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Effects of adenosine on 45Ca uptake and [3H]acetylcholine release in synaptosomal preparation from guinea-pig ileum myenteric plexus
European Journal of Pharmacology, 113 (1985) 417-424 Elsevier
417
EFFECTS OF A D E N O S I N E ON 45Ca UPTAKE AND [3H]ACETYLCHOLINE RELEASE IN S Y N A P T O S O M A L PREPARATION F R O M GUINEA-PIG ILEUM MYENTERIC PLEXUS KAZUMASA SHINOZUKA *, TOSHIO MAEDA and EI1CHI HAYASHI Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, 2 - 2 - l Oshika, Shizuoka 422, Japan Received 21 March 1985, accepted 7 May 1985
K. SHINOZUKA, T. MAEDA and E. HAYASHI. Effects of adenosine on 45Ca uptake and [-~H]acetylcholine release in svnaptosornal preparation from guinea-pig ileum myenteric plexus, European J. Pharmacol. 113 (1985) 417-424. The effects of adenosine on acetylcholine (ACh) release and calcium uptake were examined in a synaptosomal fraction prepared from guinea-pig ileum myenteric plexus-longitudinal muscle. A high concentration of potassium (40 mM) and electrical pulses (ES: 10Hz) caused a marked increase in the output of [3H]ACh from [3H]choline-preloaded crude synaptosomes. This [3H]ACh output was calcium- and temperature-dependent. Adenosine reduced the high potassium-induced release significantly, and the electrically stimulated release completely. When the preparation was depolarized by high potassium or electrical pulses, the 45Ca uptake by synaptosomes was significantly enhanced. The uptake of 45Ca induced by high potassium was significantly reduced and that induced by electrical stimulation was completely abolished by adenosine. From these results, it may be suggested that adenosine inhibits neurotransmitter release by suppressing the presynaptic influx of calcium ion during depolarization of the cholinergic nerve terminals in guinea-pig ileum. Adenosine
ACh release
Calcium influx
Synaptosome
I. Introduction There is much evidence, obtained with various preparations, suggesting that adenosine and related compounds have a presynaptic inhibitory action. We have already reported that adenosine inhibited the electrically induced contractile response in guinea-pig ileum, and that the inhibition was due to depression of acetylcholine release from intramural cholinergic nerves (Hayashi et al., 1978 a, b; 1985). It is well-known that calcium ions play an important role in neurotransmitter-releasing mechanisms in nerve endings. We had examined the influence of the extracellular calcium ion on the inhibitory action of adenosine in the twitch response of electrically stimulated guinea-pig ileum and found that calcium ion antagonized the inhibi-
* To whom all correspondence should be addressed. 0014-2999/85/$03.30 '~ 1985 Elsevier Science Publishers B.V.
Guinea-pig ileum
tory action of adenosine in a competitive manner (Hayashi et al., 1981). From such findings, it was proposed that adenosine action may be closely associated with the calcium ion movement linked to excitation-releasing coupling. However, the mechanism of action of adenosine in the intramural cholinergic nerves of the guinea-pig ileum has not been sufficiently elucidated. Difficulties were encountered with the isolation of cholinergic nerve terminals from the ileal strips, and tissues other than neuronal elements interrupt the measurement of prejunctional calcium ion movement in the nerves. Briggs and Cooper (1981) have reported a procedure for the preparation of synaptosomes from the myenteric plexus of the guinea-pig ileum. They suggested that the preparation would be useful in studying the mechanisms of action of agents modulating the release of acetylcholine. In order to further characterize the action of adenosine, therefore we examined the effects of
418
adenosine on the release of acetylcholine and calcium ion influx in the myenteric plexus synaptosome preparation.
2. Materials and methods
2.1. Preparation of crude synaptosorne Crude synaptosomes from guinea-pig ileum myenteric plexus were prepared at 0 - 4 ° C as described by Briggs and Cooper (1981). Ileal longitudinal muscle strips were prepared as described by Paton and Zar (1968). The strips were weighed, minced and homogenized for 15 s at low speed in five volumes of 0.32 M sucrose containing 3 mM sodium phosphate buffer (pH 7.2) using a polytron (PT-20-350D). The tissue was further homogenized by 10 strokes of a Teflon-glass homogenizer (type 3, Omega Electric) at 900 rpm. The homogenate was centrifuged at 1000 × g for 10 min. The supernatant was saved and the pellet was rehomogenized with the Teflon-glass homogenizer as described above. After recentrifugation at 1000 × g, the two supernatants were combined and centrifuged at 17000 × g for 20 min. The pellet (P2-fraction) was gently resuspended in 3 ml of a normal buffer having the following composition (mM): 132 NaCI, 5 KC1, 1.2 CaC12, 1.3 MgC12, 1.2 NaH2PO 4, 10 glucose, 20 Tris base. This suspension was used in "experiments on ACh release as a crude synaptosomal preparation. The normal buffer solution was buffered to pH 7.4 (at 25°C) by titration with maleic acid and saturated with 95% 02 and 5% CO 2.
2.2. Preparation of synaptosome-rich fraction A synaptosome-rich preparation was obtained from the P2 fraction by differential centrifugation and sucrose density gradient centrifugation procedure described by Gray and Whittaker (1960, 1962). The P2 fraction was resuspended in 0.32 M sucrose solution (1.5 ml) by brief, low-speed Teflon-glass homogenization and the suspension layered on a discontinuous sucrose gradient. The gradients were centrifuged in a Hitachi 70P-72
rotor for 60 min at 100000 × g. The material at the 0.8-1.2 M sucrose interface was removed, diluted 5-fold with the normal buffer and centrifuged at 5000 × g for 20 rain in the Kubota KR20000T rotor. The resultant pellet was resuspended in 1 ml of the normal buffer and used as a purified synaptosomal preparation in 45Ca uptake experiments.
2.3. [SH]A Ch release The crude synaptosomal preparation was incubated with 1 #M [3H]choline (1/~Ci/ml) for 30 rain at 37°C under 95% 02 and 5% CO 2, after a preincubation period of 30 min. The [3H]AChloaded synaptosome preparation was returned to 0-4°C, followed by centrifugation at 5000 × g for 10 min. The pellet was resuspended and washed twice in fresh normal buffer containing 10 ~M physostigmine. After the final wash, the pellet was resuspended in 1.5 ml of physostigmine-containing normal buffer saturated with 95% 02 and 5% CO 2. Aliquots (100 ~1) of the suspension were added to chilled polyethylene tubes containing 400 ~1 normal or high K buffer (mM): 88.25 NaCI, 48.75 KC1, 1.2 CaCI> 1.3 MgC12, 1.2 NaH2PO 4, 10 glucose, 20 Tris base, with or without adenosine. The final buffer solution thus obtained 5 mM or 40 mM KCI. After gentle mixing, [3H]ACh release from the preparation was initiated by transferring the tubes to a 37°C bath. Some of the preparations in the normal buffer solution were electrically stimulated during the last 5 rain of a 10 rain incubation period. After 10 rain incubation, release was terminated by chilling the tubes in an ice bath. Supernatants following centrifugation of the tubes at 5000 × g for 10 min were collected for [) H]ACh determination.
