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Pharmacological characterization of serotonin receptor induced by rat brain messenger RNA in Xenopus oocytes
Brain Research, 362 (1986) 199-203 Elsevier
199
BRE 21282
Pharmacological characterization of serotonin receptor induced by rat brain messenger RNA in Xenopusoocytes Y. SAKAI, H. KIMURA and K. OKAMOTO Departmentof Pharmacology, National DefenseMedical College, 3-2 Namiki, Tokorozawa, Saitama359 (Japan)
(Accepted August 28th, 1985) Key words: serotonin receptor - - messenger ribonucleic acid (RNA) --Xenopus oocyte - cyclic adenosine monophosphate (AMP) - - prostaglandin
Serotonin receptors incorporated in the membrane ofXenopus oocytes injected with mRNA extracted from the rat brain was investigated by intracellular recording. Serotonin elicited the membrane depolarization accompanied with membrane potential fluctuations. This serotonin action was suppressed by serotonin antagonists such as methysergide, cyproheptadine and ketanserin. Dibutyryl cyclic AMP and papaverine depolarized the membrane as seen in applying serotonin. These observations indicate that serotonin actions might involve the cAMP system.
Sumikawa et al. 17 have first generated functional nicotinic acetylcholine receptors in the oocytes of Xenopus laevis by injecting m R N A from Torpedo marmorata utilizing the technique of Gurdon et al. 7. Since then, several receptors for neurotransmitters including serotonin 3.4, glutamic acid 5, G A B A 4a2,t6 and glycine 6 have been successively generated in the Xenopus oocytes by transplanting the m R N A from the rat brain, and their properties have been investigated. As our first step of pharmacological characterization of the receptors generated in Xenopus oocytes, serotonin receptors produced by injection of m R N A extracted from neonatal rat brain were investigated by an intracellular recording technique in the present study. Two distinct types of the serotonin receptor are known. $1 receptor is specifically labeled by [3H]serotonin l, and S2 receptor is labeled by [3H]spiperone~4,~5 The neuronal action of serotonin elicited by S1 receptor was suggested to be mediated by serotonin-sensitive adenylate cyclase 14. Such serotoninsensitive adenylate cyclase was reported to exist in immature rat brain 9. Whether the cyclic nucleotide system is involved in the serotonin action mediated
by these expressed receptors is the primary question to be answered in the present study. The m R N A was extracted from whole brains of 1day-old Wistar rats using a guanidium/cesium chloride method 2. About 50 nl of 1 mg/ml poly(A)m R N A solution was injected into each Xenopus oocyte, and the oocytes were cultured for 3 days at 19 °C in modified Barth medium containing streptomycin 0.1 mg/ml and penicillin 100 U/ml. One oocyte was placed at a time on a nylon mesh in a superfusion chamber, and the bath was superfused at a rate of 2 ml/min throughout the experiments with the frog Ringer solution (control medium, composition (in mM): NaCI, 120; KCI, 2; CaCI2, 1.8; HEPES, 5; pH 7.4). Microelectrodes filled with 3 M KCI (10-20 MQ) were used for intracellular recordings in most experiments and those filled with 3 M potassium acetate (15-25 MQ) were employed only for changing the membrane potential to estimate the reversal potential of serotonin responses. Only the oocytes exhibiting resting membrane potentials o f - 3 0 to - 6 0 mV were adopted. The mean resting membrane potentials of control and mRNA-
Correspondence: Y. Sakai, Department of Pharmacology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359, Japan.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
200 injected oocytes were - 4 6 . 7 + 12.2 mV (n = 35) and -46.2 + 11.3 mV (n = 62), respectively. Serotonin was applied either by iontophoresis (50 mM solution was filled in the micropipette) on the surface of the oocyte m e m b r a n e or by superfusion (100/xM in the frog Ringer solution). Other agents following were applied only by superfusion: methysergide (10/xM), cyproheptadine (10/xM), ketansefin (50/xM), dopamine (1 mM), epinephrine (1 mM), theophylline (5 mM), dibutyryl cyclic A M P (2.5 and 0.25 mM), papaverine (1 mM), P G D 2 (50 #xM), and P G E 2 (50/xM), all being dissolved in control medium. Cyproheptadine and ketanserin were kindly presented by Dr. Y. Yamawaki, The Institute of Clinical Research, Kure National Hospital (Kure, Japan). Dibutyryl c A M P was from P-L Biochem. (WI, A
