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Independence of presynaptic bimodal actions of adrenaline in sympathetic ganglia
344
Brain Research, 265 (1983) 344- 347 Elsevier Biomedical Press
Independence of presynaptic bimodal actions of adrenaline in sympathetic ganglia EI1CHI K U M A M O T O and KENJI KUBA
Department of Physiology, Saga Medical School Saga, 840-01(Japan) (Accepted December 14th, 1982)
Key words: adrenaline - synaptic modulation - interaction of facilitation and depression - bullfrog sympathetic ganglia
Interaction of the facilitatory and inhibitory actions of adrenaline on transmitter release was studied in bullfrog sympathetic ganglia. The facilitatory action develops with a slower onset and lasts for a long time even after removal of adrenaline, while the inhibitory action appears with a negligible latency and ceases immediately after removal. The latter is unaffected by the coexistence of the former. Lowering temperature suppresses markedly the facilitation, while it increases the inhibition. These results suggest the lack of interaction of mechanisms for two different actions of adrenaline and the possibility for an involvement of a metabolic process in the mechanism of the facilitatory action.
Efficacy of synaptic transmission is regulated by various transmitters and humoral agents. This mechanism, generally called synaptic modulation, plays important roles in complexed integration ofsynaptic inputs in central neurons 4.7. Adrenaline produces two different actions on the presynaptic terminals in bullfrog sympathetic ganglia. It depresses transmitter release induced by a nerve impulse during exposure to it, while it causes sustained rises in both spontaneous and impulse-induced transmitter releases (presumably mediated by endogenous cyclic AMP) after its removal6. This investigation was undertaken to clarify whether these two opposite actions are independent of each other, and to obtain a clue for the involvement of a metabolic process in the long-lasting facilitation. Isolated ninth or tenth lumbar sympathetic ganglia of bullfrogs (Rana catesbeiana: B-type neurons) were studied, using a conventional intracellular recording techniqueL Microelectrodes were filled with 3 M KC1 (tip resistance 30100 MI2). The fast excitatory postsynaptic potential (fast EPSP) was induced at a rate of 1/3 Hz in a low Ca 2+ high Mg 2+ solution (Ca 2+ 0.7 0.9 mM, Mg 2+ 5.4-6.5 mM) and fed into a magnetic tape (NFR-3515W, Sony) for quantal
analysis with a microcomputer (MINC-I1, DEC). The quantal content of fast EPSP was calculated from 100-200 fast EPSPs by both variance and failure methods ~, and the average taken. The composition (mM) of normal Ringer was as follows: NaC1 115.5; KC1 2.0; CaC12 !.8; Na2HPO4 1.3:NaH2PO4 0.7. Experiments were carried out at room temperature except for those studying an effect of low temperature. Temperature was reduced to 11 13 °C by cooling a tube through which solution flowed from a reservoir to the tissue, while monitoring local temperature close to the ganglion by a thermister5. In a previous study~, the quantal content of fast EPSP decreased in most cells during perfusion of the ganglion with a solution containing adrenaline for 20-30 min with unchanged quantal size. In the present experiments, however, several cells (9 out of 24 cells) exhibited an increase in the quantal content during exposure to adrenaline, sometimes accompanied by a small initial reduction. This variability of adrenaline action during exposure may arise from a different intensity and time of onset of a potentiating action. Thus, if the facilitatory action takes place with a considerable delay, it may not be observable during a short period of exposure. This may
0006-8993/83/0000- 0000/$03.00 ~ 1983 Elsevier Science Publishers
345
~200 A,r.
W,s,,
~ 101] == 0
I
0
I
50
I
Time(mini
100
I
150
Fig. 1. Time course of the action of adrenaline (10 #M) on the quantal content of fast EPSPs during a long exposure to it ( 1 h). Each point is the quantal content calculated from 200 fast EPSPs. Arrows indicate the beginning and end of application.
