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Peripheral and central actions of AF64A (ethylcholine mustard aziridinium ion) on acetylcholine release, in vitro: Comparison with hemicholinium
Neuroehem. Int. Vol. 7, No. 6, pp. 1047-1053, !985 Printed in Great Britain. All rights reserved
0197-0186/85 $3.00+ 0.00 © 1985 Pergamon Press Ltd
PERIPHERAL AND CENTRAL ACTIONS OF AF64A (ETHYLCHOLINE MUSTARD AZIRIDINIUM ION) ON ACETYLCHOLINE RELEASE, IN VITRO: COMPARISON WITH HEMICHOLINIUM P. E. POTTER*, L. G. HAgS!N~3 Jr, I. KAgucsg^, G. GAAL and E. S, Vlzl Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O.K 67, H-1450 Budapest, Hungary (Received 8 February 1985; accepted 1 April 1985)
Abstract--The acute effects of ethylcholine mustard aziridinium ion (AF64A) and hemicholinium-3 (HC-3) on the release of endogenous acetyleholine (ACh) from isolated tissues were examined, Whereas addition of HC-3 (10-s-10 ~5 M) significantly reduced the output of ACh from isolated guinea-pi 8 ileum longitudinal muscle strip elicited by 10 Hz stimulation, AF64A had no effect and even enhanced the release of radiolabel elicited by 1 Hz stimulation when this tissue was pre,loaded with [3H]choline. Similarly, HC-3 (10 -s M) reduced ouabain-induced endogenous ACh release from isolated rat hippocampus, Addition of AF64A (10-s-5 × 10-s M) caused a slight increase in ACh release, In isolated rat cortex, however, AF64A did not affect ACh release. Moreover, AF64A caused a decreale in ouabain.stimniated ACh releate from striatum. The present study indicates that: (a) the/n vitro actions of AF64A differ from those of HC.3 and (b) the acute effects of AF64A on endogenous ACh release vary, depending on the tissues studied and the stimulation parameters used,
(Mantione et al., !981; Fisher et al,, 1982), In the rat, similar lesions were produced by intrahippocampal and intrastriatal injections of AF64A (Mantione et al., 1983b; Sandberg et al., 1984), and i.c.v, administration has been shown to reduce ACh release (Potter et al., 1984; Leventer et al., 1984, !985). The mechanism by which AF64A causes functional lesions of cholinergic neurons is not clear. A number of studies have documented that this compound causes an irreversible inhibition of HAChT in vitro (Rylett and Colhoun, 1980; Sandberg et al,, 1982; Mantione et al,, 1983b; Curti and Marchbanks, !984). ChAT is also inhibited /n vitro by AF64A (Sandberg et al., 1982; Mantione et al., !983b; Pedder and Prince, 1983). It seems possible that AF64A is taken up into the cholinerglc nerve terminal by HAChT system and subsequently interacts with *Address correspondence to: Pamela E. Potter, Room E-1238, Western Psychiatric Institute and Clinic, Uni- CHAT, because in synaptosomes the inhibition of versity of Pittsburgh School of Medicine, 3811 O'Hara ChAT by AF64A can be prevented by the addition Street, Pittsburgh, PA 15213, U.S.A. of hemicholinium (HC-3), in concentrations Abbreviations used: ACh: acetylcholine; AF64A: ethyl(4 × 10-6-4 x 10 ,s M) sufficient to completely inhibit choline mustard aziridinium ion; CHAT: choline acetyl- HAChT (Pedder and Prince, 1983), transferase; HAChT: high affinity choline transport; Inhibition of HAChT in vitro by AF64A would be HC-3: hemicholinium-3; HPLC: high performance expected to lead to a decline in cho!incrgic transliquid chromatography. 1047
Ethylcholine mustard aziridinium ion (AF64A) is one of a group of choline mustard analogues that has been shown to inhibit the high affinity uptake of choline into synaptosomes (Rylett and Colhoun, 1980). This compound has been proposed as a neurotoxin selective for cholinergic neurons (Mantionc et al., 1981). When injected intracerebroventricularly (i.c.v.) into the mouse, AF64A produced a longlasting, marked reduction in presynaptic cholinergic indices, such as acetylcholinc (ACh) levels, high affinity choline transport (HAChT) and choline acetyltransferase (CHAT) activity in hippocampus, striatum and cerebral cortex, but did not affect the binding of [3H]QNB, a marker for postsynaptic cholinergic muscarinic receptors in these tissues
1048
P. [-. PolffR e; al.
mission, as blockade o f choline re-uptake from the synaptic cleft should decrease ACh synthesis and reduce the ACh available for release. Indeed, A F 6 4 A has been s h o w n to inhibit cholinergic transmission in the rat phrenic nerve h e m i d i a p h r a g m preparation (Rylett and Colhoun, 1978). Furthermore, contractions o f the cat nictitating m e m b r a n e elicited by preganglionic nerve stimulation were diminished by AF64A, but the responses to postganglionic stimulation or to cholinergic agonists were unaffected ( M a n t i o n e et al., 1983a). High frequency nerve stimulation accelerated this effect (Mantione et al., 1983a), suggesting that it resulted from depletion o f A C h stores. Such an effect o f inhibiting H A C h T was d e m o n s t r a t e d more directly by Somogyi et al. (1977), who found that addition o f HC-3 to the isolated guinea-pig ileum longitudinal muscle strip markedly reduced the release o f e n d o g e n o u s ACh evoked by continuous 10 Hz stimulation and also decreased the A C h synthesis rate. The aim o f the present study was to investigate and c o m p a r e the effects o f A F 6 4 A and HC-3 on the release o f endogenous A C h from various isolated tissues. The effects o f A F 6 4 A varied depending on the tissue and the stimulation parameters used. Furthermore, the acute effects of A F 6 4 A and HC-3 differed; HC-3 reduced A C h release from the guinea pig ileum longitudinal muscle strip and rat hippocampus, whereas A F 6 4 A enhanced A C h release from these tissues. EXPERIMENTAL PROCEDURES
Preparation o f AF64A AF64A was prepared prior to each experiment by basic hydrolysis of 1 or 10mM acetylethylcholine mustard and subsequent aziridinium ion formation, as described by Fisher et al. (1982). It has been shown by both nuclear magnetic resonance and secondary ion mass spectrometry that, with the starting compound used and the conditions described below, conversion of the mustard to the deacetylated aziridinium product is in excess of 95% (Vincze et al., 1981; Fisher et al., 1982). AF64A prepared in this manner has been shown to inhibit HAChT in vitro (Sandberg et al., 1982; Mantione et al., 1983b). In addition, the AF64A prepared for these experiments was also injected i.c.v, in rats and produced the cholinergic deficits characteristic of this compound (Potter et al., 1984), indicating that the compound was active. Briefly, the pH of the solution was adjusted to l 1.3-11.7 with 10NNaOH and the solution was stirred at room temperature for 30 min, then the pH was reduced to 7.4 with concentrated HC1. AF64A was diluted immediately before use in Krebs bicarbonate buffer of the following composition (mM): NaCI 113, CaC12 2.5, KH2PO 4 1.2, MgSO4 1.2, NaHCO 3 25, glucose 11.5 and eserine sulfate 0.0062. In studies where the release of [3H]ACh was measured, no eserine sulfate was present in the Krebs solution.
