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Interferon-α,β and tumor necrosis factor-α enhance the freguency of miniature end-plate potentials at rat neuromuscular junction
Neurosc~~~~
Letters,
97
166 (1994) 97-100
0 1994 Elsevier Science Ireland
Ltd. All rights reserved
0304-3940/94/S
07.00
NSL 10133
Interferon-@ and tumor necrosis factor-a enhance the freguency of miniature end-plate potentials at rat neuromuscular junction C.G. Caratscha,*, A. Santonib.‘, F. Eusebi”,’ “Luboratorio “Diportimento
di Medicine
‘Laboratorio (Received Key wordy:
Interferon:
Tumor
electrophysiological
second
messenger
I. R. E., 1% dells ,‘Me.rs~tl’Oro 1%. I-OOIj8 Romtl. Ifcrl~,
Neuromodulation:
rat interferon-a@
techniques.
siently and with a relatively
factor:
and human
Both cytokines
Mammalian tumor
in a similar
long delay (15 to 25 min) the frequency
mechanisms
and contribute
to modulation
of mimiature
and plasticity
system [31]. At the neuromuscular junction a functional regulation of the nicotinic acetylcholine receptor channel has been established for IFN-CX$ [8,15], as well as for interleukin2 [23]. The exact mechanisms by which these effects develop have not yet been established. However, it is likely that they involve G proteins, second messenger production, or even still unknown signalling pathways [23,33]. In this study we show that rat IFN-a$ as well as human tumor necrosis factor a (TNF-a) [19,20], a cytokine also known to influence nerve cell activity in the central nervous system [3], affect the frequency of spontaneous occurring miniature end-plate potentials (m.e.p.p.s) which is an index for neurotransmitter release on the presynaptic site of the mammalian neuromuscular junction. Chemiculs. Interferon (rat interferon-@, Lot 83009, IFN-@) with a specific activity of 1.9.106 U/mg was
SSDI
author.
Riedtlistr.
0304.3940(93)E0757-M
15, CP-8006
Zurich,
necrosis
Switzerland.
1993: Accepted
neuromuscular factor-a.
way at concentrations
Cytokines are proteins involved in immune and inflammatory processes which react with own specific high affinity cell surface receptors and show very high biological potency [l]. Among these cytokines, mainly the interferons (IFNs) [5] and interleukin-2 [25], have been used in cancer immunotherapy, and neurotoxic side-effects have been reported. In clinical and experimental studies, cytokines are shown to affect both the nervous and muscular systems [3.4,6,10,16]. Moreover, recent studies have shown that the action of several cytokines may be transduced into neuronal signals in the central nervous
*Corresponding
C’iule Reginu Elentr 324. 1-00161 Rome. Ittrl~,
Universitri di Romu ‘Lu Supienzu’,
di Fisiopatologiu,
1 April 1993; Revised version received 13 October
necrosis
The effects of the two cytokines, classical
di Biofisicu, I. R. E., Via delle Messi d’Oro 156, I-00158 Romu. Iiul~,
Sperimentale.
endplate
junction;
were studied
27 October
1993)
Electroph>siolog! at the rat neuromuscular
of 2,000 and 35.000 U/ml. respectively. potentials.
junction
by using
increased
tran-
The observed effects may be related to complex
of neurotransmission.
