Jo~rrnilltfl Nrurochrmi,irv Vol 33. pp. 389 to 3Y2
Pergamon Press Lrd 1979. Printed in Great Britain 0 International Society for Neurochemistry Ltd
SHORT COMMUNICATION
A sensitive method for assaying acetylcholine synthesis in human and frog skeletal muscle
(Received 2 September 1978. Revised 9 February 1979. Accepted 26 Februury 1979)
periments it was found that the storage of the homogenate is not improved in dithioerythritol and its use is therefore not recommended. Incubation with [14C]acetyl-CoA. The composition of the mixture for incubation of frog muscle was (final con["C]acetyl-CoA (Amersham, centration in 60 $): 50,000 d.p.m.) diluted with 25 p~-unlabelled acetyl-CoA (Boehringer, Mannheim) (33.3 d.p.m./pmol); 2 mM-choline chloride; 50 mM-NaCI; 50 mM-K-phosphate (pH 7.9); 0.2 mM-physostigmine salicylate; and Triton X-I00 0.05% (w/v). For human or rat muscle the same medium was used, except that the concentration of [14C]acetyl-CoA was ~ ~ 10.4d.p.m./pmol). The mixraised to 8 0 (50,000d.p.m., tures contained 2 mg sartorius muscle, 0.2 mg human intercostal muscle or 0.02-0.1 mg rat diaphragm. The mixtures were placed in 0.4ml plastic microtubes and incubated for 10 min at 20°C for frog muscle or 30 min at 37°C for human and rat muscle. Reaction blanks were MATERIALS AND METHODS prepared by incubating tissue in the presence of 1 p g elecPreparation of homogenates from skeletal muscle. Sar- tric eel acetylcholinesterase (Boehringer, Mannheim); in torius muscles from frogs ( R a m esculenta; 25-35g) and these incubations the physostigmine was omitted (cf: HAMhemidiaphragms from Wistar rats (7G90 g) were dissected PRECHT & AMANO,1974). Isolation of [14C]ACh from the incubation mixture. The and the nerves were cut close to their entry into the muscle. Biopsies of human intercostal muscle, obtained through reaction was stopped by the addition of trichloroacetic thoracotomy, were dissected into small (3-5 mg) tendon-toacid to a final concentration of 10% (w/v). An appropriate tendon bundles (see also ITO et ul., 1976). The muscles amount (130C-1500 d.p.m.) of [3H]acetylcholine (Amerwere weighed and cut with scissors into fine pieces in a sham) was added to the mixture as an internal standard. solution containing 0.1% Triton X-100 and 100mM-K- Subsequently, protein was removed by centrifugation phosphate (pH 7.9). The pieces were subsequently hom- (5 rnin at 7000g in a Hettich mikro-rapid centrifuge). The ogenized in a glass-in-glass homogenizer (volume 1.5 ml supernatant was transferred to a 15 ml glass Corexm tube for frog and rat muscle, volume 0.25 ml for human muscle). and 5 ml 200 mM-Na-phosphate (pH 7.0), 0.1 ml H,O conEnzyme activity in sartorius and diaphragm was tested im- taining 15 pg tetramethylammonium bromide (TMA) and mediately after homogenization, but homogenates of 84 pg choline chloride, and 0.5 ml KIM, solution (3 g human muscle were frozen after addition of dithioerythri- KI + 1.8g I, in lOml water) were added. The mixture to1 (3.5 mM) and kept for 1-7 days prior to measurement was left for 10min in ice and then centrifuged at about of enzyme activity. It has been reported that sulphydryl 3000 g for 10 min. The precipitate (I;-salts of ACh, choline agents, like dithioerythritol, react spontaneously with ace- and TMA, which served as coprecipitants for ACh) was tyl-CoA, and this may inhibit the enzymic formation of dissolved at about 70°C in 5 ml 50 mM-Na-phosphate (pH ACh especially when the concentration of the sulphydryl 7.0). After the mixture was cooled the precipitation procompound exceeds 10 mM (MORRIS,1967). However, it was cedure was repeated but without a new addition of TMA found that the reaction between 2.5 m~-dithioerythritoI and choline. The purification of ACh by a series of precipiand acetyl-CoA was slow and that the synthesis of ACh tations with KI, was used earlier for mass fragmentogra1974; MILEDIer ul.. was not affected by the sulphydryl compound. In Later ex- phy of ACh (POLAK& MOLENAAR, 1977). The final precipitate was dissolved in 2 ml acetonitrile containing 10 mg tetraphenylboron (TPB) and this Abbreviations used: ChAT, choline acetyltransferase; solution was transferred to a plastic scintillation vial, to TMA, tetramethylammonium bromide; TPB, tetraphenyl- which 5 ml 50 mM-Na-phosphate (pH 7.0), 50 p1 20% (w/v) boron; EBK, ethylbutylketone; ACh, acetylcholine. NaZS03and 5 ml scintillation mixture were subsequently
IN MOST radiochemical assays of choline acetyltransferase (ChAT), radioactively labelled acetyl-CoA is used as a substrate for the enzyme. The procedure by which the radioactive acetylcholine (ACh) formed was isolated from the incubation mixture varied according to the different methods used, resulting in blanks ranging from 0.1 to 2% of the original amount of radioactive acetyl-CoA (see for instance & SHUSTER, 1967; GLOVER & FONNUM, 1969a. b ; SCHRIER GREEN,1972; HEBB,1972). While these blanks are sufficiently low for the determination of ChAT in most cholinergic systems, they may be too high when ChAT is to be assayed in tissues containing a low concentration of the enzyme, such as human and frog skeletal muscle. In the present report a very sensitive method for the determination of ACh synthesis is described, in which the blank is lowered to 0.02% due to an improved purification of the radioactive ACh.