Measuremem q/endogenous A ('h re/ea.w /}om hil,p,~, ompu~. .,triatum or jrontal cortex ~[ ra;.s Male Wistar rats weighing approx~ 150 g were kilted by decapitation and hippocampi, striata, or frontal cortices were dissected by the method of Glowinski and t~crson (1966) and weighed. The pair of hippocampi, strlata or frontal cortices from each rat were placed into a 5 ml organ bath containing 2 ml of Krebs bicarbonate buffer, thermostated at 37C and oxygenated continuously wiltz 95~';, O: 5~ CO,. ARer l h pre-incubation to allow complete inhibition of acetylcholinesterase to develop, the Krebs bicarbonate buffer was changed at 10 rain intervals. After the second 10min period, AF64A or HC-3 fin concentrations from 10 6-5 x 10 5 M) were added to the Krebs solution 1"or the remainder of the experiment. After two further resting periods in the presence of AF64A Or HC-3. ACh release was stimulated by the addition of ouabain (2 x 10 5 Hz), an inhibitor ofNa ~ -K ", activated adenosine triphosphatase (Vizi, 1978). At the end of each 10rain period, the bath contents were collected and assayed for ACh by bioassay on the guinea-pig ileum, as described by Paton and Vizi (1969). Measurement of endogenous ACh release ,fi'om guinea-pig ileum longitudinal muscle strip The in vitro effects of AF64A and HC-3 on ACh release from the guinea-pig ileum were determined as described by Somogyi et al. (1977). The longitudinal muscle strip of the guinea-pig ileum with attached Auerbach's plexus; weighing about 100mg, was prepared as described by Paton and Vizi (1969). Strips were mounted in an organ bath and perfused with eserinized Krebs solution at a rate of 0.35 ml/min. Following l h preperfusion, two 6rain fractions were collected. Then the tissue was stimulated at 10 Hz, t ms, supramaximal voltage, with square wave pulses through platinum electrodes at the top and bottom of the organ bath. Samples of perfusate were collected for three 6rain periods, then AF64A (10~-5 x 10SM) or HC-3 (10 6 10 5M) was added to the perfusion solution and fractions were collected for seven additional 6 min intervals. The ACh content of the perfusate was determined by bioassay (Paton and Vizi, 1969). Tissues taken either before or after perfusion were placed in 2 ml of l 0')~, trichloroacetic acid for 90 min then disrupted in a ground glass homogenizer. The samples were centrifuged and the supernatant was removed and extracted 3 times with ether, then the aqueous phase was neutralized with 1'~ NaHCO 3 in the presence of methylorange indicator (Vizi, 1973). ACh content was measured by bioassay (Paton and Vizi, 1969). The net synthesis of ACh was calculated as described by Viziet al. (1972). Identification o f released ACh by H P L C The longitudinal muscle strip of the guinea pig ileum was prepared as described above and perfused at a flow rate of 0.5 ml/min. Following I h preperfusion, the tissue was stimulated at 10Hz, 1 ms, supramaximal voltage, with square wave pulses through platinum electrodes at the top and bottom of the organ bath. Two tissues were used: one served as a control and 10 5M AF64A was added to the perfusion fluid of the second tissue at the beginning of the stimulation period. After 10rain, perfusate was collected from each tissue for 20 min. Samples of perfusate, or ACh standards (36.6/~M) dissolved in Krebs buffer,
In vitro effects of AF64A were frozen on dry ice and dried under vacuum. The samples and standards were redissolved in 200 #1 of 0.01 M Na acetate--citric acid, pH 4. A 20/~1 aliquot of each was injected into a Biotrouik high performance liquid chromatography (HPLC) system (BT 3020 pump, Rheodyne 7125 injector with 20/~1 loop, and 15 crn Nucleosil 5 #m Cms column; Bischoff Analysentechnik and Biotronik Wissenschaftliche Gerate GmbH, Frankfurt am Main, West Germany) for separation of ACh as described by Viziet al. (1985). Mobile phase was 0.1 M N a acetate--citric acid, pH 4, with 1 mM tetraethylammonium at a flow rate of 0.7ml/min. Twenty 1 min fractions were collected and assayed on the guinea-pig ileum preparation for ACh content. The original samples and standards were diluted 1:50 and assayed by bioassay (Paten and Vizi, 1969). The ACh content of the original samples and that eluted from the HPLC column was calculated and the percent recovery was determined. In addition, 20/zl of [~4C]ACh iodide (sp.. act. 140 MBq/mmol) was injected into the HPLC and the radioactivity in the 1 min fractions was determined by liquid scintillation spectroscopy. Recovery was calculated by comparison with the radioactivity in a 20~1 aliquot of [14C]ACh which was not injected into the HPLC.
Measurement of [~H]ACh release from guinea-pig ileum longitudinal muscle strip The release of [3H]ACh during rest and in response to electrical stimulation was determined as described by Vizi et al. (1981). Longitudinal muscle strips weighing approx. 100 mg were incubated with methyl [3H]choline chloride (sp. act. 74 KBq/ml) for 30m in in a 3 ml organ bath. Field stimulation was applied at 1Hz for 15min during the loading period. The tissues were then transferred to another organ bath and perfused at a rate of 0.4 ml/min with Krebs buffer containing 10 -5 M HC-3. After 90 rain preperfusion, 5 min fractions of perfusate were collected. The tissues were stimulated (1 Hz, l ms duration, 8 V/cm) for 5 min during the 3rd and 1lth collection periods (S~ and $2). AF64A was added to the perfusion fluid immediately after the first stimulation. Radioactivity was determined in 1 ml aliquots of perfusate in a Packard Tri-Carb liquid scintillation spectrometer with scintillation fluid of the following composition: 2 parts toluene and 1 part Triton X-100, containing 3.3 g/l PPO and 0.2 g/1 POPOP. The stimulation evoked release of [3H]ACh was calculated as described by Szerb and Somogyi (1973). The activities in samples 1-2, 9-10, and 17-18 were used for the determination of basal rates of resting ACh release. An exponential curve was fitted to these values, based on the assumption that the rate of resting release can be described exponentially. The stimulation induced release of radioactivity in samples 3~i and ll-14 was calculated by subtracting the interpolated rate of resting release from the stimulated release. The $2/S I ratios were calculated from the values for S~ and $2 calculated in this way.
Drugs and chemicals Acetylethylcholine mustard, synthesized by Dr A. Fisher, was provided by Dr I. Hauin, Pittsburgh, PA. Drugs were purchased from the following sources: acetylcholine iodide (BDH), eserine sulfate (Calbiochem), tetraethylammortium bromide (Fluka), hemicholinium-3 (Aldrich), methyl [3H]choline chloride (sp. act. 2.15 GBq/mmol; Amersham), acetyl-l-[14C]choline iodide (sp. act. 140 MBq/mmol; New
1049
England Nuclear). All other chemicals were reagent grade.
Statistics For statistical comparisons the Student's two-tailed t-test was used. RESULTS
Release o f endogenous ACh from hippocampus, striatum and frontal cortex: Effects o f AF64A and HC-3 The resting release of A C h from isolated hippocampus was 3 7 . 7 + 2 . 1 pmol/g/min (n = 20). Addition of ouabain, 2 x 10 -5 M, increased the release to 252.8 __+11.4 pmol/g/min (n = 20). The effects of A F 6 4 A and HC-3 on release of endogenous A C h from isolated hippocampus are shown in Fig. 1. HC-3, in concentrations of 10 -6 and 10 -5 M, caused significant reductions in the ouabain stimulated release o f ACh. In the presence of 1 0 - S M HC-3, A C h release was reduced by 38%. Neither concentration of HC-3 had any effect on the resting release o f ACh. Resting (4)
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Fig. 1. Effects of AF64A on HC-3 on release of endogenous ACh from isolated rat hippocampus. ACh release from isolated hippocampus was measured by bioassay on the guinea-pig ileum as described in Experimental Procedures. Hatched area indicates average of ACh release during two 10min resting periods. Total bar indicates ACh release during the second 10min of exposure to ouabain, 2 x 10 -5 M. Number of animals are indicated in parentheses. C--control. AF64A--10-6M, 10-SM, 5 x 10-SM. HC-3--10 -6 M, 10-5 M. *P < 0.05, **P < 0.01, Student's two-tailed t-test.
