Neuroscience Letters, 111 (1990) 344-350 Elsevier Scientific Publishers Ireland Ltd.
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Effect of calcitonin gene-related peptide on synaptic transmission at the neuromuscular junction of the frog C . G . C a r a t s c h I a n d F. E u s e b i 2 1Department of Pharmacology, University of Ziirich, Ziirich (Switzerland) and 2Department of Experimental Medicine, University of L'Aquila, L'Aquila (Italy)
(Received 6 November 1989; Revised version received 3 December 1989; Accepted 12 December 1989) Key words." Calcitonin gene-related peptide; Synaptic transmission; Neuromuscularjunction; Acetylcho-
line receptor regulation The effectsof calcitonin gene-related peptide (CGRP) on synaptic mechanismswere studied at the frog neuromuscular junction by using classical electrophysiological techniques. CGRP reduced the quantal content of evoked neurotransmitter release, as wellas the sensitivityof postsynaptic nicotinic acetylcholine receptors (AChRs). No effecton the frequencyof the miniature end-plate potentials or on the desensitization of the AChRs could be observed. Both the measured effects may depend on the stimulation of the cyclic AMP second messenger system.
Calcitonin gene-related peptide (CGRP) is a neuropeptide widely distributed in the nervous system, present in particular in parasympathetic cardiac ganglia [22], hypoglossal, facial and ambiguus nuclei [24], spinal cord motoneurons [23], rat trigeminal ganglia [15] and nerve terminals of neuromuscular junctions [25, 16], where it coexists with the neurotransmitter acetylcholine (ACh), and is released by mechanisms which are possibly different from those of ACh release [16]. Its presence in human neuromuscular junctions has recently also been described [19]. It has been shown that C G R P increases the level of cyclic A M P (cAMP) in isolated mouse diaphragm [26], cultured chick [14] and rat myotubes [18], by stimulating the activity of the adenylate cyclase [13]. Furthermore, it affects the desensitization of the nicotinic ACh receptor (nAChR) in cultured mouse myoblasts [20], increases both the synthesis of nAChRs [21] and the level of n A C h R ~-subunit m R N A in cultured chick myotubes [6] and regulates the synaptic nAChR-channel properties in rat muscle fibers [4]. Despite all these studies, however, it is not yet clear whether C G R P substantially affects the physiological fast transmission at adult neuromuscular junctions. The aim of the present work is to answer this question at the frog neuromuscular junction. Correspondence." C.G. Caratsch, Department of Pharmacology, University of Ziirich, Gloriastr. 32, CH8006 Z/irich, Switzerland.
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All experiments were performed on sartorius nerve-muscle preparations of the frog (Rana temporaria) as previously described [2, 5] using standard electrophysiological techniques [11]. When measuring the frequency of the miniature end-plate potentials (MEPPs) neostigmine (4.5 pM) was added to the bath in order to facilitate their recording, and tetrodotoxin (1/~M) to prevent eventually occurring action potentials. To measure the amplitude of the end-plate potentials (EPPs) (+)-tubocurarine (1-2.5 pM) was added to the bath, and the measured amplitude always corrected for the standard resting potential of - 9 0 mV using the formula described by Katz and Thesleff [12]. In other experiments, when the quantal content was calculated according to del Castillo and Katz [3] by using the ratio between the mean amplitude of the EPPs and that of the MEPPs, the calcium concentration was reduced (0.4-0.8 mM) and magnesium added (4.2-4.8 mM). To measure the ACh sensitivity of the postsynaptic membrane iontophoretic micropipettes with tip resistances of 80-120 Mr1 were filled with 1 M AChCI. On average an iontophoretic charge of ca. 60 pC was necessary to obtain a response showing an ACh sensitivity of ca. 150 mV/nC. Single pulses of ACh were applied every 20 s. In order to avoid changes in the amplitude of the ACh-induced depolarization by minute displacements, the tip of the pipette was kept a few micrometers away from the spot of maximal sensitivity. To prevent measurable depolarization when the tip of the ACh micropipette was placed near to an end-plate, braking currents of 2-5 nA were required. The desensitization of the nAChRs was tested either with longduration very weak application of ACh obtained by switching off the braking current of the micropipette, or with repetitive short iontophoretic pulses of ACh. On line data recording during the experiments were made with a PCLAB DT 2801-A Single Board Analog and Digital I/O System and their analysis with a computer (IBM PC AT03). C G R P (human ~-calcitonin gene-related peptide, Bachem, Basle, Switzerland) stock solution was 20 pM in frog Ringer solution. The drug was applied to the muscles at different final concentrations (0.1 - 1 pM) for periods of over 1 h on average (45-180 min) and at room temperature (21 + I°C). All other reagents were purchased from Sigma, St. Louis, MO (U.S.A.). In order to investigate possible effects of CGRP on the regulation of neurotransmission at the frog neuromuscular junction, we tested its influence on both preand postsynaptic mechanisms. We measured the influence of this neuropeptide on (i) the quantai content of EPPs, (ii) the frequency of MEPPs, (iii) the amplitude of EPPs, (iv) the postsynaptic sensitivity to iontophoretically applied single short pulses of ACh, and (v) the desensitization of the postsynaptic membrane. Quantal content. CGRP was proved to be able to affect the quantal content (m) of evoked release at concentrations of 0.1-1 #M. This effect was dose dependent. While at a concentration of 0.5 #M C G R P caused a decrease in m to only about 90%, at a concentration of 1 pM it reduced m on average to about 80% (79.8 _ 2.4%, + S.E.M., n = 6) of control values. The described decrease of m was faster at the beginning and took on average up to nearly 30 min to reach a plateau. Fig. 1 shows a typical example of the effect of CGRP on the m values at a single end-plate. The
346
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Fig. I. Time course of the effect of CGRP (1 ~tM) on the quantal content of a single end-plate in the sartorius muscle of the frog. The muscle was equilibrated in a low-calcium Ringer solution (0.8 mM Ca; 4.2 mM Mg) at room temperature. The resting potential of the muscle cell was - 75 mV. At the arrow CGRP was added to the bath. Abscissa: time in min. Ordinate: quantal content in percent of control values.