U.S.A.). Prostaglandin E 2 and D 2 were from Funakoshi Chem. (Tokyo, Japan). Other agents including serotonin were from Wako Pure Chem. (Tokyo, Japan.) Control Xenopus oocytes, i.e. those not injected with m R N A responded only faintly and inconsistently to serotonin applied by either iontophoresis or superfusion. In 5 out of 14 control oocytes, serotonin induced hyperpolarizations, 3 oocytes had no response to serotonin, and the remaining 6 oocytes were depolarized slightly ( 1 - 2 mV) by the application of serotonin. In contrast, as shown in Fig. 1A, all the 62 oocytes injected rat brain m R N A were depolarized by serotonin with the long latency to onset and slow fluctuations of the m e m b r a n e potential (in agreement with G u n d e r s e n et al.3). A mean value for the reversal potential of seroto-
B 2 0 nA
- - / ~
._U
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40
- ~ / ~ - ~ ,f,
O.OlmM'- c ) l ~ W l ~ e
80
_J
----~'~-~
O.Of~M- kehmeerlem 150
200
Fig. 1. A: depolarizations and potential fluctuations elicited by different iontophoretic currents of serotonin applied to an oocyte that had previously been injected with rat brain mRNA. The period of serotonin application (10 s) was indicated by bars. Serotonin was applied on the surface of the oocyte membrane with the iontophoretic current shown to the left of each record. All traces were obtained from the same oocyte. Note that responses to serotonin increased in amplitude and duration as the application current was augmented. The mean value for the latency to onset with the application current of 150 nA was 10.5 + 7.8 s (mean + S.D., n = 25). B: antagonism by methysergide, cyproheptadine and ketanserin of responses to serotonin in mRNA-injected oocytes. Serotonin was applied iontophoretically with the constant ejecting current of 150 nA for the period indicated by bars (3 s). a: methysergide applied by superfusion at a concentration of 10/xMirreversibly abolished responses to serotonin in all 3 oocytes tested. Instabilities in the membrane potential accompanied by small spikes are seen about 30 s after the start of methysergide application, b: cyproheptadine also irreversibly abolished the serotonin action in all 3 oocytes tested. Unlike methysergide no instability of the membrane potential was caused, c: ketanserin depressed the depolarization induced by serotonin but not completely even with the concentration of 50 #xMin all 3 oocytes tested. This depressant action was reversible. The middle record was obtained at about 5 min after the start of the ketanserin application. Records a, b and c were obtained from different oocytes of the same donor. The membrane potentials of records A and B were maintained at -60 mV.
201 nin induced depolarization obtained by the extrapo-
serotonin receptor antagonists (Fig. 1B-a, b). These
lation of the relation between the m a x i m u m amplitudes of depolarization and m e m b r a n e potentials was - 2 2 . 7 + 2.8 m V ( m e a n + S.D., n = 3) which corre-
blocking actions of both methysergide and cyprohep-
sponds to the chloride equilibrium potential in Xenopus oocytes3,10. The m e m b r a n e potential fluctuations were also reversed at about - 2 3 mV. Thus, both the depolarization and the oscillatory responses to serotonin are caused by an increase in the m e m b r a n e permeability to chloride ions 3. The responses of m R N A - i n j e c t e d oocytes to serotonin was abolished by superfusion with either 10 ~ M methysergide or 10/~M cyproheptadine, well k n o w n
tadine were irreversible (in agreement with G u n d e r sen et al. 4) and observed in all 3 oocytes tested. Ketanserin, a specific serotonin S2-receptor antagonist H, exhibited a rather weak and reversible antagonism to serotonin in all 3 m R N A injected oocytes tested (Fig. 1B-c). The observation that these antagonists blocked the oscillatory responses to serotonin indicates that specific receptors for serotonin were expressed in Xenopus oocytes by transplanting rat brain m R N A . Our observation of the slow onset of serotonin re-