2.4. Determination of [SH]A Ch Labeled ACh was separated from labeled choline by the method of Marchi et al. (1981, 1983). A 100 /xl sample of either supernatant was incubated with 10 mM Tris-HCl buffer (pH 8.5) 5 mM MgCI 2 1 mM ATP and 2.0 munit/ml choline kinase in a final volume of 200 /~1 at 37°C for 45 rain. The incubate was extracted with 200 t~l of 10
419 m g / m l tetraphenylboron in butyronitrile and the [3H]ACh in the organic phase was counted in an Aloka liquid scintillator and expressed as D P M / m g protein.
(1951) with fatty acid-free bovine serum albumin (Sigma) as the standard.
2.5. 45Ca uptake
A pellet of the synaptosome-rich fraction being studied was fixed in 2% glutalaldehyde and postfixed in 1% OsO 4, both in 0.2 M phosphate buffer, pH 7.4. The fixed tissue was dehydrated with ethanol and embedded in Epon. Thin sections were sliced on an ultra-microtome (LKB-8800), stained with uranylacetate and lead citrate and viewed under an electron microscope (JM-200CX).
The purified synaptosomal preparation was preincubated for 30 min at 37°C in order to return the synaptosomes to a more physiological environment. After cooling of the suspension in the ice bath, aliquots (100 ~1) were transferred to chilled polyethylene tubes containing 400 /xl normal or high K buffer with the appropriate addition and 45Ca. Thus the final buffer solution contained 5 mM or 40 mM KC1 and 1.2 mM CaC12 (45Ca: 1.0 /xCl/ml). The uptake of 45Ca by the synaptosomes was initiated by transferring the tubes to a 37°C bath and was stopped after 2 rain by adding 1 ml of ice-cold calcium-free buffer (raM): 132 NaCI, 5 KC1, 1.2 EGTA, 1.3 MgCl 2, 1.2 NaH2PO 4, 10 glucose, 20 Tris base. Electrical pulses were applied to some of the preparations in normal buffer solution during the last 30 or 60 s of a 2 min incubation period. The synaptosome suspension was immediately centrifuged at 10000 × g for 5 min. The resulting pellet was washed once with 1.0 ml of the calcium-free buffer, digested in protosol and counted in an Aloka liquid scintillator. Net 45Ca influx (zx) into synaptosomes was calculated by subtracting control values from depolarized values.
2.6. Electrical stimulation Electrical stimulation was by means of a stimulator (SEN-3201) constructed by Nihon Kohden, applied to platinum electrodes forming a curved surface in order to fit a polyethylene tube. Square-wave pulses of 20 V were used, providing a gradient of 4.0 V / m m distance between the parallel electrodes. The frequency was 10 pulses/s and the duration was 0.4 ms.
2. 7 Protein assay Synaptosomal protein concentrations were determined according to the method of Lowry et al.
2.8. Electron microscopy
2.9, Compounds Adenosine, adenosine-5'-triphosphate and choline kinase were purchased from Sigma. The other reagents were from the following sources: butyronitrate (Wako), ethylene glycol-bis[Baminoethyl ether]N,N'-tetracetic acid; E G T A (Wako), physostigmine sulfate (Merck), tetraphenylboron (Aldrich). 45CaClz, choline chloride [methyl-3H] and protosol were obtained from New England Nuclear, Boston, U.S.A.. Adenosine was dissolved in appropriate buffer solutions and the concentrations were expressed as /xM of the final concentration.
3. Results
3.1. Effects of adenosine on release of [3H]A Ch The high concentration of potassium (40 mM) produced an increase in the output of [3H]ACh from the crude synaptosomal preparation of the guinea-pig ileum myenteric plexus (table 1), The [3H]ACh release caused by the high concentration of potassium was significantly reduced in a calcium-free buffer solution containing 1.2 mM EGTA. Further, the potassium-induced release was markedly reduced at 4°C (data not shown). Adenosine at a concentration of 100 ~M significantly reduced the potassium-induced release of [3H]ACh by 34.4%. On the other hand, the spontaneous release of [3H]ACh was unaffected by adenosine. This release was unchanged in the
420 TABLE 1 Effect of adenosine on the high potassium-induced release of [3H]acetylcholine from crude synaptosomal preparations. The P, fraction of the guinea-pig ileum myenteric plexus was used for the release experiment. Condition
N ~'
[3H]ACh output b ( d p m / m g protein)
% of control ~ output
p d
Control 40 mM K 40 mM K + C a - f r e e ~
4 3 3
3365.9 ± 249.0 4974.3± 90.3 4212.7_+ 58.3
100.0 147.8 125.2
< 0.01 < 0.01 *
Control Adenosine f 40 mM K 40 mM K + adenosine
5 6 6 6
3584.5 ± 3468.5± 5586.4± 4895.5 +
159.1 89.5 127.7 147.5
100.0 96.8 155.8 136.6
NS < 0.01 < 0.01 *
Control Ca free Ice-cold g
4 4 4
3312.2 + 83.9 3328.3 ± 195.0 2535.3± 83.3
100.0 100.5 76.6
NS < 0.05
~' Number of experiments, b Data are means ± S.E.M. c Values are expressed as percentages of the control release of [3H]ACh. d Significantly different from control, Student's t-test or Cochran-Cox test. NS: not significant. * Represents a significant difference from 40 mM K condition, using a paired t-test, e The buffer with no Ca 2+ contained 1.2 mM EGTA. f 100 ~tM concentration. g Examined at 0-4°C.
calcium-free medium with 1.2 mM EGTA, but was markedly reduced when the assay temperature was lowered to 4°C. When 10 Hz electrical pulses were applied to the crude synaptosomal preparation, there was a marked increase in [3HIACh output but not with 0.1 Hz stimulation (table 2). The electrically stimulated release of [3H]ACh was abolished in calcium-free medium containing 1.2
mM EGTA or at 4°C assay temperature. Adenosine at 100 /xM completely abolished [3H]ACh release.