addition of the drug caused a reduction of m to ca. 80% of control values within a few minutes, and thereafter a further slow reduction to a steady state of 76%. MEPPs. C G R P in the range of concentrations tested (0. I-1 pM) had no effect on the frequency of the spontaneously occurring MEPPs. Their amplitude, instead, was slightly but significantly affected by the neuropeptide. For instance, in a typical experiment, while MEPP amplitude and frequency at the beginning were on average 0.27 + 0.02 mV (~ + S.E.M.) and ca. 1 Hz respectively, 60 rain after C G R P addition (1 #M) the MEPPs frequency remained substantially stable, while their amplitude decreased to 0.19 _+ 0.01 mV, i.e. to ca. 70%. Amplitude of the EPPs. At the low concentration of 0.1 #M we could not detect any effect of C G R P on the amplitude of the EPPs. At the concentration of 1 #M instead, there was on average a diminution to ca. 80% of control values (79.3 _+3.3%, Y _+ S.E.M., n = 11; values measured 1 h after application of the drug). Fig. 2 shows the time course of one of these experiments. After the application of C G R P (1 pM) the amplitude of the EPPs started to diminish and after 30 min reached a steady state of 68% of control. The time course of the decrease, however, was on average faster than that of the decrease in quantal content of the evoked release. The effect on the amplitude of the EPPs, of course, can be explained by the CGRP-induced diminution of the quantal content, which is of the same order of magnitude, or by a reduced sensitivity of the nAChR. ACh sensitivity to iontophoretically applied ACh. It has been suggested elsewhere that C G R P may act on the junctional muscle membrane [4, 20]. In order to see whether C G R P may affect nAChr function in the frog, we tested the postsynaptic membrane sensitivity to single pulses of iontophoretically applied ACh. The results of these experiments showed a rather important variation depending on the difficulty to keep the iontophoretic micropipette in position for a long time. However, on average there was a diminution of the ACh sensitivity to 55 _+ 6% of control values
347
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Fig. 2. Time course of the effect of C G R P (1 /~M) on the amplitude of EPPs at a single end-plate in the sartorius muscle of the frog. The muscle was equilibrated in normal Ringer solution with (+)-tubocurarine (2.5 #M) at room temperature. The resting potential of the muscle cell was - 8 6 mV. At the arrow C G R P was added to the bath. Abscissa: time in min. Ordinate: amplitude of EPPs in percent of control values.
(~ ___ S.E.M., n = 9) due to the addition of CGRP (1 /~M). The mean time needed to reach a steady state was around 5-10 rain and sometimes a spontaneous recovery appeared 20-30 min after addition of the drug. Fig 3 shows one of these experiments. Desensitization of the postsynaptic nA ChR. Experiments performed in order to test the effect of CGRP (1 /~M) on the desensitization of the nAChRs showed no effect at the frog neuromuscular junction (data not shown). We show here for the first time that CGRP acts on both pre- and postsynaptic sites of this synapse, on the one side by reducing the evoked ACh release (quantal Ringer
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Fig. 3. Time course of the effect o f C G R P (1/~M) on the sensitivity to iontophoretic applied A C h of an end-plate in the sartorius muscle o f the frog. The iontophoretic ACh-pulses were 30 nA × 1 ms with a braking current of 2.5 nA and the measured A C h sensitivity was of 307 mV/nC. The resting potential of the muscle fiber was - 78 mV. At the first arrow a control application of Ringer solution and at the second arrow C G R P were added to the bath. Abscissa: time in min. Ordinate: A C h sensitivity in percent of control sensitivity. Insets: single A C h potentials measured at t = 10, at t --- 30 and at t = 45 min. Upper trace: m e m b r a n e potential, lower trace: iontophoretic current.
