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Role of endogenous opioids in soman (pinacolyl methylphosphonofluoridate)-induced antinociception
Life Sciences, Vol. 41, pp. 591-596 Printed in the U.S.A.
Pergamon Journals
ROLE OF ENDOGENOUS OPIOIDS IN SOMAN (PINACOLYL METHYLPHOSPHONOFLUORIDATE)-INDUCED ANTINOCICEPTION
J. Deborah Shiloff John G. Clement
Biomedical Defence Section Defence Research Establishment Suffield Ralston, Alberta, Canada TOJ 2NO (Received in final form May 21, 1987)
Summary
The effect of soman poisoning on the levels of methionine enkephalin and B-endorphin in mice and rats were determined. Soman poisoning produced no significant effect on methionine enkephalin levels in the striatum of rats or mice or B-endorphin levels in the pituitary gland of mice. In rats B-endorphin levels were significantly reduced 24 hr post soman poisoning, but returned to control levels by 48 hr. In vitro, the hydrolysis of leucine enkephalin by aminopeptidase was virtually complete by 30 min and found to be the major route of degradation. The release of TYR-GLY-GLY in the presence or absence of puromycin (10 uM) was found to be low (~2.0%). A minor effect on TYR release in the presence of GLY-GLY-PHE-MET (50 ~M) was insignificant. Preincubation of mouse striatum homogenates with soman (I or 10 uM) did not inhibit the hydrolysis of leucine enkephalin. These results suggest that the long term antinociception following soman exposure is not due to either altered concentration of endogenous opioid-like substances or inhibition of the enzymes responsible for their degradation.
It has been reported that administration of cholinergic agonists (I-4) and anticholinesterases (5-7) to mice and rats induced an antinociceptive response. Previous results from this laboratory showed that soman (pinacolyl methylphosphonofluoridate), induced a long-lasting, naloxone-reversible antinociception in mice (5). Naloxone antagonism of the soman-induced antinociception (5) suggested that it may be due to a reduced destruction of endogenous opioid-like substances. The purpose of this investigation was to determine the effect of soman poisoning in mice on the levels of endogenous opioids, such as methionine enkephalin (met-enkephalin) and B-endorphin, and the activity of enkephalin hydrolysing enzymes in vitro. The effects of soman poisoning in rats on the levels met-enkephalin and B-endorphin were included for comparison. 0024-3205/87 $3.00 + .00
592
Soman and Endogenous Opioids
Vol. 41, No. 5, 1987
Materials and Methods Male rats (Sprague-Dawley; 275-325 g) and male mice (CD-I; 20-30 g) were obtained from Charles River Can. Ltd., St. Constant, Quebec. All animals were allowed to acclimatize in the vivarium for a period of one week prior to use. Rats were pretreated by an intraperitoneal (i.p.) injection of a combined solution of an oxime reactivator, HI-6, (1-(((4-(aminocarbonyl)pyridinio)methoxy)methyl)-2((hydroxyimino)-methyl)-pyridinium dichloride); 125 mg/kg) and a cholinolytic, atropine (17.4 mg/kg), 5 min prior to a subcutaneous (s.c.) injection of soman (287 ug/kg). Mice were pretreated with HI-6 (50 mg/kg; i.p.) and atropine (17.4 mg/kg; i.p.) 5 min prior to injection with soman (287 ~g/kg; s.c.). At various intervals the animals were sacrificed by focused microwave irradiation of the head (Metabostat, Model 4095 Gerling Moore, Palo Alto). At the 2.0 kilowatt power setting rats were irradiated for 2.0 sec and mice for 0.75 sec. Following decapitation, the striatum and pituitary gland of both species were dissected out and homogenized in 1.0 mL of 1.0 N acetic acid using a Potter-Elvehjem grinder with a teflon pestle. The homogenate was transferred to a polypropylene tube. A 1.0 mL 1.0 N acetic acid wash of the homogenizing vessel was added to the polypropylene tube. Homogenates were centrifuged at 17,000 x g for 10 min, transferred to siliconized glass test; tubes and evaporated to dryness under reduced pressure at 65°C utilizing an Evapo-mix R (Buchler Instruments). The dried samples were stored in sealed tubes at -20°C until analysed by RIA. Analysis Met-enkephalin and B-endorphin concentrations were determined by RIA (Immunonuclear Corp., Stillwater, Min., and New England Nuclear, respectively). The dried samples were reconstituted with the appropriate assay buffer to give a 10% (w/v) solution of