- Email: [email protected]
Calcium-dependent inhibition by prostaglandin E1 of spontaneous acetylcholine release from frog motor nerve
European Journal of Pharmacology, 48 (1978) 455--457 © Else~ier/North-Holland Biomedical Press
455
Short communication CALCIUM-DEPENDENT INHIBITION BY P R O S T A G L A N D I N Et OF SPONTANEOUS ACETYLCHOLINE RELEASE FROM F R O G MOTOR N E R V E PETER ILLI~S * and FRANTI~EK VYSKO~IL
Department of Pharmacology, Semmelweis University of Medicine, Budapest, Hungary, and Institute of Physiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia Received 25 January 1978, accepted 27 January 1978
P. ILLES and F. VYSKOCIL, Calcium-dependent inhibition by prostaglandin E1 of spontaneous acetylcholine release from frog motor nerve, European J. Pharmacol. 48 (1978) 455--457. PGE1 (10 -7 M) in the absence of Ca 2÷ or at low external Ca 2÷ concentrations (0.2--0.5 mM) depressed the frequency of miniature end-plate potentials (m.e.p.p.s) in frog sartorius muscle, but m.e.p.p, amplitudes were unchanged. Both an increase in Ca 2÷ concentration to 2 mM or a blockade of Ca 2+ uptake into mitochondria by metabolic inhibitors (5 × 10 -s M 2,4-dinitrophenol or 2 × 10 -s M rotenone) prevented the inhibitory action of PGE1 on m.e.p.p, frequency. We suggest that PGEI may inhibit acetylcholine release from motor nerve terminals by promoting the active uptake of Ca 2+ by mitochondria, or by facilitating the efflux of Ca2+ from the axoplasm into the extracellular medium. Prostaglandin E1
Acetylcholine
Neuromuscular transmission
1. Introduction Prostaglandins of the E t y p e (PGEs) inhibit transmitter release from peripheral noradrenergic nerve endings in many organs (see Hedqvist, 1977). PGEs were, however, reported to be inactive on cholinergic transmission in the frog neuromuscular preparation (Ginsborg and Hirst, 1971) and also in the rat diaphragm at stimulation frequencies up to 30 Hz (Gripenberg et al., 1976). Since the prejunctional inhibitory action of PGEs on noradrenaline release was found to be inversely related to the a m o u n t of calcium in the perfusing medium (Hedqvist, 1970; S t j ~ n e , 1973), we have studied the effect of prostaglandin E1 (PGEI) on the spontaneous quantal release of transmitter from cholinergic m o t o r nerves at different calcium concentra* Correspondence to P.I. at his present address: Department of Pharmacology, University of Lund, SSlvegatan 10, S-223 62 Lund, Sweden.
Calcium
tions and in the presence of metabolic inhibitots.
2. Materials and methods Isolated frog sartorius muscles from Rana temporaria were used in the summer (June to July). The muscles were immersed in Ringer solution of composition (mM): NaC1 115; KCI 2.5; CaC12 0--2 and NaHCO3 2.2; the bath temperature was 22--25°C. In the absence of Ca 2÷, ethylene glycol-bis(~-aminoethyl ether)-N,N'-tetra-acetic acid (EGTA, 10 -3 M) was present and t e t r o d o t o x i n (10 -6 M) was added to prevent spontaneous muscle twitching. Prostigmine methylsulphate (6 × 10 -6 M) was added to the bath in all cases except at a Ca 2÷ concentration of 0.5 mM, in order to increase miniature end-plate potential (m.e.p.p.) amplitude. The spontaneous quantal release of acetylcholine was estimated from the frequency of
456
P. ILLt~S, F. VYSKOCIL
m.e.p.p.s, which were recorded intracellularly by t h e conventional microelectrode technique fr o m the end-plate region of the sartorius muscle. At least 100 m.e.p.p.s were recorded on film f r o m the oscilloscope screen and used in each e x p e r i m e n t for statistical estimates o f the mean amplitude. F r e q u e n c y o f m.e.p.p.s was determined by counting at 50 potentials from each muscle fibre before and after application o f PGE,. P G E , was used at a c o n c e n t r a t i o n of 10 -7 M, and its effect was followed for 15--20 min after addition t o th e muscle bath. T he values are q u o t e d as mean + S.E.M. The statistical significance o f th e data was checked by means o f the Student's t-test.