account for the failure to observe the potentiation in the previous study. Therefore, we examined adrenaline action during a long exposure (1 h). Fig. 1 shows a typical experiment, where the quantal content of fast EPSP increased during application of adrenaline (10 #M), with a 30 min delay. Similar potentiations during a long exposure were obtained from 4 other cells. These results suggest a slow onset of the facilitatory action, which contrasts with the fast appearance of the depressant action, that reaches a maximum immediately after the addition of adrenaline. If the mechanisms for the two different actions are independent, the magnitude of depression of fast EPSPs would be unaffected by the coexistence of the potentiation. This possibility can be tested by repeating application of adrenaline. The inhibition induced by adrenaline for the first time may contain only a small component of facilitation for its slower onset, while the facilitatory action induced by the preceding application exists throughout the depression caused by the second application. As seen in Fig. 2, the magnitude of reduction of the quantal content produced by the first application was not significantly different from that produced by the subsequent application. The ratio of the magnitude of the inhibition induced by the second application to that by the first was 0.86 (n = 5, insignificant from l, P ~ 0.1), indicating the inde-
pendence of two adrenaline actions. Previous experiments6 suggested the involvement of endogenous cyclic AMP in the facilitatory action, but not in the depressant action. This, together with the slower onset of the facilitation, implies that the former mechanism may involve a sequence of metabolic events2, whereas the latter may not because of its earlier onset. If this is the case, lowering the temperature would depress the facilitation of the fast EPSP more intensely than the inhibition. Fig. 3 shows the effects of low temperature on the action of adrenaline (10 /tM). At low temperature (1113°C), the reduction and facilitation of the quantal content of fast EPSP were 58% ( _ 6 (S.E.), n = 9, during exposure for 20 rain) and 120% ( + 11, n = 5, 50-60 rain after removal)~ of the control, respectively. By contrast, they were 70% (___ 4, n = 15) and 172% ( _ 13, n = 7), respectively, at room temperature (20-24°C). Thus, the facilitatory action was markedly suppressed by lowering the temperature, consistent with the possibility of an involvement of a meta-
40Q Wash/
Adr.
~300
~200 Adr.WashJ~ `¢ t 100
I
0
I
50
I
Time(rain) 100
I
150
Fig. 2. Effect of repeated applications of adrenaline (10/~M) on the quantal content of fast EPSPs. Each point is the quantal content calculated from 200 fast EPSPs. Arrows indicate the beginning and end of application.
346 -24 "C)
(20
L~//-------/-/---/J//]
Control Adr.
(n=15) ~-/////,/~'~////////-~
Wash(n = 7)
LI/I////////~///////////~
Control
~////////////////////////////,~
i (11 - 13 "C)
Adr. (n = 9)
L//////. . . . . . . . . . //--j///////////,,,_~
Wasfl(n = 5)
L~///////////////////~--~ I
i
0
i 50
I 100
I 150
Fig. 3. Effect of low temperature on adrenaline (10/~M) action. Bars represent the quantal content (open) and size (cross-hatched) calculated from 100-200 fast EPSPs recorded before, 10-20 min after application of adrenaline, and 50-60 min after its removal. They are expressed as a percentage (mean ___ S.E.) of the value before application, n represents number of experiments.
bolic process. In contrast, the depressant action was pronounced under this condition. This must be due to disappearance, or reduction, at low temperature, of the facilitation which may coexist to some extent, albeit small, even during the first application of adrenaline. This different temperature sensitivity for two different actions is another indication of their independent mechanisms. Furthermore, our previous study~ indicated different receptor sites for these actions: the inhibition is mediated by a-receptor, and the facilitation by a receptor which is classified as neither a- nor/3-type. In support of this, the apparent sensitivity of the receptor for the facilitatory action appears to be higher than that for the inhibitory action (unpublished results), as observed for a concentration-dependent dual action of 5-hydroxytryptamine3. The mechanism for the facilitatory action of