348 content) and on the other side by decreasing the ACh sensitivity of the postsynaptic membrane. C G R P does not affect the spontaneous release (MEPP frequency) of the transmitter, while it causes a decrease in the amplitude of MEPPs which is likely related to the decreased ACh sensitivity of the postsynaptic membrane. All these CGRP-induced effects on the neurotransmission occur within a few minutes after drug addition and reach their maximum at about 20 min of exposure to the drug. The mechanisms involved in all these effects of C G R P seem to be related to its stimulating action on the adenylate cyclase, an effect demonstrated by Kobayashi et al. [13]. The maximum enzyme activation found by them was also at the concentration of 1 ~M. The CGRP-induced increase in the activity of adenylate cyclase leads to an increase of the level of cAMP as found in isolated mouse diaphragm [26], in cultured chick myotubes [14] and in rat myotubes [18]. The increase in the cAMP level with the following stimulation of the cAMP-dependent protein kinase (cAMPPK) [7] may account for both the pre- and postsynaptic effects seen at the frog neuromuscular junction in the experiments described. On the presynaptic site it is well known that cAMP-PK leads to the phosphorylation of synapsin I, a major endogenous substrate for this enzyme as well as for other protein kinases, the Ca 2+ calmodulin-dependent PK I and | h Synapsin I is strongly involved in the modulation of neurotransmitter release [7]. The increase in cAMP and, hence, the following stimulation of the cAMP-PK, may also be responsible for the postsynaptic effects, i.e. for the reduction of the ACh sensitivity demonstrated in our experiments. It has been shown by Greengard and coworkers [9, 10, 17~ 18] that the nAChR can be modulated by the cAMP-PK which phosphorylates the 7' and 6 subunits of the Torpedoreceptor. In addition it has been reported that C G R P also increases the level of the ~-subunit phosphorylation, in a way similar to forskolin, a well-known stimulator of the adenylate cyclase [18]. The transient effect of C G R P on the ACh-sensitivity sometimes observed is actually in accordance with data obtained by Eusebi et al. [4], showing that in adult muscle fibers the CGRP-induced rise in the level of intracellular cAMP decreases more rapidly with time than in myotubes. The experiments on the desensitization of the synaptic nAChR proved that C G R P had no effect in our preparation. This is in contrast with the results obtained by Mulle et al. [20] for myoblast nAChRs from a mouse cell line. However, this may depend on differences in phosphorylation sites of the cellular biochemical machinery between mammalian embryonic muscle cells and amphibian adult muscles used in the experiments reported here. Eusebi et al. [4] e.g. saw no desensitization after application of C G R P on adult rat muscle fibers, and Cachelin and Colquhoun [1] using adult dissociated frog muscle fibers proved that an increase of intracellular cAMP, either with direct application or induced by forskolin, had no effect on the time course of desensitization. According to our results and to those obtained by Cachelin and Colquhoun [I] the reduced sensitivity to ACh seems not to be connected to the desensitization of the nAChR, but rather to the fact that C G R P causes an increased probability of occurrence of two low conductance classes of the nAChR [4], which possibly corres-
349
pond to subconductance states of the receptor as described by Hamill and Sakmann
[8]. In conclusion all these results reported indicate that the CGRP action is not important for the mechanisms controlling the single fast propagation of the nerve impulse to the muscle, but may be involved in long-term modulation of the neurotransmission and in synaptic plasticity. 1 Cachelin, A.B. and Colquhoun, D., Desensitization of the acetylcholine receptor of frog end-plates measured in a vaseline-gap voltage clamp, J. Physiol. (Lond.), 415 (1989) 159.-188. 2 Caratsch, C.G., Grassi, F., Molinaro, M. and Eusebi, F., Postsynaptic effects of the phorbol ester TPA on frog end-plates, Pfl/igers Arch., 407 (1986) 409-413. 3 del Castillo, J. and Katz B., Quantal components of the end-plate potential, J. Physiol. (Lond.), 124 (1954) 560-573. 