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The inhibitory effect of adenosine and related nucleotides on the release of acetylcholine
Neuroscience, 1976. Vol. 1, pp. 391-398. Pergamon Press. Prmted m Great Britain
THE INHIBITORY EFFECT OF ADENOSINE AND RELATED NUCLEOTIDES ON THE RELEASE OF ACETYLCHOLINE E. S. VIZI and 3. KNOLL Department of Pharmacology, Semmelweis University of Medicine, H-1085 Budapest, Hungary Ah&act-Isolated Auerbach’s plexus-longitudinal muscle preparations from guinea-pig ileum and slices of the rat cerebral cortex have been used to study the effect of adenine nucleotides on the release of acetylcholine. The release of acetylcholine evoked by cholecystokinin was completely inhibited by adenosine. The effect of nucleotides on neuro-effector transmission of electrically stimulated longitudinal muscle strip was also studied. Adenosine and adenosine triphosphate reduced the release of acetylcholine provided a low frequency of stimulation was applied. While the three adenine nucleotides (adenosine mono-, di- and triphosphate) dose-dependently reduced neuro-effector transmission in Auerbach’s plexus-lon~tudinal muscle preparation, adenine, guanosine triphosphate and dibutyryl-cyclic AMP had no effect. Theophylline, an adenosine receptor antagonist, prevented the inhibitory effect of the nucleotides. In addition, theophylline alone enhanced the release of acetylcholine both from Auerbach’s plexus and from the nerve terminals of the cortical slice. This indicates that there might be a continuous control of ACh release by an adenine nucleotide. These results are discussed in relation to the release of adenosine triphosphate from purinergic nerves in the intestine and of adenosine from slices of cerebral cortex; the possibility is raised that adenine nucleotides released from nerves might act as a type of presynaptic inhibitory transmitter on cholinergic neurons. Furthermore, if some of the released nucleotide originates from the synaptic vesicles of cholinergic neurons, it might serve as a negative feed-back transmitter for acetylcholine release. The inhibitory effect of adenosine and related nucleotides on the cholecystokinin-induced release of acetylcholine from the gut might be of physiological importance since gastrointestinal polypeptides play a very important role in maintaining gastrointestinal motility. FOR MANY yearsit has been assumed that inhibition of g~troint~tinal smooth muscle motility is mediated by the release of noradrenaline from sympathetic neurones. It has been shown that noradrenaline released from sympathetic nerve terminals is able to reduce the release of acetylcholine (ACh), thereby inhibiting gastrointestinal motility (PATON & Vm, 1969; VIZI & KNOLL, 1971; VIZI, 1973). However, since 1964, pharmacological and electrophysiological evidence has been presented for a type of neurone in the intestine which is neither cholinergic nor adrenergic (see BURNSTOCK, 1972), but which causes inhibition of the gut. Evidence has accumulated in favour of the hypothesis that a purine derivative, probably adenosine triphosphate (ATP), is the transmitter released from purinergic inhibitory nerves present in Auerbach’s plexus (BURNSTOCK,CAMPBELL, SATCHELL & SMYTHE,1970). However, the role of ATP or a related nucleotide as humoral transmitter in the inhibition of intestinal motility has been questioned (KUCHII, MXYAHARA & SHIBA~A,1973). It has also been shown that adenosine is released from slices of the cerebral cortex (PULL & MCILWAIN, 1972). The present study was undertaken to analyse the effect of adenosine and closely related nucleotides on
the release of AC% from Auerbach’s plexus of the guinea-pig ileum and from isolated cortical slices of rat brain.
EXPERIMENTAL
PROCEDURES
Preparation of longitudinal muscle strip with attached bath’s plexus
Auer-
Longitudinal muscle strips of the guinea-pig ileum were prepared according to the method of PATON & VIZI (1969) and set up in an organ bath of 3.5 ml capacity in Krebs’ solution at 36”C, bubbled with 9.5% oxygen and 5% carbon dioxide. Physostigmine sulphate (2 x 10e6 g/ml) was added to the Krebs’ solution to prevent enzymic hydrolysis of acetylcholine. After 60 min preincubation time, ACh was collected for assay. In those experiments in which the contractions to electrical field stimulation were recorded, physostigmine was not added to the Krebs’ solution. fso~ured corticaI slices of rat brain
The preparation was made according to the method of VIZI (1972). Rats weighing 120-170 g were killed by stunning. The brains were immediately removed from the skull. A slice of cortex was cut from each hemisphere. Two to four slices (each weighing 70-120 mg and less than 0.8 mm thick) were incubated at 36°C in a glass chamber of 2ml containing Krebs’ solution gassed with 5% carbon dioxide Abbre~iutions: ACh, acetylcholine; ADP, adenosine in oxygen. Before the collection of the first sample, the diphosphate; AMP, adenosine monophosphate; ATP, slices were allowed to equilibrate for 6Omin in the eseradenosine triphosphate. inized Krebs solution. 391 NSC1+--c
392
E. S. VIZ.1and J. Kc01
A~tylcho~ine coltected either from lungitudinai muscle strips or from cortical slices was assayed on isolated guinea-pig ileum suspended in 3Sml Krebs solution at 33“C by the method of PATON& VIZI (1969). Since prolonged isometric contraction tended to cause spontaneous movements of the ileum, a soft spring of 0.5 g/cm compliance was interposed between the proximal end of the ileum and the transducer. The contractions were recorded on a potentiometric recorder (SERVOGOR, GOERZELECTRO). To eliminate the possibility of interference with the assay by drugs present in the assay sample, responses to standard solutions of ACh were recorded in the presence of these drugs in concentrations equivalent to those reached by addition of the sample containing the drug. When cholecystokinin was present in the bath, tetrodotoxin (5 x lo-‘M) was added to the assay bath to control for polypeptide carried over in the sample, since it has been found that ~etrodotoxin is able to inhibit completely the contractile action of gas&in-like polypeptides on guineapig ileum whiIe having no effect on the response to ACh (VIZ], BERTACCINI, IMPICCIAT~KE & KNOLL. 1973). Acetyicholine dissolved in saline in volumes of 0.05-0.3 ml was added to the assay bath. Physostigmine sulphate (5 x IO-“g/l) and morphine sulphate (1O-3 g/l) were added to the Krebs’ solution in the assay bath in order to increase the sensitivity of the ileum to ACh and to reduce endogenous ACh release. After 60min incubation time the lowest amount of ACh still was O.Sng/3.Sml (5.2 x lo-‘*M; reliably assayed 0.14 ng/ml acetylcholine iodide). Stimulution technique
the resting release of ACh (Tabie 1) to about half the control value. When the co~lcentration of ATP was increased to IO--” M, no further decrease in ACh release was seen. No tachyphyiaxis was observed to the action of ATP when it was administered repcatedly. The action of ATP was dependent on concentration below 10.’ M. In one experiment ATP in a concentration of IO’-’ M reduced the release of ACh by only IX”,. Much of the resting release of ACh was due to the spontaneous activity of neurons, since it was reduced by 601:,, in the presence of tetrodotoxin (lOeh~); under the latter conditions the residual release of ACh was not affected by adenosine (IO -a M). The inhibitory effect of ATP on the release of ACh induced by electrical stimulation depended on the frequency of stimulation applied. As shown in Table 1. the reduction in output produced by ATP was, at 0.2 Hz, 60.81,, and, at 10 Hz, only 2X,. Figures la and b show the effects of adenosine and ATP respectively on ACh release induced by 0.2 Hz stimulation. The effect of ATP was longer lasting than that of adenosine: after it had been washed out. the release of ACh in the next collection period was still significantly lower (by about 43”,,) than the initial value. When the tissue was exposed to a higher concentration of ATP (10m3 M) there was no further reduction in ACh release induced by 0.2 Hz stimulation. However, it took 30 min for the rate of release to return to about 95”‘, of the control value. ltlhihition
by &nine
nuclrorid~,s
induced
Precention
h!, cllolec~stolrinin.
by theophytline
of utlmin~ nucleotides
qf the ~rl~aibitory action
release. Theophylline, a blocker of adenosine receptors (McILWAN 1972). by itself enhanced the release of ACh induced by stimulation (Fig. 2). The release induced by either low (0.2 Hz) or high (10 Hz) frequency of stimulation was enhanced. At low frequencies of stitnulation the potentiation was more pronounced than at high frequencies. Adenosine, which otherwise reduced the release of ACh, failed to affect the release in the presence of theophylline (Fig. 2). The inhibitory effect of ATP on the ACh released by stimulation at 0.2 Hz was also partly prevented by theophylline (Table 1). on trcetylcholine
The +@ct qf adenosine and adeno,s~~~etr~phosphote the releuse~~~~etylcho~ine from
of therat
RESULTS A&nine
nucleotides
Auerbach’s Inhibitory
and
acetyicholine
e@ct of adenosine and adenosine triphos-
phate on the resting
and stimulated release of acetyiAdenosine, plexus.