389
Short communication
390
OF ACh TABLE1. ISOLATION
Isolation procedure TPB/EBK TPB/EBK + wash TPB/CH,CN TPB/CH,CN wash KI, + TPB 2 x KI, + TPB
+
FROM THE REACTION MIXTURE
Recovered ACh* (d.p.m.) (percentage) 12400 k 1010 12800 f 540 1490 f 105 1410 k 128 1390 k 184 1340 f 235
49.6 51.2 99.3 94.0 92.7 89.3
(d.p.m.) 74 f 5.9t 57 k 5.2t 210 f 982 77 f 321 20 f log 10 f 96
Blank (percentage) 0.I48 0.1 13 0.420 0.155 0.040 0.020
The reaction mixture containing 50,000 d.p.m. [14C-acetyl]CoA was incubated for 10 min at room temperature in the absence of tissue. Subsequently the mixture was extracted either with 15 mg TPB in 1 ml ethylbutylketone (TPB/EBK) or with lOmg TPB in 2 ml CH,CN + 5 ml scintillation mixture (TPB/CH3CN), essentially as described by FONNUM (19696 and 1975, respectively). Alternatively, the ACh was precipitated once or twice by KI, in the presence of carrier, and thereafter extracted by TPB (see Materials and Methods). In the other cases (TPB/EBK + wash and TPB/CH,CN + wash) the EBK or scintillator layer was shaken with 4 mg TPB in 5 ml Na-phosphate (10 mM, pH 7). * In order to determine the recovery 1500d.p.m. [3H-acetyl]choEne were added as an internal standard immediately after the incubation, except in the experiments with TPB/EBK because the EBK caused too much quenching for simultaneous measurement of ,H and I4C. In the latter experiments the recovery was determined with 25,000 d.p.m. [14C-acetyl]choline in parallel experiments without ['4C-acetyl]CoA. Although the efficiency of the TPB/EBK extraction was about 100% its recovery was only 50% because 0.5ml of the 1 ml was transferred to the scintillation vial. Mean S.D. of eight observations. t P z < 0.001 ; $ P , = 0.007; 5 P 2 = 0.052 (Welch t-test). not illustrated in Table 1). On the other hand, a two-step precipitation with KI,, in combination with TPB extraction, resulted in a substantially lower blank (0.02%) which is far below values reported by others (see for instance & SHUSTER, 1967; GWVER& GREEN,1972; FONSCHRIBR NUM,1969h. 1975) and by GOLDBEKG et al. (1969) who used a single step precipitation with KI,. Apparently a combination of KI, and TPB is more efficient than either agent alone. Table 1 further shows that the ACh is isolated with a recovery of 93 and 89% for one and two precipitations respectively, which is somewhat lower than that found in the procedures in which TPB extraction is used as the only step. Any loss of [14C]ACh is compensated for through the use of an internal standard. The results shown in Table 2 were obtained using the isolation procedure with the two K13 precipitations and TPB extraction. It can be seen that the rate of synthesis of [I4C]ACh is about 3nmol/g/h in homogenates of frog sartorius. which is low when compared to the 75 nmol/g/h synthesized by human intercostal muscle. The procedure RESULTS AND DISCUSSION appears to give a somewhat higher rate of ACh synthesis (1969a, b, 1975) that It has been reported by FONNUM in rat diaphragm (103G-1380nmol/g/h) than that found by ACh can be extracted from aqueous solutions into an HEBBer al. (1964), about 700mmol/g/h. organic phase containing tetraphenylboron (TPB). I comThe present procedure, giving very low blanks, is imporpared the efficiency and the blank of two extraction tant when assaying ChAT in tissues such as human and methods for ACh (FONNUM, 19696, 1975) with a method frog muscle where the ratio of ChAT to other enzymes in which the ACh is precipitated in the presence of KI, which