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Li+ uptake into Xenopus and Cynops oocytes injected with exogenous mRNA, observed by flame emission spectroscopy
ANALYTICAL
BICK’HEMISTRY
156,257-262
(1986)
Li+ Uptake into Xenopus and Cynops Oocytes Injected with Exogenous mRNA, Observed by Flame Emission Spectroscopy HITOSHI
AOSHIMA,
Department cf Chemistry Facnltj* qf‘Lihera1
HIRONORI
110, AND SHIGEO
and *Department of‘Biomechanics Arts, 1677-l Yoshida. Yamaguchi Received
January
KOBAYASHI* and Ph~~sio1og.v. 753, Japan
2 1, 1986
Li+ uptake into Xenopus oocytes was measured by flame emission spectroscopy. Li+ uptake into tb: oocytes increased proportionally with incubation time and was dependent on either pH or temperature. Maximum uptake of Li+ was observed around pH 7. Li+ uptake into Xenoplts oocytes increased by a factor of roughly 7 over the range 4-30°C. When mRNA prepared from electroplax of Elecm~phorzrs electricus was injected into Xelropus or Cynops oocytes, Li+ uptake into the injected oocytes increased by the addition of carbamylcholine (Garb). an agonist of the acetylcholine receptor (AChR). This increase of Li+ uptake by Carb was inhibited by d-tubocurarine, an antagonist of nicotinic AChR. Thus, a new method was established for detection of the activity of nicotinic AChR synthesized in oocytes injected with exogenous mRNA. ~tl 1986 Academic Press. Inc.
KEY WORDS: acetylcholine receptor: C)nops with mRNA: Li+ uptake: .Tenopus oocyte.
oocyte:
Xenopus laevis oocytes have been usedas a very sensitive assaysystem for the identification of different kinds of exogenous nlRNA (1). Translated proteins in oocytes injected with exogenous mRNA have been detected by various techniques such as enzyme assay, immunoprecipitation, or electrophysiological measurement (2). Recently, many neurotransmitter receptors or channels have been synthesized in X~nopus oocytes injected with exogenous mRNA and detected by the electrophysiological method (3-5). The electrophysiological method is both sensitive and sophisticated. However, other new methods are required for biochemists who do not have either electrophysiological equipment or knowledge about electrophysiology. It is reasonable to think that oocytes injected with mRNA will increase the uptake or release of ions or drugs through the membrane of the oocytes when proteins which constitute channels or transport systems are synthesized in the oocytes. One of the new ways is to measure the specific uptake or releaseof ions or drugs 257
flame emission
spectroscopy:
injection
mediated by synthesized receptors, channels, or transport systemsin the oocyte. Li+ has been used asa best monovalent ion tracer for transmembrane ion flux measurement by flame emission spectroscopy without using radioisotopes. It is the most sensitive monovalent metal ion in flame emission spectroscopy and is a rare metal ion in comparison to Na’ and K+, which minimizes contamination during the experiments. Acetylcholine receptor (AChR)‘-mediated Li’ flux was measured successfully in vitro by using membrane vesicles isolated from the electric organ of Electrophorus electricus and flame emission spectroscopy (6,7). In this paper, Li+ uptake into the oocytes injected with mRNA prepared from electroplax of E. electric-us was examined by flame emission spectroscopy. Carbamylcholine (Carb), an agonist of AChR, increasedLi+ uptake into both Xenopus and C’Jnops oocytes ’ Abbreviations used: AChR, Carb. carbamylcholine. 0003-2697/86
acetylcholine
receptor:
$3.00
Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved
258
AOSHIMA.
110,
injected with the exogenous mRNA. This increase of Li+ uptake was inhibited strongly by d-tubocurarine. an antagonist of nicotinic AChR. Thus synthesis of nicotinic AChR in the injected oocytes was confirmed by Li+ uptake measurement.