Auerbach’s from 4 x 10e5 M, and ATP, 1W4~,
choline
releuse ./iom
plexus
si~i~~~tly
reduced
oj‘ lrcetylcholine
Cholecystokinin, like other gastro-intestinal hormones (Vrzr er al.. 1973). enhanced the release of ACh from Auerbath’s plexus (Table 1). However, the ACh-releasing effect of choIecystokil]in was completely blocked by adenosine. rcktrsc,
Field stimulation was used (PATON& VIZI, 1969). Supramaximal (10 V cm- ‘) square wave pulses of 1ms duration were applied through platinum electrodes at the top and bottom of the organ bath, at a frequency of 0.1-10 Hz. The Krebs’ solution used had the following composition (mM): NaCI, 113; KCl, 4.7; CaCI,, 2.5: KH,PO,, 1.2; MgSO,, 1.2; NaHC03 25.0: and glucose, 11.5. When calcium enriched solution (5 mM) was used, NaHCO, was omitted to avoid precipitation and replaced by equimolar amounts of NaCl. The following drugs were used: acetylcholine iodide (BDH Chemicals, Poole, England), physostigmine sulphate (Burroughs Wellcome); adenosine (Reanal), adenosine monophosphate disodium (Reanal), ~holecystokinin pankreo~ymin (chemistry Dept., Karolinska Institute, Stockholm, Sweden), theophylline methanesulphonate (CIBA): (Richter); phentolamine N6-20-Dibutryl cyclic AMP (Boehringer); tetrodotoxin Sankyo); BAY-1470, 2-(2,6-xylifamino)-5,6-dihydro-4H1,3-thiazine (Bayer). The drugs were dissolved in 0.90,; w/v NaCl solution. Concentrations of the drugs are expressed in molar concentration, and sometimes given also in terms of their salts.
1
j.so~ated cortical
OS
slices
(see Tahk ?)
Adenosine, by itself, appeared to reduce the resting release of ACh, but the reduction proved to be not significantly theophylline However. significant. enhanced the release by 369;. ATP, lo-” M, also failed to affect the release. Morphine, which enhances the cyclic AMP level in nerve endings of rat cortical slices
393
Adenine nucleotides and ACh release TABLE1. THE EFFECTOF ADENOSINE NUCLEOTIDES AND THEOPHYLLINE ON ACIITYLCHOLINE RELEASEFROM ALVZRBACH'SPLEXUS-LONGITUDINAL MUSCLE PREPARATION
Acetylcholine release, S.E.M. (pm01 g-I min-‘)
Collection period (min)
Condition 1. 2. 3. 4.
Control (35) Adenosine, 4 x 10-s M (4) ATP, 1W4M (4) Cholecystokinin 10-’ M (3) 5. Cholecystokinin lo-‘M + Adenosine, 4 x lo-’ M (3)
6. 0.2 Hz (14) 7. 0.2 Hz + ATP, 1O-4 M (3) 8. 0.2 Hz + Theophylline, 1.7 X lo-4M (3) 9. 0.2 Hz + Theophylline, 1.7 x 10-4~ + ATP, 10-4~ (3) 10. 0.2 Hz, Ca’+-excess (3) 11. 0.2 Hz Cazc-excess + ATP, 1O-4 M (3) 12. 10Hz (3) 13. 10 Hz + ATP, lo+ M (3) 14. 0.2 Hz + Phentolamine, 2.5 x 10-6M (2) 15. 0.2 Hz + Phentolamine, 2.5 x 10-6~ + ATP, 10-4M (2)
Significance (P)
Resting 10 10 10
162.4 + 2.8 85.3 + 3.8 80.1 + 6.9
2:l < 0.01 3:l < 0.01
10
395.9 + 36.6
4:l < 0.01
10
96.4 + 8.1
5:4 < 0.01
Electrical stimulation 10 420.8 + 11.1 10 132.3 + 17.5
6:l < 0.01 7:6 < 0.01
10
518.4 + 24.9
8:6 < 0.05
10 10
346.5 k 30.8 461.5 f 26.8
9:7 < 0.01 10:6 > 0.05
10 1 1
159.8 + 28.3 3068.2 + 160.5 2980.0 + 310.0
11:7 > 0.5 12:l < 0.01 13:12 > 0.5
10
372.82
10
70.6
Number of experiments in brackets. Ca’+ -excess: CaClr concentration
(2.5 mM) has been doubled.
(CLOUET, GOLD & IWATSUBO,1975) did not a&t
the release. Ouabain, an inhibitor of Na+-K+-activated ATP-ase, enhanced the release in three experiments, as already shown by VIZI (1972). Adenosine, in a concentration of 3 x 10e5 M, slightly reduced the ouabain-induced release. In comparison to the reduction
Adenosim, Cx 10'M
m e
stim.
Tc ‘fG &lllJ
10 min -
%I
_--__
8,
-
:: ,o xL
0.2Hz
___-_
_____
.__._---2a).
_-__. _____
G
B
-g
ATP.
lo-& M 1stim. 0.2Hz
FIG. 1. Inhibitory effect of adenosine (a) and ATP (b) on the release of acetylcholine induced by 0.2 Hz stimulation. Longitudinal muscle strip of guinea-pig ileum. The values for acetylcholine release (pmol g-i min- I) are the means of three identical experiments. Dashed lines indicate S.E. Note the rapid offset of adenosine action.
to 92
92
10 - HZ
FIG. 2. Prevention by theophylline of the inhibitory action of adenosine on acetylcholine release from Auerbach’s plexus. Longitudinal muscle strip of the guinea-pig ileum. The values for acetylcholine release (pmol g-r min- ‘) are the means of two experiments with identical treatment schedules. Stimulation period as indicated was 1 min at 10 Hz (1 ms, 10 V/cm). Note that theophylline enhances the release of ACh and prevents the inhibitory effect of adenosine.
E. S. VIZl and J. KNOLL
394
TABLE 2. THE EFFECt OE ADENOSINE, ~-IEOPHYLLI~, OU~aN, MORPHINE AND BAY-1470 ON TIlE RESTING ACETYLCHOLINE
RELEASE FROM ISOLATED CEREBRAL CORTICAL
Collection period (min)
Drug 1. - -
2. 3. 4. 5. 6. 7. 8.
Adenosine 3 x 10-5M ATP, 10-4M Theophylline, 1.7 x 10-4M Morphine 3 x 10-SM BAY-1470, 10-SM Ouabain, 2 x 10-SM Ouabain, 2 x 10-SM + Adenosine, 3 x 10-SM 9. Ouabain, 2 x 10-SM + BAY-1470, 10 -5 M
SLICES OF THE RAT
Acetylcholine release S.E.M. (pmol g- 1 min- 1)
10 10 10 10 10 10 10
23.97 18.97 19.2 32.7 19.0 21.8 218.3
± + _ + + _ ±
10
165.5 ± 21.8(3)
10
62.8 ± 15.6(3)
Significance (p)
2.18 (8) 2.8(3) 3.1 (3) 1.8 (4) 2.1 (3) 1.1 (3) 58.2(3)
2:1 n.s. 3:1 n.s. 4:1 < 0.01 5:1 n.s. 6:1 n.s. 7:1 < 0.01 8:7
n.s.
9:7 < 0.01
Number of experiments is in brackets. produced by BAY-1470, a pure ~-adrenoceptor stimulant, however, the reduction is negligible (Table 2).
