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Acetylcholine-like activity of acetyl-l -carnityl CoA in subcellular particles of narcotized brain homogenates
ARCHIVES
OF
BIOCHEMISTRY
AND
Acety~cho~ine-hike
Activity
Particles
114, %-lo1
BIOPHYSICS
of A~ety~-L-Carnity~
of Narcotized
E. A. HOSEIN ~epart~~t
(1966)
Brain TEOW
AND
CoA
in Subcellular
Homogenates YAN
KOH
of ~io~h~~~~tr~, McGW ~~~~~r~t~, Montreal, Canada Received September 14, 1965
The acetylcholine-like activity in the subcellular particles from narcotized rat and rabbit brain was assayedwith the frog rectus preparation and found to be more than that extractable from the brains of normal animals. Chromatographic separation of this material showed that the zone identified with the butyrobetaine CoA esters oontained most of the aoetylcholine-like activity. Chemical analysis revealed the presence of mainly aoetyl-I-oarnitine in almost all the extracts prepared from narcotized brains. From other data presented, it was concluded that narcotics cause an acoumulation of acetyl-1-oarnityl CoA and trace amounts of related substances in the brain. This accumulation may have been caused by inhibition of their release from the subcellular particles.
was mainly responsible for t,he acetylcholinelike activity extractable from brain. Recently, hfaslova (14), using polarographic techniques, concluded that of the material with acetylcholine-like activity extractable from ganglia, only 20% of this was actually a~etyleholine. Hosein and Ara (6) have shown that on the basis of paper chromatographic separation of brain extracts prepared from rats narcot,ized with pentobarbital or ether, most of the increased a~etyIcholine-like activity in the extracts was present in the zone of the chromatogram (R,9.5-0.7) normally occupied by the butyrobetaine CoA esters. In that investigation it was nob possible to determine whether any particular butyrobetaine Co-4 ester could have been responsible for the increase in ~etylcholine-like activity. Hosein and Proulx (15) have shown that when brain homogenates are prepared in isotonic sucrose and separated by differential centrifugation into their particular subcellular fractions, further separation of the material(s) with acetyl~hoIine-like activity in each fraction was possible. While bio-assay of trichloroacetic acid extracts of each fraction showed that most of the acetylcholinelike activity in the original homogenate was
It is established that during narcosis, the brain content of material with acetylcholinelike activity increases (l-7). Macintosh and Oborin (8) showed that in GUO,increasing the depth of narcosis caused a diminution of the release of free acetylcholine from brain. Studies by Marley and Paton (9) also showed that narcotics inhibited the release of free acetylcholine from the perfused superior cervical ganglion. Since the total acetylcholinelike activity in the brain increases during narcosis, it is likely that this is mainly in the “bound” form Studies by Hbsein et al. (10) indicated that on the basis of results obtained from (i) partition coefficient studies, (ii) bromophenol blue complex calorimetric determination, and (iii) parallel bioassay, the material with acetyIcholine-like activity in “bound” acetylcholine fraction of brain extracts may not be solely acetylcholine. Earlier, Hosein et al. (11-13) had shown that the material with acetylcholine-like activity in brain extracts is composed of a mixture of acetyl, propionyl and butyryl choline esters and a mixture of the CoA ester derivatives of r-butyrobetaine, crotonbetaine, I-carnitine and acetyl1-carnitine. These authors also showed that the mixture of the butyrobetaine CoA esters 94
NARCOSIS
AND ACETYL-L-CARNITINE
present in the crude ~to~hondrial (Pz) fraction in augment with Whit~taker (16), paper ~h~nlat5graphy showed that the greatest percentage activity in this fraction was contributed by the butyrobetaine CoA est’ers (15). A similar distribution of activity was obtained when subcellular particles from narcotized rats were studied (15). The results presented in this paper are an extension of the work of Hosein and Ara (6) and Hosein and Proulx (15) to determine the effects of several types of narcotic drugs on t’he distribution of material(s) with acetylcholine-like activity in sub-cellular particles prepared from brain homogenates. In addit.ion, chemi(~al identi~cation of the mat,erial(s) responsible for t,he increased acetylcholine-like activity observed under these particular experimental conditions was attempted. METHODS Brain homogenates were prepared in 0.32 M sucrose from normal or narcotized rats with or without 10-S 1!4 eserine and separated into the various fractions by centrifugation as described earlier by Whittaker (16) and by Hosein and Proulx (15). The fractions were extracted with 10% trichloroacetic acid, and paper chromatographic separation in the but,anol-water system was performed as previously described by Banister et al. (17) and by Hosein and Proulx (15). Specific bands on the developed chromatograms were eluted by sha,king in methanol and the acetylcholine-like activity was determined hy bioassay
(‘3).