AND
KOBAYASHI
dition of concentrated Tris or HCI solution. Then oocytes were taken by a pipet and washed four times by 1.5 ml of amphibian Ringer solution (115 mM NaCl, 1 mM KCl, 1.8 mM CaCl,, 5 mM Tris, pH 7.2) in a loml glassbottle. The oocytes were incubated in 5 ml of 0.02 N HCl solution overnight in a 30ml clear glassbottle with a cap. After destrucMATERIALS AND METHODS tion of the oocytes by hard shaking, the mixFrogs (Xenopzls laevis), newts (Cynops p-vr- ture was filtered through Toyo filter paper (5.5 rhogaster), and electric eels (Electrophonts cm in diameter). Lithium wasdetermined with electricus) were purchased from Hamamatsu a Hitachi 508A atomic absorption spectromSeibutsukyozai, Hamamatsu, Nihon Seibut- eter used in the flame emission spectroscopy suzairyo, Tokyo, and Shimonoseki Pet Center, mode. A flame of acetylene (2.5 liters/min in Shimonoseki, respectively. All chemicals were 0.5 kg/cm’)-air (13 liters/min in 1.9 kg/cm’) of reagent grade. was used for all measurements. The flame Preparation of’oocytes and FFIRNA. Oocytes emission signal at 670.7 nm was recorded on were dissectedfrom the ovaries of adult female a Hitachi 056 recorder. The sample was asfrogs (Xenopzu laevis) or adult female newts pirated into an acetylene-air flame directly at (C’ynops pyrrhogaster) which were anesthe- a flow rate of 3.2 ml/min. Since some variatized in ice water containing 1 mg/ml MS 222 tions existed in the size of oocytes, dead oo(Sankyo, Tokyo), following a slight modificytes without negative membrane potential cation of the procedure of Kusano et al. (8). sometimesshowedextremely high Li’ uptake. The oocytes were detached manually from the and injected oocytes occasionally failed to inner ovarian epithelium and follicular en- synthesize the receptor, the highest and the velope with a forceps after incubation in col- lowest values of four measurements were lagenase(Sigma type I, 1 mg/ml) Barth solu- omitted. The two middle values of four meation (88 mM NaCl, 1 mM KCl, 2.4 mM surementswere usually averaged to determine NaHC03, 0.33 mM Ca(NO&. 0.4 1 mM the amount of Li+ uptake into the oocytes. CaC12,0.82 mM MgS04, 7.5 mM Tris, pH 7.6) The errors were given as a standard deviation of the two middle values. When the injected for about 1 h at room temperature. mRNA from electroplax of E. elecfriczo was oocytes were used in Lit uptake experiments, purified using the following procedures (9): some injected oocytes were measured electroguanidine thiocyanate homogenization, CsCl physiologically to confirm the synthesis of ultracentrifugation, phenol-chloroform ex- nicotinic AChR before use. traction and oligo(dT)cellulose chromatograLi+ release measurement. Noninjected phy. The concentration of mRNA was esti- Xenopus oocytes were incubated with a mixmated from the absorbance at 260 nm, as- ture of equal amounts of amphibian Ringer and the LiCl solution for more than 6 h and suming ,4[ Cmmgjml = 25. The collagenase-treatedXenopus or CJwzops Li’ was absorbedby the oocytes. Some oocytes oocytes were injected with the purified mRNA died when they were incubated in 100% LiCl (50- 100 ng per cell) and cultured in Barth so- solution for a long time. After being washed four times by 1.5 ml of amphibian Ringer solution at 20°C for either one or two days. Li+ uptake measurement. Oocytes were in- lution, oocytes were incubated in 2 ml amcubated with LiCl solution ( 116 mM LiCl. 1.8 phibian Ringer solution. At various times, 0.2 mM CaClz, 5 mrvr Tris, pH 7.2) for various ml amphibian Ringer solution outside the ooperiods at 20°C. When the pH of the LiCl cytes was taken and the amount of Li+ was solution was changed, it was adjusted by ad- measured by flame emission spectroscopy.
Li’
UPTAKE
INTO
Electrophysiological measurement. Oocytes were placed on the bottom of a small chamber (0.5 ml) and perfused with amphibian Ringer solution (10). The oocytes were currentclamped using a two-electrode sytem. A rectangular pulse current (10 nA, 2-s duration) was injected every 5 s to create hyperpolarizing membrane potentials (11). RESULTS
Figure la shows time dependence of Li+ uptake into noninjected Xenopus oocytes. Li+ uptake was almost proportional to the incubation time until at least 2 h. The kinetic pattern of uptake was similar among oocytes of the same stage from the same frog, though some variations in the rate of Li’ uptake existed among the oocytes from different frogs. Figure lb shows the time dependence of Li+ releasefrom the oocytes which were saturated with Li+ by preincubation in the LiCl solution. Li+ releaseincreasedwith incubation time and tended to saturate gradually. However, Li’ releasedid not follow a simple first-order kinetics (Fig. I b) and seemedto be rather complex. So quantitative analysis of Lit uptake into the oocytes is simpler than that of Li+ release. Xenupzu oocytes sometimes contain the native noradrenaline receptors which elicit smooth outward currents that are carried mainly by potassium ions (12). To detect the
AMPHIBIAN
259
OOCYTES
activity of native noradrenaline receptors in the oocytes, Li+ releasefrom the oocytes containing many electrophysiologically confirmed noradrenaline receptors was measured in the presence and the absence of 0.1 mM noradrenaline. However, no specific Li+ efflux caused by noradrenaline receptors was detected, as shown in Fig. 1b. To characterize Li+ uptake into noninjected oocytes, Li’ uptake was measured at various pH values or at various temperatures. Correlation between the pH change and induction of ion movement has already been suggested in maturing Xenopzts oocytes (2). The effect of pH on the Li+ fluxes in Xenopzts oocytes is shown in Fig. 2. Maximum flux was observed at around pH 7. However, membrane conductance as measured by the electrophysiological method increased slightly with the decrease of pH: at pH < 4, the conductance increased sharply and the membrane depolarized. Lit uptake into Xenopzrs oocytes increasedby a factor of roughly 7 over the range 4-30°C (Fig. 3). Li+ uptake into the Xenopus oocytes injected with mRNA of E. electricus was measured in the presenceof various concentrations of Carb and is summarized in Table 1. Li+ uptake into the oocytes increased with the concentration of Carb. This Li+ uptake was strongly inhibited by the addition of 0.1 mM ci-tubocurarine. Li+ uptake into noninjected IOO
t
b
6 0
Ok-
-L
150 Time
(mini
8
25 Time
0
(mln)
FIG. 1. (a) Li’ uptake into noninjected Xtwopus oocytes versus incubation time. (b) Li+ release from noninjected .k’enopzu oocytes incubated in 50% LiCl solution for 15 h. Five oocytes saturated with Li+ were incubated in 2 ml amphibian Ringer solution with (A) or without (0) 0.1 ITIM noradrenaline and 0.2 ml of the solution was taken into 2.8 ml distilled water at different times to measure Li+ amount by flame emission spectroscopy. [Li’],, Li’ amount inside the oocyte after time t; [Li+lO. Li’ amount inside the oocyte at time 0.