Inhibitory effect of adenine nucleotides on neuro-effector transmission of the electrically stimulated longitudinal muscle strip of guinea-pig ileum To study the effect of adenine nucleotides in the absence of a cholinesterase inhibitor, the contractions of the guinea-pig ileum longitudinal muscle strip were measured under conditions where the contraction is due to the release of ACh from Auerbach's plexus. Isometric contractions in response to stimulation were recorded. Adenosine (10-6-3 x 10 - s ra), A M P (10-6-10 -4M), A D P (10-6-10 -4 M) and A T P ( 1 0 - 6 - 1 0 -4 M) reduced the size of contractions of the electrically stimulated longitudinal muscle strip without influencing the sensitivity of the smooth muscle cells to added acetylcholine. The effect was rapid in onset. After washing out, a fast recovery was observed. The reduction depended on the concentrations used and the frequency of stimulation applied
(Figs. 3 and 4): the contractions induced by high frequency stimulation were not affected by the nucleotides used. Repeated exposure to adenine nucleotides did not result in a diminution of the inhibitory action. However, it was observed that at higher concentrations ( > 1 0 - 4 M ) A T P caused a contraction of the s m o o t h muscle. Theophylline, in concentrations up to 2 x 10 - 4 M, slightly enhanced the responses of the muscle strip to stimulation at a frequency of 0.2 Hz; this action proved to be dose dependent. If added before the nucleotides, it prevented their inhibitory action (Fig. 5). Since in three experiments theophylline failed to potentiate the effect of exogenous A C h on the s m o o t h muscle, it is concluded that its main site of action is o n Auerbach's plexus. This is confirmed by the observations on the effect of theophylline on ACh rain --
o
i i i ~ ~ ~ ~ z i ~
mill •
:
i
i
,
shocks 5 10 10 50 Hz 1 510 10
:
:.~
! ~
~!
510 10 50 1 5 10 10
~
:
:
0,1 HZ
slim. T
X lo'6M
30
15
7.5
3
T Adenosine
FIG. 3. The inhibitory action of adenosine on the responses of longitudinal muscle strip of guinea-pig ileum to field stimulation. Field stimulation, 1 ms, 10 V/cm, 0.1 Hz when stimulation (stim.) is indicated. T = 10Hz, 10 shocks. Adenosine was administered in different concentrations as indicated. Note the inhibitory action of adenosine on the responses of the strip to 0.1 Hz but not to 10Hz stimulation.
LATP, IO'~H J
FIG. 4. The inhibitory effect of ATP on the responses of longitudinal muscle strip of guinea-pig ileum to field stimulation with different frequencies and with different length of trains. Field stimulation, l ms, 10 V/cm. Krebs solution. 95~o 02 + 5~o CO2. Organ bath 3.5 ml. Note the inhibitory action of ATP on the responses of longitudinal muscle strip to 0.1, 1, 5 and to 10 Hz stimulation with short train (10 shocks). However, ATP had no effect when 10Hz stimulation in a longer train (50 shocks) was applied.
Adenine nucleotides and ACh release
395
min r---1
=
'
I
j I '
/ o
,
I'I
I
//
=
o
/
v
o
stim. 0,1Hz
t
~w
Adenosine (M) 1
t
o 4,
t t~w
1 2 3 .............. 1,7 x 10-4M
x10 -5 Theophyl.lJne
FIG. 5. Prevention by theophylline of the inhibitory effect of adenosine on the contractions of longitudinal muscle strip of gninea-pig ileum in response to 0.1 Hz stimulation. Field stimulation, 1 ms, 10V/cm. Krebs' solution. 95% 02 + 5% CO2. Organ bath 3.5 ml. w = wash out. Note the effect of adenosine was antagonised by theophylline. By increasing cumulatively the concentration of adenosine the control effect of adenosine was reached. release (Table 1). Recording the contractions of the strip in response to stimulation has the advantage that the effect of nucleotides can be repeatedly studied, and a dose-response curve in the absence and presence of antagonist can be obtained. The nature of the antagonism between adenosine and theophylline has also been studied (Fig. 6). It can be seen that theophylline competitively antagonised the inhibitory effect of adenosine on ACh release. Phentolamine, in a concentration of 10 -6 M did not antagonize the effect of adenosine. Guanosine triphosphate, in concentrations up to 2 × 10 -4 M, failed to affect the contractions of the strip in response to stimulation at 0.1 Hz. Unlike adenosine, the dibutyryl derivative of adenosine 3'5'-monophosphate, in concentrations ranging from 10 -6 to 10 -4 M, did not reduce the size of the contractions. In a higher concentration (10-3 M) however, it reduced both the responses to electrical stimulation, and the contraction caused by ACh added to the organ bath.
"
-3
J. log concn, of theophylline
FIG. 6. Competitive nature of the antagonism by theophylline of the depressant effect of adenosine on the contractions of stimulated longitudinal muscle strip of guinea-pig ileum. For method see Fig. 5. Each point represents three experiments. The plot of log (DR-l) against the log concentration of theophylline produced a straight line with a gradient of 0.910 and an intercept on the abscissa corresponding to log Ke. DR, the ratio of adenosine concentrations in the presence and absence of theophylline required to produce the same effect. Ke, concentration of antagonist which produces a dose ratio of 2:for theophylline 2.1 x 105 M. The exposure time for theophylline is 20 rain. Since the regression, 0.920 does not differ significantly from unity the antagonism is competitive.
Inhibitory effect of adenosine on the responses of the longitudinal muscle strip to cholecystokinin It has been shown that cholecystokinin acts indirectly on the intestinal smooth muscle by stimulating cell bodies: and causing the release of ACh from Auerbach's plexus (VIzI et al., 1973; VIzI, 1973). The contraction of the smooth muscle caused by choleoystokinin was completely inhibited by adenosine and the closely related nucleotides, AMP, A D P and ATP. The responses of the smooth muscle to ACh were, however, not affected. This result also indicates that the effect of nucleotides is on the nerve tissue. A typical experiment is shown in Fig. 7. Adenosine reduced the size of the contractions in response to stimulation with 0.1 Hz and completely inhibited the contraction induced by cholecystokinin. After washing out adenosine and cholecystokinin there was a contraction (Fig.
rain
i stim.
0.1Hz
. . . . . . .
OO
O
ACh
ACh
24
Adenosine
2
~
O CCK 1
~
O
-CCK 1
x 10-$ M
="=='===
2
1
-5
xlO
M
FIG. 7. Inhibitory effect of adenosine on the contractions of longitudinal muscle of guinea-pig ileum in response to stimulation and to cholecystokinin. Field stimulation, 0.1 Hz, 1 ms, 10 V cm -1. CCK, cholecystokinin.
396
E. S. Vu
and J.
n Adenosine ATP etc.
FIG. 8. Scheme of the site of action of ATP or related nucleotide on Auerbach’s plexus. Dotted line indicates the inhibitory effect of ATP or related nucleotides on ACh reiease, suggested by this paper, and on the smooth mu&e, suggested by BURNST~CK (1972). 7) which could be blocked by tetrodotoxin (1O-6 M), indicating that it might be caused by the polypeptides still present in the tissue. DISCUSSION The results provide evidence that adenine nucleotides are able to reduce the release of ACh from Auerbath’s plexus of the guinea-pig isolated ileum, when the release is due to firing of the nerves, but the basal release in the absence of nervous activity, i.e. when tetrodotoxin was present, was not inhibited by adenosine. However, from the isolated cortical slice of the rat the ACh release during rest, and the ouabaininduced release, were not affected by adenosine or by ATP. Theophylline, an antagonist of adenosine receptors (MCILWAIN,1972), enhanced the release of ACh from Auerbach’s plexus and from cortical slices, and prevented the inhibitory action of the nucleotides. Frequency dependence of the effect of adenine nucleotides
The reduction in ACh release from Auerbach’s plexus by adenine nucleotides was pronounced only when a low frequency of stimulation was used. At high frequencies of stimulation (10Hz) they failed to affect the release. The release of ACh induced by cholecystokinin was completely inhibited by adenosine. There was no significant difference between the potencies of the adenine nucleotides in their ability to inhibit neuro-effector transmission in the stimulated longitudinal muscle strip of guinea-pig ileum. Adenine and guanosine triphosphate were however, without any effect. The fact that theophylline enhanced the release of ACh both from Auerbach’s plexus and from the cortical slices, raises the question whether endogenous adenosine might control the release of ACh, and that the released ACh me~ured is in fact, an already reduced output. This could also explain why the output of ACh per stimulus is very low when a high
KNOLL
frequency of stimulation is applied (KNOLL & Vizr, 1971) a situation in which greater amounts of adenosine might be released. A possible explanation for the lack of effect of adenosine on the release of ACh from the cortex is that the output from cortex is continuously controlled by adenosine or by some of its related compounds. Adenosine, in fact, has been shown to be released from the cortex by stimulation (MCILWAINE, 1972: PULL & M~ILWAIN, 1972) and from purinergic nerves in smooth muscle (BURNSTOCK er ~1.. 1970). In the adrenal medulla. ATP is stored together with, and is released with, catecholamines (DOUGLAS& POISNER,1966). It has been reported that the vesicles in cholinergic nerve terminals contain besides ACh, considerable amounts of nucleotides (WHI~A~R, D~WDALL & BOYS, 1972). Therefore, it is suggested that adenine nucleotides, if rdeased from the vesicles, might inhibit the release of ACh from the nerve terminals. Another possibility is that adenosine or related nucleotides are released from specific nucleotide containing axons (Su, BEVAN& BLIRNSTOCK 1971; BURNSTOCK rf ui., 1970; cf. BUKNSTOCK, 1972). In agreement with the results reported in this paper it has been shown that adenosine reduces transmitter release from the phrenic nerve of the rat (GINSBORG & HIRST,1972). On the other band, cyclic AMP, theophylline (GOLDBERG& SINGER,1969) and adrenaline (KRNJEVIC& MILEDI, 1959) have been shown to be able to increase the frequency and amplitude of the miniature endplate potential. KENTERA& VARAGIC (1975). however, showed that at least one of the sites of action of factors known to increase the intracellular concentration of cyclic AMP is located on the effector cells.