4 Eusebi, F., Farini, D., Grassi, F., Monaco, L. and Ruzzier, F., Effects of calcitonin gene-related peptide on synaptic acetylcholine receptor-channels in rat muscle fibres, Proc. R. Soc. Lond., 234 (1988) 333-342. 5 Eusebi, F., Molinaro, M. and Caratsch, C.G., Effects of phorbol ester on spontaneous transmitter release at frog neuromuscular junction, PfliJgers Arch., 406 (I 986) 181-183. 6 Fontaine, B., Klarsfeld, A. and Changeux, J.-P., Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor ct-subunit mRNA levels by distinct intracellular pathways, J. Cell Biol., 105 (1987) 1337-1342. 7 Greengard, P., Neuronal phosphoproteins: mediators of signal transduction, Mol. Neurobiol., 1 (1987) 81 119. 8 Hamill, O.P. and Sackmann, B., Multiple conductance states of single acetylcholine receptor channels in embryonic muscle cells, Nature (Lond.), 291 (1981) 462-464. 9 Hopfield, J.F., Tank, D.W., Greengard, P. and Huganir, R.L., Functional modulation of the nicotinic acetylcholine receptor by tyrosine phosphorylation, Nature (Lond.), 336 (1988) 677~80. 10 Huganir, R.L., Regulation of the nicotinic acetylcholine receptor by protein phosphorylation, J. Rec. Res., 7 (1987) 241-256. I 1 Katz, B. and Miledi, R., Propagation of electric activity in motor nerve terminals, Proc. R. Soc. Lond. Biol., 161 (1965)453-482. 12 Katz, B. and Thesleff, S., On the factors which determine the amplitude of the m.e.p.p.s, J. Physiol., (Lond.), 137 (1957) 267 278. 13 Kobayashi, H., Hashimoto, K., Sakuma, J., Takami, K., Tohyama, M., Izumi, F. and Yoshida, H., Calcitonin gene-related peptide stimulates adenylate cyclase activity in rat striatal muscle, Experientia, 43 (1987) 314-316. 14 Laufer, R. and Changeux, J.-P., Calcitonin gene-related peptide elevates cyclic AMP levels in chick skeletal muscle: possible neurotrophic role for a coexisting neuronal messenger, EMBO J., 6 (1987) 901 906. 15 Mason, R.T., Peterfreund, R.A., Sawchenko, P.E., Corrigan, A.Z., Rivier, J. and Vale, W.W., Release of the predicted calcitonin gene-related peptide from cultured rat trigeminal ganglion cells, Nature (Lond.), 308 (1984) 653~555. 16 Matteoli, M., Haimann, C., Torri-Tarelli, F., Polak, J.M., Ceccarelli, B., and de Camilli, P., Differential effect of ct-latrotoxin on exocitosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7366-7370. 17 Miles, K., Anthony, A.D., Rubin, L.L., Greengard, P. and Huganir, R.L., Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 6591~595. 18 Miles, K., Greengard, P. and Huganir, R., Calcitonin gene-related peptide regulates phosphorylation of the nicotinic acetylcholine receptor in rat myotubes, Neuron, 2 (1989) 1517-1524.
350 19 Mora, M., Marchi, M., Polak, J.M., Gibson, S.J. and Cornelio, F., Calcitonin gene-related peptide immunoreactivity at the human neuromuscular junction, Brain Res., 492 (1989) 404-407. 20 Mulle, C., Benoit, P., Pinset, C., Roa, M. and Changeux, J.-P., Calcitonin gene-related peptide enhances the rate of desensitization of the nicotinic acetylcholine receptor in cultured mouse muscle cells, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 5728-5732. 21 New, H.V. and Mudge, A.W., Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis, Nature (Lond.), 323 (1986) 809 81 I. 22 Peng, H.B. and Chen, Q., Localization of calcitonin gene-related peptide (CGRP) at a neuronal nicotinic synapse, Neurosci. Lett., 95 (1988) 75-80. 23 Rosenfeld, M.G., Mermod, J.J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W. and Evans, R.M., Production of a novel neuropeptide encoded by the calcitonin gene via tissuespecific RNA processing, Nature (Lond.), 304 ( 1983) 129-135. 24 Takami, K., Kawai, Y., Shiosaka, S., Lee, Y., Girgis, S., Hillyard, C..I., Maclntyre, I., Emson, P.C. and Tohyama, M., Immunohistochemical evidence for the coexistence of calcitonin gene-related peptide- and choline acetyltransferase-like immunoreactivity in neurons of the rat hypoglossal, facial and ambiguus nuclei, Brain Res., 328 (1985) 386-389. 25 Takami, K., Kawai, Y., Uchida, S., Tohyama, M., Shiotani, Y., Yoshida, H., Emson, P.C., Girgis, S., Hillyard, C.J. and Maclntyre, i., Effect of calcitonin gene-related peptide on contraction of striated muscle in the mouse, Neurosci. Lett., 60 (1985) 227 230. 26 Takami, K., Hashimoto, K., Uchida, S., Tohyama, M. and Yoshida, H., Effect of calcitonin generelated peptide on cyclic AMP level of isolated mouse diaphragm, Jpn. J. Pharmacol., 42 (1986) 345 350.