260
AOSHIMA.
110. AND
KOBAYASHI TABLE
I
Li+ UPTAKE INTO .Y~ptts OOCYTES INJECTED WITH MRNA PREPARED FROM ELECT’ROPLAX OF E. &cfrrc~r.~ AFTER 30 MIN INCUBATION IN THE LiCl SOLUTION
l
Carb (mM)
A 6
9
PH
FIG. 2. The pH dependence of Li’ uptake and release compared with that of membrane conductance in noninjected .~~e~70~7us oocytes. Li+ uptake and release in the oocytes was measured after 30 min incubation. The membrane conductance in the oocytes was measured by the electrophysiological method. The value of Li’ uptake and release at about pH 7 and that of membrane conductance at pH 3.45 were taken as a standard, 100%.
oocytes was not affected by the addition of Carb (data not shown). These results can be explained by the synthesis of nicotinic AChR in the oocytes injected with exogenous mRNA of E. electricus which increased Li+ uptake into the oocytes. Time dependence of AChR-mediated specific Li+ uptake into the oocytes was examined in the presence of 2 mM Carb and is shown in Fig. 4. Lif uptake proceeded rapidly within 1 min and then continued gradually with the incubation time. This is consistent with the result that membrane vesicles
b
Li’
0 0.02 0.05 0.1 0.5 2
0.75 1.17 1.14 1.23 2.19 2.99
Drugs None 2 mM Carb 2 mM Carb and 0. I mM cl-turbocurarine
Tsmperature
I 20
l.02t0.ll
0
30
FIG. 3. Dependence of Li+ uptake into noninjected Xenopzls oocyte on temperature. Oocytes were incubated in the LiCl solution at various temperatures for 60 min and Li’ uptake was measured by flame emission spectroscopy.
0. I3 0.31 0.15 0.04 0.10 0.09
prepared from E. dectriczn show a rapid influx followed by a slow influx after completion of the equilibrium of desensitization ( 13). CJVZO~Soocytes prepared from adult female newts were used for the Li+ uptake experiments after injection of mRNA purified from E. electricus. Cyrzops oocytes also increased Li+ uptake in the presence of Carb (Table 2), which indicated that exogenous mRNA could be translated in CJVUP.Y oocytes as already observed by electrophysiological measure-
0
/
20 Time
I ‘C )
f + + f k *
0.82 + 0.2 1 2.64 + 0. I I
IO I IO
(nmol/oocyte)
n I
30
hin)
FIG. 4. Time dependence ofagonist-induced Lit uptake into Xenqxls oocytes injected with E. ekfricns mRNA. Li+ uptake into the injected oocytes was measured at various incubation times in the LiCI solution with or without 2 mM Carb. The difference between the Li’ amount with and without 2 mM Carb was taken as the value ofagonistinduced Li’ uptake mediated by AChR.
Li+
UPTAKE
INTO
AMPHIBIAN
OOCYTES
261
Morgan et al. have already succeeded in the measurement of 36Cll uptake mediated by Li’ UPTAKE INTO C~nqs OOCYTES INJECTEI) WITH mouse band 3 protein which was synthesized MRNA PREPARED FROM ELECTROPLAX OF E. electri~.~ AFTER 30 MIN INCUBATION IN THE MIXTURE OFTHE LiCl in the Xmopzu oocytes injected with mRNA SOLUTION (80%) AND AMPHIBIAN RINGER SOLUTION from the spleensof anemic mice (20). (20%). Camps oocytes (diameter 1.6-l .9 mm) were larger than Xmopzrs oocytes (diameter Drllg Lit (nmol/oocyte) 1.0-I. 1 mm) and alive longer than the latter None 0.88 f 0.06 in Barth solution, as reported in a previous 0.02 mM Carb 0.78 f 0.13 paper ( 14). Cj~rups oocytes also synthesized 0.2 mM Carb 1.05 f 0.04 nicotinic AChR and showed the AChR-me2 mM Carb I .34 f 0. I6 diated Lit translocation in the presence of 2 InM Carb and 0. I mM d-tubocurarine 0.74 f 0.02 Carb. Li’ fluxes in noninjected oocytes were maximum around pH 7 and decreasedwith the decreaseof pH. while the membrane conDISCUSSION ductance examined by electrophysiological The membrane vesicles prepared either measurement increasedgradually with the defrom the electric organ of electric fishesor from creaseof pH: at pH < 4. the conductance inrat brain have been used to measure the rate creased sharply and the membrane depolarof uptake or releaseof ions and drugs in studies ized. This conductance increase could arise from increased permeability to H+. K+, Cl-. of neurotransmitter receptors and high-affinity transport systemsof transmitters by many re- or Na+. The difference in the pH dependence searchers ( 15.