use acetyl-CoA as a substrate is extremely low. before TPB-extraction. Table 1 shows that 0.148% of the When I decreased the incubation volume (cf: FONNUM. added [14C-acetyl]CoA is measured after extraction of the 1969) or increased the incubation time, procedures which incubated mixture with TPB in ethylbutulketone, which could have been expected to result in a lower blank/ is somewhat higher than the 0.1% blank reported by FON- Ct4C]ACh ratio, the rate of [14C]ACh synthesis was no N U M (19696). In my hands the blanks were not reduced longer linearly related to time and amount of tissue. by washing with phosphate buffer. When the extraction Apparently, under these conditions appreciable amounts was carried out directly in a scintillation vial (FONNUM, of acetyl-CoA were consumed by other enzymes resulting 1975) it produced a blank of 0.42o/d, which is similar to in the accumulation of enough CoA to inhibit the activity Washing the scintillator the 0.35% reported by FONNUM. of ChAT. layer with phosphate buffer reduced the blank to 0.156%, I believe that the isolated labelled material really conbut a second wash did not further reduce the blank (0.142,. sists of ACh, and that the contamination of other labelled
added. The vials were lightly shaken, which caused the ACh to move to the organic solvent phase containing TPB and scintillator. The extraction of ACh by TPB into the (1975). scintillator layer was essentially that of FONNUM The addition of Na,SO, served to bleach the iodine originating from the I;-precipitate. The samples were counted for 20 min at room temperature in a Packard Tricarb Scintillation Spectrometer (model 2409) in a toluene based scintillation mixture. ,H and 14C were counted in two channels so that no counts from appeared in the I4C channel, and 2&25% of the I4C counts appeared in the 3H channel. Isotope overlap was checked using standards containing either I4C or ,H which were counted under the same conditions as the actual samples. The background counts were about 5 c.p.m. in both channels. The c.p.m. values were converted to d.p.m. by means of an external standard-channel ratio method.
391
Short communication TABLE2. SYNTHESIS OF ACh
BY HOMOGENATES OF SKELETAL MUSCLE ~
Enzyme source
Incubation medium
d.p.m. ~
2 mg Frog sartorius 2 mg Frog sartorius 2 mg Frog sartorius 2 rng Frog sartorius heated for 2 min at 100°C 0.2 mg Human intercostal 0.2 mg Human intercostal 0.2 mg Human intercostal 0.2 mg Human intercostal heated for 2 min at 100°C 0.02 mg Rat diaphragm 0.1 mg Rat diaphragm 0.1 mg Rat diaphragm 0.1 mg Rat diaphragm
Unchanged + I pg AChE - Physostigmine - Choline Unchanged Unchanged + 100 ng AChE - Physostigmine - Choline Unchanged Unchanged Unchanged + 1 pg AChE - Physostigmine Unchanged
~~
nmol .g - .h - I ~
~~
37 f 3(8) 1 f 2.5 (8)
3.33 0.09
6 2 3.0(8) f 2.8 (8)
0.54 - 0.09
-1
+-
78 7(8) 10 k 1.8(11)
+-
7 1.3(12) 7 & 2.6(8)
75 10 7 7
142 f 13(7) 533 _+ 47 (8) -4 & 3.2 (8)
1380 1030 -8
3 f 4.6(8)
6
heated for 2 min at 100°C Homogenates of muscle were incubated as described in Materials and Methods. Sartorius muscle (80mg) contains about 600 muscle fibres (but 1000 endplates, see MILEDIet a/., 1977), human intercostal muscle about 15,000 fibres/g of fresh tissue (cj ITO et al., 1976) & MITCHELL, 1961). Consequently, 2 mg and rat diaphragm (200 mg) 10,000 fibres (KRNJEVIC sartorius contains 15 fibres, 0.2mg human intercostal 3 fibres and 0.02mg rat diaphragm 1 fibre. Mean k S.E.M.with number of observations in brackets.