Site C$ action
of adenine nucleotides
The question
now arises as to where and how adenosine acts? Since adenine nucleotides are capable of reducing the release of ACh induced by postganglionic stimulation, their site of action has to be at least partly on the nerve terminals. However, an effect on the site -.where action potential generation takes place cannot be excluded (Fig. 8). This might explain why adenosine completely blocks the ACh-releasing effect of cholecystokinin which stimulates the cell body of the axons. The mode of action of adenine ~u~~eoiides
Two possible modes of action of the adenine nucleotides are (i) they might prevent the spread of impulses to the nerve terminal varicosities. This is unlikely since the ACh release induced by high frequency stimulation is not affected by the adenine nucleotides, (ii) they might prevent the entry of Ca2+ ions (into the nerve terminals), but their inhibitory action is not prevented by Ca2’ excess (Table 1). It has been suggested that there is a correlation between the inhibitory etfect of noradrenaline on ACh release and its stimulant action on Na+-K+-activated ATP-
Adenine nucleotides and AC%release pase (VIZ, 1972, 1975). Since adenosine and related nucleotides act in many respects in a similar way to noradrenaline, it is tempting to speculate that adenosine and other nucleotides reduce the release of ACh by stimulating membrane ATP-ase activity, a fact which has been shown biochemically for ATP (ROBW SON, 1973; YOSHIDA, NAGAT, OHASHI & NAKAGAWA, 1969). The only difference in the effect between adenosine and noradrenaline
is that the effect of adenosine
can
be blocked by theophyiline, and that of noradrenaline by phentolamine (VIZI, 1968; PATON & VIZI, 1969). MCAFEE & GREENGARD (1972) suggested that cyclic AMP
mediates dopaminergic
by modifies cholinergic
transmission
and there-
transmission
in the sympathetic ganglion. They concluded that an increase in cyclic AMP levels in the ~stgang~ionic neurons makes the post-synaptic membrane less responsive to subsequent excitatory input. Our data cannot be explained in this way since we found that theophylline increased the release of ACh and prevented the inhibitory effect of adenosine. But theophylline is an inhibitor of phosphodiesterase and therefore enhances the concentration of cyclic AMP, while adenosine is another compound that is known to increase tissue levels of cyclic AMP (MCILWAIN, 1972). It has been suggested that adenine nucleotides trol neuroeffector
transmission
at the level of the axonal impulse
generation
in Auerbach’s
membrane
conplexus
by impairing
and stimulus-secretion
397
The fact that theophylhne, an antagonist of the adenosine receptors of the cerebral cortex, enhanced the release of ACh from the nerve terminals of rat cortex, indicates that a similar mechanism might operate in the central nervous system as well. The main question now is whether endogenous nucleotides reach a sufficiently high local concentration that they can play a physiological modulatory role in cholinergic transmission by inhibiting the release presynaptically. The hypothesis is supported by the fact that theophylline, possibly by removing the inhibitory effect of endogenous adenine nucleotide(s), enhances the release of ACh both from Auerbach’s plexus and from cortical slices. Since gastrin-like hormones have been shown to play an important role in maintaining g~troint~tin~ activity by releasing ACh from Auerbach’s plexus, the inhibitory effect of adenosine and related nucleotides, as well as noradrenaline, might be important in controlling cholinergic outflow enhanced by cholecystokinin. As well as their direct action on the smooth muscle (DRURY & SZENTGY~RGYI, 1929; cf. BURNSTOCK, 1972) the nucleotides directly affect Auerbach’s plexus. The two actions would both cause a reduction in intestinal motility. However, the effect of endogenous and exogenous ACh on the smooth muscle is only slightly if at all, affected by nucleotides, which indicates that the presynaptic type of inhibition is of greater importance in reducing intestinal motility.
coupling.
REFERENCES BURNSTOCK G. (1972) Purinergic nerves. Pharmac. Rev. 24, 509-580. BURNSTOCK G., CAMPBELLG., SATCHELLD. & ANNE SMYTHE(1970) Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut. Br. J. Phnrmac. Chemother. 40, 668-688. CLOUETDORIS H., GOLD G. J. & IWATSUBOK. (1975) Effects of narcotic analgesic drugs on the cyclic adenosine 3’,5’-monophosphate-adenylate cyclase system in rat brain. Br. J. Pharmac. Chemother. 54, 541-548.
DOIJGLASW. W. C POISNERA. M. (1966) On the relation between ATP splitting and secretion in the adrenal chromaffin cell: extrusion of ATP (unhydrolysed) during release of ~techol~ines. f. Phys~o~.,Land, 183, 249-256. DRURY A. N. & SZENTGY~RGYIA. (1929) The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J. Physiol., Land. 68, 213-237. GINSEIORG B. L. & HIRST G. D. S. (1972) The effect of adenosine on the release of the transmitter from the phrenic nerve of the rat. J. Physiol., Lond. 224, 629-645. GOLDBERGA. L. & SINGERJ. J. (1969) Evidence for a role of cyclic AMP in neuromuscular transmission. Proc. natn. Acad. Sci., U.S.A. 64, 134-141.
KENT~RAD. & VARAGICV. M. (1975) The effects of cyclic N-20-dibutyryl-adenosine 3’,5’-monophosphate, adrenaline and ~inophylline on the isometric contractility of the isolated hemidiaphra~ of the rat. Br. 1. Pha~c. Ch~o~her. 54, 375-381. KRNJEVXEK. & MILEDI R. (1958) Some effects produced by adrenaline upon neuromuscular propagation in rats. J, Physiol., Land. 141, 291-304. KNOLLJ. & Vrzt E. S. (1971) Effect of frequency of stimulation on the inhibition by noradrenaline of the acetylcholine output from parasympathetic nerve terminals. Br. J. Pharmac. Chemother. 42, 263-272. KUCHII M., MIYAHARAJ. T. & SHIBATAS. (1973) [“Hladenine nucleotide and C3H]noradrenaline release evoked by electrical field stimulation, perivascular nerve stimulation and nicotine from the taenia of the guinea-pig caecm. Br. J. Pharmac. Chemother. 49, 258-266. MCAFEE D. A. & GREENGARDP. K. (1972) Adenosine 3’,S-monophosphate: el~trophysiolo~c~ evidence for a role in synaptic tr~smission. Science, N.X 178, 311r312. MCILWAI~~ H. (1972) Regulatory significance of the release and action of adenine derivatives in cerebral system. Biochem. Sot. Symp. 36, 69-85.