-18). This study examined between Li+ uptake and releasewas observed whether or not amphibian oocytes could be (Fig. 2). This difference might be caused beused for similar quantitative kinetic experi- causeuptake and releasefollowed different kiments in the transport systems through cell netics, as shown in Figs. la and b. membranes. The use of the oocytes offers the Internal volume of an oocyte where Li+ important advantage that the desiredtransport could dissolve was estimated to be about 100 systemscan be translated in the membrane of nl from the Li+ uptake after overnight incuoocytes by injection of exogenous mRNA. bation. The uptake and releaseof Li’ in the This system is useful for partially purifying oocytes followed different kinetics, as shown mRNAs ( 19) and may prove useful in cloning in Figs. la and b, because Li’ concentration the genescoding for the proteins constituting outside -the oocytes was maintained almost the transport systems. constant during the uptake measurement. In this stud.y. we succeeded in measuring while that inside the oocyte decreasedwith the Li+ fluxes mediated by nicotinic AChR which incubation time during the release measurewere synthesized in .Yerwpus oocytes by in- ment. Since oocytes are constructed of various jecting mRNA of E. dectricus. This method kinds of organelles and Lif cannot diffuse is inferior to the electrophysiological method freely in the oocytes, Li’ release possibly dein sensitivity or sophistication. However, this viated from a simple first-order kinetics. The method is more familiar to biochemists who Li+ uptake or releasewas not affected by addo not have either electrophysiological equip- dition of 0.1 mM ouabain to the Li+ solution. ment or knowledge about electrophysiology. So it was unlikely that Na/K ATPase caused Moreover, this method can be extended using the asymmetry in the transport process.Howradioisotopes, to the transport systemswhich ever, it cannot be denied that the complex kido not causemembrane current or membrane netics of Lit releasewas causedbecauseof the conductance change. From this point of view, releasemeasurement without a shaker. TABLE
2
262
AOSHIMA,
110. AND
Oocytes with many native noradrenaline receptors did not show a detectable Lit release in the presence of 0.1 mM noradrenaline. A possible reason for this result is that the K+ channels induced by noradrenaline do not have a detectable Lif permeability in our flux assay. ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Special Research on Molecular Mechanism of Bioelectrical Response (60115006) from the Japanese Ministry of Education, Science and Culture. The authors thank Mr. Michael Higgins for his valuable help with the preparation of this article.
REFERENCES 1. Gurdon, J. B., Lane. C. D.. Woodland, H. R.. and Marbaix. G. (1971) Nature (London) 233, 177-l 82. 2. Soreq, H. (1985) CRC Crit. Rev. Biochem. 18, 199238. 3. Barnard, E. A., Miledi, R., and Sumikawa, K. (1982) Proc. R. Sot. London B 215, 241-246. 4. Gundersen, C. B.. Miledi, R.. and Parker, I. (1984) Nature (London) 308, 42 I-424. 5. Houamed, K. M.. Bilbe, G., Smark, T. G., Constanti. A., Brown, P. A.. Barnard, E. A., and Richards, B. M. (1984) Nature (London) 310, 318-321. 6. Ramseyer. G. O., Morrison, G. H., Aoshima. H.. and Hess, G. P. (I 98 I) Anal. Biochem. 115. 34-4 I.
KOBAYASHI
7. Aoshima. H. (1983) J. Bwchrn~. 94, 1739- I75 I 8. Kusano. K.. Miledi, R.. and Stinnarkre. J. ( 1983) J P/z~sio/. 328, 143- 170. 9. Maniatis, T., Fristsch, E. F., and Sambrook. J. (1982) Molecular Cloning. A Laboratory Manual, pp. 196198. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York. IO. Kobayashi. S.. and Murakami, N. ( 1982) Brain RKS. Bull. 8. 12 1-726. 1 I. Kobayashi. S., and Aoshima, H. (1986) Dev. Brmz Res. 24, 2 1 l-2 16. 12. Sumikawa, K.. Parker. I.. and Miledi, R. ( 1984) Pm. R. SK. London B 223, 255-260. 13. Hess, G. P., Cash. D. J.. and Aoshima. H. ( 1979) Fllture (London) 282, 329-33 1. 14. Kobayashi. S.. Iio. H.. and Aoshima, H. (1986) ;Zfo/. Brain Re3 , in press. IS. Kasai. M., and Changeux. J. P. ( 197 I) 1. :Ifcmhr. Bit)/. 6, l-80. 16. Hess, G. P.. Cash. D. J., and Aoshima, H. (1983) .&mu. Rw BiopkyJ. Bioeng. 12, 443-473. 17. Cash, D. J.. Aoshima. H., Pasquale. E. B.. and Hess. G. P. (1985) Rev. PhJ:yiol. B~ochem. Pharrnac~ol. 102.73-I 17. 18. Fonnum. F.. Karlsen. R. L.. Sorenssen. D. M.. Sterri. S., and Walaos. I. (1980) in The Cell Surface and Neuronal Function (Cotman. C. W., Poste. G., and Nicolson. G. L.. eds.), Vol. 6. pp. 456-504. NorthHolland, Amsterdam/New York/Oxford. 19. Sumikawa. K.. Parker, I., and Miledi. R. ( 1984) Prw. Nutl. Arad. Sci. US.4 81, 7994-7999. 20. Morgan. M., Hanke. P.. Gryorczyk, R., Tintschl. A.. Fasold. H., and Passow, H. (1985) EMBBO J. 4. 1927-1931.