PGE, decreased the frequency of m.e.p.p.s to 55--65% of t he cont rol value, when no or little Ca 2+ (up to 0.5 mM) was present in the Ringer solution. No effect was found in solutions with 2 mM calcium. In anot her series o f experiments muscles were first soaked in a solution with low calcium (0.2 mM) also containing 5 X 10 -s M 2,4-dinitrophenol (DNP) or 2 × 10 -s M r o t e n o n e , in order to inhibit the active uptake of Ca 2+ into mitochondria. PGE, (10 -7 M) was t hen applied for 15--20 min. In each case, no change o f m.e.p.p, frequency was observed (table 1) in the seven muscle fibres in which continous measurements were made before and after PGE,.
3. Results
4. Discussion
PGE1 (10 -7 M) had no effect on the resting polarisation o f muscle fibres at any of the Ca 2+ concentrations tested. Similarly the drug failed to affect th e mean amplitude of the m.e.p.p.s (0.21 ± 0 . 0 2 m V in control and 0.21 + 0.02 mV after PGE,, 7 fibres, 0.5 mM
The major finding from this study is that PGE, (10 -7 M) in t he absence of Ca 2+ or at low external Ca 2+ c o n c e n t r a t i o n depressed the m.e.p.p, f r e q u e n c y in the sartorius muscle of the frog. In accordance with the results obtained by Ginsborg and Hirst (1971) we observed no effect of PGE, when the concentration of calcium was higher (2 mM). At the same time m.e.p.p, amplitudes were unchanged, an indication t hat PGE, does not influence the reactivity o f t he postsynaptic
Ca2+).
The e f f e c t o f PGE1 o n t h e f r e q u e n c y o f s p o n t a n e o u s t r a n s m i t t e r release was investigated at d i f f e r e n t c o n c e n t r a t i o n s o f external calcium. The results are summarized in table 1.
TABLE i Inhibition by PGE I (10 -7 M) of m.e.p.p, frequency in frog sartorius muscle as a function of calcium concentration (mean + S.E.M.).
Ca2+ in bathing fluid
0 2 5 2 2 2
+ EGTA 10 -3 M x 10 -4 M x 10 -4 M x 10 -3 M x 10 -4 M + DNP 5 x 10 -s M x 10 -4 M+rotenone2× 10 -s M
1 p < 0.05. 2 p < 0.001.
Number of experiments 5 5 7 4 4 3
Frequency of m.e.p.p.s (Hz) Control
PGE l 10 -7 M
0.92 3.37 2.71 9.13 7.48 11.2
0.51 1.97 1.71 8.67 7.80 10.3
+_0.13 + 0.43 _+0.45 _+1.92 + 2.44 _+2.28
+_0.09 1 _+0.19 l +_0.31 2 +_1.79 +_1.40 -+3.68
PGE 1 ON SPONTANEOUS RELEASE OF ACETYLCHOLINE
membrane to the transmitter. In peripheral tissues PGEs depress the stimulated release of noradrenaline from sympathetic nel~ce terminals b y regulating the availability of calcium for the secretory mechanism (Hedqvist, 1970). In contrast with these findings, it was reported that PGE~ only slightly reduced the amplitude of the endplate potential and its q u a n t u m content in the frog nerve-sartorius preparation, even at the low extracellular calcium concentration of 0.5 mM (Ill~s et al., 1978). It therefore seems that PGE1 has only a small, if any, effect on the influx of calcium during stimulation. According to our results, PGE~ may decrease m.e.p.p, frequency by reducing the concentration of free Ca 2÷ in the axoplasm of nerve terminals, which in turn regulates spontaneous transmitter release. The free intracellular Ca 2÷ concentration depends on a balance of calcium influx and efflux, and on intracellular calcium buffers such as mitochondria (Alnaes and Rahamimoff, 1975). PGE~ has been also reported to stimulate active sodium extrusion (Limos and Cohn, 1974) which in the nerve terminal could lead to membrane polarisation and thereby to a decrease in m.e.p.p. frequency. However, this prostaglandin is not likely to affect the resting Ca 2÷ influx, since its inhibitory effect persisted even in the absence of external calcium. Metabolic inhibitors (e.g. DNP and rotenone) inhibit the active uptake of Ca 2÷ into mitochondria (Lehninger, 1970). Pret r e a t m e n t of the preparation with these substances prevented the inhibitory action of PGE1 on spontaneous transmitter release. It seems possible that PGE~ Could affect spontaneous transmitter release from nerve terminals by promoting the active uptake of Ca 2÷ by mitochondria. This hypothesis is supported by the reported ability of PGE~ to act as a Ca 2÷ ionophore in the mitochondrial membranes o f the rat liver (Kirtland and Baum, 1972). A similar action in the mitochondria of m o t o r nerve endings could
457
promote the uptake of free calcium and in turn decrease m.e.p.p, frequency. PGE1 may also facilitate the efflux of calcium from the axoplasm across the nerve terminal under the influence of the electrochemical gradient existing during exposure to low Ca 2÷ media. A higher extra- or intracellular concentration of Ca 2÷, the latter caused by metabolic inhibitors, may counteract this effect of PGE1.