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Fig. 2. Release of endogenous A C h from perfused guineapig ileum longitudinal muscle strip. Isolated guinea-pig ileum longitudinal muscle strip was perfused at a rate of 0.35 ml/min and the A C h content of the perfusate determined by bioassay, as described in Experimental Procedures. Following 1 h pre-perfusiom two 6 min samples were collected, then the tissue was stimulated continuously at 10 Hz, with square wave pulses of 1 ms duration. After three 6 m i n periods, A F 6 4 A (10 6-10 5M) was added to the perfusion solution and fractions were collected for seven additional 6 min periods. Values shown are mean + SE. N u m b e r of tissues in parentheses. *P < 0.01. Student's two-tailed t-test.
I n c o n t r a s t to H C - 3 , A F 6 4 A d i d n o t r e d u c e A C h release at a n y c o n c e n t r a t i o n s t u d i e d . 10 - s M A F 6 4 A h a d n o effect o n A C h release, a n d 10 5 a n d 5 x 1 0 S M A F 6 4 A p r o d u c e d a s l i g h t (30--34~o) b u t s i g n i f i c a n t i n c r e a s e in A C h release e v o k e d b y o u a b a i n . T h e r e s t i n g release o f A C h w a s n o t affected. In t h e f r o n t a l c o r t e x , A F 6 4 A h a d n o effect o n A C h release. T a b l e 1 s h o w s t h e v a l u e s for A C h release
Control
AF64A, !l) :\'1
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67.7 ± ,~.7 725.8 _+84.8*
Isolated tissues were incubated and ACh release was measured by bioassay as described in Experimental Procedures. After 80rain preincubation, AF64A was added to a final concentration of 10 s M and incubation fluid was collected at 10rain intervals and assayed for ACh. Values are either the average of two 10 rain resting periods, or during the second 10 rain exposure to ouabain. 2 × 10 ~ M+ N - 4 in all groups. *P < 0.(/5. Student's two-tailed tqest
u n d e r r e s t i n g c o n d i t i o n s a n d d u r i n g e x p o s u r e to 2 × 10 5 M o u a b a i n . T h e s e v a l u e s d i d n o t differ f r o m c o n t r o l in t h e p r e s e n c e o f 10 5 A F 6 4 A . In c o n t r a s t , in t h e s t r i a t u m b o t h t h e r e s t i n g release a n d t h a t m e a s u r e d d u r i n g t h e s e c o n d 1 0 r a i n p e r i o d o f exp o s u r e to o u a b a i n were s i g n i f i c a n t l y r e d u c e d by a d d i t i o n o f 10 5 M A F 6 4 A .
Effects o f AF64A on H C - 3 on release and synthesis q[ ACh in guinea-pig ileum longitudinal muscle strip In a p r e v i o u s s t u d y ( S o m o g y i et al., 1977) it w a s shown that addition of HC-3 during continuous 10 H z s t i m u l a t i o n o f t h e i s o l a t e d p e r f u s e d g u i n e a - p i g i l e u m l o n g i t u d i n a l m u s c l e s t r i p r e s u l t e d in a m a r k e d r e d u c t i o n o f b o t h A C h release a n d s y n t h e s i s rate. In t h e p r e s e n t s t u d y , H C - 3 (10 ~ a n d 10 ~ M ) also c a u s e d a s i g n i f i c a n t r e d u c t i o n in A C h release i m m e d i ately a f t e r a d d i t i o n to t h e p e r f u s i o n fluid ( d a t a for 10 6 M H C - 3 a r e s h o w n in Fig. 2). In c o n t r a s t , A F 6 4 A , in c o n c e n t r a t i o n s o f 10.6 a n d 10 ~ M , h a d
Table 2. Effect of AF64A and HC-3 on ACh content, release and synthesis rate in guinea-pig ileum longitudinal muscle strip ACh content after perfusion (nmol/g) Control AF64A 10 ~'M 10 5M HC-3 10 -6 M 10 5M
Total release in fractions 5-12 (nmol/g/h)
Synthesis rate (nmol/g/h)
107.0 +. 15.4 (13)
50.1 ± 5.5 (13)
69.6
92.8 + 20.0(4) 93.7± 13.6(5)
45.6 + 4.6 (4) 49.2+_ 10.8(5)
50.8 55 t
113.9 _+44.7 (4) 74.8 + 11.8(4)
18.2 __ 3.4* (4) 23.2_+ 1.6"(4)
44.5 10.4
ACh synthesis rate = (Content:met r~rr,~+o,,+ Total release) - Content~+~ r~fus,o,' ACh Content before perfusion = 87.6 _+7.9 nmol/g (26). Tissues were perfused and ACh content of perfusate fractions determined by bioassay as described in Experimental Procedures. ACh synthesis rate was calculated from ACh released in fractions 5-12, during continuous electrical stimulation at 10 Hz, 1 ms, 8 V/'cm, square wave pulses. Values are means + SE. Number of tissues in parentheses. *P < 0.05, Student's two tailed t-test, compared with control.
In vitro effects of AF64A no effect whatsoever on ACh release in this tissue. Figure 2 shows results with 10 -5 M AF64A; those obtained with 10 -~ M were almost identical. The ACh contents of the guinea-pig ileum longitudinal muscle strip were determined both before and after each experiment. The ACh content determined at the beginning of the experiments was 87.6 _+ 7.9 nmol/g (n =26). The ACh content measured at the end of the experiments is shown in Table 2. The content determined at the end of perfusion did not differ from that determined at the beginning, indicating that the tissues were able to synthesize sufficient ACh to replace that which was released. The ACh synthesis rate was calculated according to Vizi et al. (1972) from the total ACh released and the ACh contents measured before and after perfusion. The synthesis rate at rest was 4.1 nmol/g/h. The total ACh released and the ACh synthesis rate is shown in Table 2. Stimulation increased the ACh synthesis rate to 69.6 nmol/g/h. AF64A did not affect the ACh synthesis rate, but in the presence of 10 -5 M HC-3, it was reduced to 10.4 nmol/g/h.
Release of [ZH]AChfrom guinea-pig ileum longitudinal muscle strip The effects of 10-5 M AF64A were also determined in guinea-pig ileum longitudinal muscle strip that had been preloaded with [3H]choline. Since a previous study (Vizi et al., 1984) showed that virtually all of the radioactivity released in response to electrical stimulation of this tissue was labelled ACh, no attempt was made to separate [3H]ACh and [3H]choline. The result of this study is shown in Table 3. When added after the first stimulation period, AF64A caused a significant increase in
Table 3. Release of [3H]ACh from perfused guinea-pigileumlongitudinalmusclestrip s2/sl Control 0.81 ± 0.04(4) AF64A, I0-5 M added after SI 1.16± 0.08*(6) Guinea-pigileumlongitudinalmusclestrip was pre.incubated with [3H]cholinefor 45 rain, and stimulatedat 1Hz for the final 15rain. Tissues were perfused at 0.4 ml/min with Krebs solution containing 10-SM HC-3, and 3 min fractions were collected. Stimulation at 1Hz, 1ms durationfor 5rain, SI and $2. AF64A, 10-5 M, was added immediately after SI. Radioactivityreleased by each stimulationwas calculated and $2/S1 ratios determined. Means+ SE. Numberof tissuesin parentheses. *P < 0.01, Student'stwo-tailedt-test.
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fraction Fig. 3. HPLC separation of ACh in perfusates and ACh standards. A 20/~1 aliquot of p4C]ACh standard (A), ACh standard (B), or perfusate from guinea-pig ileum (C and D), dissolved in 0.01 M citrate--phosphate buffer, pH 3.5, was injected into the HPLC as described in Experimental Procedures. Mobile phase was 0.1 M Na acetate--citric acid, pH 4, with I mM tetraethylammonium bromide, at a flow rate of 0.7 ml/min. Twenty 1 rain fractions were collected and assayed either for radioactivity (A) or for ACh by bioassay on the guinea-pig ileum. A--elution pattern of [14C]ACh standard. B--elution of spasmogenic activity in ACh standard. C---elution of spasmogenic activity in pcrfusate of control guinea-pig ileum. D--elution of spasmogenic activity from perfusate of guinea.pig ileum in the presence of 10-5 M AF64A.
the $2/$1 ratio, indicating an enhancement of ACh release.