Chemical identification of the materials present in tissue extra.cts was made through formation of their tetrachloroaurate or chloroplatinate salts as previously des,cribed (12, 13, 18). In each of these experiments, at least 50 rat brains were required. I-Carniti~e was extracted from Difeo beef extract as described by Krimberg (19) and this material was acetylated with acetylchloride exactly as described by Krimberg and Wittandt (20). Narcosis was induced in rats by inhalation or by intraperitoneal injection (pentobarbital 65 mg per kilogram; ethanol 15 ml per kilogram, mixture of equal volumes of absolute ethanol and 5% aqueous glucose) to produce anesthesia of 30 minutes duration. Magnesium narcosis was induced in rabbits by the subcutaneous injection of l-2 gm per kilogram magnesium sulfate, as a 25y0 solution.
95
CoA RESULTS SECTION
1
EFFECT OF VARIOUS NARCOTICS ON THE ACETYLCHOLINE-LIKE ACTIFITY OF SUBSTANCES IN SUBCELLULAR FRACTIONS OF RAT BRAIN I~OMOGENATES PREPARED IN (i) ESERINIZE~ OR (ii) ~\ToN-ESERINIZED
0.32
ikf
&JCROSE
Narcosis was induced in rats for 30 minut.es before removal of the brains. The sucrose homogenates were separated by centrifugation, and fractions P1, PZ, and Sz were prepared. Each fraction was ext,racted with trichloroa~etic acid and the extract was chromatogranhed in the water-saturated butanol system. The results obbained are shown in Tables I and II. In these experiments, attempts to remove all the sucrose in the extract by ethanol precipitation were not completely successful. This problem was partially resolved by applying the extract on several papers. The variable recoveries of activity in the eluate of the band w&h R, 0.15-0.50 may be attribut,able to this difficulty. It, can be seen that in many fractions, narcosis caused an increase in the eq~v~lent acetyl~holine-like activity extractable from brain homogenates. This increase in activity in the butyrobetaine CoA esters band (Rf 0.5-0.70) was often statistically significant; that in the acetylcholine band (R, 0.05415) because of the low activity, did not permit accurate assay and thus was of doubtful significance. The majority of the acetylcholine-like activity in the homogenates prepared from normal animals was found in the P, fraction as observed earlier by Whittaker (16) and by Hosein and Proulx (15). Narcosis caused an increase of about 75% with homogenates prepared with eserine while those without eserine showed a greater percentage increase. This indicated that in vivo narcosis in some manner facilitated the accumulation of material(s) with acetylcholine activity within these subcellular particles and that these substances were not entirely destroyed by cholinesterases. Similar experiments were performed with rabbits, which are very susceptible to magnesium narcosis. These results are shown in