1 Del Castillo, J. andKatz, B., Quantal components of the end-plate potential, J. Physiol. (Lond), 124 (1954) 560573. 2 Greengard, P., Possible role for cyclic nucleotides and phosphorylated membrane proteins in postsynaptic actions of neurotransmitters, Nature (Lond.), 260 (1976) 101 108. 3 Hirai, K. and Koketsu, K., Presynaptic regulation of the release of acetylcholine by 5-hydroxytryptamine, Brit. J. Pharmacol., 70 (1980) 499- 500. 4 Klein, M. and Kandel, E. R., Mechanism of calcium current modulation underlying presynaptic facilitation and
adrenaline is quite distinct from that for the inhibitory action and does not seem to have a common pathway with this mechanism. The facilitatory action develops slowly with a considerable delay and lasts for a long time, even after the disappearance of adrenaline from the space around the presynaptic terminal. This mode of action conforms to a likely mediation of potentiation by endogenous cyclic AMP 6 and to its high temperature sensitivity, which suggests the possibility of the involvement of a series of metabolic processes in the mechanism. Nestler and Greengard 8 recently reported clear evidence for the phosphorylation of protein I (which is a substrate for both cyclic AMP-dependent and C~2+ calmoduline-dependent protein kinases) in the presynaptic terminals by preganglionic stimulation. This process of phosphorylation, together with other, unknown, metabolic processes, may lead either to an increase in the intracellular free Ca 2+ concentration through alteration in Ca 2+buffering systems, or to an enhancement of transmitter synthesis6, eventually resulting in a long-lasting potentiation of transmitter release. By contrast, the inhibitory action of adrenaline occurs with a negligible latency and subsides as soon as adrenaline is removed from presynaptic terminals. Thus, the inhibitory action follows a local adrenaline concentration, while the facilitatory action is durable. In addition, these two actions appear completely independent in their mechanisms. We thank T. Eguchi for computer programming. This work was supported by a Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture of Japan. behavioral sensitization in Aplysia, Proc. nat. Aead. Sci. U.S.A., 77 (1980) 6912--6916. 5 Kuba, K., Release of calcium ions linked to the activation of potassium conductance in a caffeine-treated sympathetic neurone, J. Physiol. (Lond.), 298 (1980) 251269. 6 Kuba, K., Kato, E., Kumamoto, E., Koketsu, K. and Hirai, K., Sustained potentiation of transmitter release by adrenaline and dibutyryl cyclic AMP in sympathetic ganglia, Nature (Lond.), 291 ( 1981) 654-656. 7 Kupfermann, I., Modulatory actions of neurotransmitters, Ann. Rev. Neurosci., 2 (1979) 447-465.
347 8 Nestler, E. J. and Greengard, P., Nerve impulses increase the phosphorylation state of protein I in rabbit superior cervical ganglion, Nature (Lond.), 296 (1982) 452-454.
9 Nishi, S. and Koketsu, K., Electrical properties and activities of single sympathetic neurons in frogs, J. cell. comp. PhysioL, 55 (1960) 15-30.
Brain Research, 265 (1983) 344- 347 Elsevier Biomedical Press
Independence of presynaptic bimodal actions of adrenaline in sympathetic ganglia EI1CHI K U M A M O T O and KENJI KUBA
Department of Physiology, Saga Medical School Saga, 840-01(Japan) (Accepted December 14th, 1982)
Key words: adrenaline - synaptic modulation - interaction of facilitation and depression - bullfrog sympathetic ganglia
Interaction of the facilitatory and inhibitory actions of adrenaline on transmitter release was studied in bullfrog sympathetic ganglia. The facilitatory action develops with a slower onset and lasts for a long time even after removal of adrenaline, while the inhibitory action appears with a negligible latency and ceases immediately after removal. The latter is unaffected by the coexistence of the former. Lowering temperature suppresses markedly the facilitation, while it increases the inhibition. These results suggest the lack of interaction of mechanisms for two different actions of adrenaline and the possibility for an involvement of a metabolic process in the mechanism of the facilitatory action.