Identification of ACh in perfusates from guinea-pig ileum longitudinal muscle strip In order to determine the specificity of the bioassay used for the estimation of ACh contents, samples of perfusate from the guinea-pig ileum longitudinal muscle strip were lyophilized and injected into the HPLC. The pattern of elution of spasmogenic activity measured on the guinea-pig ileum was compared
with that of an ACh standard, as well as with the elution of radioactivity from the H P L C of a [~4C]ACh standard. The results are shown in Fig. 3. The majority of the radioactivity was elutcd m fraction 6 (Fig. 3A) with a recovery of 78%. When a lyophilized ACh standard was injected, the majority of the spasmogenic activity appeared in fraction 6 (Fig. 3B) and the recovery was 741~;. Elution patterns I\)r spasmogenic activity in perfusates from the guineapig ileum longitudinal muscle strip in the absence and presence of 10 s M A F 6 4 A are shown in Fig. 3C and D. The elution profiles and the recovery are identical with those of the standards: the spasmogenic activity eluted in fraction 6 and the recoveries were 751~,,in the control and 77'~i; in the sample containing AF64A. This finding strongly supports the assumption that the material measured by bioassay on the guinea-pig ileum is ACh and is not contaminated by other spasmogenic compounds. DISCUSSION Since A F 6 4 A has been shown to inhibit H A C h T in 1980; Mantione et al., 1983b) with a potency similar to HC-3, as well as to inhibit cholinergic transmission in the cat superior cervical ganglion (Mantione et al., 1983a) or rat hemidiaphragm-phrenic nerve (Rylett and Colhoun, 1978), it was expected that A F 6 4 A would act similarly to HC-3 when their in vitro effects on ACh release were compared. This, however, was not the case. Although HC-3 dramatically reduced the output and synthesis of A C h from the guinea-pig ileum longitudinal muscle strip, A F 6 4 A had no effect on endogenous A C h release during continuous 10 Hz stimulation. In order to investigate this further, the effect of A F 6 4 A on [3H]ACh release from guinea-pig ileum longitudinal muscle strip preloaded with [3H]choline was studied. In the presence of 10 S M AF64A, there was a significant enhancement of the 3H outflow induced by 1 Hz stimulation when compared to control tissues. This enhancement was not due to blockade of the re-uptake of [3H]choline, for the tissues were exposed to 10-5 M HC-3 throughout the experiment to block H A C h T . Further evidence that the action of A F 6 4 A was not due to inhibition of choline re-uptake is the observation that the ACh releasing effect of A F 6 4 A was not seen under resting conditions. Therefore, it appears that A F 6 4 A directly facilitated stimulated ACh release in the guinea-pig ileum longitudinal muscle strip. Similarly, the ouabain stimulated release of endovitro (Rylett and Colhoun,
genous ACh frmn the isolated rat h i p p o c a m p ~ was affected differently by HC3 and AF64A. Vv'hcreas there was a significant reduction in ACh outpui from hippocampus when HC-3 was added to the incubation fluid, ouabain stimulated ACh release was enhanced in the presence of AF64A. As in the guinea-pig ileum longitudinal muscle strip, the resting release of ACh was not affected by AF64A. l~here fore, A F 6 4 A appears to facilitate only the depolarization induced release of ACh from these tissues. When ACh release from the striatum or frontal cortex was measured, it was found that A F 6 4 A had no effect in the cortex and caused a slight reduction in ACh release from the striatum. In the striatum, both the ouabain stimulated and the resting release of ACh was reduced. Clearly, the mechanism by which AF64A acts, at least in citro, in these tissues is different from that in the hippocampus. In another study, in which A F 6 4 A was injected intracerebroventricularly, it was shown that tile toxic effects on cholinergic neurons are far more pronounced in the hippocampus than in the striatum or frontal cortex (Potter et al.. 1986). It is possible that the ACh releasing property of AF64A in hippocampus contributes to its toxic action on cholinergic neurons. In summary, this study has shown that the m vitro effects of A F 6 4 A on endogenous ACh release differ from those of HC-3. Whereas HC-3 causes a reduction m stimulation induced endogenous ACh release in both the hippocampus and guinea-pig ileum longitudinal muscle strip, A F 6 4 A enhanced ACh release in these tissues. The mechanism of this effect is at present unknown, but that it occurs in the presence of HC-3 indicates that it is not due to inhibition of choline re-uptake. A F 6 4 A does not act in the same manner in all tissues; in the cortex, A F 6 4 A did not affect ACh release, whereas ACh release was reduced by A F 6 4 A in the striatum. The authors wish to thank Ms Susanna Kiss and Mrs Susanna Major for their expert technical assistance and also to express their appreciation to Dr I. Hanin and Dr A. Fisher for generously providing AF64A. Acknowledgements
REFERENCES
Curti D. and Marchbanks R. M. (1984) Kinetics of irreversible inhibition of choline transport in synaptosomes by ethylcholine mustard aziridinium. J. Membr. Biol. 82, 259 268. Fisher A., Mantione C. R., Abraham D. J. and Hanin I. (1982) Long term central hypofunction induced in mice by ethylcholine aziridinium ion (AF64A) in vivo. J. Pharmac. exp. Ther. 222, t4~145.
In vitro effects of AF64A
Glowinski J. and Iversen L. L. (1966) Regional studies of cateeholamines in the rat brain--I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain. J. Neurochem. 13, 655-699. Leventer S. M., McKeag D., Clarity M., Wulfert E. and Hanin I. (1984) Intracerebroventricular AF64A also reduces acetylcholine release from rat hippocampal slices. Soc. Neurosci. Abstr. 14, 1186P. Leventer S., McKeag D., Clancy M., Wulfert E. and Hanin I. (1985) Intracerebroventricular AF64A reduces ACh release from rat hippocampal slices. Neuropharmacology 24, 453-459. Mantione C. R., DeGroat W. C., Fisher A. and Hanin I. (1983a) Selective inhibition of peripheral cholinergic transmission in the cat produced by AF64A. J. Pharmac. exp. Ther. 225, 616--622. Mantione C. R., Fisher A. and Hanin I. (1981) The AF64A treated mouse: possible model for central cholinergic hypofunction. Science 213, 579-580. Mantione C. R., Zigmond M. J., Fisher A. and Hanin I. (1983b) Selective presynaptic eholinergic neurotoxicity following intrahippocampal AF64A injection in rats. J. Neurochem. 41, 251-255. Paton W. D. M. and Vizi E. S. (1969) The inhibitory action of noradrenaline on acetylcholine output by guinea-pig longitudinal muscle strip. Br. J. Pharmac. 35, 10--28. Pedder E. K. and Prince A. K. (1983) The reaction of rat brain choline acetyltransferase (CHAT) with ethylcholine mustard aziridinium ion (ECMA) and phenoxybenzamine (PB). Br. J. Pharmac. 80, 134P. Potter P. E., Harsing L. G. Jr, Kakucska I., Gaal G., Fisher A., Hanin I. and Vizi E. S. (1984) In vitro effects of AF64A on acetylcholine release. In: Regulation of Transmitter Function: Basic and Clinical Aspects (Vizi E. S. and Magyar K., eds), pp. 295-300. Academiai Kiado, Budapest. Potter P. E., Harsing L. G. Jr, Kakucska I., Gaal G., and Vizi E. S. (1986) Selective impairment of acetylcholine release and content in the central nervous system following intracerebroventricular administration of ethylcholine mustard aziridinium ion (AF64A) in the rat. Neurochem. Int. 8.
Rylett B. J. and Colhoun H. (1978) Comparison of the effects of some choline analogues on choline transport in synaptosomes and on the in vitro rat phrenic nervehemidiaphragrn preparation. Can. Fedn Biol. Soc. 21, 2P.