3.2. Effects of adenosine on 4~Ca uptake l~v synaptosomes A synaptosome-rich preparation was isolated from the P2 fraction in order to examine the 45Ca
TABLE 2 Effect of adenosine on the electrical stimulationqnduced release of [3 H]acetylcholine from crude synaptosomal preparations. The P~ fraction of the guinea-pig ileum myenteric plexus was used for the release experiments. Condition
N ~
[3H]ACh output b ( d p m / m g protein)
% of control c output
Control Adenosine e 0.1 Hz ES f 0.1 Hz ES + adenosine 10 Hz ES 10 Hz ES + adenosine 10 Hz ES, ice-cold g 10 Hz ES + Ca-free h
6 6 6 6 6 3 3 3
3351.7 ± 3205.9± 3543.1 ± 3487.4 ± 4692.3 ± 3650.7 ± 3035.6± 3005.5 ±
100.0 96.8 105.7 104.0 140.0 108.9 90.6 89.7
122.3 89.5 62.0 210.9 240.1 242.7 39.0 232.5
pd
NS NS < < < <
0.001 0.05 * 0.01 * 0.01 *
~' Numb er of experiments, b Data are means ± S.E.M. c Values are expressed as percentages of the control release of [3H]ACh. d Significantly different from control, Student's t-test or Cochran-Cox test. NS: not significant. * Represents a significant difference from 10 Hz ES condition, Student's t-test or Cochran-Cox test. e 1 0 0 / i M concentration, f Electrical pulses (0.1 or 10 Hz, 0.4 ms, 20 V amplitude) applied during the last 5 rain of a 10 min period of incubation, g Examined at 0-4°C. h The buffer with no Ca 2+ contained 1.2 mM EGTA.
421 TABLE 3 Effect of adenosine on high potassium-stimulated 45Ca uptake by the synaptosome-rich preparation. Preparation obtained by sucrose density gradient centrifugation procedures from guinea-pig ileum myenteric plexus-longitudinal muscle homogenates. Condition
N ~
45Ca uptake b ( n m o l / m g protein)
/~ ¢
p a
Control Adenosine e
9 9
4.08_+0.14 4.26 -+ 0.20
-
NS
Control 40 mM K 40 mM K + adenosine
7 7
4.04+_0.14 5.96 _+0.35
1.92
< 0.01
7
4.97_+0.36
0.93
< 0.001 *
" Number of experiments, b Data are means + S.E.M. c Values were calculated as the difference between high K + and control data and represent the net synaptosomal 45Ca uptake in response to depolarization, d Significance of difference from control obtained by paired t-test. NS: not significant. * Represents a significant difference from 40 mM K + condition using a paired t-test, e 100 ~M concentration.
uptake. Under control conditions, the basal uptake o f 45Ca b y t h i s p r e p a r a t i o n w a s m a x i m a l 2 m i n after starting incubation
w i t h 45Ca a n d r e m a i n e d
at almost the same level for the next 3 rain. In the presence of a high concentration of potassium, synaptosomal u p t a k e o f 4SCa w a s g r e a t l y creased
(table
3). A d e n o s i n e
the in-
a t 1 0 0 I~M s i g n i f i -
Fig. 1, Electron microscopic photographs of synaptosome-rich preparation of the guinea-pig ileum myenteric plexus (upper picture 40000×, lower picture 50000×). Synaptosomes are distinguishable as thin-walled bags and small mitochondria and synaptic vesicles are visible in these bags.
TABLE 4 Effect of adenosine on electrically stimulated 45Ca uptake by the synaptosome-rich preparation. Preparation obtained by sucrose density gradient centrifugation procedures from guinea-pig ileum myenteric plexus-longitudinal muscle homogenate. Condition
N a
45Ca uptake b ( n m o l / m g protein)
Control Adenosine e
6 6
4.13 _ 0.14 4.28 _+0.20
Control 10 Hz ES f (30 s) 10 Hz ES + adenosine
7 7 7
4.11 _+0.31 4.27_+0.29 4.10 _+0.23
-
Control 10 Hz ES (60 s) 10 Hz ES + adenosine
7 7 7
3.93 _+0.17 4.94_+0.25 3.86 _+0,35
- 0.07
A c
p d
NS
0.01
NS NS
1.01
<
0.16
*
0.01 < 0.05 *
a Number of experiments, b Data are means + S.E.M. c Values were calculated as the difference between ES and control data and represent the net synaptosomal 45Ca uptake in response to electrical stimulation, d Signi.ficance of difference from control obtained by Student's t-test or Cochran-Cox test. NS: not significant. * Represents a significant difference from ES condition, Student's t-test or Cochran-Cox test., e 100 # M concentration, g Electrical pulses (10 Hz, 0.4 ms, 20 V amplitude) applied during the last 30 or 60 s of a 2 rain period of incubation.
422 cantly reduced (by 58.6%) the uptake of 45Ca induced by 40 mM potassium. Electrical pulses at 10 Hz for 60 s greatly increased the synaptosomal 45Ca uptake (table 4). Adenosine completely abolished the uptake. The basal uptake of 45Ca was not affected by adenosine at all (table 3, 4). 3. 3. Electron microscopy Fig. 1 shows typical pictures of the synaptosome-rich preparation of ileal myenteric plexus. Many synaptosomes were distinguishable as thinwalled bags, and most of them contained several small mitochondria and a large number of synaptic vesicles. Most of the vesicles were small, round agranular vesicles about 30-50 nm in diameter and among the others were some granular vesicles about 30-60 nm. It was, however, obvious that the fraction was contaminated with membranes from undefined sources. Free mitochondria were only rarely seen.