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Effect of calcitonin gene-related peptide on synaptic transmission at the neuromuscular junction of the frog C . G . C a r a t s c h I a n d F. E u s e b i 2 1Department of Pharmacology, University of Ziirich, Ziirich (Switzerland) and 2Department of Experimental Medicine, University of L'Aquila, L'Aquila (Italy)
(Received 6 November 1989; Revised version received 3 December 1989; Accepted 12 December 1989) Key words." Calcitonin gene-related peptide; Synaptic transmission; Neuromuscularjunction; Acetylcho-
line receptor regulation The effectsof calcitonin gene-related peptide (CGRP) on synaptic mechanismswere studied at the frog neuromuscular junction by using classical electrophysiological techniques. CGRP reduced the quantal content of evoked neurotransmitter release, as wellas the sensitivityof postsynaptic nicotinic acetylcholine receptors (AChRs). No effecton the frequencyof the miniature end-plate potentials or on the desensitization of the AChRs could be observed. Both the measured effects may depend on the stimulation of the cyclic AMP second messenger system.
Calcitonin gene-related peptide (CGRP) is a neuropeptide widely distributed in the nervous system, present in particular in parasympathetic cardiac ganglia [22], hypoglossal, facial and ambiguus nuclei [24], spinal cord motoneurons [23], rat trigeminal ganglia [15] and nerve terminals of neuromuscular junctions [25, 16], where it coexists with the neurotransmitter acetylcholine (ACh), and is released by mechanisms which are possibly different from those of ACh release [16]. Its presence in human neuromuscular junctions has recently also been described [19]. It has been shown that C G R P increases the level of cyclic A M P (cAMP) in isolated mouse diaphragm [26], cultured chick [14] and rat myotubes [18], by stimulating the activity of the adenylate cyclase [13]. Furthermore, it affects the desensitization of the nicotinic ACh receptor (nAChR) in cultured mouse myoblasts [20], increases both the synthesis of nAChRs [21] and the level of n A C h R ~-subunit m R N A in cultured chick myotubes [6] and regulates the synaptic nAChR-channel properties in rat muscle fibers [4]. Despite all these studies, however, it is not yet clear whether C G R P substantially affects the physiological fast transmission at adult neuromuscular junctions. The aim of the present work is to answer this question at the frog neuromuscular junction. Correspondence." C.G. Caratsch, Department of Pharmacology, University of Ziirich, Gloriastr. 32, CH8006 Z/irich, Switzerland.
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All experiments were performed on sartorius nerve-muscle preparations of the frog (Rana temporaria) as previously described [2, 5] using standard electrophysiological techniques [11]. When measuring the frequency of the miniature end-plate potentials (MEPPs) neostigmine (4.5 pM) was added to the bath in order to facilitate their recording, and tetrodotoxin (1/~M) to prevent eventually occurring action potentials. To measure the amplitude of the end-plate potentials (EPPs) (+)-tubocurarine (1-2.5 pM) was added to the bath, and the measured amplitude always corrected for the standard resting potential of - 9 0 mV using the formula described by Katz and Thesleff [12]. In other experiments, when the quantal content was calculated according to del Castillo and Katz [3] by using the ratio between the mean amplitude of the EPPs and that of the MEPPs, the calcium concentration was reduced (0.4-0.8 mM) and magnesium added (4.2-4.8 mM). To measure the ACh sensitivity of the postsynaptic membrane iontophoretic micropipettes with tip resistances of 80-120 Mr1 were filled with 1 M AChCI. On average an iontophoretic charge of ca. 60 pC was necessary to obtain a response showing an ACh sensitivity of ca. 150 mV/nC. Single pulses of ACh were applied every 20 s. In order to avoid changes in the amplitude of the ACh-induced depolarization by minute displacements, the tip of the pipette was kept a few micrometers away from the spot of maximal sensitivity. To prevent measurable depolarization when the tip of the ACh micropipette was placed near to an end-plate, braking currents of 2-5 nA were required. The desensitization of the nAChRs was tested either with longduration very weak application of ACh obtained by switching off the braking current of the micropipette, or with repetitive short iontophoretic pulses of ACh. On line data recording during the experiments were made with a PCLAB DT 2801-A Single Board Analog and Digital I/O System and their analysis with a computer (IBM PC AT03). C G R P (human ~-calcitonin gene-related peptide, Bachem, Basle, Switzerland) stock solution was 20 pM in frog Ringer solution. The drug was applied to the muscles at different final concentrations (0.1 - 1 pM) for periods of over 1 h on average (45-180 min) and at room temperature (21 + I°C). All other reagents were purchased from Sigma, St. Louis, MO (U.S.A.). In order to investigate possible effects of CGRP on the regulation of neurotransmission at the frog neuromuscular junction, we tested its influence on both preand postsynaptic mechanisms. We measured the influence of this neuropeptide on (i) the quantai content of EPPs, (ii) the frequency of MEPPs, (iii) the amplitude of EPPs, (iv) the postsynaptic sensitivity to iontophoretically applied single short pulses of ACh, and (v) the desensitization of the postsynaptic membrane. Quantal content. CGRP was proved to be able to affect the quantal content (m) of evoked release at concentrations of 0.1-1 #M. This effect was dose dependent. While at a concentration of 0.5 #M C G R P caused a decrease in m to only about 90%, at a concentration of 1 pM it reduced m on average to about 80% (79.8 _ 2.4%, + S.E.M., n = 6) of control values. The described decrease of m was faster at the beginning and took on average up to nearly 30 min to reach a plateau. Fig. 1 shows a typical example of the effect of CGRP on the m values at a single end-plate. The
346
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Fig. I. Time course of the effect of CGRP (1 ~tM) on the quantal content of a single end-plate in the sartorius muscle of the frog. The muscle was equilibrated in a low-calcium Ringer solution (0.8 mM Ca; 4.2 mM Mg) at room temperature. The resting potential of the muscle cell was - 75 mV. At the arrow CGRP was added to the bath. Abscissa: time in min. Ordinate: quantal content in percent of control values.