the original homogenate. Siliconized glass micropipettes were used throughout the RIA procedures. Radioactivity was counted using a Beckman Gamma 4000 Counter interfaced with a Beckman DP 5500 RIA computer. Statistical analyses were performed using the Student Newman Keuls test. A p~O.05 was considered significantly different. Enkephalin Endopeptidase and Aminopeptidase Assay Male CD-I mice (20-30 g) were decapitated and striatal tissues rapidly removed. A 5% (w/v) homogenate in O.05M piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 6.8) was made from the tissues of 5 mice. An aliquot of the homogenate was placed in a boiling water bath for 10-15 mln and was utilized as a blank for the enzyme assay. The incubation mixtures contained 0.05 M PIPES (pH 6.8), 5-10 ~g striatal tissue (based on protein) and inhibitors such as puromycin (10 ~M), GLY-GLY-PHE-MET, (GGFM; 50 ~M) or soman (I and 10 ~M) in a final volume of 200 uL. After a 10 min preincubation at 25°C, the reaction was started by the addition of 3H-Leu-enkephalin (20 nM) and incubated for an additional 10 min. The reaction was stopped by the addition of 50 ~L 0.1 N HCI and placing the tubes in a boiling water bath for 15 min. A 5 ~L aliquot of incubation mixtures were applied to Whatman LK6D Linear K silica gel plates (20 x 20 cm, 250 ~ thickness, glass, prescored into I cm tracks). All plates were chromatographed in a pre-equilibrated tank for 2-2½ hrs using a solvent system consisting of 2-propanol: ethyl acetate: acetic acid: water (40:40:1:19). A standard mixture containing approx I ug of Leu-enkephalin, tyrosine (Tyr), tyrosine-glycine-glycine (Tyr-Gly-Gly) was co-chromatographed with the
Vol. 41, No. 5, 1987
Soman and Endogenous Opioids
593
incubation mixtures and yielded Rf values of 0.70, 0.55 and 0.32, respectively. Upon completion of the chromatographic run, the plates were air dried and the marker spots visualized with ninhydrin (0.2% in ethanol) and gentle heating. Three sections of the plate were identified for each incubation mixture according to how the standard mixture for the plate had migrated. The silica gel within those sections was scraped off and transferred to a scintillation vial containing 800 ~L of water. 10 mL of scintillation cocktail (10 g PPO, I g bis-MSB, 633 mLs Triton X-100 to 2000 mLs with toluene) was then added to the vials and counted in a Beckman LS 9800 liquid scintillation counter.
Materials Soman and HI-6 (batch # DRES-32) were prepared by the Organic Chemistry Group, Defence Research Establishment Suffield. GGFM (Peninsula Labs, Ca) and Tyr-Tyr-Gly were kindly supplied by Dr. P. Wood, Douglas Hospital Research Centre, Verdun, Quebec. All other chemicals used were reagent grade and obtained from standard commercial sources.
Results and Discussion Enkephalins are thought to be metabolized (Fig I) by the action of a membrane bound aminopeptidase which results in appearance of Tyr (8) and/or by the action of an endopeptidase which results in appearance of Tyr-Gly-Gly (8,9). The time-course of the hydrolysis of Leu-enkephalin by a homogenate of mouse striatum and the effect of an aminopeptidase and endopeptidase inhibitor are shown in Fig 2. Hydrolysis of leu-enkephalin, mainly by aminopeptidase action, is virtually complete within 30 min under the experimental conditions used. Puromycin (10 uM), an aminopeptidase inhibitor, prevented the hydrolysis of leu-enkephalin, whereas GGFM (50 BM) an endopeptidase inhibitor (9) had a very minor inhibitory effect on Tyr release. Tyr-Gly-Gly release was extremely low (~2.0%) in the presence or absence of puromycin indicating that hydrolysis of leu-enkephalin at the GIy-PhE bond was not a major route of degradation. Preincubation of mouse striatum homogenate with soman (I or 10 ~M) had no significant effect on the hydrolysis of leu-enkephalin (Table I). The lack of an inhibitory effect by soman, which inhibits serine containing enzymes, is supported by results of Marchner (10). Similarily, it has been shown that neither DFP (Swerts, unpublished observation cited in 8), sarin (10) or tabun (10) inhibited enkephalin degradation.
TABLE I In Vitro Hydrolysis of Leu-Enkephalin by Mouse Brain Striatum: Effect of Soman DPM Group Blank Control Soman (I uM) Soman (10 uM) IMean ± SEM
L-Tyr-Gly-Gly 228 370 324 351
± 55 i ± 131 ± 63 ± 84
(N = 3-5 observations).