4. Discussion
Electron microscopic photographs of a pelleted, enriched synaptosomal fractionof the guinea-pig ileum myenteric plexus showed synaptosomes as thin-walled bags. Most synaptosomes contained a large number of small, round agranular vesicles about 30-50 nm in diameter and a few granular vesicles about 30-60 nm in diameter, contrary to Briggs and Cooper's report (1981) that the synaptosomes contained large (90-140 nm) vesicles with an electron-opaque core and an absent or ill-defined 'halo'. Cook and Burnstock (1976) have examined the ultrastructural features of neurons within the myenteric plexus in the ileum of guineapig and identified eight morphologically distinct types of axon profile on the basis of vesicular size, shape and content. Two of them, type 2 and 3 as described by Cook and Burnstock contained many small, round agranular vesicles. Generally, axons which are known to be cholinergic, such as axons at the motor end-plate, preganglionic axons in sympathetic ganglia and axons supplying the adrenal medulla, contain mainly small round agranular vesicles. Furness and Costa (1980) sug-
gested that the axons with the numerous small round agranular vesicles may be cholinergic nerves in the intestine. Such axons have a distribution consistent with this suggestion. They are very numerous, form synapses in the enteric plexuses and also supply the plexuses and the smooth muscle (Gabella, 1972a, b). The electron microscopic profile of synaptosomes obtained in our experiments was similar to such an axon profile, suggesting that the synaptosomes may originate in the cholinergic nerve varicosities. In the present study, high potassium and electrical square-wave pulses were used as stimulants of excitation-release coupling of the ileal synaptosomes. A high concentration of potassium caused an increase in [3H]ACh output from the crude synaptosomal preparation and the release was both calcium- and temperature-dependent. Electrical pulses (10 Hz) to the crude synaptosomal preparation also caused a release of [3H]ACh which was calcium- and temperature-dependent. Osborne et al. (1973) had reported that glycine was released by electrical stimulation from spinal cord synaptosomes and that the release which was accompanied by the release of other putative amino acid transmitters was calcium-dependent. It is estimated that the [~H]ACh released from the ileal synaptosomes originated in the cholinergic nerve terminals. Low frequency electrical stimulation (0.1 Hz) could not have caused the release of [3H]ACh from the crude synaptosomes. It is difficult to explain why low frequency stimulation did not increase the [3H]ACh output. As a possibility, it is assumed that the excitability of the synaptosomes may be relatively low and electrical pulses at 0.1 Hz may be insufficient to trigger depolarization of the membrane. Ikushima et al. (1981) suggested that electrical stimulation with a short pulse duration might excite the nerve fibers, but that the longer pulse might excite the presynaptic terminals in addition to the nerve fibers. Possibly, more intense conditions e.g. high frequency and longer pulses might be required to generate the excitation of nerve terminals by electrical stimulation. In this study, adenosine inhibited the high potassium-induced [3 H]ACh release from the crude synaptosomes significantly. The electrically in-
423 duced [3H]ACh release was also markedly depressed by adenosine at the same concentration and the depression was more intense than that in the case of high potassium stimulation. Reese and Cooper (1982) suggested that the 50 mM potassium-induced [3H]ACh release was relatively insensitive to adenosine as compared with nicotinically induced release. The ACh releasing mechanism activated by electrical stimulation and nicotinic stimulation seemed to be more susceptible to the inhibitory action of adenosine than was the mechanism activated by high potassium. Generally, it is recognized that extracellular calcium ions flowing in the nerve terminals play an important role in the neurotransmitter releasing mechanisms. Previously, we reported that external calcium ions antagonized the inhibitory effect of adenosine in a competitive manner, suggesting that the action of adenosine might be closely associated with the extracellular calcium movement in the nerve terminals (Hayashi et al., 1981). As the present results showed, adenosine inhibited significantly the high potassium-induced 45Ca uptake by the synaptosome-rich preparations, and completely abolished the electrically stimulated uptake. The increase of 45Ca uptake induced by high potassium or electrical pulses was temperature-dependent (data not shown). Probably, such 45Ca uptake may reflect the calcium ion influx through the voltage-dependent calcium channel activated by membrane depolarization, and adenosine will be able to inhibit such an influx of calcium ion into the synaptosomes. From these results, it is suggested that the inhibitory effect of adenosine on the ACh releasing system may result from inhibition of calcium influx. Reese and Cooper (1982) reported that adenosine reduced the basal release of [3H]ACh from ileal synaptosomes, but in the present study the spontaneous [3H]ACh release was not affected by adenosine. Further, the basal 45Ca uptake was also independent of adenosine. Alnaes and Rahamimoff (1975) suggested that the spontaneous release of the transmitter depended on the resting level of intracellular calcium ion which was regulated for the greater part by the mitochondria or by nonmitochondrial structures. Therefore, it is proposed that adenosine could not have an influence on the
intracellular calcium ion movement in unstimulated synaptosomes, and might affect only the extracellular calcium ion influx triggered by membrane excitation. Ribeiro et al. (1975) demonstrated that in rat brain synasptosomes, adenosine decreased the uptake of 45Ca stimulated by potassium. Also, Kuroda (1983) reported that the high potassiumdependent calcium accumulation was decreased by 2-chloroadenosine in synaptosomes from guineapig cerebral cortex. These inhibition rates for the potassium-stimulated calcium ion uptake were almost similar to our data, and they were relatively small as compared to the value obtained with electrical stimulation in our study. The reason for this insensitivity is unknown, but two possible explanations should be considered: (1) the ability of potassium depolarization to stimulate the calcium ion influx may be intense, thus overcoming the inhibitory action of adenosine; (2) potassium-induced depolarization may stimulate another mechanism of calcium entry in addition to the adenosine-sensitive calcium influx mechanism, which is independent of adenosine. Although it is generally accepted that potassium at a high concentration produces membrane depolarization by potassium cation influx, the development of such depolarization seems to be essentially different from the electrically induced excitation, and from the physiological excitatory response. In an intact ileal myenteric plexus-longitudinal muscle preparation, adenosine at 100 /~M depressed the electrically (10 Hz) induced cholinergic contractile response completely (Hayashi et al., 1985). In the present study, adenosine at the same concentration also completely inhibited the calcium influx elicited by electrical stimulation at 10 Hz. These results suggested that the calcium channel coupled with the depolarization of presynaptic membrane induced by electrical stimulation may be sensitive to adenosine. Furthermore, it is proposed that the adenosine receptor may be linked to such a channel directly or indirectly, and may regulate ACh release in the nerve terminals of guinea-pig ileum physiologically, in agreement with our previous suggestions (Hayashi et al., 1978a, 1985).
424
Acknowledgements The authors are deeply indebted to Prof. M. Sano and Dr. A. lshii of Hamamastu University School of Medicine for their considerable assistance in performing the electron microscopy.