addition of the drug caused a reduction of m to ca. 80% of control values within a few minutes, and thereafter a further slow reduction to a steady state of 76%. MEPPs. C G R P in the range of concentrations tested (0. I-1 pM) had no effect on the frequency of the spontaneously occurring MEPPs. Their amplitude, instead, was slightly but significantly affected by the neuropeptide. For instance, in a typical experiment, while MEPP amplitude and frequency at the beginning were on average 0.27 + 0.02 mV (~ + S.E.M.) and ca. 1 Hz respectively, 60 rain after C G R P addition (1 #M) the MEPPs frequency remained substantially stable, while their amplitude decreased to 0.19 _+ 0.01 mV, i.e. to ca. 70%. Amplitude of the EPPs. At the low concentration of 0.1 #M we could not detect any effect of C G R P on the amplitude of the EPPs. At the concentration of 1 #M instead, there was on average a diminution to ca. 80% of control values (79.3 _+3.3%, Y _+ S.E.M., n = 11; values measured 1 h after application of the drug). Fig. 2 shows the time course of one of these experiments. After the application of C G R P (1 pM) the amplitude of the EPPs started to diminish and after 30 min reached a steady state of 68% of control. The time course of the decrease, however, was on average faster than that of the decrease in quantal content of the evoked release. The effect on the amplitude of the EPPs, of course, can be explained by the CGRP-induced diminution of the quantal content, which is of the same order of magnitude, or by a reduced sensitivity of the nAChR. ACh sensitivity to iontophoretically applied ACh. It has been suggested elsewhere that C G R P may act on the junctional muscle membrane [4, 20]. In order to see whether C G R P may affect nAChr function in the frog, we tested the postsynaptic membrane sensitivity to single pulses of iontophoretically applied ACh. The results of these experiments showed a rather important variation depending on the difficulty to keep the iontophoretic micropipette in position for a long time. However, on average there was a diminution of the ACh sensitivity to 55 _+ 6% of control values
347
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Fig. 2. Time course of the effect of C G R P (1 /~M) on the amplitude of EPPs at a single end-plate in the sartorius muscle of the frog. The muscle was equilibrated in normal Ringer solution with (+)-tubocurarine (2.5 #M) at room temperature. The resting potential of the muscle cell was - 8 6 mV. At the arrow C G R P was added to the bath. Abscissa: time in min. Ordinate: amplitude of EPPs in percent of control values.
(~ ___ S.E.M., n = 9) due to the addition of CGRP (1 /~M). The mean time needed to reach a steady state was around 5-10 rain and sometimes a spontaneous recovery appeared 20-30 min after addition of the drug. Fig 3 shows one of these experiments. Desensitization of the postsynaptic nA ChR. Experiments performed in order to test the effect of CGRP (1 /~M) on the desensitization of the nAChRs showed no effect at the frog neuromuscular junction (data not shown). We show here for the first time that CGRP acts on both pre- and postsynaptic sites of this synapse, on the one side by reducing the evoked ACh release (quantal Ringer
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Time (mini
Fig. 3. Time course of the effect o f C G R P (1/~M) on the sensitivity to iontophoretic applied A C h of an end-plate in the sartorius muscle o f the frog. The iontophoretic ACh-pulses were 30 nA × 1 ms with a braking current of 2.5 nA and the measured A C h sensitivity was of 307 mV/nC. The resting potential of the muscle fiber was - 78 mV. At the first arrow a control application of Ringer solution and at the second arrow C G R P were added to the bath. Abscissa: time in min. Ordinate: A C h sensitivity in percent of control sensitivity. Insets: single A C h potentials measured at t = 10, at t --- 30 and at t = 45 min. Upper trace: m e m b r a n e potential, lower trace: iontophoretic current.