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Rylett B. J. and Colhoun H. (1980) Kinetic data on the inhibition of high-affinity choline transport into rat forebrain synaptosomes by choline-like compounds and nitrogen mustard analogues. J. Neurochem. 34, 713-719. Sandberg K., Hanin I., Fisher A. and Coyle J. (1984) Selective cholinergic neurotoxin: AF64A's effects in rat striatum. Brain Res. 293, 49-55. Sandberg K., Sehnaar R. L., Hanin I. and Coyle J. T. (1982) Effects of AF64A on neuroblastoma x glioma hybrid cell line NG-108-15: a neurotoxin selective for cholinergic cells. Soc. Neurosci. Abstr. 8, 516P. Somogyi G. T., Vizi E. S. and Knoll J. (1977) Effect of hemicolinium-3 on the release and net synthesis of acetylcholine in Auerbach's plexus of guinea-pig ileum. Neuroscience 2, 791-796. Szerb J. C. and Somogyi G. T. (1973) Depression of acetylcholine release from cerebral cortical slices by cholinesterase inhibition and by oxotremorine. Nature, New Biol. 241, 121-123. Vincze A., Cooks R. G., Hanin I. and Fisher A. (1981) Secondary ion mass spectrometry for identification of neuroactive choline aziridinium ion analogs. Proc. Am. Soc. Mass Spectr., 29th Annual Conference.
Vizi E. S. (1973) Acetylcholine release from guinea pig ileum by parasympathetic ganglion stimulant and gastrin like polypeptides. Br. J. Pharmac. 47, 765-777. Vizi E. S. (1978) NA+-K+-activated adenosinetriphosphatase as a trigger in neurotransmitter release. Neuroscience 3, 367-384. Vizi E. S., Harsing L. G. Jr, Duncalf D., Nagashima H., Potter P. E. and Foldes F. F. (1985) A simple and sensitive method of acetylcholine identification and assay: bioassay combined with mini-column gel filtration or high performance liquid chromatography. J. Pharmac. Meth. 13, 201-211. Vizi E. S., Illes P., Ronai A. and Knoll J. (1972) The effect of lithium on acetylcholine release and synthesis. Neuropharmacology 11, 521-530. Vizi E. S., Ono K., Adam-Vizi V., Duncalf D. and Foldes F. F. (1984) Presynaptic inhibitory effect of metenkephalin on [14C]acetylcholine release from the myenteric plexus and its interaction with muscarinic negative feedback inhibition. J. Pharmac. exp. Ther. 230, 493--499. Vizi E. S., Somogyi G. T. and Magyar K. (1981) Evidence that morphine and opioid peptides do not share a common pathway with adenosine in inhibiting acetylcholine release from isolated intestine. J. Auton. Pharmac. 1, 413-419.
0197-0186/85 $3.00+ 0.00 © 1985 Pergamon Press Ltd
PERIPHERAL AND CENTRAL ACTIONS OF AF64A (ETHYLCHOLINE MUSTARD AZIRIDINIUM ION) ON ACETYLCHOLINE RELEASE, IN VITRO: COMPARISON WITH HEMICHOLINIUM P. E. POTTER*, L. G. HAgS!N~3 Jr, I. KAgucsg^, G. GAAL and E. S, Vlzl Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O.K 67, H-1450 Budapest, Hungary (Received 8 February 1985; accepted 1 April 1985)
Abstract--The acute effects of ethylcholine mustard aziridinium ion (AF64A) and hemicholinium-3 (HC-3) on the release of endogenous acetyleholine (ACh) from isolated tissues were examined, Whereas addition of HC-3 (10-s-10 ~5 M) significantly reduced the output of ACh from isolated guinea-pi 8 ileum longitudinal muscle strip elicited by 10 Hz stimulation, AF64A had no effect and even enhanced the release of radiolabel elicited by 1 Hz stimulation when this tissue was pre,loaded with [3H]choline. Similarly, HC-3 (10 -s M) reduced ouabain-induced endogenous ACh release from isolated rat hippocampus, Addition of AF64A (10-s-5 × 10-s M) caused a slight increase in ACh release, In isolated rat cortex, however, AF64A did not affect ACh release. Moreover, AF64A caused a decreale in ouabain.stimniated ACh releate from striatum. The present study indicates that: (a) the/n vitro actions of AF64A differ from those of HC.3 and (b) the acute effects of AF64A on endogenous ACh release vary, depending on the tissues studied and the stimulation parameters used,
(Mantione et al., !981; Fisher et al,, 1982), In the rat, similar lesions were produced by intrahippocampal and intrastriatal injections of AF64A (Mantione et al., 1983b; Sandberg et al., 1984), and i.c.v, administration has been shown to reduce ACh release (Potter et al., 1984; Leventer et al., 1984, !985). The mechanism by which AF64A causes functional lesions of cholinergic neurons is not clear. A number of studies have documented that this compound causes an irreversible inhibition of HAChT in vitro (Rylett and Colhoun, 1980; Sandberg et al,, 1982; Mantione et al,, 1983b; Curti and Marchbanks, !984). ChAT is also inhibited /n vitro by AF64A (Sandberg et al., 1982; Mantione et al., !983b; Pedder and Prince, 1983). It seems possible that AF64A is taken up into the cholinerglc nerve terminal by HAChT system and subsequently interacts with *Address correspondence to: Pamela E. Potter, Room E-1238, Western Psychiatric Institute and Clinic, Uni- CHAT, because in synaptosomes the inhibition of versity of Pittsburgh School of Medicine, 3811 O'Hara ChAT by AF64A can be prevented by the addition Street, Pittsburgh, PA 15213, U.S.A. of hemicholinium (HC-3), in concentrations Abbreviations used: ACh: acetylcholine; AF64A: ethyl(4 × 10-6-4 x 10 ,s M) sufficient to completely inhibit choline mustard aziridinium ion; CHAT: choline acetyl- HAChT (Pedder and Prince, 1983), transferase; HAChT: high affinity choline transport; Inhibition of HAChT in vitro by AF64A would be HC-3: hemicholinium-3; HPLC: high performance expected to lead to a decline in cho!incrgic transliquid chromatography. 1047
Ethylcholine mustard aziridinium ion (AF64A) is one of a group of choline mustard analogues that has been shown to inhibit the high affinity uptake of choline into synaptosomes (Rylett and Colhoun, 1980). This compound has been proposed as a neurotoxin selective for cholinergic neurons (Mantionc et al., 1981). When injected intracerebroventricularly (i.c.v.) into the mouse, AF64A produced a longlasting, marked reduction in presynaptic cholinergic indices, such as acetylcholinc (ACh) levels, high affinity choline transport (HAChT) and choline acetyltransferase (CHAT) activity in hippocampus, striatum and cerebral cortex, but did not affect the binding of [3H]QNB, a marker for postsynaptic cholinergic muscarinic receptors in these tissues