BICK’HEMISTRY
156,257-262
(1986)
Li+ Uptake into Xenopus and Cynops Oocytes Injected with Exogenous mRNA, Observed by Flame Emission Spectroscopy HITOSHI
AOSHIMA,
Department cf Chemistry Facnltj* qf‘Lihera1
HIRONORI
110, AND SHIGEO
and *Department of‘Biomechanics Arts, 1677-l Yoshida. Yamaguchi Received
January
KOBAYASHI* and Ph~~sio1og.v. 753, Japan
2 1, 1986
Li+ uptake into Xenopus oocytes was measured by flame emission spectroscopy. Li+ uptake into tb: oocytes increased proportionally with incubation time and was dependent on either pH or temperature. Maximum uptake of Li+ was observed around pH 7. Li+ uptake into Xenoplts oocytes increased by a factor of roughly 7 over the range 4-30°C. When mRNA prepared from electroplax of Elecm~phorzrs electricus was injected into Xelropus or Cynops oocytes, Li+ uptake into the injected oocytes increased by the addition of carbamylcholine (Garb). an agonist of the acetylcholine receptor (AChR). This increase of Li+ uptake by Carb was inhibited by d-tubocurarine, an antagonist of nicotinic AChR. Thus, a new method was established for detection of the activity of nicotinic AChR synthesized in oocytes injected with exogenous mRNA. ~tl 1986 Academic Press. Inc.
KEY WORDS: acetylcholine receptor: C)nops with mRNA: Li+ uptake: .Tenopus oocyte.
oocyte:
Xenopus laevis oocytes have been usedas a very sensitive assaysystem for the identification of different kinds of exogenous nlRNA (1). Translated proteins in oocytes injected with exogenous mRNA have been detected by various techniques such as enzyme assay, immunoprecipitation, or electrophysiological measurement (2). Recently, many neurotransmitter receptors or channels have been synthesized in X~nopus oocytes injected with exogenous mRNA and detected by the electrophysiological method (3-5). The electrophysiological method is both sensitive and sophisticated. However, other new methods are required for biochemists who do not have either electrophysiological equipment or knowledge about electrophysiology. It is reasonable to think that oocytes injected with mRNA will increase the uptake or release of ions or drugs through the membrane of the oocytes when proteins which constitute channels or transport systems are synthesized in the oocytes. One of the new ways is to measure the specific uptake or releaseof ions or drugs 257
flame emission
spectroscopy:
injection
mediated by synthesized receptors, channels, or transport systemsin the oocyte. Li+ has been used asa best monovalent ion tracer for transmembrane ion flux measurement by flame emission spectroscopy without using radioisotopes. It is the most sensitive monovalent metal ion in flame emission spectroscopy and is a rare metal ion in comparison to Na’ and K+, which minimizes contamination during the experiments. Acetylcholine receptor (AChR)‘-mediated Li’ flux was measured successfully in vitro by using membrane vesicles isolated from the electric organ of Electrophorus electricus and flame emission spectroscopy (6,7). In this paper, Li+ uptake into the oocytes injected with mRNA prepared from electroplax of E. electric-us was examined by flame emission spectroscopy. Carbamylcholine (Carb), an agonist of AChR, increasedLi+ uptake into both Xenopus and C’Jnops oocytes ’ Abbreviations used: AChR, Carb. carbamylcholine. 0003-2697/86
acetylcholine
receptor:
$3.00
Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved
258
AOSHIMA.
110,
injected with the exogenous mRNA. This increase of Li+ uptake was inhibited strongly by d-tubocurarine. an antagonist of nicotinic AChR. Thus synthesis of nicotinic AChR in the injected oocytes was confirmed by Li+ uptake measurement.