398
E. S. Vm and .r. i(wx.r
PATONW. D. M. & VIZI E. S. (1969) The inhibitory action of ~loradrenalille and adre~~aline on acctylchoiine output by guinea-pig ileum longitudinal muscle strip. Br. J. P~a~~?~u~. ~.~~~,~~f~r~?~~. 35, 10 18. PULL L. & MCILWAI~JH. (1972) Metabolism of [‘?I’]adenosine and derivatives by cerebral tissues superfused and electrically stimulated. Bioche~n. J. 126, 965-972. ROBINKJNJ. D. (1973) Cation sites of Na ‘-K”-ATP-ase: Mechanisms for Na’ induced changes in K” affinity of the phosphatase activity. Biochem. hiophys. Acru 321, 662-670. SU C., BEVAN J. & BUXNSTOC~G. (1971) [‘H]Adenosine release during stimulation of enteric nerves, S<~OIU~,N.Y: 173, 337-339. VIZI E. S. (1968) The inhibitory action of noradrenaline on release of acetylcholine from guinea-pig ifeum longitudinal muscle strips. Arch. mp. ~~ur~~~. 259, 199.-200. Vrzt E. S. (1972) Stimulation, by inhibition of Na*-K+-ME’*-activated ATP-ase, of acetyicholine release in cortical slices from rat brain. J. Ph~siol., Lo&. 226, 95-l 17. VZI E. E. (1973) Acetylcholine release from guinea-pig ileum by par~ympathetic ganglion stimulants and gastrin-like polypeptides. Br. J. Phurmuc. Chemother. 47, 76-777. Vrzr E. S. (197.5) Refease mechanisms of acetylcholine and the role of Na’-K+-activated ATP-ase. In: Cholineryic~ Mechanisms (ed. WAD P. G.) pp. 199-21 I. Raven Press, New York. Vtzr E. S., BERTACCINI G., IMPICCIATORE M. & KNOLL J. (1973) Evidence that acetylcholine released by gastrin and related potypeptides contributes to their effect on gastrointestinal motility. ~f~srroenterul~g~ 64, 26827’7. Vtzt E. S. & Klriot,t. J. (1971) The effect ofs~mpatbetic nerve stimulation and guanethidine on par~y~npathetic neuroeffector transmission; the inhibition of acetylcholine release. J. Planurn. ~~f~rrn~~. 23, 918-925. WHITTAKERV. P., DOWDALL M. J. & BOU;UEA. F. (1972) The storage and release of acetylchohne by cholinergic nerve terminals: recent results with non-mamm~ian preparations. Biuchem. Sot. sSymp. 36, ‘79-68. YOSHIDA H., NACA~ K,, OHASHI T. & NAXAGAWA Y. (1969) K’-dependent phosphatase activ,ity observed in the presence of both adenosine triphosphate and Na’. Biochim. hio&s. Ar~ir 171, 178 -185. (:4rcep& 29 Mtax 1976)
THE INHIBITORY EFFECT OF ADENOSINE AND RELATED NUCLEOTIDES ON THE RELEASE OF ACETYLCHOLINE E. S. VIZI and 3. KNOLL Department of Pharmacology, Semmelweis University of Medicine, H-1085 Budapest, Hungary Ah&act-Isolated Auerbach’s plexus-longitudinal muscle preparations from guinea-pig ileum and slices of the rat cerebral cortex have been used to study the effect of adenine nucleotides on the release of acetylcholine. The release of acetylcholine evoked by cholecystokinin was completely inhibited by adenosine. The effect of nucleotides on neuro-effector transmission of electrically stimulated longitudinal muscle strip was also studied. Adenosine and adenosine triphosphate reduced the release of acetylcholine provided a low frequency of stimulation was applied. While the three adenine nucleotides (adenosine mono-, di- and triphosphate) dose-dependently reduced neuro-effector transmission in Auerbach’s plexus-lon~tudinal muscle preparation, adenine, guanosine triphosphate and dibutyryl-cyclic AMP had no effect. Theophylline, an adenosine receptor antagonist, prevented the inhibitory effect of the nucleotides. In addition, theophylline alone enhanced the release of acetylcholine both from Auerbach’s plexus and from the nerve terminals of the cortical slice. This indicates that there might be a continuous control of ACh release by an adenine nucleotide. These results are discussed in relation to the release of adenosine triphosphate from purinergic nerves in the intestine and of adenosine from slices of cerebral cortex; the possibility is raised that adenine nucleotides released from nerves might act as a type of presynaptic inhibitory transmitter on cholinergic neurons. Furthermore, if some of the released nucleotide originates from the synaptic vesicles of cholinergic neurons, it might serve as a negative feed-back transmitter for acetylcholine release. The inhibitory effect of adenosine and related nucleotides on the cholecystokinin-induced release of acetylcholine from the gut might be of physiological importance since gastrointestinal polypeptides play a very important role in maintaining gastrointestinal motility. FOR MANY yearsit has been assumed that inhibition of g~troint~tinal smooth muscle motility is mediated by the release of noradrenaline from sympathetic neurones. It has been shown that noradrenaline released from sympathetic nerve terminals is able to reduce the release of acetylcholine (ACh), thereby inhibiting gastrointestinal motility (PATON & Vm, 1969; VIZI & KNOLL, 1971; VIZI, 1973). However, since 1964, pharmacological and electrophysiological evidence has been presented for a type of neurone in the intestine which is neither cholinergic nor adrenergic (see BURNSTOCK, 1972), but which causes inhibition of the gut. Evidence has accumulated in favour of the hypothesis that a purine derivative, probably adenosine triphosphate (ATP), is the transmitter released from purinergic inhibitory nerves present in Auerbach’s plexus (BURNSTOCK,CAMPBELL, SATCHELL & SMYTHE,1970). However, the role of ATP or a related nucleotide as humoral transmitter in the inhibition of intestinal motility has been questioned (KUCHII, MXYAHARA & SHIBA~A,1973). It has also been shown that adenosine is released from slices of the cerebral cortex (PULL & MCILWAIN, 1972). The present study was undertaken to analyse the effect of adenosine and closely related nucleotides on
the release of AC% from Auerbach’s plexus of the guinea-pig ileum and from isolated cortical slices of rat brain.
EXPERIMENTAL
PROCEDURES
Preparation of longitudinal muscle strip with attached bath’s plexus
Auer-
Longitudinal muscle strips of the guinea-pig ileum were prepared according to the method of PATON & VIZI (1969) and set up in an organ bath of 3.5 ml capacity in Krebs’ solution at 36”C, bubbled with 9.5% oxygen and 5% carbon dioxide. Physostigmine sulphate (2 x 10e6 g/ml) was added to the Krebs’ solution to prevent enzymic hydrolysis of acetylcholine. After 60 min preincubation time, ACh was collected for assay. In those experiments in which the contractions to electrical field stimulation were recorded, physostigmine was not added to the Krebs’ solution. fso~ured corticaI slices of rat brain
The preparation was made according to the method of VIZI (1972). Rats weighing 120-170 g were killed by stunning. The brains were immediately removed from the skull. A slice of cortex was cut from each hemisphere. Two to four slices (each weighing 70-120 mg and less than 0.8 mm thick) were incubated at 36°C in a glass chamber of 2ml containing Krebs’ solution gassed with 5% carbon dioxide Abbre~iutions: ACh, acetylcholine; ADP, adenosine in oxygen. Before the collection of the first sample, the diphosphate; AMP, adenosine monophosphate; ATP, slices were allowed to equilibrate for 6Omin in the eseradenosine triphosphate. inized Krebs solution. 391 NSC1+--c
392
E. S. VIZ.1and J. Kc01
A~tylcho~ine coltected either from lungitudinai muscle strips or from cortical slices was assayed on isolated guinea-pig ileum suspended in 3Sml Krebs solution at 33“C by the method of PATON& VIZI (1969). Since prolonged isometric contraction tended to cause spontaneous movements of the ileum, a soft spring of 0.5 g/cm compliance was interposed between the proximal end of the ileum and the transducer. The contractions were recorded on a potentiometric recorder (SERVOGOR, GOERZELECTRO). To eliminate the possibility of interference with the assay by drugs present in the assay sample, responses to standard solutions of ACh were recorded in the presence of these drugs in concentrations equivalent to those reached by addition of the sample containing the drug. When cholecystokinin was present in the bath, tetrodotoxin (5 x lo-‘M) was added to the assay bath to control for polypeptide carried over in the sample, since it has been found that ~etrodotoxin is able to inhibit completely the contractile action of gas&in-like polypeptides on guineapig ileum whiIe having no effect on the response to ACh (VIZ], BERTACCINI, IMPICCIAT~KE & KNOLL. 1973). Acetyicholine dissolved in saline in volumes of 0.05-0.3 ml was added to the assay bath. Physostigmine sulphate (5 x IO-“g/l) and morphine sulphate (1O-3 g/l) were added to the Krebs’ solution in the assay bath in order to increase the sensitivity of the ileum to ACh and to reduce endogenous ACh release. After 60min incubation time the lowest amount of ACh still was O.Sng/3.Sml (5.2 x lo-‘*M; reliably assayed 0.14 ng/ml acetylcholine iodide). Stimulution technique
the resting release of ACh (Tabie 1) to about half the control value. When the co~lcentration of ATP was increased to IO--” M, no further decrease in ACh release was seen. No tachyphyiaxis was observed to the action of ATP when it was administered repcatedly. The action of ATP was dependent on concentration below 10.’ M. In one experiment ATP in a concentration of IO’-’ M reduced the release of ACh by only IX”,. Much of the resting release of ACh was due to the spontaneous activity of neurons, since it was reduced by 601:,, in the presence of tetrodotoxin (lOeh~); under the latter conditions the residual release of ACh was not affected by adenosine (IO -a M). The inhibitory effect of ATP on the release of ACh induced by electrical stimulation depended on the frequency of stimulation applied. As shown in Table 1. the reduction in output produced by ATP was, at 0.2 Hz, 60.81,, and, at 10 Hz, only 2X,. Figures la and b show the effects of adenosine and ATP respectively on ACh release induced by 0.2 Hz stimulation. The effect of ATP was longer lasting than that of adenosine: after it had been washed out. the release of ACh in the next collection period was still significantly lower (by about 43”,,) than the initial value. When the tissue was exposed to a higher concentration of ATP (10m3 M) there was no further reduction in ACh release induced by 0.2 Hz stimulation. However, it took 30 min for the rate of release to return to about 95”‘, of the control value. ltlhihition
by &nine
nuclrorid~,s
induced
Precention
h!, cllolec~stolrinin.