A b-1 O.lmll- m
a-2
b-2 lmM-d~amme
lmlt-m
c-1
C
0.01~
llql~
0.O111111- I~GO 7 1¢1~O
Fig. 2. A: depolarizations induced by bath applied serotonin, dibutyryl cAMP and papaverine in mRNA-injected oocytes, a: the typical feature of responses to bath applied serotonin. This type of depolarization accompanied by membrane potential fluctuations was seen in all 6 oocytes superfused with serotonin, b: similar depolarizations and membrane fluctuations induced by bath applied dibutyryl cAMP as serotonin, which were observed in all 3 oocytes tested. Although the magnitude of the depolarization induced by the second application of dibutyryl cAMP was smaller than that by the first one, the membrane potential fluctuations showed the same feature in both cases, c: bath applied papaverine induced the smooth depolarization which was followed by a plateau and the oscillatory nature as in the case of either serotonin or dibutyryl cAMP. Similar responses to papaverine were observed in all 4 oocytes tested. Records a, b and c were obtained from different oocytes. B: antagonism of dopamine, epinephrine and theophylline to the serotonin action in mRNA-injected oocytes. Serotonin was applied by iontophoresis with the constant ejecting current of 150 nA for the period indicated by bars (20 s). a: a-l, before, and a-2, 2 min after the application of dopamine. Superfusion of dopamine (1 mM) irreversibly depressed serotonin-induced depolarizations in all 4 oocytes tested, b: irreversible depressant action of epinephrine on serotonin action. b-l, before, and b-2, 2 min after the application of epinephrine. This depressant action of epinephrine was seen in all 4 oocytes tested, c: reversible depressant action of theophylline on responses to serotonin, c-l, before, c-2, 2 min after adding theophylline, and c-3, 3 min after returning the control Ringer solution. As exemplified in c-3, in 2 out of 5 oocytes tested serotonin-induced depolarization was increased in amplitude after switching back to control medium. Records a, b and c were obtained from different oocytes. C: blocking actions of PGE 2 and D 2 on responses to iontophoretically applied serotonin in mRNA-injected oocytes, a: application of PGE2 by superfusion gradually depressed the depolarization induced by serotonin and irreversibly abolished it about 3 min after adding PGE2 in all 3 oocytes tested, b: PGD2 also gradually suppressed the responses to serotonin and irreversibly abolished it after 3 min continuous application of PGD2 in all 3 oocytes tested. Records a and b were obtained from different oocytes. The membrane potentials of records A, B and C were maintained at -60 inV.
202 sponse and the early finding 9 that the immature rat (1-3-days-old) brain contained serotonin sensitive adenylate cyclase urged us to examine whether cAMP contributes to serotonin action in Xenopus oocytes injected with rat brain mRNA. In all 4 oocytes injected with mRNA, superfusion of dibutyryl cAMP (2.5 raM) for 20-60 s elicited the depolarization accompanied by membrane potential fluctuations which resembled the slow oscillatory responses to serotonin (Fig. 2A-a, b). The response appeared after a delay of 30-40 s and lasted about 3 min after switching back to normal Ringer solution. In two out of 8 control oocytes the protracted application for 5 min of dibutyryl cAMP elicited oscillatory responses, but their amplitudes were smaller than those of mRNA-injected oocytes and no background depolarization was observed. Six remaining oocytes exhibited little or no response to dibutyryl cAMP. Papaverine (1 mM), a phosphodiesterase inhibitor which is expected to raise the intracellular concentration of cAMP, also depolarized the membrane of all 4 oocytes injected with rat brain m R N A (Fig. 2A-c). The falling phase of depolarization induced by papaverine to the resting potential showed oscillatory responses similar to the action of serotonin or dibutyryl cAMP. In 3 out of 7 control oocytes, papaverine caused slight depolarizations (2-6 mV) which were sometimes accompanied by membrane potential fluctuations. In the remaining 4 control oocytes, no response to papaverine was observed. In spite of being mediated by Cl-channel as in the case of the serotonin action, responses