None (Controls) Ether Pentobarbital Ethanol Nitrous oxide
Treatment
DISTRIBUTION
None (controls) Ether Pentobarbital Ethanol Nitrous oxide
Treatment
DISTRIBUTION
0.02 0.01 0.03 0.02 0.02
ACh R/ 0.05-0.15
ACTIVITY
PI
f zk It f f
0.16 0.13 0.12 0.04 0.12
0.24 0.48 0.39 0.2 0.21
f f f & f
0.14 0.10 0.18 0.03 0.08
0.22 0.16 0.24 0.07 0.11
f f. f f f
TABLE
I
0.11 0.02 0.12 0.05 0.04
0.26 0.22 0.17 0.11 0.15
f f f f f
TABLE
0.02 0.01 0.03 0.03 0.05
FROM RAT BRAIN
II
0.19 0.56 0.50 0.67 0.46
z!z zk f f zk
0.07 0.23 0.21 0.10 0.16
Substances in band with R/ 0.15-0.50
Pa
0.44 0.84 1.13 1.12 0.89
0.12 0.14 0.13 0.23 0.10
ISOLATED
i f f f f
Butyrobetaine CoA esters Rj 0.504.70
0.02 0.02 0.04 0.04 0.06
0.26 0.82 0.39 0.65 0.55
zk f f f f
0.10 0.35 0.22 0.20 0.20
Substances in band with R/ 15.0450
P2
0.95 1.71 1.61 1.71 1.41
f f f f f
0.14 0.16 0.11 0.22 0.12
Butyrobetaine CoA esters R/ 0.5GO.70
sz
RAT
0.01 0.01 0.01 0.04 0.04
ACh Rf 0.05415
assay)
FROM
0.01 0.01 0.01 0.04 0.03
f f f f f
0.09 0.09 0.07 0.35 0.27
f f f f f
0.12 0.25 0.21 0.30 0.29
52
0.01 0.03 0.01 0.03 0.11
f f f f f
0.02 0.01 0.05 0.04 0.07
0.34 0.50 0.48 0.41 0.61
f f f f f
0.04 0.04 0.07 0.02 0.11
Butyrobetaine CoA esters R/ [email protected]
HOMOGENATES
0.02 0.01 0.02 0.05 0.03
Substances in band with Rj0.15-0.50
BRAIN
0.07 0.06 0.07 0.24 0.16
IN
Butyrobetaine CoA esters R, 0.X-0.70
HOMOGENATES
ACh Substances in band RI 0.05-0.15 with RI 0.150.50
in pg ACh-Cl per half rat brain; mean and SD (frog reck
ACh RI 0.05-0.15
activity
0.13 0.12 0.12 0.03 0.04
Butyrobetaine CoA esters RI 0.50-0.70
Equivalent
ISOLATED
in pg ACh-Cl per half rat brain; mean and SD (frog rectus assay)
ACh Rf 0.05-0.15
activity
IN THE P,, Pz, AND SZ FRACTIONS ABSENCE OF ESERINE
OF SUBSTANCES IN THE Pl, Pz, AND SZ FRACTIONS IN THE PRESENCE OF ESERINE
Substances in band with R~0.15-0.50
PI
ACTIVITY
0.20 0.51 0.29 0.17 0.23
Butyrobetaine CoA esters Rj 0.S3-0.70
Equivalent
OF SUBSTANCES
Substances in band with Rf 0.15-0.50
OF ACh-LIKE
0.01 0.01 0.03 0.04 0.01
ACh RfO.OS-0.15
OF ACh-Lrm
5
s
g g
NARCOSIS
AND
A~ETYL-L-CAR,NITI~E TABLE
97
CoA
III
SUBCELLKLAR DISTRIBUTION OF SUBSTANCES WITH ACh-LIKE ACTIVITY ISOLATED IN ESERINIZFJD 0.32 ICI SUCROSE
IN RABBIT
BRAIN
-
IiOlllXilS
Magnesiumnarcosis
Probable substances
. R, 0.05-0.15 (ACh) Substances in band with Rf 0.15.-0.50 Ilf 0.50-0.70 (butyrobetaine CoA esters) Total a Equivalent
activity
Pz
Pl
ss
0.02” 0.06*0.04
0.02 0.08f0.02
O.l.3*0.04
0.?11.0.07/0.27*0.02
0.21~0.05~0.81~0.07 in pg ACh-Cl
0.02 0.10rt0.03
P-2
PI
0.03 0.05 o.07zko.05jo.15io.03
sz 0.05 0.03zk0.01
O.ZO*O.Ol
0.39f0.02~0.30~0.03
per gm; mean and SD (frog rectus
assay).