References Alnaes, E. and R. RahamimofL 1975, On the role of mitochondria in transmitter release from motor nerve terminals, J. Physiol. 248, 285. Briggs, C.A. and J.R. Cooper, 1981, A synaptosomal preparation from the guinea pig ileum myenteric plexus, J. Neurochem. 36, 1097. Cook, R.D. and G. Burnstock, 1976, The ultrastructure of Auerbach's plexus in the guinea-pig. I. Neuronal elements, J. Neurocytol. 5, 171. Furness, J.B. and M. Costa, 1980, Types of nerves in the enteric nervous system, Neuroscience 5, 1. Gabella, G., 1972a, Fine structure of the myenteric plexus in the guinea pig ileum, J. Anat. 111, 69. Gabella, G., 1972b, Innervation of the intestinal muscular coat, J. Neurocytol. 1, 341. Gray, E.G. and V.P. Whittaker, 1960. The isolation of synaptic vesicles from the central nervous system, J. Physiol. 153, 35. Gray, E.G. and V.P. Whittaker, 1962, The isolation of nerve endings from brain: An electron-microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat. 96, 79. Hayashi, E., M. Kunitomo, M. Mori, K. Shinozuka and S. Yamada, 1978a, The development of tachyphylaxis to electrical stimulation in guinea pig ileal longitudinal muscle and the possible participation of adenosine and adenine nucleotides, Br. J. Pharmacol. 63, 457. Hayashi, E., T. Maeda and K. Shinozuka, 1985, Adenosine and dipyridamole: actions and interactions on the contractile response of guinea-pig ileum to high frequency electrical field stimulation, Br. J. Pharmacol. (in press)
Hayashi, E., M. Mori, S. Yamada and M. Kunitomo, 1978b, Effects of purine compounds on cholinergic nerves. Specificity of adenosine and related compounds on acetylcholine release in electrically stimulated guinea pig ileum, European J. Pharmacol, 48, 297. Hayashi, E., S. Yamada and K. Shinozuka, 1981, The influence of extracellular Ca 2 ~ concentration on the inhibitory effect of adenosine in guinea-pig ileal longitudinal muscles, Jap. J. Pharmacoh 31. 141. lkushima, S., 1. Muramatsu and M. Fujiwara, 1981, Effects c~f 4-aminopyridine on the adrenergic nerve terminals of rabbit arteries, J. Pharmacol. Exp. Ther. 219, 792. Kuroda, Y., 1983, Neuronal plasticity and adenosine derivatives in mammalian brain, in: Physiology and pharmacology of adenosine derivatives, eds. J.W. Daly, Y. Kuroda, J.W. Phyllis and M. Ui, Raven Press, New York, p. 245. Lowry, O.H., N.J. Rosenbrough, A.E. Farr and R.J. Randall. 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193. 265. Marchi, M., D.W. Hoffman, E. Giacobini and R. Volle, 1981, Development and aging of cholinergic synapses: V1 Mechani,,ms of acetylcholine biosynthesis in the chick iris. Dev. Neurosci. 4, 442. Marchi, M., P. Paudice, A. Cavigila and M. Raiteri, 1983, Is acetylcholine release from striatal nerve endings regulated by muscarinic autoreceptors? European J. Pharmacol. 91, 63. Osborne, R.H., H.F. Bradford and D.G. Jones, 1973, Patterns of amino acid release from nerve-endings isolated from spinal cord and medulla, J. Neurochem. 21,407. Paton, W.D.M. and M.A. Zar, 1968, The origin of acetylcholine release from guinea-pig intestine and longitudinal muscle strips, J. Physiol. 194, 13. Reese, J.H. and J.R. Cooper, 1982, Modulation of release of acetylcholine from ileal synaptosomes by adenosine and adenosine 5'-triphosphate, J. Pharmacol. Exp. Ther. 223, 612. Ribeiro, J.A., A,M. Sa-Alneida and J.M. Namorado, 1979, Adenosine and adenosine triphosphate decrease 45Ca uptake by synaptosomes stimulated by potassium, Biochem, Pharmacol. 28, 1297.
417
EFFECTS OF A D E N O S I N E ON 45Ca UPTAKE AND [3H]ACETYLCHOLINE RELEASE IN S Y N A P T O S O M A L PREPARATION F R O M GUINEA-PIG ILEUM MYENTERIC PLEXUS KAZUMASA SHINOZUKA *, TOSHIO MAEDA and EI1CHI HAYASHI Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, 2 - 2 - l Oshika, Shizuoka 422, Japan Received 21 March 1985, accepted 7 May 1985
K. SHINOZUKA, T. MAEDA and E. HAYASHI. Effects of adenosine on 45Ca uptake and [-~H]acetylcholine release in svnaptosornal preparation from guinea-pig ileum myenteric plexus, European J. Pharmacol. 113 (1985) 417-424. The effects of adenosine on acetylcholine (ACh) release and calcium uptake were examined in a synaptosomal fraction prepared from guinea-pig ileum myenteric plexus-longitudinal muscle. A high concentration of potassium (40 mM) and electrical pulses (ES: 10Hz) caused a marked increase in the output of [3H]ACh from [3H]choline-preloaded crude synaptosomes. This [3H]ACh output was calcium- and temperature-dependent. Adenosine reduced the high potassium-induced release significantly, and the electrically stimulated release completely. When the preparation was depolarized by high potassium or electrical pulses, the 45Ca uptake by synaptosomes was significantly enhanced. The uptake of 45Ca induced by high potassium was significantly reduced and that induced by electrical stimulation was completely abolished by adenosine. From these results, it may be suggested that adenosine inhibits neurotransmitter release by suppressing the presynaptic influx of calcium ion during depolarization of the cholinergic nerve terminals in guinea-pig ileum. Adenosine
ACh release
Calcium influx
Synaptosome
I. Introduction There is much evidence, obtained with various preparations, suggesting that adenosine and related compounds have a presynaptic inhibitory action. We have already reported that adenosine inhibited the electrically induced contractile response in guinea-pig ileum, and that the inhibition was due to depression of acetylcholine release from intramural cholinergic nerves (Hayashi et al., 1978 a, b; 1985). It is well-known that calcium ions play an important role in neurotransmitter-releasing mechanisms in nerve endings. We had examined the influence of the extracellular calcium ion on the inhibitory action of adenosine in the twitch response of electrically stimulated guinea-pig ileum and found that calcium ion antagonized the inhibi-
* To whom all correspondence should be addressed. 0014-2999/85/$03.30 '~ 1985 Elsevier Science Publishers B.V.
Guinea-pig ileum
tory action of adenosine in a competitive manner (Hayashi et al., 1981). From such findings, it was proposed that adenosine action may be closely associated with the calcium ion movement linked to excitation-releasing coupling. However, the mechanism of action of adenosine in the intramural cholinergic nerves of the guinea-pig ileum has not been sufficiently elucidated. Difficulties were encountered with the isolation of cholinergic nerve terminals from the ileal strips, and tissues other than neuronal elements interrupt the measurement of prejunctional calcium ion movement in the nerves. Briggs and Cooper (1981) have reported a procedure for the preparation of synaptosomes from the myenteric plexus of the guinea-pig ileum. They suggested that the preparation would be useful in studying the mechanisms of action of agents modulating the release of acetylcholine. In order to further characterize the action of adenosine, therefore we examined the effects of
418
adenosine on the release of acetylcholine and calcium ion influx in the myenteric plexus synaptosome preparation.
2. Materials and methods
2.1. Preparation of crude synaptosorne Crude synaptosomes from guinea-pig ileum myenteric plexus were prepared at 0 - 4 ° C as described by Briggs and Cooper (1981). Ileal longitudinal muscle strips were prepared as described by Paton and Zar (1968). The strips were weighed, minced and homogenized for 15 s at low speed in five volumes of 0.32 M sucrose containing 3 mM sodium phosphate buffer (pH 7.2) using a polytron (PT-20-350D). The tissue was further homogenized by 10 strokes of a Teflon-glass homogenizer (type 3, Omega Electric) at 900 rpm. The homogenate was centrifuged at 1000 × g for 10 min. The supernatant was saved and the pellet was rehomogenized with the Teflon-glass homogenizer as described above. After recentrifugation at 1000 × g, the two supernatants were combined and centrifuged at 17000 × g for 20 min. The pellet (P2-fraction) was gently resuspended in 3 ml of a normal buffer having the following composition (mM): 132 NaCI, 5 KC1, 1.2 CaC12, 1.3 MgC12, 1.2 NaH2PO 4, 10 glucose, 20 Tris base. This suspension was used in "experiments on ACh release as a crude synaptosomal preparation. The normal buffer solution was buffered to pH 7.4 (at 25°C) by titration with maleic acid and saturated with 95% 02 and 5% CO 2.