Efficacy of synaptic transmission is regulated by various transmitters and humoral agents. This mechanism, generally called synaptic modulation, plays important roles in complexed integration ofsynaptic inputs in central neurons 4.7. Adrenaline produces two different actions on the presynaptic terminals in bullfrog sympathetic ganglia. It depresses transmitter release induced by a nerve impulse during exposure to it, while it causes sustained rises in both spontaneous and impulse-induced transmitter releases (presumably mediated by endogenous cyclic AMP) after its removal6. This investigation was undertaken to clarify whether these two opposite actions are independent of each other, and to obtain a clue for the involvement of a metabolic process in the long-lasting facilitation. Isolated ninth or tenth lumbar sympathetic ganglia of bullfrogs (Rana catesbeiana: B-type neurons) were studied, using a conventional intracellular recording techniqueL Microelectrodes were filled with 3 M KC1 (tip resistance 30100 MI2). The fast excitatory postsynaptic potential (fast EPSP) was induced at a rate of 1/3 Hz in a low Ca 2+ high Mg 2+ solution (Ca 2+ 0.7 0.9 mM, Mg 2+ 5.4-6.5 mM) and fed into a magnetic tape (NFR-3515W, Sony) for quantal
analysis with a microcomputer (MINC-I1, DEC). The quantal content of fast EPSP was calculated from 100-200 fast EPSPs by both variance and failure methods ~, and the average taken. The composition (mM) of normal Ringer was as follows: NaC1 115.5; KC1 2.0; CaC12 !.8; Na2HPO4 1.3:NaH2PO4 0.7. Experiments were carried out at room temperature except for those studying an effect of low temperature. Temperature was reduced to 11 13 °C by cooling a tube through which solution flowed from a reservoir to the tissue, while monitoring local temperature close to the ganglion by a thermister5. In a previous study~, the quantal content of fast EPSP decreased in most cells during perfusion of the ganglion with a solution containing adrenaline for 20-30 min with unchanged quantal size. In the present experiments, however, several cells (9 out of 24 cells) exhibited an increase in the quantal content during exposure to adrenaline, sometimes accompanied by a small initial reduction. This variability of adrenaline action during exposure may arise from a different intensity and time of onset of a potentiating action. Thus, if the facilitatory action takes place with a considerable delay, it may not be observable during a short period of exposure. This may
0006-8993/83/0000- 0000/$03.00 ~ 1983 Elsevier Science Publishers
345
~200 A,r.
W,s,,
~ 101] == 0
I
0
I
50
I
Time(mini
100
I
150
Fig. 1. Time course of the action of adrenaline (10 #M) on the quantal content of fast EPSPs during a long exposure to it ( 1 h). Each point is the quantal content calculated from 200 fast EPSPs. Arrows indicate the beginning and end of application.
account for the failure to observe the potentiation in the previous study. Therefore, we examined adrenaline action during a long exposure (1 h). Fig. 1 shows a typical experiment, where the quantal content of fast EPSP increased during application of adrenaline (10 #M), with a 30 min delay. Similar potentiations during a long exposure were obtained from 4 other cells. These results suggest a slow onset of the facilitatory action, which contrasts with the fast appearance of the depressant action, that reaches a maximum immediately after the addition of adrenaline. If the mechanisms for the two different actions are independent, the magnitude of depression of fast EPSPs would be unaffected by the coexistence of the potentiation. This possibility can be tested by repeating application of adrenaline. The inhibition induced by adrenaline for the first time may contain only a small component of facilitation for its slower onset, while the facilitatory action induced by the preceding application exists throughout the depression caused by the second application. As seen in Fig. 2, the magnitude of reduction of the quantal content produced by the first application was not significantly different from that produced by the subsequent application. The ratio of the magnitude of the inhibition induced by the second application to that by the first was 0.86 (n = 5, insignificant from l, P ~ 0.1), indicating the inde-
pendence of two adrenaline actions. Previous experiments6 suggested the involvement of endogenous cyclic AMP in the facilitatory action, but not in the depressant action. This, together with the slower onset of the facilitation, implies that the former mechanism may involve a sequence of metabolic events2, whereas the latter may not because of its earlier onset. If this is the case, lowering the temperature would depress the facilitation of the fast EPSP more intensely than the inhibition. Fig. 3 shows the effects of low temperature on the action of adrenaline (10 /tM). At low temperature (1113°C), the reduction and facilitation of the quantal content of fast EPSP were 58% ( _ 6 (S.E.), n = 9, during exposure for 20 rain) and 120% ( + 11, n = 5, 50-60 rain after removal)~ of the control, respectively. By contrast, they were 70% (___ 4, n = 15) and 172% ( _ 13, n = 7), respectively, at room temperature (20-24°C). Thus, the facilitatory action was markedly suppressed by lowering the temperature, consistent with the possibility of an involvement of a meta-
40Q Wash/
Adr.
~300
~200 Adr.WashJ~ `¢ t 100
I
0
I
50
I
Time(rain) 100
I
150
Fig. 2. Effect of repeated applications of adrenaline (10/~M) on the quantal content of fast EPSPs. Each point is the quantal content calculated from 200 fast EPSPs. Arrows indicate the beginning and end of application.
346 -24 "C)
(20
L~//-------/-/---/J//]
Control Adr.