The various compounds of eluates of the latter band were separated by fractional crystallization and identified by their melting points and mixed melting points (12, 13). Extracts from normal untreated control rats contained choline and acetylcholine at at Rf 0.25-0.50 Rj 0.05-0.X; butyrylcholine and a mixt.ure of ~-buty~betaine, crotonbeSECTION 2 taine, 1-~arnitine and aeetyl-l-ca~itine at CHEMICAL II)E~TI~~~TION OF SUBSTA~VCE~ RI 0.5-0.7, as described earlier by Hosein et aE. (12, 13). Similar extracts from t,he narWITH ACEXTLCHOLINE-LIKE ACTIVITY cotized rats contained acetyl-1-carnitine IN NARCOTIZED RAT BRAIN (122-126’) at both R, 0.05-0.15 (small (a) Isolation and Identification of Quatenury amount) and R, 0.5-0.7 (large amount). In Ammonium Compounds addition, in this latter zone there was a small In the foregoing experiments, it was con- amount, of I-carnitine (148-150’). In these firmed that narcosis caused an accumulation experiments, it was not possible to weigh of material(s) with acetylcholine-like activthe crystals formed from the material in each ity in rat and rabbit brain. Chromatographeluate as the amount was small. ically, it appeared likely that. these subTo test the efficiency of the method to restances could be identified with the CoA cover and identify added a~etyl~hol~e chloesters of the butyrobetaines. It was therefore ride, an aliquot of an eluate from narcotized necessary to determine chemically the iden- brain extract (Ri 0.5-0.7) was assayed for tity of the material(s) in the eluates of the acetylcholine-like activity with the frog recband of the chromatogram (Rf 0.5-0.7) tus preparation and the remainder was diwhich possessed t,he highest acetylcholinevided into two equal parts. A 200 pg amount like activity. of pure acetylcholine chloride equivalent to (i) Forma,tion of tetrachloroaurate derivathe contained acetylcholine-like activity was tives. One hundred rats were narcotized with added to one part and both samples were pentobarbital for 30 minutes and their exthen converted to their tetrachloroaurate cised brains were extracted with 10 % TCA. derivatives. The added acetylcholine (162After Reinecke salt precipitation at pH 8.5 166”) was recovered and couid be identified (21), the residue was chromatography in the butanol-wat,er system as previously de- with a~etyl-l-carnitine in the mixture whiIe alone was found in the scribed (12, 13). The substances in the elu- acetyl-1-~arnitine control sample. ates of the bands with Rf 0.050.15; 0.15 (ii) Formation of ch~or~l~~nate derivatives. 0.25; 0.25-0.50; and 0.50-0.70 were conOne hnndred rats were narcotized with penverted to their tetrachloroaurate derivatives. Table III. Again, a similar pattern of distribution of act’ivity is obtained as described above with rats when other narcotics were used. The increased activity which was statistically significant (p < 0.05) was again accounted for by the butyrobetaine CoA esters in the PZ fraction.