AND
KOBAYASHI
dition of concentrated Tris or HCI solution. Then oocytes were taken by a pipet and washed four times by 1.5 ml of amphibian Ringer solution (115 mM NaCl, 1 mM KCl, 1.8 mM CaCl,, 5 mM Tris, pH 7.2) in a loml glassbottle. The oocytes were incubated in 5 ml of 0.02 N HCl solution overnight in a 30ml clear glassbottle with a cap. After destrucMATERIALS AND METHODS tion of the oocytes by hard shaking, the mixFrogs (Xenopzls laevis), newts (Cynops p-vr- ture was filtered through Toyo filter paper (5.5 rhogaster), and electric eels (Electrophonts cm in diameter). Lithium wasdetermined with electricus) were purchased from Hamamatsu a Hitachi 508A atomic absorption spectromSeibutsukyozai, Hamamatsu, Nihon Seibut- eter used in the flame emission spectroscopy suzairyo, Tokyo, and Shimonoseki Pet Center, mode. A flame of acetylene (2.5 liters/min in Shimonoseki, respectively. All chemicals were 0.5 kg/cm’)-air (13 liters/min in 1.9 kg/cm’) of reagent grade. was used for all measurements. The flame Preparation of’oocytes and FFIRNA. Oocytes emission signal at 670.7 nm was recorded on were dissectedfrom the ovaries of adult female a Hitachi 056 recorder. The sample was asfrogs (Xenopzu laevis) or adult female newts pirated into an acetylene-air flame directly at (C’ynops pyrrhogaster) which were anesthe- a flow rate of 3.2 ml/min. Since some variatized in ice water containing 1 mg/ml MS 222 tions existed in the size of oocytes, dead oo(Sankyo, Tokyo), following a slight modificytes without negative membrane potential cation of the procedure of Kusano et al. (8). sometimesshowedextremely high Li’ uptake. The oocytes were detached manually from the and injected oocytes occasionally failed to inner ovarian epithelium and follicular en- synthesize the receptor, the highest and the velope with a forceps after incubation in col- lowest values of four measurements were lagenase(Sigma type I, 1 mg/ml) Barth solu- omitted. The two middle values of four meation (88 mM NaCl, 1 mM KCl, 2.4 mM surementswere usually averaged to determine NaHC03, 0.33 mM Ca(NO&. 0.4 1 mM the amount of Li+ uptake into the oocytes. CaC12,0.82 mM MgS04, 7.5 mM Tris, pH 7.6) The errors were given as a standard deviation of the two middle values. When the injected for about 1 h at room temperature. mRNA from electroplax of E. elecfriczo was oocytes were used in Lit uptake experiments, purified using the following procedures (9): some injected oocytes were measured electroguanidine thiocyanate homogenization, CsCl physiologically to confirm the synthesis of ultracentrifugation, phenol-chloroform ex- nicotinic AChR before use. traction and oligo(dT)cellulose chromatograLi+ release measurement. Noninjected phy. The concentration of mRNA was esti- Xenopus oocytes were incubated with a mixmated from the absorbance at 260 nm, as- ture of equal amounts of amphibian Ringer and the LiCl solution for more than 6 h and suming ,4[ Cmmgjml = 25. The collagenase-treatedXenopus or CJwzops Li’ was absorbedby the oocytes. Some oocytes oocytes were injected with the purified mRNA died when they were incubated in 100% LiCl (50- 100 ng per cell) and cultured in Barth so- solution for a long time. After being washed four times by 1.5 ml of amphibian Ringer solution at 20°C for either one or two days. Li+ uptake measurement. Oocytes were in- lution, oocytes were incubated in 2 ml amcubated with LiCl solution ( 116 mM LiCl. 1.8 phibian Ringer solution. At various times, 0.2 mM CaClz, 5 mrvr Tris, pH 7.2) for various ml amphibian Ringer solution outside the ooperiods at 20°C. When the pH of the LiCl cytes was taken and the amount of Li+ was solution was changed, it was adjusted by ad- measured by flame emission spectroscopy.
Li’
UPTAKE
INTO
Electrophysiological measurement. Oocytes were placed on the bottom of a small chamber (0.5 ml) and perfused with amphibian Ringer solution (10). The oocytes were currentclamped using a two-electrode sytem. A rectangular pulse current (10 nA, 2-s duration) was injected every 5 s to create hyperpolarizing membrane potentials (11). RESULTS
Figure la shows time dependence of Li+ uptake into noninjected Xenopus oocytes. Li+ uptake was almost proportional to the incubation time until at least 2 h. The kinetic pattern of uptake was similar among oocytes of the same stage from the same frog, though some variations in the rate of Li’ uptake existed among the oocytes from different frogs. Figure lb shows the time dependence of Li+ releasefrom the oocytes which were saturated with Li+ by preincubation in the LiCl solution. Li+ releaseincreasedwith incubation time and tended to saturate gradually. However, Li’ releasedid not follow a simple first-order kinetics (Fig. I b) and seemedto be rather complex. So quantitative analysis of Lit uptake into the oocytes is simpler than that of Li+ release. Xenupzu oocytes sometimes contain the native noradrenaline receptors which elicit smooth outward currents that are carried mainly by potassium ions (12). To detect the
AMPHIBIAN
259
OOCYTES
activity of native noradrenaline receptors in the oocytes, Li+ releasefrom the oocytes containing many electrophysiologically confirmed noradrenaline receptors was measured in the presence and the absence of 0.1 mM noradrenaline. However, no specific Li+ efflux caused by noradrenaline receptors was detected, as shown in Fig. 1b. To characterize Li+ uptake into noninjected oocytes, Li’ uptake was measured at various pH values or at various temperatures. Correlation between the pH change and induction of ion movement has already been suggested in maturing Xenopzts oocytes (2). The effect of pH on the Li+ fluxes in Xenopzts oocytes is shown in Fig. 2. Maximum flux was observed at around pH 7. However, membrane conductance as measured by the electrophysiological method increased slightly with the decrease of pH: at pH < 4, the conductance increased sharply and the membrane depolarized. Lit uptake into Xenopzrs oocytes increasedby a factor of roughly 7 over the range 4-30°C (Fig. 3). Li+ uptake into the Xenopus oocytes injected with mRNA of E. electricus was measured in the presenceof various concentrations of Carb and is summarized in Table 1. Li+ uptake into the oocytes increased with the concentration of Carb. This Li+ uptake was strongly inhibited by the addition of 0.1 mM ci-tubocurarine. Li+ uptake into noninjected IOO
t
b
6 0
Ok-
-L
150 Time
(mini
8
25 Time
0
(mln)
FIG. 1. (a) Li’ uptake into noninjected Xtwopus oocytes versus incubation time. (b) Li+ release from noninjected .k’enopzu oocytes incubated in 50% LiCl solution for 15 h. Five oocytes saturated with Li+ were incubated in 2 ml amphibian Ringer solution with (A) or without (0) 0.1 ITIM noradrenaline and 0.2 ml of the solution was taken into 2.8 ml distilled water at different times to measure Li+ amount by flame emission spectroscopy. [Li’],, Li’ amount inside the oocyte after time t; [Li+lO. Li’ amount inside the oocyte at time 0.