L-Tyr 598 6012 5818 5855
± ± ± ±
178 299 363 218
594
Soman and Endogenous Opioids
AM
Vol. 41, No. 5, 1987
IN OPEPTIDASE
(-)
H2N
PUROMYCIN
- TYR 1 - GLY 2 - GLY 3 - PHE 4-
LEU - OH
( - ) GGFM
LEU-ENKEPHALIN
ENDOPEPTIDASE
FIG. I Proposed Hydrolysis
of Leu-Enkephalin
46 ¸
= ×
345
a
•
•
•
-v
•
•
NO I N H I B I T O R , GGFM + GGFM
+
PURO~,'IYCIN
.J 115-
TIME
(minutes}
FIG. 2 Time-Course
of the Hydrolysis of Leu-Enkephalin Homogenate of Mouse Striatum
by
Vol. 41, No. 5, 1987
Soman and Endogenous Opioids
595
MOUSE
40
• -•
MET E N K E P H A L I N j¢ ENOORPHtN
,7, 32
32
T
~
CO
24 Z;
24
I- -I
2 Z -.i
16
~
\ 2 i.u
,\
2 ,\ 03
TIME
~
(hours)
FIG. 3 Effect of Soman Poisoning Met-Enkephalin and B-Endorphin
(287 ~g/kg; sc) in Mice on the Levels of in the Striatum and Pituitary, Respectively.
RAT
150
2500
O----O M E T ~ E N K E P H A U N • • j ENOORPHIN
-2100
130
CO
7-
,
110
t
t
1700
(.9 Q.
2 .J
.... z I
~ 90
1300
C
.~ 2 uJ
2 w /
L
70
I
05
I
10
30
I
24
60 TIME
48
72
r
500
96
(hours)
FIG. 4 Effect of Soman Poisoning Met-Enkephalin and B-Endorphin
(287 pg/kg; sc) in Rats on the Levels of in the Striatum and Pituitary, Respectively.
596
Soman and Endogenous Opioids
Vol. 41, No. 5, 1987
The effect of poisoning mice and rats with soman on met-enkephalin and B-endorphin levels was determined. In mice, soman poisoning had no significant effect on levels of met-enkephalin in striatum or B-endorphin in the pituitary (Fig 3). Similar results were found in the rat (Fig 4). Although levels of met-enkephalin in striatum tended to increase numerically, no significant differences were observed. ~-endorphin levels in pituitary decreased significantly (p~O.05) by 24 hr then returned to control levels by 48 hr after soman administration. The results suggest that the long term antinociception following soman exposure (5) was due to neither altered concentrations of endogenous opioidlike substances nor inhibition of the enzymes responsible for their degradation. However, this does not preclude the possibility that the long-lasting soman-induced naloxone-reversible antinociceptive response (5) could result from on effect in the levels of endorphins and enkephalins in an isolated area directly concerned with antinociception and/or another endogenous opioid-like substance such as dynorphin B (11).
References
I.
J.E. TAYLOR, T.L. YAKSH and E. RICHELSON, J. Neurochem. 39 521-524 (1982). 2. N.W. PEDIGO, W.L. DEWEY and L.S. HARRIS, J. Pharmacol. Expt. Therapy. 193(3) 845-852 (1975). 3. A.G. KARCZMAR, In: Biology of Cholinergic Function, A.M, Goldberg and I. Hanin (Eds), pp. 395-449, Raven Press, New York (1976). 4. G.B. LESLIE, J. Pharm. Pharmacol. 21 248-250 (1969). 5. J.G. CLEMENT and H.T. COPEMAN, Life Sci. 34 1415-1422 (1984). 6. G.L. KOEHN and A.G. KARCZMAR, Prog. Neuro-Psychopharmac. 2 169-177 (1978). 7. G.L. KOEHN, G. HENDERSON and A.G.KARCZMAR, Eur. J. Pharmacol. 61 167-173 (1980). 8. J.C. SCHWARTZ, B. MALFROY and S. DE LA BAUME, Life Sci. 29 1715-1740 (1981). 9. R.L. HUDGIN, SG. CHARLESON, M. ZIMMERMAN, R. MUMFORD and P.L. WOOD, Life Sei. 29 2593-2601 (1981). 10. H. MARCHNER, S. HARALDSSON and S. LUNDBERG, Life Sci. 38, 1317-1321 (1986). 11. J.S. HAN, G.X. XIE and A. GOLDSTEIN, Life Sol. 34 1573-1579 (1984).