purchased from Lee Biomolecular Research Laboratories Inc., San Diego, Ca., USA. The IFN contained a not exactly characterized mixture of the forms c1 and p only. without the form. After dialysis performed in order to remove glycine, a stock solution (2. 1Oi U/ml) was prepared in mammalian Ringer solution, which contained (in mM): NaCl 142, KC1 5. MgCl, I, CaCI, 1.5, glucose 5, N-2-hydroxyl-piperazine-N’-2-ethanesulfonic acid (Hepes) 2 (pH 7.2). The tested antiviral activity of IFNcr,/? was 3.8.10” IU/mg. Human recombinant tumor necrosis factor (SWG 001 TNF r AMT-0101. Batch 81041) was obtained from Boehringer. Ingelheim. Germany. Stock solutions were 1 and IOpM in mammalian Ringer. The biological activity of TNF-a was evaluated as its ability to induce intracellular adhesion molecules-1 (ICAM-I) on human umbilical vein endothelial cells [ 121 and 10 ng of TNF-a were found to correspond to 200 U of activity. All other reagents were of the highest purity available. Elec~troph~siolo~i~al rrcordings. All electrophysiological experiments were performed at room temperature (23-28°C). Conventional intracellular recordings were performed on isolated hemidiaphragms of younger adult rats killed by decapitation (Sprague -Dawley. 150&l 70 g of weight) in order to measure either the frequency of the spontaneous occurring m.e.p.p.s. the quanta1 content (117) of the evoked neurotransmitter release or the amplitude of the end-plate potentials. The bath medium was oxygenated throughout the experiment. When measuring tn. the calcium concentration was reduced (0.5 mM) and the
98
magnesium concentration increased (6-8 mM), as necessary [l 11. Control experiments of 1 h with application of Ringer solution instead of the cytokine medium had no effects on the measured parameters. IFN-c@ and TNF-cr were added to the bath after a control period of 10 to 15 min and a control application of Ringer solution alone, and the fluid stirred vigorously. Fibers with unstable m.e.p.p.‘s frequency and/or resting potential were discarded. Application of IFN-a,/? (2,000 U/ml) in 6 out of 8 experiments led to a marked increase in the frequency of the m.e.p.p.s, on average about 40 times control values (46 _+14, X _+S.E.M., n = 6). In 2 experiments, instead, IFN-a$ had no obvious effect. Lower concentrations of IFN-a,/? (200-1,000 U/ml) had a very weak effect (3 experiments), and increasing the concentration (up to 10,000 U/ml) did not substantially potentiate it (2 experiments). The onset of this IFN-Q-induced frequency increase had a mean delay of about 25 min (25.3 + 2.3, X + S.E.M., n = 6) after application of the cytokine to the bath, was marked by a sharp peak sometimes followed by smaller oscillations, and was transient. About 20 to 40 min after beginning of the effect, the frequency fell again to control values. Fig. 1 shows a typical example of the effect of IFN-a& (2,000 U/ml) on the frequency of m.e.p.p.s in our preparations. 20 min after application of IFN-a# the m.e.p.p.‘s frequency increased from about 1.5 Hz to a peak of 82 Hz. After this first peak followed by a longer period at about 10 Hz, the frequency attained again control values, which were then kept up to the end of the experiment (30 min later). IFN-a,/? did not influence the resting potential of the muscle fibers or the amplitude of the m.e.p.p.s, as well as the amplitude of the evoked end-plate potentials or their m-value. Similar experiments were performed with human TNF-a. This cytokine mimicked the effects of IFN-a,/?, but with a clearly reduced efficacy. At the concentration of 35,000 U/ml in 5 out of 10 experiments, it increased the m.e.p.p.‘s frequency on average up to 20 times the control values (23 + 5, X + S.E.M., n = 5). Lower concentrations (3,500 U/ml) had only weak effects. The mean delay was also about 25 min (26.2 f 3.7. X _+S.E.M., n = 5), and again no obvious effects on the resting potentials or on the evoked release could be observed. Fig. 2 shows an experiment performed with TNF-a (35,000 U/ml) with a frequency increase of 20fold control values and a delay of 24 min. The reason for the need of relatively high TNF concentrations to influence the m.e.p.p.‘s frequency derives from the fact that human TNF is not much effective on the rat cell system ]351.
Time
(min)
Fig. I. Time course of the effect of IFN-a& (final concentration 2,000 U/ml) on the frequency of miniature end-plate potentials (m.e.p.p.s) in an isolated rat hemidiaphragm. Muscle fiber resting potential: -60 mV. At the arrow the cytokine was added to the bath and the fluid stirred. Abscissa: time in min. Ordinate: m.e.p.p.‘s frequency in Hz. Note the delay between addition of the drug and the onset of the effect.