substances, such as ['4C]acetylcarnitine (cf: HAMPRECHTmuscle contains an enzyme other than ChAT which is able & AMANO. 1974), is small. Thus, it is unlikely that to synthesize ACh. This may explain why I found a much ['4C]acetylcarnitine is isolated by the present procedure, lower value for ACh synthesis in frog muscle (3.3 nmol/g/h) because acetylcarnitine does not precipitate with K13 at compared with that (29.4 nmol/g/h) reported by TUEEKet pH 7 (POLAK er al., 1977) and acetylcarnitine is not al. (1978). These authors used 12.5 mw-choline in the incuextracted by TPB at pH 7 (FONNUM, 1975). Moreover, as bation medium, whereas I used only 2 mM-choline. In fact, shown in Table 2, a low blank is obtained, regardless of using 10mM-choline I found ACh synthesizing activities whether tissue is incubated in the presence of acetylcho- of 3 W O nmol/g/h (not illustrated in Table 2). linesterase, in the absence of choline, or after inactivation In conclusion, the present method is very sensitive by heat. Apparently, practically all labelled material con- because of its low blank. It allows the analysis of ACh sists of ACh and the formation of ACh is dependent on synthesis in tissues with low ChAT activities. About 50 enzyme activity. samples can be assayed on a single day. However, the It should be borne in mind that in muscle, enzymes present method is more cumbersome (two precipitation other than ChAT are present which may be responsible steps with losses of ACh necessitating internal standardizafor part of the formed [14C]ACh. For instance, although tion and dual label counting) than the simple and rapid choline is a poor substrate for carnitine acetyltransferase procedure, without significant losses of ACh, described by (see for instance WHITE& Wu, 1973), this enzyme might FONNUM (19696, 1975). FONNUM'Smethods in my view synthesize ACh in appreciable amounts because in striated remain the methods of choice when the concentration of muscle carnitine acetyltransferase must be present in large ChAT in the tissue is not as low as that found in human excess to ChAT. Carnitine acetyltransferase is contained and frog muscle. in mitochondria of nerve and muscle, whereas ChAT is Acknowledgements-Financial support by the Foundation probably located only in the nerve terminals, which consti- FUNGO/ZWO is gratefully acknowledged. I thank Dr. tute less than 1:1O,OOO of the total muscle mass. In fact P. C. MOLENAAR for helpful discussions and suggestions. TUEEKet a!. (1978) have reported that 1 mM-4-(l-naphtylvinyl)pyridine, which blocks about 97% of the ChAT ac- Leiden University P. BRAGGAAR-SCHAAP tivity in frog sciatic nerve, inhibited ACh only about 60% Medical Centre, of synthesis in homogenates of frog sartorius muscle. The Department of Pharmacology, remaining 40% of ACh synthesizing activity may have been Wassenaarseweg 12, due to enzymes other than ChAT. In this connection it is 2333 A1 Leiden, of interest to note that frog sartorius muscle and human The Netherlands intercostal muscle show an appreciable amount of ACh REFERENCES synthesizing enzyme activity (which is not saturated at higher choline concentrations) in addition to a saturable activity ( K , i0.3 mM) only present in the endplate containing parts of the muscles (P. C. Molenaar & R. L. Polak, personal communication). These findings also indicate that
FONNUMF. (1969~)Isolation of choline esters from aqueous solutions by extraction with sodium tetraphenylboron in organic solvents. Eiochem. J . 113, 291-298.
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Short communication
FONNUM F. (1969b) Radiochemical microassays for the determination of choline acetyltransferase and acetylcholinesterase activities. Biochem. J . 115, 465472. FONNUM F. (1975) A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24, 407409. GLOVER V. & GREEND. P. L. (1972) A simple quick microassay for choline acetyltransferase. J . Neurochem. 19, 2465-2466. GOLDBERG A. M.. KAITAA. A. & MCCAMAN R. E. (1969) Microdetermination of choline acetyltransferase : a comparison of reineckate vs. periodide precipitation. J . Neurochem. 16, 823-824. HAMPRECHT B. & AMANOT. (1974) Differential assay for choline acetyltransferase. Analyt. Biochem. 57, 162-172. HERBC. 0. (1972) Biosynthesis of acetylcholine in nervous tissue. Physiol. Rev. 52, 918-943. HEBBC. O., KRNJEVIC K. & SILVER A. (1964) Acetylcholine and choline acetyltransferase in the diaphragm of the rat. J . Physiol., Lond. 171, 504513. ITO Y., MILEDIR., MOLENAAR P. C., VINCENTA., POLAK M. & NEWSOM DAVISJ. (1976) AcetR. L., VAN GELDER ylcholine in human muscle. Proc. R. Soc. 8192, 475480. KR NJ EVI K. ~& MITCHELL J. F. (1961)The release of acetylcholine in the isolated rat diaphragm. J . Physiol., Lond. 155, 246262.