by theophytline
of utlmin~ nucleotides
qf the ~rl~aibitory action
release. Theophylline, a blocker of adenosine receptors (McILWAN 1972). by itself enhanced the release of ACh induced by stimulation (Fig. 2). The release induced by either low (0.2 Hz) or high (10 Hz) frequency of stimulation was enhanced. At low frequencies of stitnulation the potentiation was more pronounced than at high frequencies. Adenosine, which otherwise reduced the release of ACh, failed to affect the release in the presence of theophylline (Fig. 2). The inhibitory effect of ATP on the ACh released by stimulation at 0.2 Hz was also partly prevented by theophylline (Table 1). on trcetylcholine
The +@ct qf adenosine and adeno,s~~~etr~phosphote the releuse~~~~etylcho~ine from
of therat
RESULTS A&nine
nucleotides
Auerbach’s Inhibitory
and
acetyicholine
e@ct of adenosine and adenosine triphos-
phate on the resting
and stimulated release of acetyiAdenosine, plexus.
Auerbach’s from 4 x 10e5 M, and ATP, 1W4~,
choline
releuse ./iom
plexus
si~i~~~tly
reduced
oj‘ lrcetylcholine
Cholecystokinin, like other gastro-intestinal hormones (Vrzr er al.. 1973). enhanced the release of ACh from Auerbath’s plexus (Table 1). However, the ACh-releasing effect of choIecystokil]in was completely blocked by adenosine. rcktrsc,
Field stimulation was used (PATON& VIZI, 1969). Supramaximal (10 V cm- ‘) square wave pulses of 1ms duration were applied through platinum electrodes at the top and bottom of the organ bath, at a frequency of 0.1-10 Hz. The Krebs’ solution used had the following composition (mM): NaCI, 113; KCl, 4.7; CaCI,, 2.5: KH,PO,, 1.2; MgSO,, 1.2; NaHC03 25.0: and glucose, 11.5. When calcium enriched solution (5 mM) was used, NaHCO, was omitted to avoid precipitation and replaced by equimolar amounts of NaCl. The following drugs were used: acetylcholine iodide (BDH Chemicals, Poole, England), physostigmine sulphate (Burroughs Wellcome); adenosine (Reanal), adenosine monophosphate disodium (Reanal), ~holecystokinin pankreo~ymin (chemistry Dept., Karolinska Institute, Stockholm, Sweden), theophylline methanesulphonate (CIBA): (Richter); phentolamine N6-20-Dibutryl cyclic AMP (Boehringer); tetrodotoxin Sankyo); BAY-1470, 2-(2,6-xylifamino)-5,6-dihydro-4H1,3-thiazine (Bayer). The drugs were dissolved in 0.90,; w/v NaCl solution. Concentrations of the drugs are expressed in molar concentration, and sometimes given also in terms of their salts.
1
j.so~ated cortical
OS
slices
(see Tahk ?)
Adenosine, by itself, appeared to reduce the resting release of ACh, but the reduction proved to be not significantly theophylline However. significant. enhanced the release by 369;. ATP, lo-” M, also failed to affect the release. Morphine, which enhances the cyclic AMP level in nerve endings of rat cortical slices
393
Adenine nucleotides and ACh release TABLE1. THE EFFECTOF ADENOSINE NUCLEOTIDES AND THEOPHYLLINE ON ACIITYLCHOLINE RELEASEFROM ALVZRBACH'SPLEXUS-LONGITUDINAL MUSCLE PREPARATION
Acetylcholine release, S.E.M. (pm01 g-I min-‘)
Collection period (min)
Condition 1. 2. 3. 4.
Control (35) Adenosine, 4 x 10-s M (4) ATP, 1W4M (4) Cholecystokinin 10-’ M (3) 5. Cholecystokinin lo-‘M + Adenosine, 4 x lo-’ M (3)
6. 0.2 Hz (14) 7. 0.2 Hz + ATP, 1O-4 M (3) 8. 0.2 Hz + Theophylline, 1.7 X lo-4M (3) 9. 0.2 Hz + Theophylline, 1.7 x 10-4~ + ATP, 10-4~ (3) 10. 0.2 Hz, Ca’+-excess (3) 11. 0.2 Hz Cazc-excess + ATP, 1O-4 M (3) 12. 10Hz (3) 13. 10 Hz + ATP, lo+ M (3) 14. 0.2 Hz + Phentolamine, 2.5 x 10-6M (2) 15. 0.2 Hz + Phentolamine, 2.5 x 10-6~ + ATP, 10-4M (2)
Significance (P)
Resting 10 10 10
162.4 + 2.8 85.3 + 3.8 80.1 + 6.9
2:l < 0.01 3:l < 0.01
10
395.9 + 36.6
4:l < 0.01
10
96.4 + 8.1
5:4 < 0.01
Electrical stimulation 10 420.8 + 11.1 10 132.3 + 17.5
6:l < 0.01 7:6 < 0.01
10
518.4 + 24.9
8:6 < 0.05
10 10
346.5 k 30.8 461.5 f 26.8
9:7 < 0.01 10:6 > 0.05
10 1 1
159.8 + 28.3 3068.2 + 160.5 2980.0 + 310.0
11:7 > 0.5 12:l < 0.01 13:12 > 0.5
10
372.82
10
70.6
Number of experiments in brackets. Ca’+ -excess: CaClr concentration
(2.5 mM) has been doubled.
(CLOUET, GOLD & IWATSUBO,1975) did not a&t
the release. Ouabain, an inhibitor of Na+-K+-activated ATP-ase, enhanced the release in three experiments, as already shown by VIZI (1972). Adenosine, in a concentration of 3 x 10e5 M, slightly reduced the ouabain-induced release. In comparison to the reduction
Adenosim, Cx 10'M
m e
stim.
Tc ‘fG &lllJ
10 min -
%I
_--__
8,
-
:: ,o xL
0.2Hz
___-_
_____
.__._---2a).
_-__. _____
G
B
-g
ATP.
lo-& M 1stim. 0.2Hz
FIG. 1. Inhibitory effect of adenosine (a) and ATP (b) on the release of acetylcholine induced by 0.2 Hz stimulation. Longitudinal muscle strip of guinea-pig ileum. The values for acetylcholine release (pmol g-i min- I) are the means of three identical experiments. Dashed lines indicate S.E. Note the rapid offset of adenosine action.
to 92
92
10 - HZ
FIG. 2. Prevention by theophylline of the inhibitory action of adenosine on acetylcholine release from Auerbach’s plexus. Longitudinal muscle strip of the guinea-pig ileum. The values for acetylcholine release (pmol g-r min- ‘) are the means of two experiments with identical treatment schedules. Stimulation period as indicated was 1 min at 10 Hz (1 ms, 10 V/cm). Note that theophylline enhances the release of ACh and prevents the inhibitory effect of adenosine.
