intracellularly shifted calcium was shown to gather at the inner surface of the cell membrane by an examination using an ion shower milling machine and calcium measurement using a flameless atomic analyzer '~'1. Moreover, the binding state change of calcium occurred at the same membrane site '5. These experimental facts prompted us to seek the substances to which the shifted calcium binds. The present experiment reveals that the substances are most likely the 5-7 and 10-15 kdalton proteins. The detailed characteristics of these proteins, however, are difficult to determine at this stage because of the insufficient qua~.tity of the samples. The high molecular weight part of more than 30 kdalton of the protein showed an almost unchanged pattern with some fluctuation in PTZ-treated and normal PTZ-sensitive neurons. The significance of the higher molecular part in the manifestation of BA, however, is still obscure and requires further investigation. Gainer and his colleagues performed an extensive study on the protein synthesis of a single identified neuron of Otala and Aplysia 8,u. They concluded that the 5 and 12.5 kdalton proteins are specific to spontaneously bursting cells, cell 10 in Otala and R I 5 in Aplysia. Thereafter, in neurosecretory cells of Aplysia, conversion from higher molecular weight protein to low molecular weight protein or peptide was demonstrated1-a,14,19; for example, from 29 to 6 and 3 kdaltons protein 14, and from 18 to 3 kdaltons protein a. Strumwasser and Wilson demonstrated a temporal processing of 12 to 6-9 kdalton protein in the R 15 neuron of Aplysia using a doublelabel technique 19, All of these processes from higher molecular weight protein to lower molecular weight protein required at least 2 h. In our present experiment, 15 min incubation in PTZ resulted in the appearance of the 5-10 kdalton protein and the change in the 10-15 kdalton protein accompanied by calcium incorporation. Since 15 min incubation in PTZ is not sufficient time to show the synthesis of
new proteins, it would be more natural to imagine a shift from one cell compartment to another. The calcium binding to these two proteins prominently increased after PTZ treatment. The fact that the specific calcium binding to PTZ-treated samples after rather drastic treatment such as that with flmercaptoethanol, dithiothreitol and SDS suggests that calcium binding to these two proteins might not be labile. Burgess et al. observed calcium binding to calmodulin in the presence of SDS 4. The calcium binding of our protein is probably of a similar nature. Since a marked release of stored calcium from lysosomes with clear morphological changes has been observed previously 2a and 10-15 kdalton protein most likely originated from lysosomes and microsomes in the present experiment using fractionated materials, we assume that the calcium release from lysosomes will be accompanied by 10-15 kdalton protein. The previous experiment showed calcium gathering at the inner surface of the cell membrane after PTZ treatment 22 with a calcium binding state change 2s. The substance to which the shifted calcium binds is most likely 5-7 kdalton protein. In any case, PTZ-induced BA required calcium incorporation to 10-15 kdalton protein which is related to lysosome and also to 5-7 kdalton protein which is related to plasma membrane. The relationship between 10-15 and 5-7 kdalton proteins, however, is a subject of further investigation. The calcium shift to the 5-7 kdalton protein occurred earlier than the complete electrophysiological manifestation of BA. Even in calcium free medium, the 45Ca intracellularly incorporated in advance shifted to the 5 7 kdalton protein and 10-15 kdalton protein. The study by computer controlled electron probe X-ray microanalysis showed that the shifted calcium was mainly of intracellular origin z2. Electrophysiological observation of PTZ induced BA with cobalt chloride-containing medium to eliminate the calcium influx also showed that the manifestation of BA did not essentially require calcium influx (tmpublished observation). These experimental facts lead us to conclude that the shifted calcium is mainly of intracellular origin, and occurs prior to BA manifestation. In the examination using a computer-controlled electron probe Xray microanalyzer, the calcium binding state after PTZ treatment of PTZ-sensitive neurons was found
279 to be different f r o m the n o r m a l state 25. In the e x a m i n a t i o n using the freeze fracture technique, the i n t r a m e m b r a n e o u s particles were f o u n d to be c h a n g e d b y P T Z t r e a t m e n t 26. The p r o m i n e n t increase o f 45Ca r a d i o a c t i v i t y b y P T Z t r e a t m e n t in 5-7 k d a l t o n p r o t e i n suggests that the shifted intracellular calcium binds with the intracellular cell memb r a n e related protein. Calcium and calcium-dependent regulatory proteins are i m p o r t a n t for the regulation o f various aspects o f cell functions 5,6. B A is a p a t h o l o g i c a l change which is characteristic o f seizure discharge.
A b n o r m a l b e h a v i o r o f the intracellular calcium a n d t h a t o f the related proteins might induce seizure discharge. ACKNOWLEDGEMENTS The a u t h o r s are grateful to Prof. S. H i r a n o , Prof. K. U e m u r a a n d Dr. A. A s o for advice a n d criticism d u r i n g the course o f this experiment. This research was s u p p o r t e d in p a r t by a grant f r o m the J a p a n e s e M i n i s t r y o f Education, Science a n d Culture, a n d by a g r a n t f r o m the Prof. Genichi K a t o M e m o r i a l F u n d for P h y s i o l o g y a n d Medicine.
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