MILEDIR., MOLENAAR P. C. & POLAKR. L. (1977) An analysis of acetylcholine in frog muscle by mass fragrnentography. Proc. R. Soc. B197, 285-297. MORRISD.(1967) The effect of sulphydryl and othcr disulphide reducing agents on choline acetyltransferase activity estimated with synthetic acetyl-CoA. J . Neurochem. 14, 19-27. POLAKR. L. & MOLENAAR P. C. (1974) Pitfalls in determination of acetylcholine from brain by pyrolysis-gas chromatography/mass spectrometry. J . Neurochem. 23, 1295-1297. POLAK R. L.. MOLENAAR P. c. & BKAGGAAR-SCHAAP P. (1977) Regulation of acetylcholine synthesis in rat brain. in Cholinergic Mechanisms und Psychopharrnucolog). (JENDEN D. J., ed.) pp. 511-524. Plenum Press, New York. SCHRIERB. K. & SHUSTER L. (1967) A simplified radiochemical assay for choline acetyltransferase. J . Nruruchem. 14, 977-985. TUEEKS., ZELENAJ., GE I. & VYSKOEILF. (1978) Choline acetyltransferase in transected nerves, denervated muscles and Schwann cells of the frog: correlation of biochemical, electron microscopical and electrophysiological observations. Neuroscience 3, 709-724. WHITEH. L. & Wu J. C. (1973) Choline and carnitine acetyltransferase of heart. Biochemistry 12, 841-846.
Pergamon Press Lrd 1979. Printed in Great Britain 0 International Society for Neurochemistry Ltd
SHORT COMMUNICATION
A sensitive method for assaying acetylcholine synthesis in human and frog skeletal muscle
(Received 2 September 1978. Revised 9 February 1979. Accepted 26 Februury 1979)
periments it was found that the storage of the homogenate is not improved in dithioerythritol and its use is therefore not recommended. Incubation with [14C]acetyl-CoA. The composition of the mixture for incubation of frog muscle was (final con["C]acetyl-CoA (Amersham, centration in 60 $): 50,000 d.p.m.) diluted with 25 p~-unlabelled acetyl-CoA (Boehringer, Mannheim) (33.3 d.p.m./pmol); 2 mM-choline chloride; 50 mM-NaCI; 50 mM-K-phosphate (pH 7.9); 0.2 mM-physostigmine salicylate; and Triton X-I00 0.05% (w/v). For human or rat muscle the same medium was used, except that the concentration of [14C]acetyl-CoA was ~ ~ 10.4d.p.m./pmol). The mixraised to 8 0 (50,000d.p.m., tures contained 2 mg sartorius muscle, 0.2 mg human intercostal muscle or 0.02-0.1 mg rat diaphragm. The mixtures were placed in 0.4ml plastic microtubes and incubated for 10 min at 20°C for frog muscle or 30 min at 37°C for human and rat muscle. Reaction blanks were MATERIALS AND METHODS prepared by incubating tissue in the presence of 1 p g elecPreparation of homogenates from skeletal muscle. Sar- tric eel acetylcholinesterase (Boehringer, Mannheim); in torius muscles from frogs ( R a m esculenta; 25-35g) and these incubations the physostigmine was omitted (cf: HAMhemidiaphragms from Wistar rats (7G90 g) were dissected PRECHT & AMANO,1974). Isolation of [14C]ACh from the incubation mixture. The and the nerves were cut close to their entry into the muscle. Biopsies of human intercostal muscle, obtained through reaction was stopped by the addition of trichloroacetic thoracotomy, were dissected into small (3-5 mg) tendon-toacid to a final concentration of 10% (w/v). An appropriate tendon bundles (see also ITO et ul., 1976). The muscles amount (130C-1500 d.p.m.) of [3H]acetylcholine (Amerwere weighed and cut with scissors into fine pieces in a sham) was added to the mixture as an internal standard. solution containing 0.1% Triton X-100 and 100mM-K- Subsequently, protein was removed by centrifugation phosphate (pH 7.9). The pieces were subsequently hom- (5 rnin at 7000g in a Hettich mikro-rapid centrifuge). The ogenized in a glass-in-glass homogenizer (volume 1.5 ml supernatant was transferred to a 15 ml glass Corexm tube for frog and rat muscle, volume 0.25 ml for human muscle). and 5 ml 200 mM-Na-phosphate (pH 7.0), 0.1 ml H,O conEnzyme activity in sartorius and diaphragm was tested im- taining 15 pg tetramethylammonium bromide (TMA) and mediately after homogenization, but homogenates of 84 pg choline chloride, and 0.5 ml KIM, solution (3 g human muscle were frozen after addition of dithioerythri- KI + 1.8g I, in lOml water) were added. The mixture to1 (3.5 mM) and kept for 1-7 days prior to measurement was left for 10min in ice and then centrifuged at about of enzyme activity. It has been reported that sulphydryl 3000 g for 10 min. The precipitate (I;-salts of ACh, choline agents, like dithioerythritol, react spontaneously with ace- and TMA, which served as coprecipitants for ACh) was tyl-CoA, and this may