HOSEIN AND KOH
98
tobarbital, the brains were extracted with TCA, and Reinecke salt precipitation was carried out at# pH 8.5. Both the chloroplatinate (18) and tetrachloroa~ate derivatives were prepared from the Reinecke salt precipitable material. Acetyl-I-carnitine was identified by the melting point of its tetrachloroa~ate (122-126’) and of its chloroplatinate (185”). These values compare favorably with 128” and 187“ described by Krimberg and Wittandt (20) and were confirmed in prelill~inary experilnent,s with synthetic acetyl-1-carnitine. The tetrachloroaurate derivative of acetyl1-carnitine isolated from brain extract was di~c~tly soluble in water, insoluble in ether and soluble in alcohol. The ~hloroplat,inate derivative also was difficultly soluble in water (20). (iii) F~~~~o~ of cro~onbe~~~ne.To confirm further t,he identity of acetyl-l-carnitine in the extract, Reinecke salt precipitable material from the extract prepared from one hundred narcotized rats was heated with concent~ted sulphuric acid to destroy the ester linkage and dehydrate the carnitine to crotonbetaine (22). The product of the reaction was converted to its tekachloroaurate derivative, which mehed at 193-195”. Using this react,ion, Carter et cd. (22) obtained 192-200” for crotonbetaine. These values are lower than those for pure crotonbetaine t,etrachloroa~ate (212-214°) reported by Linneweh (23). This was likely due to the fact that after treatment with the sulphuric acid the crotonbetaine formed was not isolated before father treatment was carried out. (b) IdentiJcation
of Acyl CoA Estem
The results from the experin~ents described above indicated that in extracts prepared from narcotized rat brain, acetyl-lcarnitine and I-narnitine were found on the chro~~a~gran~. These substances were found pre~omin~tly in the band w&h Rf 0.5-0.7 which earlier work (12, 13) showed to contain the CoA esters of the butyrobetaines. These quaternary ammonium compounds identified in the narcotized brain extracts originally were precipitated as reineckates at pH 8.5. Bregoff et al. (21) have shown that at this pH, betaine esters, choline and ace-
tylcholine are precipitable as reineckntes while at pH 1.2 the unesterified betaines are precipitable. Since the mat,erial(s) identified above were obt.ained by precipitation at pH 8.5, it is likely that they were betaine esters rather than unesterified betaines. This was further supported by chromatographie data. Acetyl-1-~arnit,ine and I-carnitir~e have RI 0.05-0.15 in the butanol-water system. In the experiments described above, these substances were identified in eluates of the band with R.f 0.5-0.7. ~st.erifi~ation of gammabutyrobetaine either with an alkyl radicle (24) or with CoA-SH (25) increases it’s mobility in this chromatographic system. Since Hosein et al. (12, 13) had earlier identified the CoA est,ers of several butyrobetaines in this band of the chromatogram, similar experiments were performed on extracts from the brains of narcotized rats. Lynen (26), Stadtman and Soadtman (27), and Mahler et al. (28) have shown that COA esters can be identified by measuring the difference in optical density at 232 and 260 rnp before and after alkaline hydrolysis. When this experiment was performed on an eluate of the band with Rf 0.5-0.7 from narcotized brain extract a difference in optical density was observed at 232 but not at, 260; indicative of thiol ester destruction (26-28). These result’s were similar to those described earIier by Hosein and Proulx (15). It now appeared likely t~hat acetyl-I-carnitine and 1-carnitine were present in the eluate as CoA esters. To check this point further, the mother liquor after tetrachloroa~ate forl~ation of acetyl-1-carnitine and 1-carnitine (Section 2, a, i) was treated with HZS to remove excess gold and the clear filtrate was further analysed. Ribose (29), adenine (26) and phosphate (30) were found to be present. These substances are components of several nucleotides including CoA-SH. Since all such nucIeotides have little or no mobilit,y in the butanol-water system, it is likely that their presence in this band of the chromatogram with R, 0.5--Q.7 was in conjugation with other material. These results are similar to those described earlier by Hosein et al. (12, 13, 25) for other butyrobetaine CoA esters and suggest that the mat,eriaI with acetylcholine-like activity
NARCOSIS
AND ACETYL-I,-CAI-LNITINE
in the band of the chromatogram with RI 0.5-0.7 was acet’yl-1-carnityl CoA and lcarnityl CoA. Since acetyl-1-carnitine rather than 1-carnitine possesses acetyl~holine-like activity (31,32), it would appear that acetylI-carnityl CoA is mainly responsible for the acetylcholine-like activity in narcotized rat brain. DISCUSSION