to G A B A and ACh following injection of m R N A extracted from chick brain 16 and from cat muscle 13, respectively, have a rather abrupt onset and a smooth change in the membrane potential. Therefore, the oscillatory responses are characteristic to the actions of serotonin, cAMP and papaverine. Moreover, responses to these agents have a long latency to onset. These findings suggest that the cAMP system may be involved in the responses to both serotonin and papaverine. It is somewhat surprising that another phosphodiesterase inhibitor, theophylline (5 mM), suppressed the responses to serotonin (Fig. 2B-c). Dopamine (1 mM) and epinephrine (1 mM) also de-
pressed the serotonin action (Fig. 2B-a, b). These agents, i.e. theophylline, dopamine and epinephrine, however, hyperpolarized by themselves the membrane of both m R N A injected and non-injected oocytes. According to Kusano et al. 10, both epinephrine and dopamine caused the hyperpolarization of the oocyte membrane by increasing the permeability for K ions. Although the mechanism is still unclear, the reduction of serotonin action by these agents may have at least some relationship with an increase in K effiux. In all 3 mRNA-injected oocytes, either PGE 2 or D 2 (50/~M, superfused) blocked the responses to iontophoretically-applied serotonin (Fig. 2C). This antagonism began to take place 10-20 s after commencing the application of PGs and became stronger as the time elapsed. With application for 3 rain, PGs completely and irreversibly blocked the serotonin-induced depolarization. On the other hand, spontaneous depolarizing membrane potential fluctuations, which were frequently observed in control oocytes and are also mediated by a Cl-conductance l0 as in the case of the serotonin action, were unaffected in all 5 oocytes tested, even by the continuous application of PGE 2 or D2 up to 5 min. Therefore, the blocking actions of PGD 2 and E 2 o n serotonin responses were not due to the deterioration of the membrane by PGD 2 and E 2 but the effects on the serotonin action. Hoffer et al. 8 demonstrated that PGE1 and E 2 suppressed the accumulation of intracellular cAMP in cerebellar Purkinje cells. Taking this observation into account, it is inferable that observed blockade of serotonin action by PGE 2 and D 2 in the present study might also have a relationship with the cAMP system. In conclusion, the serotonin receptors generated in Xenopus oocytes by the injection of rat brain m R N A respond to serotonin with a long latency to onset and oscillatory changes in the membrane potential. These responses are possibly mediated by intracellular cAMP systems.
We would like to express our sincere thanks to Dr. Tetsuo Sudo, Basic Research Laboratories, Toray Industries, Inc., Kamakura, Japan for his technical instruction and advice on the isolation of mRNA.
203 1 Bennett, J.P., Jr. and Snyder, S.H., Serotonin and lysergic acid diethylamide binding in rat brain membranes: relationship to postsynaptic serotonin receptors, MoL Pharmacol., 12 (1976) 373-389. 2 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299. 3 Gundersen, C.B., Miledi, R. and Parker, I., Serotonin receptors induced by exogenous messenger RNA in Xenopus oocytes, Proc. R. Soc. London Ser. B., 219 (1983) 103-109. 4 Gundersen, C.B., Miledi, R. and Parker, I., Messenger RNA from human brain induces drug- and voltage-operated channels in Xenopus oocytes, Nature (London), 308 (1984) 421-424. 5 Gundersen, C.B., Miledi, R. and Parker, I., Glutamate and kainate receptors induced by rat brain messenger RNA in Xenopus oocytes, Proc. R. Soc. London Ser. B., 221 (1984) 127-143. 6 Gundersen, C.B., Miledi, R. and Parker, I., Properties of human brain glycine receptors expressed in Xenopus oocytes, Proc. R. Soc. London Set. B., 221 (1984) 235-244. 7 Gurdon, J.B., Lane, C.D., Woodland, H.R. and Marbaix, G., Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells, Nature (London), 233 (1971) 177-182. 8 Hoffer, B.J., Siggins, G.R. and Bloom, F.E., Prostaglandins E 1 and E 2 antagonize norepinephrine effects on cerebellar Purkinje cells: microelectrophoretic study, Science, 166 (1969) 1418-1420. 9 Hungen, K.V., Roberts, S. and Hill, D.F., Serotonin-sensi-