2.2. Preparation of synaptosome-rich fraction A synaptosome-rich preparation was obtained from the P2 fraction by differential centrifugation and sucrose density gradient centrifugation procedure described by Gray and Whittaker (1960, 1962). The P2 fraction was resuspended in 0.32 M sucrose solution (1.5 ml) by brief, low-speed Teflon-glass homogenization and the suspension layered on a discontinuous sucrose gradient. The gradients were centrifuged in a Hitachi 70P-72
rotor for 60 min at 100000 × g. The material at the 0.8-1.2 M sucrose interface was removed, diluted 5-fold with the normal buffer and centrifuged at 5000 × g for 20 rain in the Kubota KR20000T rotor. The resultant pellet was resuspended in 1 ml of the normal buffer and used as a purified synaptosomal preparation in 45Ca uptake experiments.
2.3. [SH]A Ch release The crude synaptosomal preparation was incubated with 1 #M [3H]choline (1/~Ci/ml) for 30 rain at 37°C under 95% 02 and 5% CO 2, after a preincubation period of 30 min. The [3H]AChloaded synaptosome preparation was returned to 0-4°C, followed by centrifugation at 5000 × g for 10 min. The pellet was resuspended and washed twice in fresh normal buffer containing 10 ~M physostigmine. After the final wash, the pellet was resuspended in 1.5 ml of physostigmine-containing normal buffer saturated with 95% 02 and 5% CO 2. Aliquots (100 ~1) of the suspension were added to chilled polyethylene tubes containing 400 ~1 normal or high K buffer (mM): 88.25 NaCI, 48.75 KC1, 1.2 CaCI> 1.3 MgC12, 1.2 NaH2PO 4, 10 glucose, 20 Tris base, with or without adenosine. The final buffer solution thus obtained 5 mM or 40 mM KCI. After gentle mixing, [3H]ACh release from the preparation was initiated by transferring the tubes to a 37°C bath. Some of the preparations in the normal buffer solution were electrically stimulated during the last 5 rain of a 10 rain incubation period. After 10 rain incubation, release was terminated by chilling the tubes in an ice bath. Supernatants following centrifugation of the tubes at 5000 × g for 10 min were collected for [) H]ACh determination.
2.4. Determination of [SH]A Ch Labeled ACh was separated from labeled choline by the method of Marchi et al. (1981, 1983). A 100 /xl sample of either supernatant was incubated with 10 mM Tris-HCl buffer (pH 8.5) 5 mM MgCI 2 1 mM ATP and 2.0 munit/ml choline kinase in a final volume of 200 /~1 at 37°C for 45 rain. The incubate was extracted with 200 t~l of 10
419 m g / m l tetraphenylboron in butyronitrile and the [3H]ACh in the organic phase was counted in an Aloka liquid scintillator and expressed as D P M / m g protein.
(1951) with fatty acid-free bovine serum albumin (Sigma) as the standard.
2.5. 45Ca uptake
A pellet of the synaptosome-rich fraction being studied was fixed in 2% glutalaldehyde and postfixed in 1% OsO 4, both in 0.2 M phosphate buffer, pH 7.4. The fixed tissue was dehydrated with ethanol and embedded in Epon. Thin sections were sliced on an ultra-microtome (LKB-8800), stained with uranylacetate and lead citrate and viewed under an electron microscope (JM-200CX).
The purified synaptosomal preparation was preincubated for 30 min at 37°C in order to return the synaptosomes to a more physiological environment. After cooling of the suspension in the ice bath, aliquots (100 ~1) were transferred to chilled polyethylene tubes containing 400 /xl normal or high K buffer with the appropriate addition and 45Ca. Thus the final buffer solution contained 5 mM or 40 mM KC1 and 1.2 mM CaC12 (45Ca: 1.0 /xCl/ml). The uptake of 45Ca by the synaptosomes was initiated by transferring the tubes to a 37°C bath and was stopped after 2 rain by adding 1 ml of ice-cold calcium-free buffer (raM): 132 NaCI, 5 KC1, 1.2 EGTA, 1.3 MgCl 2, 1.2 NaH2PO 4, 10 glucose, 20 Tris base. Electrical pulses were applied to some of the preparations in normal buffer solution during the last 30 or 60 s of a 2 min incubation period. The synaptosome suspension was immediately centrifuged at 10000 × g for 5 min. The resulting pellet was washed once with 1.0 ml of the calcium-free buffer, digested in protosol and counted in an Aloka liquid scintillator. Net 45Ca influx (zx) into synaptosomes was calculated by subtracting control values from depolarized values.
2.6. Electrical stimulation Electrical stimulation was by means of a stimulator (SEN-3201) constructed by Nihon Kohden, applied to platinum electrodes forming a curved surface in order to fit a polyethylene tube. Square-wave pulses of 20 V were used, providing a gradient of 4.0 V / m m distance between the parallel electrodes. The frequency was 10 pulses/s and the duration was 0.4 ms.
2. 7 Protein assay Synaptosomal protein concentrations were determined according to the method of Lowry et al.
2.8. Electron microscopy
2.9, Compounds Adenosine, adenosine-5'-triphosphate and choline kinase were purchased from Sigma. The other reagents were from the following sources: butyronitrate (Wako), ethylene glycol-bis[Baminoethyl ether]N,N'-tetracetic acid; E G T A (Wako), physostigmine sulfate (Merck), tetraphenylboron (Aldrich). 45CaClz, choline chloride [methyl-3H] and protosol were obtained from New England Nuclear, Boston, U.S.A.. Adenosine was dissolved in appropriate buffer solutions and the concentrations were expressed as /xM of the final concentration.
3. Results
3.1. Effects of adenosine on release of [3H]A Ch The high concentration of potassium (40 mM) produced an increase in the output of [3H]ACh from the crude synaptosomal preparation of the guinea-pig ileum myenteric plexus (table 1), The [3H]ACh release caused by the high concentration of potassium was significantly reduced in a calcium-free buffer solution containing 1.2 mM EGTA. Further, the potassium-induced release was markedly reduced at 4°C (data not shown). Adenosine at a concentration of 100 ~M significantly reduced the potassium-induced release of [3H]ACh by 34.4%. On the other hand, the spontaneous release of [3H]ACh was unaffected by adenosine. This release was unchanged in the
420 TABLE 1 Effect of adenosine on the high potassium-induced release of [3H]acetylcholine from crude synaptosomal preparations. The P, fraction of the guinea-pig ileum myenteric plexus was used for the release experiment. Condition
N ~'
[3H]ACh output b ( d p m / m g protein)
% of control ~ output
p d
Control 40 mM K 40 mM K + C a - f r e e ~
4 3 3
3365.9 ± 249.0 4974.3± 90.3 4212.7_+ 58.3
100.0 147.8 125.2
< 0.01 < 0.01 *
Control Adenosine f 40 mM K 40 mM K + adenosine
5 6 6 6
3584.5 ± 3468.5± 5586.4± 4895.5 +
159.1 89.5 127.7 147.5
100.0 96.8 155.8 136.6
NS < 0.01 < 0.01 *
Control Ca free Ice-cold g
4 4 4
3312.2 + 83.9 3328.3 ± 195.0 2535.3± 83.3
100.0 100.5 76.6
NS < 0.05
~' Number of experiments, b Data are means ± S.E.M. c Values are expressed as percentages of the control release of [3H]ACh. d Significantly different from control, Student's t-test or Cochran-Cox test. NS: not significant. * Represents a significant difference from 40 mM K condition, using a paired t-test, e The buffer with no Ca 2+ contained 1.2 mM EGTA. f 100 ~tM concentration. g Examined at 0-4°C.