1048
P. [-. PolffR e; al.
mission, as blockade o f choline re-uptake from the synaptic cleft should decrease ACh synthesis and reduce the ACh available for release. Indeed, A F 6 4 A has been s h o w n to inhibit cholinergic transmission in the rat phrenic nerve h e m i d i a p h r a g m preparation (Rylett and Colhoun, 1978). Furthermore, contractions o f the cat nictitating m e m b r a n e elicited by preganglionic nerve stimulation were diminished by AF64A, but the responses to postganglionic stimulation or to cholinergic agonists were unaffected ( M a n t i o n e et al., 1983a). High frequency nerve stimulation accelerated this effect (Mantione et al., 1983a), suggesting that it resulted from depletion o f A C h stores. Such an effect o f inhibiting H A C h T was d e m o n s t r a t e d more directly by Somogyi et al. (1977), who found that addition o f HC-3 to the isolated guinea-pig ileum longitudinal muscle strip markedly reduced the release o f e n d o g e n o u s ACh evoked by continuous 10 Hz stimulation and also decreased the A C h synthesis rate. The aim o f the present study was to investigate and c o m p a r e the effects o f A F 6 4 A and HC-3 on the release o f endogenous A C h from various isolated tissues. The effects o f A F 6 4 A varied depending on the tissue and the stimulation parameters used. Furthermore, the acute effects of A F 6 4 A and HC-3 differed; HC-3 reduced A C h release from the guinea pig ileum longitudinal muscle strip and rat hippocampus, whereas A F 6 4 A enhanced A C h release from these tissues. EXPERIMENTAL PROCEDURES
Preparation o f AF64A AF64A was prepared prior to each experiment by basic hydrolysis of 1 or 10mM acetylethylcholine mustard and subsequent aziridinium ion formation, as described by Fisher et al. (1982). It has been shown by both nuclear magnetic resonance and secondary ion mass spectrometry that, with the starting compound used and the conditions described below, conversion of the mustard to the deacetylated aziridinium product is in excess of 95% (Vincze et al., 1981; Fisher et al., 1982). AF64A prepared in this manner has been shown to inhibit HAChT in vitro (Sandberg et al., 1982; Mantione et al., 1983b). In addition, the AF64A prepared for these experiments was also injected i.c.v, in rats and produced the cholinergic deficits characteristic of this compound (Potter et al., 1984), indicating that the compound was active. Briefly, the pH of the solution was adjusted to l 1.3-11.7 with 10NNaOH and the solution was stirred at room temperature for 30 min, then the pH was reduced to 7.4 with concentrated HC1. AF64A was diluted immediately before use in Krebs bicarbonate buffer of the following composition (mM): NaCI 113, CaC12 2.5, KH2PO 4 1.2, MgSO4 1.2, NaHCO 3 25, glucose 11.5 and eserine sulfate 0.0062. In studies where the release of [3H]ACh was measured, no eserine sulfate was present in the Krebs solution.
Measuremem q/endogenous A ('h re/ea.w /}om hil,p,~, ompu~. .,triatum or jrontal cortex ~[ ra;.s Male Wistar rats weighing approx~ 150 g were kilted by decapitation and hippocampi, striata, or frontal cortices were dissected by the method of Glowinski and t~crson (1966) and weighed. The pair of hippocampi, strlata or frontal cortices from each rat were placed into a 5 ml organ bath containing 2 ml of Krebs bicarbonate buffer, thermostated at 37C and oxygenated continuously wiltz 95~';, O: 5~ CO,. ARer l h pre-incubation to allow complete inhibition of acetylcholinesterase to develop, the Krebs bicarbonate buffer was changed at 10 rain intervals. After the second 10min period, AF64A or HC-3 fin concentrations from 10 6-5 x 10 5 M) were added to the Krebs solution 1"or the remainder of the experiment. After two further resting periods in the presence of AF64A Or HC-3. ACh release was stimulated by the addition of ouabain (2 x 10 5 Hz), an inhibitor ofNa ~ -K ", activated adenosine triphosphatase (Vizi, 1978). At the end of each 10rain period, the bath contents were collected and assayed for ACh by bioassay on the guinea-pig ileum, as described by Paton and Vizi (1969). Measurement of endogenous ACh release ,fi'om guinea-pig ileum longitudinal muscle strip The in vitro effects of AF64A and HC-3 on ACh release from the guinea-pig ileum were determined as described by Somogyi et al. (1977). The longitudinal muscle strip of the guinea-pig ileum with attached Auerbach's plexus; weighing about 100mg, was prepared as described by Paton and Vizi (1969). Strips were mounted in an organ bath and perfused with eserinized Krebs solution at a rate of 0.35 ml/min. Following l h preperfusion, two 6rain fractions were collected. Then the tissue was stimulated at 10 Hz, t ms, supramaximal voltage, with square wave pulses through platinum electrodes at the top and bottom of the organ bath. Samples of perfusate were collected for three 6rain periods, then AF64A (10~-5 x 10SM) or HC-3 (10 6 10 5M) was added to the perfusion solution and fractions were collected for seven additional 6 min intervals. The ACh content of the perfusate was determined by bioassay (Paton and Vizi, 1969). Tissues taken either before or after perfusion were placed in 2 ml of l 0')~, trichloroacetic acid for 90 min then disrupted in a ground glass homogenizer. The samples were centrifuged and the supernatant was removed and extracted 3 times with ether, then the aqueous phase was neutralized with 1'~ NaHCO 3 in the presence of methylorange indicator (Vizi, 1973). ACh content was measured by bioassay (Paton and Vizi, 1969). The net synthesis of ACh was calculated as described by Viziet al. (1972). Identification o f released ACh by H P L C The longitudinal muscle strip of the guinea pig ileum was prepared as described above and perfused at a flow rate of 0.5 ml/min. Following I h preperfusion, the tissue was stimulated at 10Hz, 1 ms, supramaximal voltage, with square wave pulses through platinum electrodes at the top and bottom of the organ bath. Two tissues were used: one served as a control and 10 5M AF64A was added to the perfusion fluid of the second tissue at the beginning of the stimulation period. After 10rain, perfusate was collected from each tissue for 20 min. Samples of perfusate, or ACh standards (36.6/~M) dissolved in Krebs buffer,
In vitro effects of AF64A were frozen on dry ice and dried under vacuum. The samples and standards were redissolved in 200 #1 of 0.01 M Na acetate--citric acid, pH 4. A 20/~1 aliquot of each was injected into a Biotrouik high performance liquid chromatography (HPLC) system (BT 3020 pump, Rheodyne 7125 injector with 20/~1 loop, and 15 crn Nucleosil 5 #m Cms column; Bischoff Analysentechnik and Biotronik Wissenschaftliche Gerate GmbH, Frankfurt am Main, West Germany) for separation of ACh as described by Viziet al. (1985). Mobile phase was 0.1 M N a acetate--citric acid, pH 4, with 1 mM tetraethylammonium at a flow rate of 0.7ml/min. Twenty 1 min fractions were collected and assayed on the guinea-pig ileum preparation for ACh content. The original samples and standards were diluted 1:50 and assayed by bioassay (Paten and Vizi, 1969). The ACh content of the original samples and that eluted from the HPLC column was calculated and the percent recovery was determined. In addition, 20/zl of [~4C]ACh iodide (sp.. act. 140 MBq/mmol) was injected into the HPLC and the radioactivity in the 1 min fractions was determined by liquid scintillation spectroscopy. Recovery was calculated by comparison with the radioactivity in a 20~1 aliquot of [14C]ACh which was not injected into the HPLC.
Measurement of [~H]ACh release from guinea-pig ileum longitudinal muscle strip The release of [3H]ACh during rest and in response to electrical stimulation was determined as described by Vizi et al. (1981). Longitudinal muscle strips weighing approx. 100 mg were incubated with methyl [3H]choline chloride (sp. act. 74 KBq/ml) for 30m in in a 3 ml organ bath. Field stimulation was applied at 1Hz for 15min during the loading period. The tissues were then transferred to another organ bath and perfused at a rate of 0.4 ml/min with Krebs buffer containing 10 -5 M HC-3. After 90 rain preperfusion, 5 min fractions of perfusate were collected. The tissues were stimulated (1 Hz, l ms duration, 8 V/cm) for 5 min during the 3rd and 1lth collection periods (S~ and $2). AF64A was added to the perfusion fluid immediately after the first stimulation. Radioactivity was determined in 1 ml aliquots of perfusate in a Packard Tri-Carb liquid scintillation spectrometer with scintillation fluid of the following composition: 2 parts toluene and 1 part Triton X-100, containing 3.3 g/l PPO and 0.2 g/1 POPOP. The stimulation evoked release of [3H]ACh was calculated as described by Szerb and Somogyi (1973). The activities in samples 1-2, 9-10, and 17-18 were used for the determination of basal rates of resting ACh release. An exponential curve was fitted to these values, based on the assumption that the rate of resting release can be described exponentially. The stimulation induced release of radioactivity in samples 3~i and ll-14 was calculated by subtracting the interpolated rate of resting release from the stimulated release. The $2/S I ratios were calculated from the values for S~ and $2 calculated in this way.