Our results demonstrate that the cytokines IFN-o$ and TNF-a cause a delayed and transient increase of the m.e.p.p.‘s frequency. This result may suggest a regulatory role of these cytokines for the spontaneous neurotransmitter release at the neuromuscular junction, but not for the evoked one. This role is possibly relevant for synaptic efficacy. The effect seemed to follow a rather sharp dose-relation curve since, as seen for IFN-a,/?, concentrations below 2,000 U/ml proved to be scarsely effective and those above to poorly increase the effect. The actual concentration of the cytokines at the membrane of the recorded cells, however, is not sure. Diffusion barrier due to connective tissue still present on the surface of the cells and which could absorbe drug molecules because of the presence of receptors, may considerably alter the calculated value. This diffusion barrier could also contribute to the relatively long delay measured between application of the drug and onset of the effect. Involvement of a second messenger production usually implies a certain delay. It is possible that cytokines may directly influence intracellular signalling rather than alter gene expression [26]. Some hypotheses about possible mechanisms of action can be made. IFNs for instance are known to cause an intracellular increase in 1,2-diacylgiycerol and cyclic AMP [24,29,34,36,37] and therefore to stimulate the protein kinases C and A (PKC, PKA). Stimulation of PKC through phorbol esters has already been shown to increase the spontaneous release of neurotransmitter at amphibian neuromuscular junctions [7,9,13]. In particular IFN-a has also been shown to activate the PKC through phosphatidylcholine hydrolysis, thus providing a transient source of diacylglycerol without increasing the intracellular calcium [27,28]. The sharp dose-re-
Y9
and thus a contribution neurotransmission. pathological probably
to modulation
and plasticity
This modulation
relevance
during
be one of the main
of
may have a physio-
immune
responses
reasons
for neurotoxic
symptoms as weakness. fatigue or asthenia ing cancer immunotherapy [4].
and
observed
dur-
The present work has been supported by a grant from Fidia (to F.E.) and by a grant from the Consiglio Nazi0
1
I
20
I
40 Time
t
onale delle Ricerche. P.F. Applicazioni Oncologica (to F.E. and A.S.).
60
(min)
Fig. 2. Time course of the effect of TNF-a
(final concentration
-65
mV. Arrow.
Ricerca
network.
Immunol.
35.000
U/ml) on the frequency of m.e.p.p.s in an isolated rat hemidiaphragm. Muscle fiber resting potential:
Cliniche
I
abscissa and ordinate
as in Fig. 1.
Balkwill, Today.
F.R.
and Burke.
F.. The cqtokinc
IO ( 19X9) 299 304.
2 Baud. L.. Perez. J.. Friedlander.
G. and Ardaillou.
crosis factor stimulates prostaglandin
sponse curve supports the view that cytokines act on the m.e.p.p.‘s frequency through mechanisms involving protein kinases. For istance, in muscle cells the stimulation of PKC by the phorbol ester TPA are scarcely dose dependent [ 141. Stimulation of PKA may lead to phosphorylation of the synaptic vesicle-associated phosphoprotein synapsin I, which (i) undergoes multisite phosphorylations [17], (ii) is a substrate for PKA [17], and (iii) seems to have a prominent role in the release of neurotransmitter into the synaptic cleft following calcium influx triggered by depolarization of the nerve terminal [21]. another possible mechanism of signal Finally, transduction for IFN-cr is through arachidonic acid metabolism. IFN-a has been proved to induce a transient activation of phospholipase A1 in 3T3 fibroblasts and hydrolysis of arachidonic acid [18]. which in turn is also able to stimulate the PKC [22]. As for IFN-a$, also for TNF-ar, the exact mechanisms of signal transduction is still far from being understood. However. some recent observations may give some clues for the phenomenon we described. TNF-a has been proved to increase basal neurotransmission in rat hippocampus slices [32], and to induce in U 937 cells, a human histiocytic cell line, a rapid production of 1,2diacylglycerol [30]. In addition, TNF-a is known to enhance CAMP levels in some cell systems, as e.g. in rat cultured mesangial cells [2] and in human fibroblasts, where also a transient increase in the PKA activity could be measured [38]. Therefore also for TNF-cl the observed increase of spontaneous neurotransmitter release could possibly be related to the activation of the PKC, to phosphorylations of synapsin I or to still unknown pathways. In conclusion both IFN-cl,/? and TNF-ol enhance the frequency of m.e.p.p.s suggesting a possible regulatory role for spontaneous synaptic neurotransmitter release
production
R.. Tumor
ne-
and cyclic AMP
levels in rat cultured mesangial cells. FEBS Left.. 239 (1988) 50 54. 3 Blatteis. C.M..