inhibit the enzymic formation of dissolved at about 70°C in 5 ml 50 mM-Na-phosphate (pH ACh especially when the concentration of the sulphydryl 7.0). After the mixture was cooled the precipitation procompound exceeds 10 mM (MORRIS,1967). However, it was cedure was repeated but without a new addition of TMA found that the reaction between 2.5 m~-dithioerythritoI and choline. The purification of ACh by a series of precipiand acetyl-CoA was slow and that the synthesis of ACh tations with KI, was used earlier for mass fragmentogra1974; MILEDIer ul.. was not affected by the sulphydryl compound. In Later ex- phy of ACh (POLAK& MOLENAAR, 1977). The final precipitate was dissolved in 2 ml acetonitrile containing 10 mg tetraphenylboron (TPB) and this Abbreviations used: ChAT, choline acetyltransferase; solution was transferred to a plastic scintillation vial, to TMA, tetramethylammonium bromide; TPB, tetraphenyl- which 5 ml 50 mM-Na-phosphate (pH 7.0), 50 p1 20% (w/v) boron; EBK, ethylbutylketone; ACh, acetylcholine. NaZS03and 5 ml scintillation mixture were subsequently
IN MOST radiochemical assays of choline acetyltransferase (ChAT), radioactively labelled acetyl-CoA is used as a substrate for the enzyme. The procedure by which the radioactive acetylcholine (ACh) formed was isolated from the incubation mixture varied according to the different methods used, resulting in blanks ranging from 0.1 to 2% of the original amount of radioactive acetyl-CoA (see for instance & SHUSTER, 1967; GLOVER & FONNUM, 1969a. b ; SCHRIER GREEN,1972; HEBB,1972). While these blanks are sufficiently low for the determination of ChAT in most cholinergic systems, they may be too high when ChAT is to be assayed in tissues containing a low concentration of the enzyme, such as human and frog skeletal muscle. In the present report a very sensitive method for the determination of ACh synthesis is described, in which the blank is lowered to 0.02% due to an improved purification of the radioactive ACh.
389
Short communication
390
OF ACh TABLE1. ISOLATION
Isolation procedure TPB/EBK TPB/EBK + wash TPB/CH,CN TPB/CH,CN wash KI, + TPB 2 x KI, + TPB
+
FROM THE REACTION MIXTURE
Recovered ACh* (d.p.m.) (percentage) 12400 k 1010 12800 f 540 1490 f 105 1410 k 128 1390 k 184 1340 f 235
49.6 51.2 99.3 94.0 92.7 89.3
(d.p.m.) 74 f 5.9t 57 k 5.2t 210 f 982 77 f 321 20 f log 10 f 96
Blank (percentage) 0.I48 0.1 13 0.420 0.155 0.040 0.020
The reaction mixture containing 50,000 d.p.m. [14C-acetyl]CoA was incubated for 10 min at room temperature in the absence of tissue. Subsequently the mixture was extracted either with 15 mg TPB in 1 ml ethylbutylketone (TPB/EBK) or with lOmg TPB in 2 ml CH,CN + 5 ml scintillation mixture (TPB/CH3CN), essentially as described by FONNUM (19696 and 1975, respectively). Alternatively, the ACh was precipitated once or twice by KI, in the presence of carrier, and thereafter extracted by TPB (see Materials and Methods). In the other cases (TPB/EBK + wash and TPB/CH,CN + wash) the EBK or scintillator layer was shaken with 4 mg TPB in 5 ml Na-phosphate (10 mM, pH 7). * In order to determine the recovery 1500d.p.m. [3H-acetyl]choEne were added as an internal standard immediately after the incubation, except in the experiments with TPB/EBK because the EBK caused too much quenching for simultaneous measurement of ,H and I4C. In the latter experiments the recovery was determined with 25,000 d.p.m. [14C-acetyl]choline in parallel experiments without ['4C-acetyl]CoA. Although the efficiency of the TPB/EBK extraction was about 100% its recovery was only 50% because 0.5ml of the 1 ml was transferred to the scintillation vial. Mean S.D. of eight observations. t P z < 0.001 ; $ P , = 0.007; 5 P 2 = 0.052 (Welch t-test). not illustrated in Table 1). On the other hand, a two-step precipitation with KI,, in combination with TPB extraction, resulted in a substantially lower blank (0.02%) which is far below values reported by others (see for instance & SHUSTER, 1967; GWVER& GREEN,1972; FONSCHRIBR NUM,1969h. 1975) and by GOLDBEKG et al. (1969) who used a single step precipitation with KI,. Apparently a combination of KI, and TPB is more efficient than either agent alone. Table 1 further shows that the ACh is isolated with a recovery of 93 and 89% for one and two precipitations respectively, which is somewhat lower than that found in the procedures in which TPB extraction is used as the only step. Any loss of [14C]ACh is compensated for through the use of an internal standard. The