348 content) and on the other side by decreasing the ACh sensitivity of the postsynaptic membrane. C G R P does not affect the spontaneous release (MEPP frequency) of the transmitter, while it causes a decrease in the amplitude of MEPPs which is likely related to the decreased ACh sensitivity of the postsynaptic membrane. All these CGRP-induced effects on the neurotransmission occur within a few minutes after drug addition and reach their maximum at about 20 min of exposure to the drug. The mechanisms involved in all these effects of C G R P seem to be related to its stimulating action on the adenylate cyclase, an effect demonstrated by Kobayashi et al. [13]. The maximum enzyme activation found by them was also at the concentration of 1 ~M. The CGRP-induced increase in the activity of adenylate cyclase leads to an increase of the level of cAMP as found in isolated mouse diaphragm [26], in cultured chick myotubes [14] and in rat myotubes [18]. The increase in the cAMP level with the following stimulation of the cAMP-dependent protein kinase (cAMPPK) [7] may account for both the pre- and postsynaptic effects seen at the frog neuromuscular junction in the experiments described. On the presynaptic site it is well known that cAMP-PK leads to the phosphorylation of synapsin I, a major endogenous substrate for this enzyme as well as for other protein kinases, the Ca 2+ calmodulin-dependent PK I and | h Synapsin I is strongly involved in the modulation of neurotransmitter release [7]. The increase in cAMP and, hence, the following stimulation of the cAMP-PK, may also be responsible for the postsynaptic effects, i.e. for the reduction of the ACh sensitivity demonstrated in our experiments. It has been shown by Greengard and coworkers [9, 10, 17~ 18] that the nAChR can be modulated by the cAMP-PK which phosphorylates the 7' and 6 subunits of the Torpedoreceptor. In addition it has been reported that C G R P also increases the level of the ~-subunit phosphorylation, in a way similar to forskolin, a well-known stimulator of the adenylate cyclase [18]. The transient effect of C G R P on the ACh-sensitivity sometimes observed is actually in accordance with data obtained by Eusebi et al. [4], showing that in adult muscle fibers the CGRP-induced rise in the level of intracellular cAMP decreases more rapidly with time than in myotubes. The experiments on the desensitization of the synaptic nAChR proved that C G R P had no effect in our preparation. This is in contrast with the results obtained by Mulle et al. [20] for myoblast nAChRs from a mouse cell line. However, this may depend on differences in phosphorylation sites of the cellular biochemical machinery between mammalian embryonic muscle cells and amphibian adult muscles used in the experiments reported here. Eusebi et al. [4] e.g. saw no desensitization after application of C G R P on adult rat muscle fibers, and Cachelin and Colquhoun [1] using adult dissociated frog muscle fibers proved that an increase of intracellular cAMP, either with direct application or induced by forskolin, had no effect on the time course of desensitization. According to our results and to those obtained by Cachelin and Colquhoun [I] the reduced sensitivity to ACh seems not to be connected to the desensitization of the nAChR, but rather to the fact that C G R P causes an increased probability of occurrence of two low conductance classes of the nAChR [4], which possibly corres-
349
pond to subconductance states of the receptor as described by Hamill and Sakmann
[8]. In conclusion all these results reported indicate that the CGRP action is not important for the mechanisms controlling the single fast propagation of the nerve impulse to the muscle, but may be involved in long-term modulation of the neurotransmission and in synaptic plasticity. 1 Cachelin, A.B. and Colquhoun, D., Desensitization of the acetylcholine receptor of frog end-plates measured in a vaseline-gap voltage clamp, J. Physiol. (Lond.), 415 (1989) 159.-188. 2 Caratsch, C.G., Grassi, F., Molinaro, M. and Eusebi, F., Postsynaptic effects of the phorbol ester TPA on frog end-plates, Pfl/igers Arch., 407 (1986) 409-413. 3 del Castillo, J. and Katz B., Quantal components of the end-plate potential, J. Physiol. (Lond.), 124 (1954) 560-573. 