Pergamon Journals
ROLE OF ENDOGENOUS OPIOIDS IN SOMAN (PINACOLYL METHYLPHOSPHONOFLUORIDATE)-INDUCED ANTINOCICEPTION
J. Deborah Shiloff John G. Clement
Biomedical Defence Section Defence Research Establishment Suffield Ralston, Alberta, Canada TOJ 2NO (Received in final form May 21, 1987)
Summary
The effect of soman poisoning on the levels of methionine enkephalin and B-endorphin in mice and rats were determined. Soman poisoning produced no significant effect on methionine enkephalin levels in the striatum of rats or mice or B-endorphin levels in the pituitary gland of mice. In rats B-endorphin levels were significantly reduced 24 hr post soman poisoning, but returned to control levels by 48 hr. In vitro, the hydrolysis of leucine enkephalin by aminopeptidase was virtually complete by 30 min and found to be the major route of degradation. The release of TYR-GLY-GLY in the presence or absence of puromycin (10 uM) was found to be low (~2.0%). A minor effect on TYR release in the presence of GLY-GLY-PHE-MET (50 ~M) was insignificant. Preincubation of mouse striatum homogenates with soman (I or 10 uM) did not inhibit the hydrolysis of leucine enkephalin. These results suggest that the long term antinociception following soman exposure is not due to either altered concentration of endogenous opioid-like substances or inhibition of the enzymes responsible for their degradation.
It has been reported that administration of cholinergic agonists (I-4) and anticholinesterases (5-7) to mice and rats induced an antinociceptive response. Previous results from this laboratory showed that soman (pinacolyl methylphosphonofluoridate), induced a long-lasting, naloxone-reversible antinociception in mice (5). Naloxone antagonism of the soman-induced antinociception (5) suggested that it may be due to a reduced destruction of endogenous opioid-like substances. The purpose of this investigation was to determine the effect of soman poisoning in mice on the levels of endogenous opioids, such as methionine enkephalin (met-enkephalin) and B-endorphin, and the activity of enkephalin hydrolysing enzymes in vitro. The effects of soman poisoning in rats on the levels met-enkephalin and B-endorphin were included for comparison. 0024-3205/87 $3.00 + .00
592
Soman and Endogenous Opioids
Vol. 41, No. 5, 1987
Materials and Methods Male rats (Sprague-Dawley; 275-325 g) and male mice (CD-I; 20-30 g) were obtained from Charles River Can. Ltd., St. Constant, Quebec. All animals were allowed to acclimatize in the vivarium for a period of one week prior to use. Rats were pretreated by an intraperitoneal (i.p.) injection of a combined solution of an oxime reactivator, HI-6, (1-(((4-(aminocarbonyl)pyridinio)methoxy)methyl)-2((hydroxyimino)-methyl)-pyridinium dichloride); 125 mg/kg) and a cholinolytic, atropine (17.4 mg/kg), 5 min prior to a subcutaneous (s.c.) injection of soman (287 ug/kg). Mice were pretreated with HI-6 (50 mg/kg; i.p.) and atropine (17.4 mg/kg; i.p.) 5 min prior to injection with soman (287 ~g/kg; s.c.). At various intervals the animals were sacrificed by focused microwave irradiation of the head (Metabostat, Model 4095 Gerling Moore, Palo Alto). At the 2.0 kilowatt power setting rats were irradiated for 2.0 sec and mice for 0.75 sec. Following decapitation, the striatum and pituitary gland of both species were dissected out and homogenized in 1.0 mL of 1.0 N acetic acid using a Potter-Elvehjem grinder with a teflon pestle. The homogenate was transferred to a polypropylene tube. A 1.0 mL 1.0 N acetic acid wash of the homogenizing vessel was added to the polypropylene tube. Homogenates were centrifuged at 17,000 x g for 10 min, transferred to siliconized glass test; tubes and evaporated to dryness under reduced pressure at 65°C utilizing an Evapo-mix R (Buchler Instruments). The dried samples were stored in sealed tubes at -20°C until analysed by RIA. Analysis Met-enkephalin and B-endorphin concentrations were determined by RIA (Immunonuclear Corp., Stillwater, Min., and New England Nuclear, respectively). The dried samples were reconstituted with the appropriate assay buffer to give a 10% (w/v) solution of the original homogenate. Siliconized glass micropipettes were used throughout the RIA procedures. Radioactivity