E. S. VIZl and J. KNOLL
394
TABLE 2. THE EFFECt OE ADENOSINE, ~-IEOPHYLLI~, OU~aN, MORPHINE AND BAY-1470 ON TIlE RESTING ACETYLCHOLINE
RELEASE FROM ISOLATED CEREBRAL CORTICAL
Collection period (min)
Drug 1. - -
2. 3. 4. 5. 6. 7. 8.
Adenosine 3 x 10-5M ATP, 10-4M Theophylline, 1.7 x 10-4M Morphine 3 x 10-SM BAY-1470, 10-SM Ouabain, 2 x 10-SM Ouabain, 2 x 10-SM + Adenosine, 3 x 10-SM 9. Ouabain, 2 x 10-SM + BAY-1470, 10 -5 M
SLICES OF THE RAT
Acetylcholine release S.E.M. (pmol g- 1 min- 1)
10 10 10 10 10 10 10
23.97 18.97 19.2 32.7 19.0 21.8 218.3
± + _ + + _ ±
10
165.5 ± 21.8(3)
10
62.8 ± 15.6(3)
Significance (p)
2.18 (8) 2.8(3) 3.1 (3) 1.8 (4) 2.1 (3) 1.1 (3) 58.2(3)
2:1 n.s. 3:1 n.s. 4:1 < 0.01 5:1 n.s. 6:1 n.s. 7:1 < 0.01 8:7
n.s.
9:7 < 0.01
Number of experiments is in brackets. produced by BAY-1470, a pure ~-adrenoceptor stimulant, however, the reduction is negligible (Table 2).
Inhibitory effect of adenine nucleotides on neuro-effector transmission of the electrically stimulated longitudinal muscle strip of guinea-pig ileum To study the effect of adenine nucleotides in the absence of a cholinesterase inhibitor, the contractions of the guinea-pig ileum longitudinal muscle strip were measured under conditions where the contraction is due to the release of ACh from Auerbach's plexus. Isometric contractions in response to stimulation were recorded. Adenosine (10-6-3 x 10 - s ra), A M P (10-6-10 -4M), A D P (10-6-10 -4 M) and A T P ( 1 0 - 6 - 1 0 -4 M) reduced the size of contractions of the electrically stimulated longitudinal muscle strip without influencing the sensitivity of the smooth muscle cells to added acetylcholine. The effect was rapid in onset. After washing out, a fast recovery was observed. The reduction depended on the concentrations used and the frequency of stimulation applied
(Figs. 3 and 4): the contractions induced by high frequency stimulation were not affected by the nucleotides used. Repeated exposure to adenine nucleotides did not result in a diminution of the inhibitory action. However, it was observed that at higher concentrations ( > 1 0 - 4 M ) A T P caused a contraction of the s m o o t h muscle. Theophylline, in concentrations up to 2 x 10 - 4 M, slightly enhanced the responses of the muscle strip to stimulation at a frequency of 0.2 Hz; this action proved to be dose dependent. If added before the nucleotides, it prevented their inhibitory action (Fig. 5). Since in three experiments theophylline failed to potentiate the effect of exogenous A C h on the s m o o t h muscle, it is concluded that its main site of action is o n Auerbach's plexus. This is confirmed by the observations on the effect of theophylline on ACh rain --
o
i i i ~ ~ ~ ~ z i ~
mill •
:
i
i
,
shocks 5 10 10 50 Hz 1 510 10
:
:.~
! ~
~!
510 10 50 1 5 10 10
~
:
:
0,1 HZ
slim. T
X lo'6M
30
15
7.5
3
T Adenosine
FIG. 3. The inhibitory action of adenosine on the responses of longitudinal muscle strip of guinea-pig ileum to field stimulation. Field stimulation, 1 ms, 10 V/cm, 0.1 Hz when stimulation (stim.) is indicated. T = 10Hz, 10 shocks. Adenosine was administered in different concentrations as indicated. Note the inhibitory action of adenosine on the responses of the strip to 0.1 Hz but not to 10Hz stimulation.
LATP, IO'~H J
FIG. 4. The inhibitory effect of ATP on the responses of longitudinal muscle strip of guinea-pig ileum to field stimulation with different frequencies and with different length of trains. Field stimulation, l ms, 10 V/cm. Krebs solution. 95~o 02 + 5~o CO2. Organ bath 3.5 ml. Note the inhibitory action of ATP on the responses of longitudinal muscle strip to 0.1, 1, 5 and to 10 Hz stimulation with short train (10 shocks). However, ATP had no effect when 10Hz stimulation in a longer train (50 shocks) was applied.
Adenine nucleotides and ACh release
395
min r---1
=
'
I
j I '
/ o
,
I'I
I
//
=
o
/
v
o
stim. 0,1Hz
t
~w
Adenosine (M) 1
t
o 4,
t t~w
1 2 3 .............. 1,7 x 10-4M
x10 -5 Theophyl.lJne
FIG. 5. Prevention by theophylline of the inhibitory effect of adenosine on the contractions of longitudinal muscle strip of gninea-pig ileum in response to 0.1 Hz stimulation. Field stimulation, 1 ms, 10V/cm. Krebs' solution. 95% 02 + 5% CO2. Organ bath 3.5 ml. w = wash out. Note the effect of adenosine was antagonised by theophylline. By increasing cumulatively the concentration of adenosine the control effect of adenosine was reached. release (Table 1). Recording the contractions of the strip in response to stimulation has the advantage that the effect of nucleotides can be repeatedly studied, and a dose-response curve in the absence and presence of antagonist can be obtained. The nature of the antagonism between adenosine and theophylline has also been studied (Fig. 6). It can be seen that theophylline competitively antagonised the inhibitory effect of adenosine on ACh release. Phentolamine, in a concentration of 10 -6 M did not antagonize the effect of adenosine. Guanosine triphosphate, in concentrations up to 2 × 10 -4 M, failed to affect the contractions of the strip in response to stimulation at 0.1 Hz. Unlike adenosine, the dibutyryl derivative of adenosine 3'5'-monophosphate, in concentrations ranging from 10 -6 to 10 -4 M, did not reduce the size of the contractions. In a higher concentration (10-3 M) however, it reduced both the responses to electrical stimulation, and the contraction caused by ACh added to the organ bath.
"
-3
J. log concn, of theophylline
FIG. 6. Competitive nature of the antagonism by theophylline of the depressant effect of adenosine on the contractions of stimulated longitudinal muscle strip of guinea-pig ileum. For method see Fig. 5. Each point represents three experiments. The plot of log (DR-l) against the log concentration of theophylline produced a straight line with a gradient of 0.910 and an intercept on the abscissa corresponding to log Ke. DR, the ratio of adenosine concentrations in the presence and absence of theophylline required to produce the same effect. Ke, concentration of antagonist which produces a dose ratio of 2:for theophylline 2.1 x 105 M. The exposure time for theophylline is 20 rain. Since the regression, 0.920 does not differ significantly from unity the antagonism is competitive.
Inhibitory effect of adenosine on the responses of the longitudinal muscle strip to cholecystokinin It has been shown that cholecystokinin acts indirectly on the intestinal smooth muscle by stimulating cell bodies: and causing the release of ACh from Auerbach's plexus (VIzI et al., 1973; VIzI, 1973). The contraction of the smooth muscle caused by choleoystokinin was completely inhibited by adenosine and the closely related nucleotides, AMP, A D P and ATP. The responses of the smooth muscle to ACh were, however, not affected. This result also indicates that the effect of nucleotides is on the nerve tissue. A typical experiment is shown in Fig. 7. Adenosine reduced the size of the contractions in response to stimulation with 0.1 Hz and completely inhibited the contraction induced by cholecystokinin. After washing out adenosine and cholecystokinin there was a contraction (Fig.
rain
i stim.
0.1Hz
. . . . . . .