tive adenylate cyclase activity in immature rat brain, Brain Research, 84 (1975) 257-267. 10 Kusano, K., Miledi, R. and Stinnakre, J., Cholinergic and catecholaminergic receptors in the Xenopus oocyte membrane, J. Physiol. (London), 328 (1982) 143-170. 11 Leysen, J.E., Niemegeers, C.J.E., Van Nueten, J.M. and Laduron, P.M., [3H] Ketanserin (R 41 468), a selective 3Hligand for serotonin S2receptor binding sites, Mol. Pharmacol., 21 (1981) 301-314. 12 Miledi, R., Parker, I. and Sumikawa, K., Recording of single y-aminobutyrate- and acetylcholine-activated receptor channels translated by exogenous mRNA in Xenopus oocytes, Proc. R. Soc. London Ser. B., 218 (1983) 481-484. 13 Miledi, R. and Sumikawa, K., Synthesis of cat muscle acetylcholine receptors by Xenopus oocytes, Biomed. Res., 3 (1982) 390-399. 14 Peroutka, S.J., Lebovitz, R.M. and Snyder, S.H., Two distinct central serotonin receptors with different physiological functions, Science, 212 (1981) 827-829. 15 Peroutka, S.J. and Snyder, S.H., Multiple serotonin receptors differential binding of [3H]-5-hydroxytryptamine, [3H]lysergic acid diethylamide, and [3H]-spiroperidol, Mol. Pharmacol., 16 (1979) 687-699, 16 Smart, T.G., Constanti, A., Bilbe, G., Brown, D.A. and Barnard, E.A., Synthesis of functional chick brain GABAbenzodiazepine-barbiturate/receptor complexes in mRNAinjected Xenopus oocytes, Neurosci. Lett., 40 (1983) 55-59. 17 Sumikawa, K., Houghton, M., Emtage, J.S., Richards, B.M. and Barnard, E.A., Active multi-subunit ACh receptor assembled by translation of heterologous mRNA in Xenopus oocytes, Nature (London), 292 (1981) 862-864.
199
BRE 21282
Pharmacological characterization of serotonin receptor induced by rat brain messenger RNA in Xenopusoocytes Y. SAKAI, H. KIMURA and K. OKAMOTO Departmentof Pharmacology, National DefenseMedical College, 3-2 Namiki, Tokorozawa, Saitama359 (Japan)
(Accepted August 28th, 1985) Key words: serotonin receptor - - messenger ribonucleic acid (RNA) --Xenopus oocyte - cyclic adenosine monophosphate (AMP) - - prostaglandin
Serotonin receptors incorporated in the membrane ofXenopus oocytes injected with mRNA extracted from the rat brain was investigated by intracellular recording. Serotonin elicited the membrane depolarization accompanied with membrane potential fluctuations. This serotonin action was suppressed by serotonin antagonists such as methysergide, cyproheptadine and ketanserin. Dibutyryl cyclic AMP and papaverine depolarized the membrane as seen in applying serotonin. These observations indicate that serotonin actions might involve the cAMP system.
Sumikawa et al. 17 have first generated functional nicotinic acetylcholine receptors in the oocytes of Xenopus laevis by injecting m R N A from Torpedo marmorata utilizing the technique of Gurdon et al. 7. Since then, several receptors for neurotransmitters including serotonin 3.4, glutamic acid 5, G A B A 4a2,t6 and glycine 6 have been successively generated in the Xenopus oocytes by transplanting the m R N A from the rat brain, and their properties have been investigated. As our first step of pharmacological characterization of the receptors generated in Xenopus oocytes, serotonin receptors produced by injection of m R N A extracted from neonatal rat brain were investigated by an intracellular recording technique in the present study. Two distinct types of the serotonin receptor are known. $1 receptor is specifically labeled by [3H]serotonin l, and S2 receptor is labeled by [3H]spiperone~4,~5 The neuronal action of serotonin elicited by S1 receptor was suggested to be mediated by serotonin-sensitive adenylate cyclase 14. Such serotoninsensitive adenylate cyclase was reported to exist in immature rat brain 9. Whether the cyclic nucleotide system is involved in the serotonin action mediated