Neuromodulati\e
actions ofcytokines.
4 Bocci. V.. Central
nervous system toxicity of intcrfcrons
cytokines. J. Biol. Reg. Homeo. 5 Borden. E.C.,
Edivards.
Plenum. NCM York.
M.J. and Mcrrit.
enhances the excitability
and Eusebi. F.. Regulatron
of pcripheric
by the C-kinasc system. In R. Verna (Ed.).
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Raven. Neu York.
C.G.,
Narahashi
(Ed.).
I. 01
rynaptic
Membrane
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Grassi. F. and Eusebi. F.. Functional
the nicotinic acetylcholine
York.
J.A.. In-
278 (1979) 55X 560.
transmission
8 Caratsch,
and other
118.
BiologIcal responses in cancer. Vol.
and Gresser, 1.. Interferon
C.G.
I07
lY83. pp. 169-218.
cultured neurones. Nature, 7 Caratsch.
Agents. 7 (1988)
B.S.. Hawkins.
tcrfcronb. In E. Mihich (Ed.),
6 Calbet, M.C.
Yale J. Biol.
133 146.
Med., 63 (1990)
regulation
of
receptor channel in the muscle cell. In T.
Ion Channels.
Vol. 3. Plenum.
London
and Neu
1992. pp. 177 306.
9 Caratsch.
C.G..
Schumacher.
S.. Grasai.
cncc of protein kinase C-stimulation transmitter
rcleasc
Arch. Pharmacol.. IO d‘Arcangclo.
at frog
by
end-plate\.
phorbot cstcr on neuro-
Natmyn
Schmiedeberg’s
337 (198X) 9 12.
G.. Grdssi. F.. RagoLzino.
V. and Eusebl. F.. Interferon hlppocampus,
I:. and Euacbi. F.. Influ21
D.. Santonl.
inhibits synaptic
A.. Iancrcdi.
potcntiation
in rat
Brain Res.. 564 (I 99 1 ) 245 24X.
I I del Castillo. J. and Katz. B.. Quanta1 content ofthe end-plate potcntial. J. Physlol.. 124. 560 573. I2
Dustln. M.L. antigen-l
and Sprlngcr. T.A..
(LFA-I)
cules-l (ICAM-
interaction
t ) is one
Lymphocytes hith
function absociated
intracellular
adhesion
mole-
of at least three mechanisms for limphocyte
adhesion to cultured endothclian
cell\. J. Cell Blot.. 107 (tYX8) 321
331. I3
Eusebi. F., Molinaro.
M. and Caratsch.
ester on spontaneous
transmitter
junction.
Pliiigers Arch.. 406
14 Eusebi. F., Molinaro,
(1986)IXI
M. and Zani. B.M..
tcin kinase C reduce acetylcholinc J. Cell Biol.. 100 (1985) I5
Euscbi.
F.. Farini.
muscle
acetylcholine
l33Y
C.G.,
t:ffects 01‘ phorbol
rclcasc at frog neuromuscular 183. Agents that actibatc pro-
sensitivity in cultured myotubes,
1342.
D.. GrassI.
F. and Santoni,
receptor-channel
A., Regulation
function
by
of
interferon.
PAtigers Arch., 415 (19X9) 150 155. 16 Fent, K. and Zbinden, Trends Pharmcol. 17 Greengard. transduction.
G.. Toxicity
Sci.. 8 (1987)
P.. Neuronal
of intcrfcron
phosphoprotcins:
Mol. Neurobiol..
and interleukin.