calcium-free medium with 1.2 mM EGTA, but was markedly reduced when the assay temperature was lowered to 4°C. When 10 Hz electrical pulses were applied to the crude synaptosomal preparation, there was a marked increase in [3HIACh output but not with 0.1 Hz stimulation (table 2). The electrically stimulated release of [3H]ACh was abolished in calcium-free medium containing 1.2
mM EGTA or at 4°C assay temperature. Adenosine at 100 /xM completely abolished [3H]ACh release.
3.2. Effects of adenosine on 4~Ca uptake l~v synaptosomes A synaptosome-rich preparation was isolated from the P2 fraction in order to examine the 45Ca
TABLE 2 Effect of adenosine on the electrical stimulationqnduced release of [3 H]acetylcholine from crude synaptosomal preparations. The P~ fraction of the guinea-pig ileum myenteric plexus was used for the release experiments. Condition
N ~
[3H]ACh output b ( d p m / m g protein)
% of control c output
Control Adenosine e 0.1 Hz ES f 0.1 Hz ES + adenosine 10 Hz ES 10 Hz ES + adenosine 10 Hz ES, ice-cold g 10 Hz ES + Ca-free h
6 6 6 6 6 3 3 3
3351.7 ± 3205.9± 3543.1 ± 3487.4 ± 4692.3 ± 3650.7 ± 3035.6± 3005.5 ±
100.0 96.8 105.7 104.0 140.0 108.9 90.6 89.7
122.3 89.5 62.0 210.9 240.1 242.7 39.0 232.5
pd
NS NS < < < <
0.001 0.05 * 0.01 * 0.01 *
~' Numb er of experiments, b Data are means ± S.E.M. c Values are expressed as percentages of the control release of [3H]ACh. d Significantly different from control, Student's t-test or Cochran-Cox test. NS: not significant. * Represents a significant difference from 10 Hz ES condition, Student's t-test or Cochran-Cox test. e 1 0 0 / i M concentration, f Electrical pulses (0.1 or 10 Hz, 0.4 ms, 20 V amplitude) applied during the last 5 rain of a 10 min period of incubation, g Examined at 0-4°C. h The buffer with no Ca 2+ contained 1.2 mM EGTA.
421 TABLE 3 Effect of adenosine on high potassium-stimulated 45Ca uptake by the synaptosome-rich preparation. Preparation obtained by sucrose density gradient centrifugation procedures from guinea-pig ileum myenteric plexus-longitudinal muscle homogenates. Condition
N ~
45Ca uptake b ( n m o l / m g protein)
/~ ¢
p a
Control Adenosine e
9 9
4.08_+0.14 4.26 -+ 0.20
-
NS
Control 40 mM K 40 mM K + adenosine
7 7
4.04+_0.14 5.96 _+0.35
1.92
< 0.01
7
4.97_+0.36
0.93
< 0.001 *
" Number of experiments, b Data are means + S.E.M. c Values were calculated as the difference between high K + and control data and represent the net synaptosomal 45Ca uptake in response to depolarization, d Significance of difference from control obtained by paired t-test. NS: not significant. * Represents a significant difference from 40 mM K + condition using a paired t-test, e 100 ~M concentration.
uptake. Under control conditions, the basal uptake o f 45Ca b y t h i s p r e p a r a t i o n w a s m a x i m a l 2 m i n after starting incubation
w i t h 45Ca a n d r e m a i n e d
at almost the same level for the next 3 rain. In the presence of a high concentration of potassium, synaptosomal u p t a k e o f 4SCa w a s g r e a t l y creased
(table
3). A d e n o s i n e
the in-
a t 1 0 0 I~M s i g n i f i -
Fig. 1, Electron microscopic photographs of synaptosome-rich preparation of the guinea-pig ileum myenteric plexus (upper picture 40000×, lower picture 50000×). Synaptosomes are distinguishable as thin-walled bags and small mitochondria and synaptic vesicles are visible in these bags.
TABLE 4 Effect of adenosine on electrically stimulated 45Ca uptake by the synaptosome-rich preparation. Preparation obtained by sucrose density gradient centrifugation procedures from guinea-pig ileum myenteric plexus-longitudinal muscle homogenate. Condition
N a
45Ca uptake b ( n m o l / m g protein)
Control Adenosine e
6 6
4.13 _ 0.14 4.28 _+0.20
Control 10 Hz ES f (30 s) 10 Hz ES + adenosine
7 7 7
4.11 _+0.31 4.27_+0.29 4.10 _+0.23
-
Control 10 Hz ES (60 s) 10 Hz ES + adenosine
7 7 7
3.93 _+0.17 4.94_+0.25 3.86 _+0,35
- 0.07
A c
p d
NS
0.01
NS NS
1.01
<
0.16
*
0.01 < 0.05 *
a Number of experiments, b Data are means + S.E.M. c Values were calculated as the difference between ES and control data and represent the net synaptosomal 45Ca uptake in response to electrical stimulation, d Signi.ficance of difference from control obtained by Student's t-test or Cochran-Cox test. NS: not significant. * Represents a significant difference from ES condition, Student's t-test or Cochran-Cox test., e 100 # M concentration, g Electrical pulses (10 Hz, 0.4 ms, 20 V amplitude) applied during the last 30 or 60 s of a 2 rain period of incubation.
422 cantly reduced (by 58.6%) the uptake of 45Ca induced by 40 mM potassium. Electrical pulses at 10 Hz for 60 s greatly increased the synaptosomal 45Ca uptake (table 4). Adenosine completely abolished the uptake. The basal uptake of 45Ca was not affected by adenosine at all (table 3, 4). 3. 3. Electron microscopy Fig. 1 shows typical pictures of the synaptosome-rich preparation of ileal myenteric plexus. Many synaptosomes were distinguishable as thinwalled bags, and most of them contained several small mitochondria and a large number of synaptic vesicles. Most of the vesicles were small, round agranular vesicles about 30-50 nm in diameter and among the others were some granular vesicles about 30-60 nm. It was, however, obvious that the fraction was contaminated with membranes from undefined sources. Free mitochondria were only rarely seen.