260
AOSHIMA.
110. AND
KOBAYASHI TABLE
I
Li+ UPTAKE INTO .Y~ptts OOCYTES INJECTED WITH MRNA PREPARED FROM ELECT’ROPLAX OF E. &cfrrc~r.~ AFTER 30 MIN INCUBATION IN THE LiCl SOLUTION
l
Carb (mM)
A 6
9
PH
FIG. 2. The pH dependence of Li’ uptake and release compared with that of membrane conductance in noninjected .~~e~70~7us oocytes. Li+ uptake and release in the oocytes was measured after 30 min incubation. The membrane conductance in the oocytes was measured by the electrophysiological method. The value of Li’ uptake and release at about pH 7 and that of membrane conductance at pH 3.45 were taken as a standard, 100%.
oocytes was not affected by the addition of Carb (data not shown). These results can be explained by the synthesis of nicotinic AChR in the oocytes injected with exogenous mRNA of E. electricus which increased Li+ uptake into the oocytes. Time dependence of AChR-mediated specific Li+ uptake into the oocytes was examined in the presence of 2 mM Carb and is shown in Fig. 4. Lif uptake proceeded rapidly within 1 min and then continued gradually with the incubation time. This is consistent with the result that membrane vesicles
b
Li’
0 0.02 0.05 0.1 0.5 2
0.75 1.17 1.14 1.23 2.19 2.99
Drugs None 2 mM Carb 2 mM Carb and 0. I mM cl-turbocurarine
Tsmperature
I 20
l.02t0.ll
0
30
FIG. 3. Dependence of Li+ uptake into noninjected Xenopzls oocyte on temperature. Oocytes were incubated in the LiCl solution at various temperatures for 60 min and Li’ uptake was measured by flame emission spectroscopy.
0. I3 0.31 0.15 0.04 0.10 0.09
prepared from E. dectriczn show a rapid influx followed by a slow influx after completion of the equilibrium of desensitization ( 13). CJVZO~Soocytes prepared from adult female newts were used for the Li+ uptake experiments after injection of mRNA purified from E. electricus. Cyrzops oocytes also increased Li+ uptake in the presence of Carb (Table 2), which indicated that exogenous mRNA could be translated in CJVUP.Y oocytes as already observed by electrophysiological measure-
0
/
20 Time
I ‘C )
f + + f k *
0.82 + 0.2 1 2.64 + 0. I I
IO I IO
(nmol/oocyte)
n I
30
hin)
FIG. 4. Time dependence ofagonist-induced Lit uptake into Xenqxls oocytes injected with E. ekfricns mRNA. Li+ uptake into the injected oocytes was measured at various incubation times in the LiCI solution with or without 2 mM Carb. The difference between the Li’ amount with and without 2 mM Carb was taken as the value ofagonistinduced Li’ uptake mediated by AChR.