by these expressed receptors is the primary question to be answered in the present study. The m R N A was extracted from whole brains of 1day-old Wistar rats using a guanidium/cesium chloride method 2. About 50 nl of 1 mg/ml poly(A)m R N A solution was injected into each Xenopus oocyte, and the oocytes were cultured for 3 days at 19 °C in modified Barth medium containing streptomycin 0.1 mg/ml and penicillin 100 U/ml. One oocyte was placed at a time on a nylon mesh in a superfusion chamber, and the bath was superfused at a rate of 2 ml/min throughout the experiments with the frog Ringer solution (control medium, composition (in mM): NaCI, 120; KCI, 2; CaCI2, 1.8; HEPES, 5; pH 7.4). Microelectrodes filled with 3 M KCI (10-20 MQ) were used for intracellular recordings in most experiments and those filled with 3 M potassium acetate (15-25 MQ) were employed only for changing the membrane potential to estimate the reversal potential of serotonin responses. Only the oocytes exhibiting resting membrane potentials o f - 3 0 to - 6 0 mV were adopted. The mean resting membrane potentials of control and mRNA-
Correspondence: Y. Sakai, Department of Pharmacology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359, Japan.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
200 injected oocytes were - 4 6 . 7 + 12.2 mV (n = 35) and -46.2 + 11.3 mV (n = 62), respectively. Serotonin was applied either by iontophoresis (50 mM solution was filled in the micropipette) on the surface of the oocyte m e m b r a n e or by superfusion (100/xM in the frog Ringer solution). Other agents following were applied only by superfusion: methysergide (10/xM), cyproheptadine (10/xM), ketansefin (50/xM), dopamine (1 mM), epinephrine (1 mM), theophylline (5 mM), dibutyryl cyclic A M P (2.5 and 0.25 mM), papaverine (1 mM), P G D 2 (50 #xM), and P G E 2 (50/xM), all being dissolved in control medium. Cyproheptadine and ketanserin were kindly presented by Dr. Y. Yamawaki, The Institute of Clinical Research, Kure National Hospital (Kure, Japan). Dibutyryl c A M P was from P-L Biochem. (WI, A
U.S.A.). Prostaglandin E 2 and D 2 were from Funakoshi Chem. (Tokyo, Japan). Other agents including serotonin were from Wako Pure Chem. (Tokyo, Japan.) Control Xenopus oocytes, i.e. those not injected with m R N A responded only faintly and inconsistently to serotonin applied by either iontophoresis or superfusion. In 5 out of 14 control oocytes, serotonin induced hyperpolarizations, 3 oocytes had no response to serotonin, and the remaining 6 oocytes were depolarized slightly ( 1 - 2 mV) by the application of serotonin. In contrast, as shown in Fig. 1A, all the 62 oocytes injected rat brain m R N A were depolarized by serotonin with the long latency to onset and slow fluctuations of the m e m b r a n e potential (in agreement with G u n d e r s e n et al.3). A mean value for the reversal potential of seroto-
B 2 0 nA
- - / ~
._U
lOmV lOs4~
40
- ~ / ~ - ~ ,f,
O.OlmM'- c ) l ~ W l ~ e
80
_J
----~'~-~
O.Of~M- kehmeerlem 150
200
Fig. 1. A: depolarizations and potential fluctuations elicited by different iontophoretic currents of serotonin applied to an oocyte that had previously been injected with rat brain mRNA. The period of serotonin application (10 s) was indicated by bars. Serotonin was applied on the surface of the oocyte membrane with the iontophoretic current shown to the left of each record. All traces were obtained from the same oocyte. Note that responses to serotonin increased in amplitude and duration as the application current was augmented. The mean value for the latency to onset with the application current of 150 nA was 10.5 + 7.8 s (mean + S.D., n = 25). B: antagonism by methysergide, cyproheptadine and ketanserin of responses to serotonin in mRNA-injected oocytes. Serotonin was applied iontophoretically with the constant ejecting current of 150 nA for the period indicated by bars (3 s). a: methysergide applied by superfusion at a concentration of 10/xMirreversibly abolished responses to serotonin in all 3 oocytes tested. Instabilities in the membrane potential accompanied by small spikes are seen about 30 s after the start of methysergide application, b: cyproheptadine also irreversibly abolished the serotonin action in all 3 oocytes tested. Unlike methysergide no instability of the membrane potential was caused, c: ketanserin depressed the depolarization induced by serotonin but not completely even with the concentration of 50 #xMin all 3 oocytes tested. This depressant action was reversible. The middle record was obtained at about 5 min after the start of the ketanserin application. Records a, b and c were obtained from different oocytes of the same donor. The membrane potentials of records A and B were maintained at -60 mV.