Drugs and chemicals Acetylethylcholine mustard, synthesized by Dr A. Fisher, was provided by Dr I. Hauin, Pittsburgh, PA. Drugs were purchased from the following sources: acetylcholine iodide (BDH), eserine sulfate (Calbiochem), tetraethylammortium bromide (Fluka), hemicholinium-3 (Aldrich), methyl [3H]choline chloride (sp. act. 2.15 GBq/mmol; Amersham), acetyl-l-[14C]choline iodide (sp. act. 140 MBq/mmol; New
1049
England Nuclear). All other chemicals were reagent grade.
Statistics For statistical comparisons the Student's two-tailed t-test was used. RESULTS
Release o f endogenous ACh from hippocampus, striatum and frontal cortex: Effects o f AF64A and HC-3 The resting release of A C h from isolated hippocampus was 3 7 . 7 + 2 . 1 pmol/g/min (n = 20). Addition of ouabain, 2 x 10 -5 M, increased the release to 252.8 __+11.4 pmol/g/min (n = 20). The effects of A F 6 4 A and HC-3 on release of endogenous A C h from isolated hippocampus are shown in Fig. 1. HC-3, in concentrations of 10 -6 and 10 -5 M, caused significant reductions in the ouabain stimulated release o f ACh. In the presence of 1 0 - S M HC-3, A C h release was reduced by 38%. Neither concentration of HC-3 had any effect on the resting release o f ACh. Resting (4)
~] Ouabaln 2 x 10-5M
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I
100
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6
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Fig. 1. Effects of AF64A on HC-3 on release of endogenous ACh from isolated rat hippocampus. ACh release from isolated hippocampus was measured by bioassay on the guinea-pig ileum as described in Experimental Procedures. Hatched area indicates average of ACh release during two 10min resting periods. Total bar indicates ACh release during the second 10min of exposure to ouabain, 2 x 10 -5 M. Number of animals are indicated in parentheses. C--control. AF64A--10-6M, 10-SM, 5 x 10-SM. HC-3--10 -6 M, 10-5 M. *P < 0.05, **P < 0.01, Student's two-tailed t-test.
1051l
P. [" P~} H R
7
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64A
or HC-3
ACh release (pmol/g/mim
1200-
~ooo-
Frontal cortex Resting Stimulated
~.~:
Striatum Resting Stimulated
E
<~
al. Iablc I In t,itro effect or' l0 M AF64A tm \~, !~ release from frontal cortex and striatum
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,+
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60
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Fig. 2. Release of endogenous A C h from perfused guineapig ileum longitudinal muscle strip. Isolated guinea-pig ileum longitudinal muscle strip was perfused at a rate of 0.35 ml/min and the A C h content of the perfusate determined by bioassay, as described in Experimental Procedures. Following 1 h pre-perfusiom two 6 min samples were collected, then the tissue was stimulated continuously at 10 Hz, with square wave pulses of 1 ms duration. After three 6 m i n periods, A F 6 4 A (10 6-10 5M) was added to the perfusion solution and fractions were collected for seven additional 6 min periods. Values shown are mean + SE. N u m b e r of tissues in parentheses. *P < 0.01. Student's two-tailed t-test.
I n c o n t r a s t to H C - 3 , A F 6 4 A d i d n o t r e d u c e A C h release at a n y c o n c e n t r a t i o n s t u d i e d . 10 - s M A F 6 4 A h a d n o effect o n A C h release, a n d 10 5 a n d 5 x 1 0 S M A F 6 4 A p r o d u c e d a s l i g h t (30--34~o) b u t s i g n i f i c a n t i n c r e a s e in A C h release e v o k e d b y o u a b a i n . T h e r e s t i n g release o f A C h w a s n o t affected. In t h e f r o n t a l c o r t e x , A F 6 4 A h a d n o effect o n A C h release. T a b l e 1 s h o w s t h e v a l u e s for A C h release
Control
AF64A, !l) :\'1
28.8 + 2.4 131.7±2~4
36.2 ~ 5.3 1271-171
90.5 + 13.3 t 173.8 + 92.6
67.7 ± ,~.7 725.8 _+84.8*
Isolated tissues were incubated and ACh release was measured by bioassay as described in Experimental Procedures. After 80rain preincubation, AF64A was added to a final concentration of 10 s M and incubation fluid was collected at 10rain intervals and assayed for ACh. Values are either the average of two 10 rain resting periods, or during the second 10 rain exposure to ouabain. 2 × 10 ~ M+ N - 4 in all groups. *P < 0.(/5. Student's two-tailed tqest
u n d e r r e s t i n g c o n d i t i o n s a n d d u r i n g e x p o s u r e to 2 × 10 5 M o u a b a i n . T h e s e v a l u e s d i d n o t differ f r o m c o n t r o l in t h e p r e s e n c e o f 10 5 A F 6 4 A . In c o n t r a s t , in t h e s t r i a t u m b o t h t h e r e s t i n g release a n d t h a t m e a s u r e d d u r i n g t h e s e c o n d 1 0 r a i n p e r i o d o f exp o s u r e to o u a b a i n were s i g n i f i c a n t l y r e d u c e d by a d d i t i o n o f 10 5 M A F 6 4 A .
Effects o f AF64A on H C - 3 on release and synthesis q[ ACh in guinea-pig ileum longitudinal muscle strip In a p r e v i o u s s t u d y ( S o m o g y i et al., 1977) it w a s shown that addition of HC-3 during continuous 10 H z s t i m u l a t i o n o f t h e i s o l a t e d p e r f u s e d g u i n e a - p i g i l e u m l o n g i t u d i n a l m u s c l e s t r i p r e s u l t e d in a m a r k e d r e d u c t i o n o f b o t h A C h release a n d s y n t h e s i s rate. In t h e p r e s e n t s t u d y , H C - 3 (10 ~ a n d 10 ~ M ) also c a u s e d a s i g n i f i c a n t r e d u c t i o n in A C h release i m m e d i ately a f t e r a d d i t i o n to t h e p e r f u s i o n fluid ( d a t a for 10 6 M H C - 3 a r e s h o w n in Fig. 2). In c o n t r a s t , A F 6 4 A , in c o n c e n t r a t i o n s o f 10.6 a n d 10 ~ M , h a d
Table 2. Effect of AF64A and HC-3 on ACh content, release and synthesis rate in guinea-pig ileum longitudinal muscle strip ACh content after perfusion (nmol/g) Control AF64A 10 ~'M 10 5M HC-3 10 -6 M 10 5M
Total release in fractions 5-12 (nmol/g/h)
Synthesis rate (nmol/g/h)
107.0 +. 15.4 (13)
50.1 ± 5.5 (13)
69.6
92.8 + 20.0(4) 93.7± 13.6(5)
45.6 + 4.6 (4) 49.2+_ 10.8(5)
50.8 55 t
113.9 _+44.7 (4) 74.8 + 11.8(4)
18.2 __ 3.4* (4) 23.2_+ 1.6"(4)
44.5 10.4
ACh synthesis rate = (Content:met r~rr,~+o,,+ Total release) - Content~+~ r~fus,o,' ACh Content before perfusion = 87.6 _+7.9 nmol/g (26). Tissues were perfused and ACh content of perfusate fractions determined by bioassay as described in Experimental Procedures. ACh synthesis rate was calculated from ACh released in fractions 5-12, during continuous electrical stimulation at 10 Hz, 1 ms, 8 V/'cm, square wave pulses. Values are means + SE. Number of tissues in parentheses. *P < 0.05, Student's two tailed t-test, compared with control.