Acknowledgement We are grateful to Professor C. Edwards for this valuable criticism of the manuscript.
References Alnaes, E. and R. Rahamimoff, 1975, On the role of mitochondria in transmitter release from motor nerve terminals, J. Physiol. (London) 248,285. Ginsborg, B.L. and G.D.S. Hirst, 1971, Prostaglandin E 1 and noradrenaline at the neuromuscular ]unction, Brit. J. Pharmacol. 42, 153. Gripenberg, J., S.E. Jansson, V. Hein~/nen, E. Heinonen, J. Hyv~rinen and E.M. T o l p p a n e n , 1976, Effect of prostaglandin E1 on neuromuscular transmission in the rat, Brit. J. Pharmacol. 5 7 , 3 8 7 . Hedqvist, P., 1970, Antagonism by calcium of the inhibitory action of prostaglandin E2 on sympathetic neurotransmission in the cat spleen, Acta Physiol. Scand. 80, 269. Hedqvist, P., 1977, Basic mechanisms of prostaglandin action on autonomic neurotransmission, Ann. Rev. Pharmacol. 17,259. Ill,s, P., L.G. Magazanik and J. Knoll, 1978, The effect of PGE 1 and PGF2~ on neuromuscular transmission in the frog sartorius muscle, Acta Physiol. Acad. Sci. Hung. (in press). Kirtland, S.J. and H. Baum, 1972, Prostaglandin E1 may act as a "calcium ionophore", Nature (New Biology) 236, 47. Lehninger, A.L., 1970, Mitochondria and calcium ion transport, Biochem. J. 1 1 9 , 1 2 9 . Limos, C.J. and J.N. Cohn, 1974, Stimulation of vascular smooth muscle Na+,K+-ATPase by vasodilators, Circulation Res. 3 5 , 6 0 1 . Stj~rne, L., 1973, Kinetics of secretion of sympathetic neurotransmitter as a function of external calcium; mechanism of inhibitory effect of prostaglandin E, Acta Physiol. Scand. 8 7 , 4 2 8 .
455
Short communication CALCIUM-DEPENDENT INHIBITION BY P R O S T A G L A N D I N Et OF SPONTANEOUS ACETYLCHOLINE RELEASE FROM F R O G MOTOR N E R V E PETER ILLI~S * and FRANTI~EK VYSKO~IL
Department of Pharmacology, Semmelweis University of Medicine, Budapest, Hungary, and Institute of Physiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia Received 25 January 1978, accepted 27 January 1978
P. ILLES and F. VYSKOCIL, Calcium-dependent inhibition by prostaglandin E1 of spontaneous acetylcholine release from frog motor nerve, European J. Pharmacol. 48 (1978) 455--457. PGE1 (10 -7 M) in the absence of Ca 2÷ or at low external Ca 2÷ concentrations (0.2--0.5 mM) depressed the frequency of miniature end-plate potentials (m.e.p.p.s) in frog sartorius muscle, but m.e.p.p, amplitudes were unchanged. Both an increase in Ca 2÷ concentration to 2 mM or a blockade of Ca 2+ uptake into mitochondria by metabolic inhibitors (5 × 10 -s M 2,4-dinitrophenol or 2 × 10 -s M rotenone) prevented the inhibitory action of PGE1 on m.e.p.p, frequency. We suggest that PGEI may inhibit acetylcholine release from motor nerve terminals by promoting the active uptake of Ca 2+ by mitochondria, or by facilitating the efflux of Ca2+ from the axoplasm into the extracellular medium. Prostaglandin E1
Acetylcholine
Neuromuscular transmission
1. Introduction Prostaglandins of the E t y p e (PGEs) inhibit transmitter release from peripheral noradrenergic nerve endings in many organs (see Hedqvist, 1977). PGEs were, however, reported to be inactive on cholinergic transmission in the frog neuromuscular preparation (Ginsborg and Hirst, 1971) and also in the rat diaphragm at stimulation frequencies up to 30 Hz (Gripenberg et al., 1976). Since the prejunctional inhibitory action of PGEs on noradrenaline release was found to be inversely related to the a m o u n t of calcium in the perfusing medium (Hedqvist, 1970; S t j ~ n e , 1973), we have studied the effect of prostaglandin E1 (PGEI) on the spontaneous quantal release of transmitter from cholinergic m o t o r nerves at different calcium concentra* Correspondence to P.I. at his present address: Department of Pharmacology, University of Lund, SSlvegatan 10, S-223 62 Lund, Sweden.