201 nin induced depolarization obtained by the extrapo-
serotonin receptor antagonists (Fig. 1B-a, b). These
lation of the relation between the m a x i m u m amplitudes of depolarization and m e m b r a n e potentials was - 2 2 . 7 + 2.8 m V ( m e a n + S.D., n = 3) which corre-
blocking actions of both methysergide and cyprohep-
sponds to the chloride equilibrium potential in Xenopus oocytes3,10. The m e m b r a n e potential fluctuations were also reversed at about - 2 3 mV. Thus, both the depolarization and the oscillatory responses to serotonin are caused by an increase in the m e m b r a n e permeability to chloride ions 3. The responses of m R N A - i n j e c t e d oocytes to serotonin was abolished by superfusion with either 10 ~ M methysergide or 10/~M cyproheptadine, well k n o w n
tadine were irreversible (in agreement with G u n d e r sen et al. 4) and observed in all 3 oocytes tested. Ketanserin, a specific serotonin S2-receptor antagonist H, exhibited a rather weak and reversible antagonism to serotonin in all 3 m R N A injected oocytes tested (Fig. 1B-c). The observation that these antagonists blocked the oscillatory responses to serotonin indicates that specific receptors for serotonin were expressed in Xenopus oocytes by transplanting rat brain m R N A . Our observation of the slow onset of serotonin re-
A b-1 O.lmll- m
a-2
b-2 lmM-d~amme
lmlt-m
c-1
C
0.01~
llql~
0.O111111- I~GO 7 1¢1~O
Fig. 2. A: depolarizations induced by bath applied serotonin, dibutyryl cAMP and papaverine in mRNA-injected oocytes, a: the typical feature of responses to bath applied serotonin. This type of depolarization accompanied by membrane potential fluctuations was seen in all 6 oocytes superfused with serotonin, b: similar depolarizations and membrane fluctuations induced by bath applied dibutyryl cAMP as serotonin, which were observed in all 3 oocytes tested. Although the magnitude of the depolarization induced by the second application of dibutyryl cAMP was smaller than that by the first one, the membrane potential fluctuations showed the same feature in both cases, c: bath applied papaverine induced the smooth depolarization which was followed by a plateau and the oscillatory nature as in the case of either serotonin or dibutyryl cAMP. Similar responses to papaverine were observed in all 4 oocytes tested. Records a, b and c were obtained from different oocytes. B: antagonism of dopamine, epinephrine and theophylline to the serotonin action in mRNA-injected oocytes. Serotonin was applied by iontophoresis with the constant ejecting current of 150 nA for the period indicated by bars (20 s). a: a-l, before, and a-2, 2 min after the application of dopamine. Superfusion of dopamine (1 mM) irreversibly depressed serotonin-induced depolarizations in all 4 oocytes tested, b: irreversible depressant action of epinephrine on serotonin action. b-l, before, and b-2, 2 min after the application of epinephrine. This depressant action of epinephrine was seen in all 4 oocytes tested, c: reversible depressant action of theophylline on responses to serotonin, c-l, before, c-2, 2 min after adding theophylline, and c-3, 3 min after returning the control Ringer solution. As exemplified in c-3, in 2 out of 5 oocytes tested serotonin-induced depolarization was increased in amplitude after switching back to control medium. Records a, b and c were obtained from different oocytes. C: blocking actions of PGE 2 and D 2 on responses to iontophoretically applied serotonin in mRNA-injected oocytes, a: application of PGE2 by superfusion gradually depressed the depolarization induced by serotonin and irreversibly abolished it about 3 min after adding PGE2 in all 3 oocytes tested, b: PGD2 also gradually suppressed the responses to serotonin and irreversibly abolished it after 3 min continuous application of PGD2 in all 3 oocytes tested. Records a and b were obtained from different oocytes. The membrane potentials of records A, B and C were maintained at -60 inV.