100 105. mediators
I (1987) 81 II9
of signal
100
18 Hannigan, G.E. and Williams, B.R.G., Signal transduction by interferon-u through arachidonic acid metabolism, Science, 251 (1991) 204-207. 19 Jaattela, M., Biological activities and mechanisms of action of tumor necrosis factor-a/cachectin, Lab. Invest., 64 (1991) 724-742. 20 Jones, E.Y., Stuart, D.I. and Walker, N.P.C., Structure of tumour necrosis factor, Nature, 338 (1989) 225-228. 21 Kennedy, M.B., Experimental approaches to understanding the role of protein phosphorylation in the regulation of neuronal function, Annu. Rev. Neurosci., 6 (1983) 493-525. 22 Kikkawa, V., Kishimoto, A. and Nishizuka, Y., The protein kinase C family: heterogeneity and its implications, Annu. Rev. Biochem.. 58 (1989) 31-44. 23 Lorenzon, P., Ruzzier, F., Caratsch, C.G., Giovannelli, A.. Velotti, F.. Santoni, A. and Eusebi, F., Interleukin-2 lengthens extrajunctional acetylcholine receptor-channel open time in mammalian muscle cells, Plhigers Arch., 419 (1991) 380-385. 24 Meldolesi, M.F., Friedmann, R.M. and Kohn, L.D., An interferoninduced increase in cyclic AMP levels precedes the establishment of the antiviral state, Biochem. Biophys. Res. Commun.. 79 (1977) 239-246. 25 Parkinson, D.R., Interleukin-2 in cancer therapy, Seminars Oncol., 15 (1988) 10-26. 26 Patterson, P.H. and Nawa, H., Neuronal differentiation factors/ cytokines and synaptic plasticity, Cell/Neuron Rev., Suppl., 37 (1993) 1233137. 27 Pfeffer, L.M., Strulovici, B. and Saltiel, A.R., Interferon-cl selectively activates thep isoform of protein kinase C through phosphatidyl hydrolysis, Proc. Natl. Acad. Sci. USA, 87 (1990) 6537.-6541, 28 Reich, N.C. and Pfeffer, L.M., Evidence for involvement of protein kinase C in the cellular response to interferon a, Proc. Natl. Acad. Sci. USA, 87 (1990) 8761-8765. 29 Schneck. J., Rager-Zisman, B., Rosen, O.M. and Bloom, B.R., Ge-
30
31 32
33
34
35
36
37
38
netic analysis of the role of CAMP in mediating effects of interferon. Proc. Natl. Acad. Sci. USA, 79 (1982) 1879-1883. Schtitze, S., Berkovic, D., Tomsing, O., Unger, C. and Kronze, M., Tumor necrosis factor induces rapid production of l,2-diacylglycerol by a phosphatidylcholine-specific phospholipase C. J. Exp. Med., 174 (1991) 975-988. Shibata, M., Hypothalamic neuronal responses to cytokines, Yale J. Biol. Med., 63 (1990) 147-156. Tancredi, V., d’Arcangelo, G., Grassi, F., Tarroni, P., Palmieri, G.. Santoni, A. and Eusebi, F., Tumor necrosis factor alters synaptic transmission in rat hippocampus, Neurosci. Lett., 146 (1992) 176 178. Tigges, M.A., Casey, L.S. and Koshland, ME.. Mechanism of interleukin-2 signaling: mediation of different outcomes by a single receptor and transduction pathway, Science, 243 (1989) 781.-786. Tovey, M.G., Rochette-Egly, C. and Castagna, M., Effect of interferon on concentration of cyclic nucleotides in cultured cells, Proc. Natl. Acad. Sci. USA, 76 (1979) 3890-3896. Vandenaebeele, H., Declercq, W., Vercammen, D., Van-de-Craen, M., Grooten, J., Loetscher, H.. Brockhaus, M., Lesslauer and Fiers, W., Functional characterization of the human tumor necrosis factor receptor ~75 in a transferred rat/mouse T cell hybridoma, J. Exp. Med., 176, 1015-1024. Yap, W.H., Teo, T.S., McCoy, E. and Tan, Y.H.. Rapid and transient rise in diacylglycerol concentration in Daudi cells exposed to interferon, Proc. Natl. Acad. Sci. USA, 83 (1986) 7765-7769. Yap, W.H., Teo, T.S. and Tan, Y.H., An early event in the interferon-induced transmembrane signaling process, Science, 234 (1986) 355.-358. Zhang, Y., Lin, J.X., Yip, Y.K. and Vilcek, J., Enhancement of CAMP levels and of protein kinase activity by tumor necrosis factor and interleukin 1 in human fibroblasts: role on the induction of interleukin 6, Proc. Natl. Acad. Sci. USA, 85 (1988) 680226805.