In vitro effects of AF64A no effect whatsoever on ACh release in this tissue. Figure 2 shows results with 10 -5 M AF64A; those obtained with 10 -~ M were almost identical. The ACh contents of the guinea-pig ileum longitudinal muscle strip were determined both before and after each experiment. The ACh content determined at the beginning of the experiments was 87.6 _+ 7.9 nmol/g (n =26). The ACh content measured at the end of the experiments is shown in Table 2. The content determined at the end of perfusion did not differ from that determined at the beginning, indicating that the tissues were able to synthesize sufficient ACh to replace that which was released. The ACh synthesis rate was calculated according to Vizi et al. (1972) from the total ACh released and the ACh contents measured before and after perfusion. The synthesis rate at rest was 4.1 nmol/g/h. The total ACh released and the ACh synthesis rate is shown in Table 2. Stimulation increased the ACh synthesis rate to 69.6 nmol/g/h. AF64A did not affect the ACh synthesis rate, but in the presence of 10 -5 M HC-3, it was reduced to 10.4 nmol/g/h.
Release of [ZH]AChfrom guinea-pig ileum longitudinal muscle strip The effects of 10-5 M AF64A were also determined in guinea-pig ileum longitudinal muscle strip that had been preloaded with [3H]choline. Since a previous study (Vizi et al., 1984) showed that virtually all of the radioactivity released in response to electrical stimulation of this tissue was labelled ACh, no attempt was made to separate [3H]ACh and [3H]choline. The result of this study is shown in Table 3. When added after the first stimulation period, AF64A caused a significant increase in
Table 3. Release of [3H]ACh from perfused guinea-pigileumlongitudinalmusclestrip s2/sl Control 0.81 ± 0.04(4) AF64A, I0-5 M added after SI 1.16± 0.08*(6) Guinea-pigileumlongitudinalmusclestrip was pre.incubated with [3H]cholinefor 45 rain, and stimulatedat 1Hz for the final 15rain. Tissues were perfused at 0.4 ml/min with Krebs solution containing 10-SM HC-3, and 3 min fractions were collected. Stimulation at 1Hz, 1ms durationfor 5rain, SI and $2. AF64A, 10-5 M, was added immediately after SI. Radioactivityreleased by each stimulationwas calculated and $2/S1 ratios determined. Means+ SE. Numberof tissuesin parentheses. *P < 0.01, Student'stwo-tailedt-test.
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fraction Fig. 3. HPLC separation of ACh in perfusates and ACh standards. A 20/~1 aliquot of p4C]ACh standard (A), ACh standard (B), or perfusate from guinea-pig ileum (C and D), dissolved in 0.01 M citrate--phosphate buffer, pH 3.5, was injected into the HPLC as described in Experimental Procedures. Mobile phase was 0.1 M Na acetate--citric acid, pH 4, with I mM tetraethylammonium bromide, at a flow rate of 0.7 ml/min. Twenty 1 rain fractions were collected and assayed either for radioactivity (A) or for ACh by bioassay on the guinea-pig ileum. A--elution pattern of [14C]ACh standard. B--elution of spasmogenic activity in ACh standard. C---elution of spasmogenic activity in pcrfusate of control guinea-pig ileum. D--elution of spasmogenic activity from perfusate of guinea.pig ileum in the presence of 10-5 M AF64A.
the $2/$1 ratio, indicating an enhancement of ACh release.
Identification of ACh in perfusates from guinea-pig ileum longitudinal muscle strip In order to determine the specificity of the bioassay used for the estimation of ACh contents, samples of perfusate from the guinea-pig ileum longitudinal muscle strip were lyophilized and injected into the HPLC. The pattern of elution of spasmogenic activity measured on the guinea-pig ileum was compared
with that of an ACh standard, as well as with the elution of radioactivity from the H P L C of a [~4C]ACh standard. The results are shown in Fig. 3. The majority of the radioactivity was elutcd m fraction 6 (Fig. 3A) with a recovery of 78%. When a lyophilized ACh standard was injected, the majority of the spasmogenic activity appeared in fraction 6 (Fig. 3B) and the recovery was 741~;. Elution patterns I\)r spasmogenic activity in perfusates from the guineapig ileum longitudinal muscle strip in the absence and presence of 10 s M A F 6 4 A are shown in Fig. 3C and D. The elution profiles and the recovery are identical with those of the standards: the spasmogenic activity eluted in fraction 6 and the recoveries were 751~,,in the control and 77'~i; in the sample containing AF64A. This finding strongly supports the assumption that the material measured by bioassay on the guinea-pig ileum is ACh and is not contaminated by other spasmogenic compounds. DISCUSSION Since A F 6 4 A has been shown to inhibit H A C h T in 1980; Mantione et al., 1983b) with a potency similar to HC-3, as well as to inhibit cholinergic transmission in the cat superior cervical ganglion (Mantione et al., 1983a) or rat hemidiaphragm-phrenic nerve (Rylett and Colhoun, 1978), it was expected that A F 6 4 A would act similarly to HC-3 when their in vitro effects on ACh release were compared. This, however, was not the case. Although HC-3 dramatically reduced the output and synthesis of A C h from the guinea-pig ileum longitudinal muscle strip, A F 6 4 A had no effect on endogenous A C h release during continuous 10 Hz stimulation. In order to investigate this further, the effect of A F 6 4 A on [3H]ACh release from guinea-pig ileum longitudinal muscle strip preloaded with [3H]choline was studied. In the presence of 10 S M AF64A, there was a significant enhancement of the 3H outflow induced by 1 Hz stimulation when compared to control tissues. This enhancement was not due to blockade of the re-uptake of [3H]choline, for the tissues were exposed to 10-5 M HC-3 throughout the experiment to block H A C h T . Further evidence that the action of A F 6 4 A was not due to inhibition of choline re-uptake is the observation that the ACh releasing effect of A F 6 4 A was not seen under resting conditions. Therefore, it appears that A F 6 4 A directly facilitated stimulated ACh release in the guinea-pig ileum longitudinal muscle strip. Similarly, the ouabain stimulated release of endovitro (Rylett and Colhoun,
genous ACh frmn the isolated rat h i p p o c a m p ~ was affected differently by HC3 and AF64A. Vv'hcreas there was a significant reduction in ACh outpui from hippocampus when HC-3 was added to the incubation fluid, ouabain stimulated ACh release was enhanced in the presence of AF64A. As in the guinea-pig ileum longitudinal muscle strip, the resting release of ACh was not affected by AF64A. l~here fore, A F 6 4 A appears to facilitate only the depolarization induced release of ACh from these tissues. When ACh release from the striatum or frontal cortex was measured, it was found that A F 6 4 A had no effect in the cortex and caused a slight reduction in ACh release from the striatum. In the striatum, both the ouabain stimulated and the resting release of ACh was reduced. Clearly, the mechanism by which AF64A acts, at least in citro, in these tissues is different from that in the hippocampus. In another study, in which A F 6 4 A was injected intracerebroventricularly, it was shown that tile toxic effects on cholinergic neurons are far more pronounced in the hippocampus than in the striatum or frontal cortex (Potter et al.. 1986). It is possible that the ACh releasing property of AF64A in hippocampus contributes to its toxic action on cholinergic neurons. In summary, this study has shown that the m vitro effects of A F 6 4 A on endogenous ACh release differ from those of HC-3. Whereas HC-3 causes a reduction m stimulation induced endogenous ACh release in both the hippocampus and guinea-pig ileum longitudinal muscle strip, A F 6 4 A enhanced ACh release in these tissues. The mechanism of this effect is at present unknown, but that it occurs in the presence of HC-3 indicates that it is not due to inhibition of choline re-uptake. A F 6 4 A does not act in the same manner in all tissues; in the cortex, A F 6 4 A did not affect ACh release, whereas ACh release was reduced by A F 6 4 A in the striatum. The authors wish to thank Ms Susanna Kiss and Mrs Susanna Major for their expert technical assistance and also to express their appreciation to Dr I. Hanin and Dr A. Fisher for generously providing AF64A. Acknowledgements
REFERENCES
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