4. Discussion
Electron microscopic photographs of a pelleted, enriched synaptosomal fractionof the guinea-pig ileum myenteric plexus showed synaptosomes as thin-walled bags. Most synaptosomes contained a large number of small, round agranular vesicles about 30-50 nm in diameter and a few granular vesicles about 30-60 nm in diameter, contrary to Briggs and Cooper's report (1981) that the synaptosomes contained large (90-140 nm) vesicles with an electron-opaque core and an absent or ill-defined 'halo'. Cook and Burnstock (1976) have examined the ultrastructural features of neurons within the myenteric plexus in the ileum of guineapig and identified eight morphologically distinct types of axon profile on the basis of vesicular size, shape and content. Two of them, type 2 and 3 as described by Cook and Burnstock contained many small, round agranular vesicles. Generally, axons which are known to be cholinergic, such as axons at the motor end-plate, preganglionic axons in sympathetic ganglia and axons supplying the adrenal medulla, contain mainly small round agranular vesicles. Furness and Costa (1980) sug-
gested that the axons with the numerous small round agranular vesicles may be cholinergic nerves in the intestine. Such axons have a distribution consistent with this suggestion. They are very numerous, form synapses in the enteric plexuses and also supply the plexuses and the smooth muscle (Gabella, 1972a, b). The electron microscopic profile of synaptosomes obtained in our experiments was similar to such an axon profile, suggesting that the synaptosomes may originate in the cholinergic nerve varicosities. In the present study, high potassium and electrical square-wave pulses were used as stimulants of excitation-release coupling of the ileal synaptosomes. A high concentration of potassium caused an increase in [3H]ACh output from the crude synaptosomal preparation and the release was both calcium- and temperature-dependent. Electrical pulses (10 Hz) to the crude synaptosomal preparation also caused a release of [3H]ACh which was calcium- and temperature-dependent. Osborne et al. (1973) had reported that glycine was released by electrical stimulation from spinal cord synaptosomes and that the release which was accompanied by the release of other putative amino acid transmitters was calcium-dependent. It is estimated that the [~H]ACh released from the ileal synaptosomes originated in the cholinergic nerve terminals. Low frequency electrical stimulation (0.1 Hz) could not have caused the release of [3H]ACh from the crude synaptosomes. It is difficult to explain why low frequency stimulation did not increase the [3H]ACh output. As a possibility, it is assumed that the excitability of the synaptosomes may be relatively low and electrical pulses at 0.1 Hz may be insufficient to trigger depolarization of the membrane. Ikushima et al. (1981) suggested that electrical stimulation with a short pulse duration might excite the nerve fibers, but that the longer pulse might excite the presynaptic terminals in addition to the nerve fibers. Possibly, more intense conditions e.g. high frequency and longer pulses might be required to generate the excitation of nerve terminals by electrical stimulation. In this study, adenosine inhibited the high potassium-induced [3 H]ACh release from the crude synaptosomes significantly. The electrically in-
423 duced [3H]ACh release was also markedly depressed by adenosine at the same concentration and the depression was more intense than that in the case of high potassium stimulation. Reese and Cooper (1982) suggested that the 50 mM potassium-induced [3H]ACh release was relatively insensitive to adenosine as compared with nicotinically induced release. The ACh releasing mechanism activated by electrical stimulation and nicotinic stimulation seemed to be more susceptible to the inhibitory action of adenosine than was the mechanism activated by high potassium. Generally, it is recognized that extracellular calcium ions flowing in the nerve terminals play an important role in the neurotransmitter releasing mechanisms. Previously, we reported that external calcium ions antagonized the inhibitory effect of adenosine in a competitive manner, suggesting that the action of adenosine might be closely associated with the extracellular calcium movement in the nerve terminals (Hayashi et al., 1981). As the present results showed, adenosine inhibited significantly the high potassium-induced 45Ca uptake by the synaptosome-rich preparations, and completely abolished the electrically stimulated uptake. The increase of 45Ca uptake induced by high potassium or electrical pulses was temperature-dependent (data not shown). Probably, such 45Ca uptake may reflect the calcium ion influx through the voltage-dependent calcium channel activated by membrane depolarization, and adenosine will be able to inhibit such an influx of calcium ion into the synaptosomes. From these results, it is suggested that the inhibitory effect of adenosine on the ACh releasing system may result from inhibition of calcium influx. Reese and Cooper (1982) reported that adenosine reduced the basal release of [3H]ACh from ileal synaptosomes, but in the present study the spontaneous [3H]ACh release was not affected by adenosine. Further, the basal 45Ca uptake was also independent of adenosine. Alnaes and Rahamimoff (1975) suggested that the spontaneous release of the transmitter depended on the resting level of intracellular calcium ion which was regulated for the greater part by the mitochondria or by nonmitochondrial structures. Therefore, it is proposed that adenosine could not have an influence on the
intracellular calcium ion movement in unstimulated synaptosomes, and might affect only the extracellular calcium ion influx triggered by membrane excitation. Ribeiro et al. (1975) demonstrated that in rat brain synasptosomes, adenosine decreased the uptake of 45Ca stimulated by potassium. Also, Kuroda (1983) reported that the high potassiumdependent calcium accumulation was decreased by 2-chloroadenosine in synaptosomes from guineapig cerebral cortex. These inhibition rates for the potassium-stimulated calcium ion uptake were almost similar to our data, and they were relatively small as compared to the value obtained with electrical stimulation in our study. The reason for this insensitivity is unknown, but two possible explanations should be considered: (1) the ability of potassium depolarization to stimulate the calcium ion influx may be intense, thus overcoming the inhibitory action of adenosine; (2) potassium-induced depolarization may stimulate another mechanism of calcium entry in addition to the adenosine-sensitive calcium influx mechanism, which is independent of adenosine. Although it is generally accepted that potassium at a high concentration produces membrane depolarization by potassium cation influx, the development of such depolarization seems to be essentially different from the electrically induced excitation, and from the physiological excitatory response. In an intact ileal myenteric plexus-longitudinal muscle preparation, adenosine at 100 /~M depressed the electrically (10 Hz) induced cholinergic contractile response completely (Hayashi et al., 1985). In the present study, adenosine at the same concentration also completely inhibited the calcium influx elicited by electrical stimulation at 10 Hz. These results suggested that the calcium channel coupled with the depolarization of presynaptic membrane induced by electrical stimulation may be sensitive to adenosine. Furthermore, it is proposed that the adenosine receptor may be linked to such a channel directly or indirectly, and may regulate ACh release in the nerve terminals of guinea-pig ileum physiologically, in agreement with our previous suggestions (Hayashi et al., 1978a, 1985).
424
Acknowledgements The authors are deeply indebted to Prof. M. Sano and Dr. A. lshii of Hamamastu University School of Medicine for their considerable assistance in performing the electron microscopy.
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