Letters,
97
166 (1994) 97-100
0 1994 Elsevier Science Ireland
Ltd. All rights reserved
0304-3940/94/S
07.00
NSL 10133
Interferon-@ and tumor necrosis factor-a enhance the freguency of miniature end-plate potentials at rat neuromuscular junction C.G. Caratscha,*, A. Santonib.‘, F. Eusebi”,’ “Luboratorio “Diportimento
di Medicine
‘Laboratorio (Received Key wordy:
Interferon:
Tumor
electrophysiological
second
messenger
I. R. E., 1% dells ,‘Me.rs~tl’Oro 1%. I-OOIj8 Romtl. Ifcrl~,
Neuromodulation:
rat interferon-a@
techniques.
siently and with a relatively
factor:
and human
Both cytokines
Mammalian tumor
in a similar
long delay (15 to 25 min) the frequency
mechanisms
and contribute
to modulation
of mimiature
and plasticity
system [31]. At the neuromuscular junction a functional regulation of the nicotinic acetylcholine receptor channel has been established for IFN-CX$ [8,15], as well as for interleukin2 [23]. The exact mechanisms by which these effects develop have not yet been established. However, it is likely that they involve G proteins, second messenger production, or even still unknown signalling pathways [23,33]. In this study we show that rat IFN-a$ as well as human tumor necrosis factor a (TNF-a) [19,20], a cytokine also known to influence nerve cell activity in the central nervous system [3], affect the frequency of spontaneous occurring miniature end-plate potentials (m.e.p.p.s) which is an index for neurotransmitter release on the presynaptic site of the mammalian neuromuscular junction. Chemiculs. Interferon (rat interferon-@, Lot 83009, IFN-@) with a specific activity of 1.9.106 U/mg was
SSDI
author.
Riedtlistr.
0304.3940(93)E0757-M
15, CP-8006
Zurich,
necrosis
Switzerland.
1993: Accepted
neuromuscular factor-a.
way at concentrations
Cytokines are proteins involved in immune and inflammatory processes which react with own specific high affinity cell surface receptors and show very high biological potency [l]. Among these cytokines, mainly the interferons (IFNs) [5] and interleukin-2 [25], have been used in cancer immunotherapy, and neurotoxic side-effects have been reported. In clinical and experimental studies, cytokines are shown to affect both the nervous and muscular systems [3.4,6,10,16]. Moreover, recent studies have shown that the action of several cytokines may be transduced into neuronal signals in the central nervous
*Corresponding
C’iule Reginu Elentr 324. 1-00161 Rome. Ittrl~,
Universitri di Romu ‘Lu Supienzu’,
di Fisiopatologiu,
1 April 1993; Revised version received 13 October
necrosis
The effects of the two cytokines, classical
di Biofisicu, I. R. E., Via delle Messi d’Oro 156, I-00158 Romu. Iiul~,
Sperimentale.
endplate
junction;
were studied
27 October
1993)
Electroph>siolog! at the rat neuromuscular
of 2,000 and 35.000 U/ml. respectively. potentials.
junction
by using
increased
tran-
The observed effects may be related to complex
of neurotransmission.