Calcium
tions and in the presence of metabolic inhibitots.
2. Materials and methods Isolated frog sartorius muscles from Rana temporaria were used in the summer (June to July). The muscles were immersed in Ringer solution of composition (mM): NaC1 115; KCI 2.5; CaC12 0--2 and NaHCO3 2.2; the bath temperature was 22--25°C. In the absence of Ca 2÷, ethylene glycol-bis(~-aminoethyl ether)-N,N'-tetra-acetic acid (EGTA, 10 -3 M) was present and t e t r o d o t o x i n (10 -6 M) was added to prevent spontaneous muscle twitching. Prostigmine methylsulphate (6 × 10 -6 M) was added to the bath in all cases except at a Ca 2÷ concentration of 0.5 mM, in order to increase miniature end-plate potential (m.e.p.p.) amplitude. The spontaneous quantal release of acetylcholine was estimated from the frequency of
456
P. ILLt~S, F. VYSKOCIL
m.e.p.p.s, which were recorded intracellularly by t h e conventional microelectrode technique fr o m the end-plate region of the sartorius muscle. At least 100 m.e.p.p.s were recorded on film f r o m the oscilloscope screen and used in each e x p e r i m e n t for statistical estimates o f the mean amplitude. F r e q u e n c y o f m.e.p.p.s was determined by counting at 50 potentials from each muscle fibre before and after application o f PGE,. P G E , was used at a c o n c e n t r a t i o n of 10 -7 M, and its effect was followed for 15--20 min after addition t o th e muscle bath. T he values are q u o t e d as mean + S.E.M. The statistical significance o f th e data was checked by means o f the Student's t-test.
PGE, decreased the frequency of m.e.p.p.s to 55--65% of t he cont rol value, when no or little Ca 2+ (up to 0.5 mM) was present in the Ringer solution. No effect was found in solutions with 2 mM calcium. In anot her series o f experiments muscles were first soaked in a solution with low calcium (0.2 mM) also containing 5 X 10 -s M 2,4-dinitrophenol (DNP) or 2 × 10 -s M r o t e n o n e , in order to inhibit the active uptake of Ca 2+ into mitochondria. PGE, (10 -7 M) was t hen applied for 15--20 min. In each case, no change o f m.e.p.p, frequency was observed (table 1) in the seven muscle fibres in which continous measurements were made before and after PGE,.
3. Results
4. Discussion
PGE1 (10 -7 M) had no effect on the resting polarisation o f muscle fibres at any of the Ca 2+ concentrations tested. Similarly the drug failed to affect th e mean amplitude of the m.e.p.p.s (0.21 ± 0 . 0 2 m V in control and 0.21 + 0.02 mV after PGE,, 7 fibres, 0.5 mM
The major finding from this study is that PGE, (10 -7 M) in t he absence of Ca 2+ or at low external Ca 2+ c o n c e n t r a t i o n depressed the m.e.p.p, f r e q u e n c y in the sartorius muscle of the frog. In accordance with the results obtained by Ginsborg and Hirst (1971) we observed no effect of PGE, when the concentration of calcium was higher (2 mM). At the same time m.e.p.p, amplitudes were unchanged, an indication t hat PGE, does not influence the reactivity o f t he postsynaptic
Ca2+).
The e f f e c t o f PGE1 o n t h e f r e q u e n c y o f s p o n t a n e o u s t r a n s m i t t e r release was investigated at d i f f e r e n t c o n c e n t r a t i o n s o f external calcium. The results are summarized in table 1.
TABLE i Inhibition by PGE I (10 -7 M) of m.e.p.p, frequency in frog sartorius muscle as a function of calcium concentration (mean + S.E.M.).
Ca2+ in bathing fluid
0 2 5 2 2 2
+ EGTA 10 -3 M x 10 -4 M x 10 -4 M x 10 -3 M x 10 -4 M + DNP 5 x 10 -s M x 10 -4 M+rotenone2× 10 -s M
1 p < 0.05. 2 p < 0.001.