(n=15) ~-/////,/~'~////////-~
Wash(n = 7)
LI/I////////~///////////~
Control
~////////////////////////////,~
i (11 - 13 "C)
Adr. (n = 9)
L//////. . . . . . . . . . //--j///////////,,,_~
Wasfl(n = 5)
L~///////////////////~--~ I
i
0
i 50
I 100
I 150
Fig. 3. Effect of low temperature on adrenaline (10/~M) action. Bars represent the quantal content (open) and size (cross-hatched) calculated from 100-200 fast EPSPs recorded before, 10-20 min after application of adrenaline, and 50-60 min after its removal. They are expressed as a percentage (mean ___ S.E.) of the value before application, n represents number of experiments.
bolic process. In contrast, the depressant action was pronounced under this condition. This must be due to disappearance, or reduction, at low temperature, of the facilitation which may coexist to some extent, albeit small, even during the first application of adrenaline. This different temperature sensitivity for two different actions is another indication of their independent mechanisms. Furthermore, our previous study~ indicated different receptor sites for these actions: the inhibition is mediated by a-receptor, and the facilitation by a receptor which is classified as neither a- nor/3-type. In support of this, the apparent sensitivity of the receptor for the facilitatory action appears to be higher than that for the inhibitory action (unpublished results), as observed for a concentration-dependent dual action of 5-hydroxytryptamine3. The mechanism for the facilitatory action of
1 Del Castillo, J. andKatz, B., Quantal components of the end-plate potential, J. Physiol. (Lond), 124 (1954) 560573. 2 Greengard, P., Possible role for cyclic nucleotides and phosphorylated membrane proteins in postsynaptic actions of neurotransmitters, Nature (Lond.), 260 (1976) 101 108. 3 Hirai, K. and Koketsu, K., Presynaptic regulation of the release of acetylcholine by 5-hydroxytryptamine, Brit. J. Pharmacol., 70 (1980) 499- 500. 4 Klein, M. and Kandel, E. R., Mechanism of calcium current modulation underlying presynaptic facilitation and
adrenaline is quite distinct from that for the inhibitory action and does not seem to have a common pathway with this mechanism. The facilitatory action develops slowly with a considerable delay and lasts for a long time, even after the disappearance of adrenaline from the space around the presynaptic terminal. This mode of action conforms to a likely mediation of potentiation by endogenous cyclic AMP 6 and to its high temperature sensitivity, which suggests the possibility of the involvement of a series of metabolic processes in the mechanism. Nestler and Greengard 8 recently reported clear evidence for the phosphorylation of protein I (which is a substrate for both cyclic AMP-dependent and C~2+ calmoduline-dependent protein kinases) in the presynaptic terminals by preganglionic stimulation. This process of phosphorylation, together with other, unknown, metabolic processes, may lead either to an increase in the intracellular free Ca 2+ concentration through alteration in Ca 2+buffering systems, or to an enhancement of transmitter synthesis6, eventually resulting in a long-lasting potentiation of transmitter release. By contrast, the inhibitory action of adrenaline occurs with a negligible latency and subsides as soon as adrenaline is removed from presynaptic terminals. Thus, the inhibitory action follows a local adrenaline concentration, while the facilitatory action is durable. In addition, these two actions appear completely independent in their mechanisms. We thank T. Eguchi for computer programming. This work was supported by a Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture of Japan. behavioral sensitization in Aplysia, Proc. nat. Aead. Sci. U.S.A., 77 (1980) 6912--6916. 5 Kuba, K., Release of calcium ions linked to the activation of potassium conductance in a caffeine-treated sympathetic neurone, J. Physiol. (Lond.), 298 (1980) 251269. 6 Kuba, K., Kato, E., Kumamoto, E., Koketsu, K. and Hirai, K., Sustained potentiation of transmitter release by adrenaline and dibutyryl cyclic AMP in sympathetic ganglia, Nature (Lond.), 291 ( 1981) 654-656. 7 Kupfermann, I., Modulatory actions of neurotransmitters, Ann. Rev. Neurosci., 2 (1979) 447-465.
347 8 Nestler, E. J. and Greengard, P., Nerve impulses increase the phosphorylation state of protein I in rabbit superior cervical ganglion, Nature (Lond.), 296 (1982) 452-454.
9 Nishi, S. and Koketsu, K., Electrical properties and activities of single sympathetic neurons in frogs, J. cell. comp. PhysioL, 55 (1960) 15-30.