purchased from Lee Biomolecular Research Laboratories Inc., San Diego, Ca., USA. The IFN contained a not exactly characterized mixture of the forms c1 and p only. without the form. After dialysis performed in order to remove glycine, a stock solution (2. 1Oi U/ml) was prepared in mammalian Ringer solution, which contained (in mM): NaCl 142, KC1 5. MgCl, I, CaCI, 1.5, glucose 5, N-2-hydroxyl-piperazine-N’-2-ethanesulfonic acid (Hepes) 2 (pH 7.2). The tested antiviral activity of IFNcr,/? was 3.8.10” IU/mg. Human recombinant tumor necrosis factor (SWG 001 TNF r AMT-0101. Batch 81041) was obtained from Boehringer. Ingelheim. Germany. Stock solutions were 1 and IOpM in mammalian Ringer. The biological activity of TNF-a was evaluated as its ability to induce intracellular adhesion molecules-1 (ICAM-I) on human umbilical vein endothelial cells [ 121 and 10 ng of TNF-a were found to correspond to 200 U of activity. All other reagents were of the highest purity available. Elec~troph~siolo~i~al rrcordings. All electrophysiological experiments were performed at room temperature (23-28°C). Conventional intracellular recordings were performed on isolated hemidiaphragms of younger adult rats killed by decapitation (Sprague -Dawley. 150&l 70 g of weight) in order to measure either the frequency of the spontaneous occurring m.e.p.p.s. the quanta1 content (117) of the evoked neurotransmitter release or the amplitude of the end-plate potentials. The bath medium was oxygenated throughout the experiment. When measuring tn. the calcium concentration was reduced (0.5 mM) and the
98
magnesium concentration increased (6-8 mM), as necessary [l 11. Control experiments of 1 h with application of Ringer solution instead of the cytokine medium had no effects on the measured parameters. IFN-c@ and TNF-cr were added to the bath after a control period of 10 to 15 min and a control application of Ringer solution alone, and the fluid stirred vigorously. Fibers with unstable m.e.p.p.‘s frequency and/or resting potential were discarded. Application of IFN-a,/? (2,000 U/ml) in 6 out of 8 experiments led to a marked increase in the frequency of the m.e.p.p.s, on average about 40 times control values (46 _+14, X _+S.E.M., n = 6). In 2 experiments, instead, IFN-a$ had no obvious effect. Lower concentrations of IFN-a,/? (200-1,000 U/ml) had a very weak effect (3 experiments), and increasing the concentration (up to 10,000 U/ml) did not substantially potentiate it (2 experiments). The onset of this IFN-Q-induced frequency increase had a mean delay of about 25 min (25.3 + 2.3, X + S.E.M., n = 6) after application of the cytokine to the bath, was marked by a sharp peak sometimes followed by smaller oscillations, and was transient. About 20 to 40 min after beginning of the effect, the frequency fell again to control values. Fig. 1 shows a typical example of the effect of IFN-a& (2,000 U/ml) on the frequency of m.e.p.p.s in our preparations. 20 min after application of IFN-a# the m.e.p.p.‘s frequency increased from about 1.5 Hz to a peak of 82 Hz. After this first peak followed by a longer period at about 10 Hz, the frequency attained again control values, which were then kept up to the end of the experiment (30 min later). IFN-a,/? did not influence the resting potential of the muscle fibers or the amplitude of the m.e.p.p.s, as well as the amplitude of the evoked end-plate potentials or their m-value. Similar experiments were performed with human TNF-a. This cytokine mimicked the effects of IFN-a,/?, but with a clearly reduced efficacy. At the concentration of 35,000 U/ml in 5 out of 10 experiments, it increased the m.e.p.p.‘s frequency on average up to 20 times the control values (23 + 5, X + S.E.M., n = 5). Lower concentrations (3,500 U/ml) had only weak effects. The mean delay was also about 25 min (26.2 f 3.7. X _+S.E.M., n = 5), and again no obvious effects on the resting potentials or on the evoked release could be observed. Fig. 2 shows an experiment performed with TNF-a (35,000 U/ml) with a frequency increase of 20fold control values and a delay of 24 min. The reason for the need of relatively high TNF concentrations to influence the m.e.p.p.‘s frequency derives from the fact that human TNF is not much effective on the rat cell system ]351.
Time
(min)
Fig. I. Time course of the effect of IFN-a& (final concentration 2,000 U/ml) on the frequency of miniature end-plate potentials (m.e.p.p.s) in an isolated rat hemidiaphragm. Muscle fiber resting potential: -60 mV. At the arrow the cytokine was added to the bath and the fluid stirred. Abscissa: time in min. Ordinate: m.e.p.p.‘s frequency in Hz. Note the delay between addition of the drug and the onset of the effect.