OO
O
ACh
ACh
24
Adenosine
2
~
O CCK 1
~
O
-CCK 1
x 10-$ M
="=='===
2
1
-5
xlO
M
FIG. 7. Inhibitory effect of adenosine on the contractions of longitudinal muscle of guinea-pig ileum in response to stimulation and to cholecystokinin. Field stimulation, 0.1 Hz, 1 ms, 10 V cm -1. CCK, cholecystokinin.
396
E. S. Vu
and J.
n Adenosine ATP etc.
FIG. 8. Scheme of the site of action of ATP or related nucleotide on Auerbach’s plexus. Dotted line indicates the inhibitory effect of ATP or related nucleotides on ACh reiease, suggested by this paper, and on the smooth mu&e, suggested by BURNST~CK (1972). 7) which could be blocked by tetrodotoxin (1O-6 M), indicating that it might be caused by the polypeptides still present in the tissue. DISCUSSION The results provide evidence that adenine nucleotides are able to reduce the release of ACh from Auerbath’s plexus of the guinea-pig isolated ileum, when the release is due to firing of the nerves, but the basal release in the absence of nervous activity, i.e. when tetrodotoxin was present, was not inhibited by adenosine. However, from the isolated cortical slice of the rat the ACh release during rest, and the ouabaininduced release, were not affected by adenosine or by ATP. Theophylline, an antagonist of adenosine receptors (MCILWAIN,1972), enhanced the release of ACh from Auerbach’s plexus and from cortical slices, and prevented the inhibitory action of the nucleotides. Frequency dependence of the effect of adenine nucleotides
The reduction in ACh release from Auerbach’s plexus by adenine nucleotides was pronounced only when a low frequency of stimulation was used. At high frequencies of stimulation (10Hz) they failed to affect the release. The release of ACh induced by cholecystokinin was completely inhibited by adenosine. There was no significant difference between the potencies of the adenine nucleotides in their ability to inhibit neuro-effector transmission in the stimulated longitudinal muscle strip of guinea-pig ileum. Adenine and guanosine triphosphate were however, without any effect. The fact that theophylline enhanced the release of ACh both from Auerbach’s plexus and from the cortical slices, raises the question whether endogenous adenosine might control the release of ACh, and that the released ACh me~ured is in fact, an already reduced output. This could also explain why the output of ACh per stimulus is very low when a high
KNOLL
frequency of stimulation is applied (KNOLL & Vizr, 1971) a situation in which greater amounts of adenosine might be released. A possible explanation for the lack of effect of adenosine on the release of ACh from the cortex is that the output from cortex is continuously controlled by adenosine or by some of its related compounds. Adenosine, in fact, has been shown to be released from the cortex by stimulation (MCILWAINE, 1972: PULL & M~ILWAIN, 1972) and from purinergic nerves in smooth muscle (BURNSTOCK er ~1.. 1970). In the adrenal medulla. ATP is stored together with, and is released with, catecholamines (DOUGLAS& POISNER,1966). It has been reported that the vesicles in cholinergic nerve terminals contain besides ACh, considerable amounts of nucleotides (WHI~A~R, D~WDALL & BOYS, 1972). Therefore, it is suggested that adenine nucleotides, if rdeased from the vesicles, might inhibit the release of ACh from the nerve terminals. Another possibility is that adenosine or related nucleotides are released from specific nucleotide containing axons (Su, BEVAN& BLIRNSTOCK 1971; BURNSTOCK rf ui., 1970; cf. BUKNSTOCK, 1972). In agreement with the results reported in this paper it has been shown that adenosine reduces transmitter release from the phrenic nerve of the rat (GINSBORG & HIRST,1972). On the other band, cyclic AMP, theophylline (GOLDBERG& SINGER,1969) and adrenaline (KRNJEVIC& MILEDI, 1959) have been shown to be able to increase the frequency and amplitude of the miniature endplate potential. KENTERA& VARAGIC (1975). however, showed that at least one of the sites of action of factors known to increase the intracellular concentration of cyclic AMP is located on the effector cells.
Site C$ action
of adenine nucleotides
The question
now arises as to where and how adenosine acts? Since adenine nucleotides are capable of reducing the release of ACh induced by postganglionic stimulation, their site of action has to be at least partly on the nerve terminals. However, an effect on the site -.where action potential generation takes place cannot be excluded (Fig. 8). This might explain why adenosine completely blocks the ACh-releasing effect of cholecystokinin which stimulates the cell body of the axons. The mode of action of adenine ~u~~eoiides
Two possible modes of action of the adenine nucleotides are (i) they might prevent the spread of impulses to the nerve terminal varicosities. This is unlikely since the ACh release induced by high frequency stimulation is not affected by the adenine nucleotides, (ii) they might prevent the entry of Ca2+ ions (into the nerve terminals), but their inhibitory action is not prevented by Ca2’ excess (Table 1). It has been suggested that there is a correlation between the inhibitory etfect of noradrenaline on ACh release and its stimulant action on Na+-K+-activated ATP-
Adenine nucleotides and AC%release pase (VIZ, 1972, 1975). Since adenosine and related nucleotides act in many respects in a similar way to noradrenaline, it is tempting to speculate that adenosine and other nucleotides reduce the release of ACh by stimulating membrane ATP-ase activity, a fact which has been shown biochemically for ATP (ROBW SON, 1973; YOSHIDA, NAGAT, OHASHI & NAKAGAWA, 1969). The only difference in the effect between adenosine and noradrenaline
is that the effect of adenosine
can
be blocked by theophyiline, and that of noradrenaline by phentolamine (VIZI, 1968; PATON & VIZI, 1969). MCAFEE & GREENGARD (1972) suggested that cyclic AMP
mediates dopaminergic
by modifies cholinergic
transmission
and there-
transmission
in the sympathetic ganglion. They concluded that an increase in cyclic AMP levels in the ~stgang~ionic neurons makes the post-synaptic membrane less responsive to subsequent excitatory input. Our data cannot be explained in this way since we found that theophylline increased the release of ACh and prevented the inhibitory effect of adenosine. But theophylline is an inhibitor of phosphodiesterase and therefore enhances the concentration of cyclic AMP, while adenosine is another compound that is known to increase tissue levels of cyclic AMP (MCILWAIN, 1972). It has been suggested that adenine nucleotides trol neuroeffector
transmission
at the level of the axonal impulse
generation
in Auerbach’s
membrane
conplexus
by impairing
and stimulus-secretion
397
The fact that theophylhne, an antagonist of the adenosine receptors of the cerebral cortex, enhanced the release of ACh from the nerve terminals of rat cortex, indicates that a similar mechanism might operate in the central nervous system as well. The main question now is whether endogenous nucleotides reach a sufficiently high local concentration that they can play a physiological modulatory role in cholinergic transmission by inhibiting the release presynaptically. The hypothesis is supported by the fact that theophylline, possibly by removing the inhibitory effect of endogenous adenine nucleotide(s), enhances the release of ACh both from Auerbach’s plexus and from cortical slices. Since gastrin-like hormones have been shown to play an important role in maintaining g~troint~tin~ activity by releasing ACh from Auerbach’s plexus, the inhibitory effect of adenosine and related nucleotides, as well as noradrenaline, might be important in controlling cholinergic outflow enhanced by cholecystokinin. As well as their direct action on the smooth muscle (DRURY & SZENTGY~RGYI, 1929; cf. BURNSTOCK, 1972) the nucleotides directly affect Auerbach’s plexus. The two actions would both cause a reduction in intestinal motility. However, the effect of endogenous and exogenous ACh on the smooth muscle is only slightly if at all, affected by nucleotides, which indicates that the presynaptic type of inhibition is of greater importance in reducing intestinal motility.
coupling.
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DOIJGLASW. W. C POISNERA. M. (1966) On the relation between ATP splitting and secretion in the adrenal chromaffin cell: extrusion of ATP (unhydrolysed) during release of ~techol~ines. f. Phys~o~.,Land, 183, 249-256. DRURY A. N. & SZENTGY~RGYIA. (1929) The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J. Physiol., Land. 68, 213-237. GINSEIORG B. L. & HIRST G. D. S. (1972) The effect of adenosine on the release of the transmitter from the phrenic nerve of the rat. J. Physiol., Lond. 224, 629-645. GOLDBERGA. L. & SINGERJ. J. (1969) Evidence for a role of cyclic AMP in neuromuscular transmission. Proc. natn. Acad. Sci., U.S.A. 64, 134-141.
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E. S. Vm and .r. i(wx.r
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