was counted using a Beckman Gamma 4000 Counter interfaced with a Beckman DP 5500 RIA computer. Statistical analyses were performed using the Student Newman Keuls test. A p~O.05 was considered significantly different. Enkephalin Endopeptidase and Aminopeptidase Assay Male CD-I mice (20-30 g) were decapitated and striatal tissues rapidly removed. A 5% (w/v) homogenate in O.05M piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 6.8) was made from the tissues of 5 mice. An aliquot of the homogenate was placed in a boiling water bath for 10-15 mln and was utilized as a blank for the enzyme assay. The incubation mixtures contained 0.05 M PIPES (pH 6.8), 5-10 ~g striatal tissue (based on protein) and inhibitors such as puromycin (10 ~M), GLY-GLY-PHE-MET, (GGFM; 50 ~M) or soman (I and 10 ~M) in a final volume of 200 uL. After a 10 min preincubation at 25°C, the reaction was started by the addition of 3H-Leu-enkephalin (20 nM) and incubated for an additional 10 min. The reaction was stopped by the addition of 50 ~L 0.1 N HCI and placing the tubes in a boiling water bath for 15 min. A 5 ~L aliquot of incubation mixtures were applied to Whatman LK6D Linear K silica gel plates (20 x 20 cm, 250 ~ thickness, glass, prescored into I cm tracks). All plates were chromatographed in a pre-equilibrated tank for 2-2½ hrs using a solvent system consisting of 2-propanol: ethyl acetate: acetic acid: water (40:40:1:19). A standard mixture containing approx I ug of Leu-enkephalin, tyrosine (Tyr), tyrosine-glycine-glycine (Tyr-Gly-Gly) was co-chromatographed with the
Vol. 41, No. 5, 1987
Soman and Endogenous Opioids
593
incubation mixtures and yielded Rf values of 0.70, 0.55 and 0.32, respectively. Upon completion of the chromatographic run, the plates were air dried and the marker spots visualized with ninhydrin (0.2% in ethanol) and gentle heating. Three sections of the plate were identified for each incubation mixture according to how the standard mixture for the plate had migrated. The silica gel within those sections was scraped off and transferred to a scintillation vial containing 800 ~L of water. 10 mL of scintillation cocktail (10 g PPO, I g bis-MSB, 633 mLs Triton X-100 to 2000 mLs with toluene) was then added to the vials and counted in a Beckman LS 9800 liquid scintillation counter.
Materials Soman and HI-6 (batch # DRES-32) were prepared by the Organic Chemistry Group, Defence Research Establishment Suffield. GGFM (Peninsula Labs, Ca) and Tyr-Tyr-Gly were kindly supplied by Dr. P. Wood, Douglas Hospital Research Centre, Verdun, Quebec. All other chemicals used were reagent grade and obtained from standard commercial sources.
Results and Discussion Enkephalins are thought to be metabolized (Fig I) by the action of a membrane bound aminopeptidase which results in appearance of Tyr (8) and/or by the action of an endopeptidase which results in appearance of Tyr-Gly-Gly (8,9). The time-course of the hydrolysis of Leu-enkephalin by a homogenate of mouse striatum and the effect of an aminopeptidase and endopeptidase inhibitor are shown in Fig 2. Hydrolysis of leu-enkephalin, mainly by aminopeptidase action, is virtually complete within 30 min under the experimental conditions used. Puromycin (10 uM), an aminopeptidase inhibitor, prevented the hydrolysis of leu-enkephalin, whereas GGFM (50 BM) an endopeptidase inhibitor (9) had a very minor inhibitory effect on Tyr release. Tyr-Gly-Gly release was extremely low (~2.0%) in the presence or absence of puromycin indicating that hydrolysis of leu-enkephalin at the GIy-PhE bond was not a major route of degradation. Preincubation of mouse striatum homogenate with soman (I or 10 ~M) had no significant effect on the hydrolysis of leu-enkephalin (Table I). The lack of an inhibitory effect by soman, which inhibits serine containing enzymes, is supported by results of Marchner (10). Similarily, it has been shown that neither DFP (Swerts, unpublished observation cited in 8), sarin (10) or tabun (10) inhibited enkephalin degradation.
TABLE I In Vitro Hydrolysis of Leu-Enkephalin by Mouse Brain Striatum: Effect of Soman DPM Group Blank Control Soman (I uM) Soman (10 uM) IMean ± SEM
L-Tyr-Gly-Gly 228 370 324 351
± 55 i ± 131 ± 63 ± 84
(N = 3-5 observations).