Our results demonstrate that the cytokines IFN-o$ and TNF-a cause a delayed and transient increase of the m.e.p.p.‘s frequency. This result may suggest a regulatory role of these cytokines for the spontaneous neurotransmitter release at the neuromuscular junction, but not for the evoked one. This role is possibly relevant for synaptic efficacy. The effect seemed to follow a rather sharp dose-relation curve since, as seen for IFN-a,/?, concentrations below 2,000 U/ml proved to be scarsely effective and those above to poorly increase the effect. The actual concentration of the cytokines at the membrane of the recorded cells, however, is not sure. Diffusion barrier due to connective tissue still present on the surface of the cells and which could absorbe drug molecules because of the presence of receptors, may considerably alter the calculated value. This diffusion barrier could also contribute to the relatively long delay measured between application of the drug and onset of the effect. Involvement of a second messenger production usually implies a certain delay. It is possible that cytokines may directly influence intracellular signalling rather than alter gene expression [26]. Some hypotheses about possible mechanisms of action can be made. IFNs for instance are known to cause an intracellular increase in 1,2-diacylgiycerol and cyclic AMP [24,29,34,36,37] and therefore to stimulate the protein kinases C and A (PKC, PKA). Stimulation of PKC through phorbol esters has already been shown to increase the spontaneous release of neurotransmitter at amphibian neuromuscular junctions [7,9,13]. In particular IFN-a has also been shown to activate the PKC through phosphatidylcholine hydrolysis, thus providing a transient source of diacylglycerol without increasing the intracellular calcium [27,28]. The sharp dose-re-
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and thus a contribution neurotransmission. pathological probably
to modulation
and plasticity
This modulation
relevance
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immune
responses
reasons
for neurotoxic
symptoms as weakness. fatigue or asthenia ing cancer immunotherapy [4].
and
observed
dur-
The present work has been supported by a grant from Fidia (to F.E.) and by a grant from the Consiglio Nazi0
1
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20
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40 Time
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onale delle Ricerche. P.F. Applicazioni Oncologica (to F.E. and A.S.).
60
(min)
Fig. 2. Time course of the effect of TNF-a
(final concentration
-65
mV. Arrow.
Ricerca
network.
Immunol.
35.000
U/ml) on the frequency of m.e.p.p.s in an isolated rat hemidiaphragm. Muscle fiber resting potential:
Cliniche
I
abscissa and ordinate
as in Fig. 1.
Balkwill, Today.
F.R.
and Burke.
F.. The cqtokinc
IO ( 19X9) 299 304.
2 Baud. L.. Perez. J.. Friedlander.
G. and Ardaillou.
crosis factor stimulates prostaglandin
sponse curve supports the view that cytokines act on the m.e.p.p.‘s frequency through mechanisms involving protein kinases. For istance, in muscle cells the stimulation of PKC by the phorbol ester TPA are scarcely dose dependent [ 141. Stimulation of PKA may lead to phosphorylation of the synaptic vesicle-associated phosphoprotein synapsin I, which (i) undergoes multisite phosphorylations [17], (ii) is a substrate for PKA [17], and (iii) seems to have a prominent role in the release of neurotransmitter into the synaptic cleft following calcium influx triggered by depolarization of the nerve terminal [21]. another possible mechanism of signal Finally, transduction for IFN-cr is through arachidonic acid metabolism. IFN-a has been proved to induce a transient activation of phospholipase A1 in 3T3 fibroblasts and hydrolysis of arachidonic acid [18]. which in turn is also able to stimulate the PKC [22]. As for IFN-a$, also for TNF-ar, the exact mechanisms of signal transduction is still far from being understood. However. some recent observations may give some clues for the phenomenon we described. TNF-a has been proved to increase basal neurotransmission in rat hippocampus slices [32], and to induce in U 937 cells, a human histiocytic cell line, a rapid production of 1,2diacylglycerol [30]. In addition, TNF-a is known to enhance CAMP levels in some cell systems, as e.g. in rat cultured mesangial cells [2] and in human fibroblasts, where also a transient increase in the PKA activity could be measured [38]. Therefore also for TNF-cl the observed increase of spontaneous neurotransmitter release could possibly be related to the activation of the PKC, to phosphorylations of synapsin I or to still unknown pathways. In conclusion both IFN-cl,/? and TNF-ol enhance the frequency of m.e.p.p.s suggesting a possible regulatory role for spontaneous synaptic neurotransmitter release
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