Number of experiments 5 5 7 4 4 3
Frequency of m.e.p.p.s (Hz) Control
PGE l 10 -7 M
0.92 3.37 2.71 9.13 7.48 11.2
0.51 1.97 1.71 8.67 7.80 10.3
+_0.13 + 0.43 _+0.45 _+1.92 + 2.44 _+2.28
+_0.09 1 _+0.19 l +_0.31 2 +_1.79 +_1.40 -+3.68
PGE 1 ON SPONTANEOUS RELEASE OF ACETYLCHOLINE
membrane to the transmitter. In peripheral tissues PGEs depress the stimulated release of noradrenaline from sympathetic nel~ce terminals b y regulating the availability of calcium for the secretory mechanism (Hedqvist, 1970). In contrast with these findings, it was reported that PGE~ only slightly reduced the amplitude of the endplate potential and its q u a n t u m content in the frog nerve-sartorius preparation, even at the low extracellular calcium concentration of 0.5 mM (Ill~s et al., 1978). It therefore seems that PGE1 has only a small, if any, effect on the influx of calcium during stimulation. According to our results, PGE~ may decrease m.e.p.p, frequency by reducing the concentration of free Ca 2÷ in the axoplasm of nerve terminals, which in turn regulates spontaneous transmitter release. The free intracellular Ca 2÷ concentration depends on a balance of calcium influx and efflux, and on intracellular calcium buffers such as mitochondria (Alnaes and Rahamimoff, 1975). PGE~ has been also reported to stimulate active sodium extrusion (Limos and Cohn, 1974) which in the nerve terminal could lead to membrane polarisation and thereby to a decrease in m.e.p.p. frequency. However, this prostaglandin is not likely to affect the resting Ca 2÷ influx, since its inhibitory effect persisted even in the absence of external calcium. Metabolic inhibitors (e.g. DNP and rotenone) inhibit the active uptake of Ca 2÷ into mitochondria (Lehninger, 1970). Pret r e a t m e n t of the preparation with these substances prevented the inhibitory action of PGE1 on spontaneous transmitter release. It seems possible that PGE~ Could affect spontaneous transmitter release from nerve terminals by promoting the active uptake of Ca 2÷ by mitochondria. This hypothesis is supported by the reported ability of PGE~ to act as a Ca 2÷ ionophore in the mitochondrial membranes o f the rat liver (Kirtland and Baum, 1972). A similar action in the mitochondria of m o t o r nerve endings could
457
promote the uptake of free calcium and in turn decrease m.e.p.p, frequency. PGE1 may also facilitate the efflux of calcium from the axoplasm across the nerve terminal under the influence of the electrochemical gradient existing during exposure to low Ca 2÷ media. A higher extra- or intracellular concentration of Ca 2÷, the latter caused by metabolic inhibitors, may counteract this effect of PGE1.
Acknowledgement We are grateful to Professor C. Edwards for this valuable criticism of the manuscript.
References Alnaes, E. and R. Rahamimoff, 1975, On the role of mitochondria in transmitter release from motor nerve terminals, J. Physiol. (London) 248,285. Ginsborg, B.L. and G.D.S. Hirst, 1971, Prostaglandin E 1 and noradrenaline at the neuromuscular ]unction, Brit. J. Pharmacol. 42, 153. Gripenberg, J., S.E. Jansson, V. Hein~/nen, E. Heinonen, J. Hyv~rinen and E.M. T o l p p a n e n , 1976, Effect of prostaglandin E1 on neuromuscular transmission in the rat, Brit. J. Pharmacol. 5 7 , 3 8 7 . Hedqvist, P., 1970, Antagonism by calcium of the inhibitory action of prostaglandin E2 on sympathetic neurotransmission in the cat spleen, Acta Physiol. Scand. 80, 269. Hedqvist, P., 1977, Basic mechanisms of prostaglandin action on autonomic neurotransmission, Ann. Rev. Pharmacol. 17,259. Ill,s, P., L.G. Magazanik and J. Knoll, 1978, The effect of PGE 1 and PGF2~ on neuromuscular transmission in the frog sartorius muscle, Acta Physiol. Acad. Sci. Hung. (in press). Kirtland, S.J. and H. Baum, 1972, Prostaglandin E1 may act as a "calcium ionophore", Nature (New Biology) 236, 47. Lehninger, A.L., 1970, Mitochondria and calcium ion transport, Biochem. J. 1 1 9 , 1 2 9 . Limos, C.J. and J.N. Cohn, 1974, Stimulation of vascular smooth muscle Na+,K+-ATPase by vasodilators, Circulation Res. 3 5 , 6 0 1 . Stj~rne, L., 1973, Kinetics of secretion of sympathetic neurotransmitter as a function of external calcium; mechanism of inhibitory effect of prostaglandin E, Acta Physiol. Scand. 8 7 , 4 2 8 .