results shown in Table 2 were obtained using the isolation procedure with the two K13 precipitations and TPB extraction. It can be seen that the rate of synthesis of [I4C]ACh is about 3nmol/g/h in homogenates of frog sartorius. which is low when compared to the 75 nmol/g/h synthesized by human intercostal muscle. The procedure RESULTS AND DISCUSSION appears to give a somewhat higher rate of ACh synthesis (1969a, b, 1975) that It has been reported by FONNUM in rat diaphragm (103G-1380nmol/g/h) than that found by ACh can be extracted from aqueous solutions into an HEBBer al. (1964), about 700mmol/g/h. organic phase containing tetraphenylboron (TPB). I comThe present procedure, giving very low blanks, is imporpared the efficiency and the blank of two extraction tant when assaying ChAT in tissues such as human and methods for ACh (FONNUM, 19696, 1975) with a method frog muscle where the ratio of ChAT to other enzymes in which the ACh is precipitated in the presence of KI, which use acetyl-CoA as a substrate is extremely low. before TPB-extraction. Table 1 shows that 0.148% of the When I decreased the incubation volume (cf: FONNUM. added [14C-acetyl]CoA is measured after extraction of the 1969) or increased the incubation time, procedures which incubated mixture with TPB in ethylbutulketone, which could have been expected to result in a lower blank/ is somewhat higher than the 0.1% blank reported by FON- Ct4C]ACh ratio, the rate of [14C]ACh synthesis was no N U M (19696). In my hands the blanks were not reduced longer linearly related to time and amount of tissue. by washing with phosphate buffer. When the extraction Apparently, under these conditions appreciable amounts was carried out directly in a scintillation vial (FONNUM, of acetyl-CoA were consumed by other enzymes resulting 1975) it produced a blank of 0.42o/d, which is similar to in the accumulation of enough CoA to inhibit the activity Washing the scintillator the 0.35% reported by FONNUM. of ChAT. layer with phosphate buffer reduced the blank to 0.156%, I believe that the isolated labelled material really conbut a second wash did not further reduce the blank (0.142,. sists of ACh, and that the contamination of other labelled
added. The vials were lightly shaken, which caused the ACh to move to the organic solvent phase containing TPB and scintillator. The extraction of ACh by TPB into the (1975). scintillator layer was essentially that of FONNUM The addition of Na,SO, served to bleach the iodine originating from the I;-precipitate. The samples were counted for 20 min at room temperature in a Packard Tricarb Scintillation Spectrometer (model 2409) in a toluene based scintillation mixture. ,H and 14C were counted in two channels so that no counts from appeared in the I4C channel, and 2&25% of the I4C counts appeared in the 3H channel. Isotope overlap was checked using standards containing either I4C or ,H which were counted under the same conditions as the actual samples. The background counts were about 5 c.p.m. in both channels. The c.p.m. values were converted to d.p.m. by means of an external standard-channel ratio method.
391
Short communication TABLE2. SYNTHESIS OF ACh
BY HOMOGENATES OF SKELETAL MUSCLE ~
Enzyme source
Incubation medium
d.p.m. ~
2 mg Frog sartorius 2 mg Frog sartorius 2 mg Frog sartorius 2 rng Frog sartorius heated for 2 min at 100°C 0.2 mg Human intercostal 0.2 mg Human intercostal 0.2 mg Human intercostal 0.2 mg Human intercostal heated for 2 min at 100°C 0.02 mg Rat diaphragm 0.1 mg Rat diaphragm 0.1 mg Rat diaphragm 0.1 mg Rat diaphragm
Unchanged + I pg AChE - Physostigmine - Choline Unchanged Unchanged + 100 ng AChE - Physostigmine - Choline Unchanged Unchanged Unchanged + 1 pg AChE - Physostigmine Unchanged
~~
nmol .g - .h - I ~
~~
37 f 3(8) 1 f 2.5 (8)
3.33 0.09
6 2 3.0(8) f 2.8 (8)
0.54 - 0.09
-1
+-
78 7(8) 10 k 1.8(11)
+-
7 1.3(12) 7 & 2.6(8)
75 10 7 7
142 f 13(7) 533 _+ 47 (8) -4 & 3.2 (8)
1380 1030 -8
3 f 4.6(8)
6
heated for 2 min at 100°C Homogenates of muscle were incubated as described in Materials and Methods. Sartorius muscle (80mg) contains about 600 muscle fibres (but 1000 endplates, see MILEDIet a/., 1977), human intercostal muscle about 15,000 fibres/g of fresh tissue (cj ITO et al., 1976) & MITCHELL, 1961). Consequently, 2 mg and rat diaphragm (200 mg) 10,000 fibres (KRNJEVIC sartorius contains 15 fibres, 0.2mg human intercostal 3 fibres and 0.02mg rat diaphragm 1 fibre. Mean k S.E.M.with number of observations in brackets.