L-Tyr 598 6012 5818 5855
± ± ± ±
178 299 363 218
594
Soman and Endogenous Opioids
AM
Vol. 41, No. 5, 1987
IN OPEPTIDASE
(-)
H2N
PUROMYCIN
- TYR 1 - GLY 2 - GLY 3 - PHE 4-
LEU - OH
( - ) GGFM
LEU-ENKEPHALIN
ENDOPEPTIDASE
FIG. I Proposed Hydrolysis
of Leu-Enkephalin
46 ¸
= ×
345
a
•
•
•
-v
•
•
NO I N H I B I T O R , GGFM + GGFM
+
PURO~,'IYCIN
.J 115-
TIME
(minutes}
FIG. 2 Time-Course
of the Hydrolysis of Leu-Enkephalin Homogenate of Mouse Striatum
by
Vol. 41, No. 5, 1987
Soman and Endogenous Opioids
595
MOUSE
40
• -•
MET E N K E P H A L I N j¢ ENOORPHtN
,7, 32
32
T
~
CO
24 Z;
24
I- -I
2 Z -.i
16
~
\ 2 i.u
,\
2 ,\ 03
TIME
~
(hours)
FIG. 3 Effect of Soman Poisoning Met-Enkephalin and B-Endorphin
(287 ~g/kg; sc) in Mice on the Levels of in the Striatum and Pituitary, Respectively.
RAT
150
2500
O----O M E T ~ E N K E P H A U N • • j ENOORPHIN
-2100
130
CO
7-
,
110
t
t
1700
(.9 Q.
2 .J
.... z I
~ 90
1300
C
.~ 2 uJ
2 w /
L
70
I
05
I
10
30
I
24
60 TIME
48
72
r
500
96
(hours)
FIG. 4 Effect of Soman Poisoning Met-Enkephalin and B-Endorphin
(287 pg/kg; sc) in Rats on the Levels of in the Striatum and Pituitary, Respectively.
596
Soman and Endogenous Opioids
Vol. 41, No. 5, 1987
The effect of poisoning mice and rats with soman on met-enkephalin and B-endorphin levels was determined. In mice, soman poisoning had no significant effect on levels of met-enkephalin in striatum or B-endorphin in the pituitary (Fig 3). Similar results were found in the rat (Fig 4). Although levels of met-enkephalin in striatum tended to increase numerically, no significant differences were observed. ~-endorphin levels in pituitary decreased significantly (p~O.05) by 24 hr then returned to control levels by 48 hr after soman administration. The results suggest that the long term antinociception following soman exposure (5) was due to neither altered concentrations of endogenous opioidlike substances nor inhibition of the enzymes responsible for their degradation. However, this does not preclude the possibility that the long-lasting soman-induced naloxone-reversible antinociceptive response (5) could result from on effect in the levels of endorphins and enkephalins in an isolated area directly concerned with antinociception and/or another endogenous opioid-like substance such as dynorphin B (11).
References
I.
J.E. TAYLOR, T.L. YAKSH and E. RICHELSON, J. Neurochem. 39 521-524 (1982). 2. N.W. PEDIGO, W.L. DEWEY and L.S. HARRIS, J. Pharmacol. Expt. Therapy. 193(3) 845-852 (1975). 3. A.G. KARCZMAR, In: Biology of Cholinergic Function, A.M, Goldberg and I. Hanin (Eds), pp. 395-449, Raven Press, New York (1976). 4. G.B. LESLIE, J. Pharm. Pharmacol. 21 248-250 (1969). 5. J.G. CLEMENT and H.T. COPEMAN, Life Sci. 34 1415-1422 (1984). 6. G.L. KOEHN and A.G. KARCZMAR, Prog. Neuro-Psychopharmac. 2 169-177 (1978). 7. G.L. KOEHN, G. HENDERSON and A.G.KARCZMAR, Eur. J. Pharmacol. 61 167-173 (1980). 8. J.C. SCHWARTZ, B. MALFROY and S. DE LA BAUME, Life Sci. 29 1715-1740 (1981). 9. R.L. HUDGIN, SG. CHARLESON, M. ZIMMERMAN, R. MUMFORD and P.L. WOOD, Life Sei. 29 2593-2601 (1981). 10. H. MARCHNER, S. HARALDSSON and S. LUNDBERG, Life Sci. 38, 1317-1321 (1986). 11. J.S. HAN, G.X. XIE and A. GOLDSTEIN, Life Sol. 34 1573-1579 (1984).