Li+
UPTAKE
INTO
AMPHIBIAN
OOCYTES
261
Morgan et al. have already succeeded in the measurement of 36Cll uptake mediated by Li’ UPTAKE INTO C~nqs OOCYTES INJECTEI) WITH mouse band 3 protein which was synthesized MRNA PREPARED FROM ELECTROPLAX OF E. electri~.~ AFTER 30 MIN INCUBATION IN THE MIXTURE OFTHE LiCl in the Xmopzu oocytes injected with mRNA SOLUTION (80%) AND AMPHIBIAN RINGER SOLUTION from the spleensof anemic mice (20). (20%). Camps oocytes (diameter 1.6-l .9 mm) were larger than Xmopzrs oocytes (diameter Drllg Lit (nmol/oocyte) 1.0-I. 1 mm) and alive longer than the latter None 0.88 f 0.06 in Barth solution, as reported in a previous 0.02 mM Carb 0.78 f 0.13 paper ( 14). Cj~rups oocytes also synthesized 0.2 mM Carb 1.05 f 0.04 nicotinic AChR and showed the AChR-me2 mM Carb I .34 f 0. I6 diated Lit translocation in the presence of 2 InM Carb and 0. I mM d-tubocurarine 0.74 f 0.02 Carb. Li’ fluxes in noninjected oocytes were maximum around pH 7 and decreasedwith the decreaseof pH. while the membrane conDISCUSSION ductance examined by electrophysiological The membrane vesicles prepared either measurement increasedgradually with the defrom the electric organ of electric fishesor from creaseof pH: at pH < 4. the conductance inrat brain have been used to measure the rate creased sharply and the membrane depolarof uptake or releaseof ions and drugs in studies ized. This conductance increase could arise from increased permeability to H+. K+, Cl-. of neurotransmitter receptors and high-affinity transport systemsof transmitters by many re- or Na+. The difference in the pH dependence searchers ( 15.-18). This study examined between Li+ uptake and releasewas observed whether or not amphibian oocytes could be (Fig. 2). This difference might be caused beused for similar quantitative kinetic experi- causeuptake and releasefollowed different kiments in the transport systems through cell netics, as shown in Figs. la and b. membranes. The use of the oocytes offers the Internal volume of an oocyte where Li+ important advantage that the desiredtransport could dissolve was estimated to be about 100 systemscan be translated in the membrane of nl from the Li+ uptake after overnight incuoocytes by injection of exogenous mRNA. bation. The uptake and releaseof Li’ in the This system is useful for partially purifying oocytes followed different kinetics, as shown mRNAs ( 19) and may prove useful in cloning in Figs. la and b, because Li’ concentration the genescoding for the proteins constituting outside -the oocytes was maintained almost the transport systems. constant during the uptake measurement. In this stud.y. we succeeded in measuring while that inside the oocyte decreasedwith the Li+ fluxes mediated by nicotinic AChR which incubation time during the release measurewere synthesized in .Yerwpus oocytes by in- ment. Since oocytes are constructed of various jecting mRNA of E. dectricus. This method kinds of organelles and Lif cannot diffuse is inferior to the electrophysiological method freely in the oocytes, Li’ release possibly dein sensitivity or sophistication. However, this viated from a simple first-order kinetics. The method is more familiar to biochemists who Li+ uptake or releasewas not affected by addo not have either electrophysiological equip- dition of 0.1 mM ouabain to the Li+ solution. ment or knowledge about electrophysiology. So it was unlikely that Na/K ATPase caused Moreover, this method can be extended using the asymmetry in the transport process.Howradioisotopes, to the transport systemswhich ever, it cannot be denied that the complex kido not causemembrane current or membrane netics of Lit releasewas causedbecauseof the conductance change. From this point of view, releasemeasurement without a shaker. TABLE
2
262
AOSHIMA,
110. AND
Oocytes with many native noradrenaline receptors did not show a detectable Lit release in the presence of 0.1 mM noradrenaline. A possible reason for this result is that the K+ channels induced by noradrenaline do not have a detectable Lif permeability in our flux assay. ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Special Research on Molecular Mechanism of Bioelectrical Response (60115006) from the Japanese Ministry of Education, Science and Culture. The authors thank Mr. Michael Higgins for his valuable help with the preparation of this article.
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7. Aoshima. H. (1983) J. Bwchrn~. 94, 1739- I75 I 8. Kusano. K.. Miledi, R.. and Stinnarkre. J. ( 1983) J P/z~sio/. 328, 143- 170. 9. Maniatis, T., Fristsch, E. F., and Sambrook. J. (1982) Molecular Cloning. A Laboratory Manual, pp. 196198. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York. IO. Kobayashi. S.. and Murakami, N. ( 1982) Brain RKS. Bull. 8. 12 1-726. 1 I. Kobayashi. S., and Aoshima, H. (1986) Dev. Brmz Res. 24, 2 1 l-2 16. 12. Sumikawa, K.. Parker. I.. and Miledi, R. ( 1984) Pm. R. SK. London B 223, 255-260. 13. Hess, G. P., Cash. D. J.. and Aoshima. H. ( 1979) Fllture (London) 282, 329-33 1. 14. Kobayashi. S.. Iio. H.. and Aoshima, H. (1986) ;Zfo/. Brain Re3 , in press. IS. Kasai. M., and Changeux. J. P. ( 197 I) 1. :Ifcmhr. Bit)/. 6, l-80. 16. Hess, G. P.. Cash. D. J., and Aoshima, H. (1983) .&mu. Rw BiopkyJ. Bioeng. 12, 443-473. 17. Cash, D. J.. Aoshima. H., Pasquale. E. B.. and Hess. G. P. (1985) Rev. PhJ:yiol. B~ochem. Pharrnac~ol. 102.73-I 17. 18. Fonnum. F.. Karlsen. R. L.. Sorenssen. D. M.. Sterri. S., and Walaos. I. (1980) in The Cell Surface and Neuronal Function (Cotman. C. W., Poste. G., and Nicolson. G. L.. eds.), Vol. 6. pp. 456-504. NorthHolland, Amsterdam/New York/Oxford. 19. Sumikawa. K.. Parker, I., and Miledi. R. ( 1984) Prw. Nutl. Arad. Sci. US.4 81, 7994-7999. 20. Morgan. M., Hanke. P.. Gryorczyk, R., Tintschl. A.. Fasold. H., and Passow, H. (1985) EMBBO J. 4. 1927-1931.