substances, such as ['4C]acetylcarnitine (cf: HAMPRECHTmuscle contains an enzyme other than ChAT which is able & AMANO. 1974), is small. Thus, it is unlikely that to synthesize ACh. This may explain why I found a much ['4C]acetylcarnitine is isolated by the present procedure, lower value for ACh synthesis in frog muscle (3.3 nmol/g/h) because acetylcarnitine does not precipitate with K13 at compared with that (29.4 nmol/g/h) reported by TUEEKet pH 7 (POLAK er al., 1977) and acetylcarnitine is not al. (1978). These authors used 12.5 mw-choline in the incuextracted by TPB at pH 7 (FONNUM, 1975). Moreover, as bation medium, whereas I used only 2 mM-choline. In fact, shown in Table 2, a low blank is obtained, regardless of using 10mM-choline I found ACh synthesizing activities whether tissue is incubated in the presence of acetylcho- of 3 W O nmol/g/h (not illustrated in Table 2). linesterase, in the absence of choline, or after inactivation In conclusion, the present method is very sensitive by heat. Apparently, practically all labelled material con- because of its low blank. It allows the analysis of ACh sists of ACh and the formation of ACh is dependent on synthesis in tissues with low ChAT activities. About 50 enzyme activity. samples can be assayed on a single day. However, the It should be borne in mind that in muscle, enzymes present method is more cumbersome (two precipitation other than ChAT are present which may be responsible steps with losses of ACh necessitating internal standardizafor part of the formed [14C]ACh. For instance, although tion and dual label counting) than the simple and rapid choline is a poor substrate for carnitine acetyltransferase procedure, without significant losses of ACh, described by (see for instance WHITE& Wu, 1973), this enzyme might FONNUM (19696, 1975). FONNUM'Smethods in my view synthesize ACh in appreciable amounts because in striated remain the methods of choice when the concentration of muscle carnitine acetyltransferase must be present in large ChAT in the tissue is not as low as that found in human excess to ChAT. Carnitine acetyltransferase is contained and frog muscle. in mitochondria of nerve and muscle, whereas ChAT is Acknowledgements-Financial support by the Foundation probably located only in the nerve terminals, which consti- FUNGO/ZWO is gratefully acknowledged. I thank Dr. tute less than 1:1O,OOO of the total muscle mass. In fact P. C. MOLENAAR for helpful discussions and suggestions. TUEEKet a!. (1978) have reported that 1 mM-4-(l-naphtylvinyl)pyridine, which blocks about 97% of the ChAT ac- Leiden University P. BRAGGAAR-SCHAAP tivity in frog sciatic nerve, inhibited ACh only about 60% Medical Centre, of synthesis in homogenates of frog sartorius muscle. The Department of Pharmacology, remaining 40% of ACh synthesizing activity may have been Wassenaarseweg 12, due to enzymes other than ChAT. In this connection it is 2333 A1 Leiden, of interest to note that frog sartorius muscle and human The Netherlands intercostal muscle show an appreciable amount of ACh REFERENCES synthesizing enzyme activity (which is not saturated at higher choline concentrations) in addition to a saturable activity ( K , i0.3 mM) only present in the endplate containing parts of the muscles (P. C. Molenaar & R. L. Polak, personal communication). These findings also indicate that
FONNUMF. (1969~)Isolation of choline esters from aqueous solutions by extraction with sodium tetraphenylboron in organic solvents. Eiochem. J . 113, 291-298.
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MILEDIR., MOLENAAR P. C. & POLAKR. L. (1977) An analysis of acetylcholine in frog muscle by mass fragrnentography. Proc. R. Soc. B197, 285-297. MORRISD.(1967) The effect of sulphydryl and othcr disulphide reducing agents on choline acetyltransferase activity estimated with synthetic acetyl-CoA. J . Neurochem. 14, 19-27. POLAKR. L. & MOLENAAR P. C. (1974) Pitfalls in determination of acetylcholine from brain by pyrolysis-gas chromatography/mass spectrometry. J . Neurochem. 23, 1295-1297. POLAK R. L.. MOLENAAR P. c. & BKAGGAAR-SCHAAP P. (1977) Regulation of acetylcholine synthesis in rat brain. in Cholinergic Mechanisms und Psychopharrnucolog). (JENDEN D. J., ed.) pp. 511-524. Plenum Press, New York. SCHRIERB. K. & SHUSTER L. (1967) A simplified radiochemical assay for choline acetyltransferase. J . Nruruchem. 14, 977-985. TUEEKS., ZELENAJ., GE I. & VYSKOEILF. (1978) Choline acetyltransferase in transected nerves, denervated muscles and Schwann cells of the frog: correlation of biochemical, electron microscopical and electrophysiological observations. Neuroscience 3, 709-724. WHITEH. L. & Wu J. C. (1973) Choline and carnitine acetyltransferase of heart. Biochemistry 12, 841-846.