4 Eusebi, F., Farini, D., Grassi, F., Monaco, L. and Ruzzier, F., Effects of calcitonin gene-related peptide on synaptic acetylcholine receptor-channels in rat muscle fibres, Proc. R. Soc. Lond., 234 (1988) 333-342. 5 Eusebi, F., Molinaro, M. and Caratsch, C.G., Effects of phorbol ester on spontaneous transmitter release at frog neuromuscular junction, PfliJgers Arch., 406 (I 986) 181-183. 6 Fontaine, B., Klarsfeld, A. and Changeux, J.-P., Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor ct-subunit mRNA levels by distinct intracellular pathways, J. Cell Biol., 105 (1987) 1337-1342. 7 Greengard, P., Neuronal phosphoproteins: mediators of signal transduction, Mol. Neurobiol., 1 (1987) 81 119. 8 Hamill, O.P. and Sackmann, B., Multiple conductance states of single acetylcholine receptor channels in embryonic muscle cells, Nature (Lond.), 291 (1981) 462-464. 9 Hopfield, J.F., Tank, D.W., Greengard, P. and Huganir, R.L., Functional modulation of the nicotinic acetylcholine receptor by tyrosine phosphorylation, Nature (Lond.), 336 (1988) 677~80. 10 Huganir, R.L., Regulation of the nicotinic acetylcholine receptor by protein phosphorylation, J. Rec. Res., 7 (1987) 241-256. I 1 Katz, B. and Miledi, R., Propagation of electric activity in motor nerve terminals, Proc. R. Soc. Lond. Biol., 161 (1965)453-482. 12 Katz, B. and Thesleff, S., On the factors which determine the amplitude of the m.e.p.p.s, J. Physiol., (Lond.), 137 (1957) 267 278. 13 Kobayashi, H., Hashimoto, K., Sakuma, J., Takami, K., Tohyama, M., Izumi, F. and Yoshida, H., Calcitonin gene-related peptide stimulates adenylate cyclase activity in rat striatal muscle, Experientia, 43 (1987) 314-316. 14 Laufer, R. and Changeux, J.-P., Calcitonin gene-related peptide elevates cyclic AMP levels in chick skeletal muscle: possible neurotrophic role for a coexisting neuronal messenger, EMBO J., 6 (1987) 901 906. 15 Mason, R.T., Peterfreund, R.A., Sawchenko, P.E., Corrigan, A.Z., Rivier, J. and Vale, W.W., Release of the predicted calcitonin gene-related peptide from cultured rat trigeminal ganglion cells, Nature (Lond.), 308 (1984) 653~555. 16 Matteoli, M., Haimann, C., Torri-Tarelli, F., Polak, J.M., Ceccarelli, B., and de Camilli, P., Differential effect of ct-latrotoxin on exocitosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7366-7370. 17 Miles, K., Anthony, A.D., Rubin, L.L., Greengard, P. and Huganir, R.L., Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 6591~595. 18 Miles, K., Greengard, P. and Huganir, R., Calcitonin gene-related peptide regulates phosphorylation of the nicotinic acetylcholine receptor in rat myotubes, Neuron, 2 (1989) 1517-1524.
350 19 Mora, M., Marchi, M., Polak, J.M., Gibson, S.J. and Cornelio, F., Calcitonin gene-related peptide immunoreactivity at the human neuromuscular junction, Brain Res., 492 (1989) 404-407. 20 Mulle, C., Benoit, P., Pinset, C., Roa, M. and Changeux, J.-P., Calcitonin gene-related peptide enhances the rate of desensitization of the nicotinic acetylcholine receptor in cultured mouse muscle cells, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 5728-5732. 21 New, H.V. and Mudge, A.W., Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis, Nature (Lond.), 323 (1986) 809 81 I. 22 Peng, H.B. and Chen, Q., Localization of calcitonin gene-related peptide (CGRP) at a neuronal nicotinic synapse, Neurosci. Lett., 95 (1988) 75-80. 23 Rosenfeld, M.G., Mermod, J.J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W. and Evans, R.M., Production of a novel neuropeptide encoded by the calcitonin gene via tissuespecific RNA processing, Nature (Lond.), 304 ( 1983) 129-135. 24 Takami, K., Kawai, Y., Shiosaka, S., Lee, Y., Girgis, S., Hillyard, C..I., Maclntyre, I., Emson, P.C. and Tohyama, M., Immunohistochemical evidence for the coexistence of calcitonin gene-related peptide- and choline acetyltransferase-like immunoreactivity in neurons of the rat hypoglossal, facial and ambiguus nuclei, Brain Res., 328 (1985) 386-389. 25 Takami, K., Kawai, Y., Uchida, S., Tohyama, M., Shiotani, Y., Yoshida, H., Emson, P.C., Girgis, S., Hillyard, C.J. and Maclntyre, i., Effect of calcitonin gene-related peptide on contraction of striated muscle in the mouse, Neurosci. Lett., 60 (1985) 227 230. 26 Takami, K., Hashimoto, K., Uchida, S., Tohyama, M. and Yoshida, H., Effect of calcitonin generelated peptide on cyclic AMP level of isolated mouse diaphragm, Jpn. J. Pharmacol., 42 (1986) 345 350.