202 sponse and the early finding 9 that the immature rat (1-3-days-old) brain contained serotonin sensitive adenylate cyclase urged us to examine whether cAMP contributes to serotonin action in Xenopus oocytes injected with rat brain mRNA. In all 4 oocytes injected with mRNA, superfusion of dibutyryl cAMP (2.5 raM) for 20-60 s elicited the depolarization accompanied by membrane potential fluctuations which resembled the slow oscillatory responses to serotonin (Fig. 2A-a, b). The response appeared after a delay of 30-40 s and lasted about 3 min after switching back to normal Ringer solution. In two out of 8 control oocytes the protracted application for 5 min of dibutyryl cAMP elicited oscillatory responses, but their amplitudes were smaller than those of mRNA-injected oocytes and no background depolarization was observed. Six remaining oocytes exhibited little or no response to dibutyryl cAMP. Papaverine (1 mM), a phosphodiesterase inhibitor which is expected to raise the intracellular concentration of cAMP, also depolarized the membrane of all 4 oocytes injected with rat brain m R N A (Fig. 2A-c). The falling phase of depolarization induced by papaverine to the resting potential showed oscillatory responses similar to the action of serotonin or dibutyryl cAMP. In 3 out of 7 control oocytes, papaverine caused slight depolarizations (2-6 mV) which were sometimes accompanied by membrane potential fluctuations. In the remaining 4 control oocytes, no response to papaverine was observed. In spite of being mediated by Cl-channel as in the case of the serotonin action, responses to G A B A and ACh following injection of m R N A extracted from chick brain 16 and from cat muscle 13, respectively, have a rather abrupt onset and a smooth change in the membrane potential. Therefore, the oscillatory responses are characteristic to the actions of serotonin, cAMP and papaverine. Moreover, responses to these agents have a long latency to onset. These findings suggest that the cAMP system may be involved in the responses to both serotonin and papaverine. It is somewhat surprising that another phosphodiesterase inhibitor, theophylline (5 mM), suppressed the responses to serotonin (Fig. 2B-c). Dopamine (1 mM) and epinephrine (1 mM) also de-
pressed the serotonin action (Fig. 2B-a, b). These agents, i.e. theophylline, dopamine and epinephrine, however, hyperpolarized by themselves the membrane of both m R N A injected and non-injected oocytes. According to Kusano et al. 10, both epinephrine and dopamine caused the hyperpolarization of the oocyte membrane by increasing the permeability for K ions. Although the mechanism is still unclear, the reduction of serotonin action by these agents may have at least some relationship with an increase in K effiux. In all 3 mRNA-injected oocytes, either PGE 2 or D 2 (50/~M, superfused) blocked the responses to iontophoretically-applied serotonin (Fig. 2C). This antagonism began to take place 10-20 s after commencing the application of PGs and became stronger as the time elapsed. With application for 3 rain, PGs completely and irreversibly blocked the serotonin-induced depolarization. On the other hand, spontaneous depolarizing membrane potential fluctuations, which were frequently observed in control oocytes and are also mediated by a Cl-conductance l0 as in the case of the serotonin action, were unaffected in all 5 oocytes tested, even by the continuous application of PGE 2 or D2 up to 5 min. Therefore, the blocking actions of PGD 2 and E 2 o n serotonin responses were not due to the deterioration of the membrane by PGD 2 and E 2 but the effects on the serotonin action. Hoffer et al. 8 demonstrated that PGE1 and E 2 suppressed the accumulation of intracellular cAMP in cerebellar Purkinje cells. Taking this observation into account, it is inferable that observed blockade of serotonin action by PGE 2 and D 2 in the present study might also have a relationship with the cAMP system. In conclusion, the serotonin receptors generated in Xenopus oocytes by the injection of rat brain m R N A respond to serotonin with a long latency to onset and oscillatory changes in the membrane potential. These responses are possibly mediated by intracellular cAMP systems.
We would like to express our sincere thanks to Dr. Tetsuo Sudo, Basic Research Laboratories, Toray Industries, Inc., Kamakura, Japan for his technical instruction and advice on the isolation of mRNA.
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