The increased acetylcholine-like activity in the narcotized brain could be due to the non-utilization of t,he transmitter substance for nervous activit,y. Indeed, in most of the instances studied, the animals were invariably fully anest,hetized prior to killing. Nonutilization of the transmitter substances may be attribut’able t’o failure of release of these materials from subcellular particles. The I’2 fraction which Whitt,aker (15) and others (33) have shown to be a crude mitochondrial contains t,he subfraction with fraction, pinched-off nerve endings and the synaptic vesicles. S&e these subfractions in turn contain most of the aeetyl~holine~like activity (15,16) it appears that the narcotic agent may in some manner affect the release of these substances from the subcellular particles. The acet,ylcholine-like activity of the material in these particles has been identified exclusively with acetylcholine (16). However, failure to identify a~etylcholine itself with more than 20% of the acetylcholinelike activity in ganglia (14) suggests that the bulk of the acetylcholine-like activity was due to some other material. Hosein et al. (12, 13) have shown by microchemical analyses of Reinecke salt precipitable material from extra&s of large numbers of fresh rat brain, that the acetyl~holine-like activity in such extracts comprises a mixture of choline esters and butyrobet8aine CoA esters. Several investigators (34-37) have implied that they have repeated these experiments. Examination of t,heir data reveals that they did not, attempt the complete procedure as described by Hosein er’ al. (12) but rather they merely performed ~hrol~~atographic separation in butanol-wat,er of tri~hloroaceti~ acid extracts prepared from a small number of rat brains. Using the ehromatographic system originally described by Banister et al. (17) for the
CoA
99
separation of choline esters in ox spleen as applied by Hosein et! al. (12) for trichloroacetic acid extracts of brain, Szerb (34) obtained peak a~etyl~holine-like activity in a brain extract in the band with R, 0.05-0.15; Pepeu et al. (35) between 0.2 and 0.6, and McLennan et al. (36) in two zones, 0.1 and 0.6. Crossland and Redfern (37), on the other hand, could fmd no activit,y below 0.5, but found peak activity between 0.5 and 0.9. l’epeu et al. (35) also performed chromatographic separation of Reinecke salt precipitable material and found peak activity at R, 0.12-0.36. These authors (35) and Crossland and Redfern (37) added acetylcholine chloride to their extracts and found that the added acetylcholine had Rf 0.12-0.36 (35) and 0.5-0.9 (37), respectively, rather than 0.05-0.15 for pure acetylcholine (12, 17). Despite the wide discrepancy between the Rf of pure ~cetylcholi~e and those in the various experiments, these authors concluded that the active material present was solely acetylcholine. This discrepancy between t#he Rf of pure a~etyleholi~e and that added to brain extracts was not observed by Hosein et al. (lo), who obtained Rf 0.05-0.15 for acetylcholine chloride in both systems. Richter and Crossland (2), Szerb (34), McLennan et aE. (36), Crossland and Redfern (37), and Ryall et al. (38) have used the method of parallel bio-assay to support the identit.y of the acetylcholine-like activity in brain extracts with acetylcholine. In all these instances, these authors assumed that the material with acetylcholine-like activity in the crude brain extract was acetylcholine because of the close correspondence of assay values between several test preparations for the same brain extract (39). This method has never been proven for mixtures of substances with acet,ylcholine-like activity. Hosein and Koh (40) have shown that this method of parallel bio-assay failed to identify acetylcholine in mixtures of materials with acetylcholine-like activity. In other work it was shown that ratios close to unity could be obt#ained with several test preparations when samples prepared without acetylcholine but containiI~g mixtures of non-natural substances with acetylcholine-like activity were tested (10). The reliability of the method of parallel bioassay for t,he identification of
100
HOSEIN
acetylcholine in crude tissue extracts is also of questionable validity. If the methods of chromatographi~ separation and parallel bio-assay fail to provide proof of the identity of the mat#erial(s) with acetylcholine-like activity found in tissue extracts, then other methods must8 be sought. Hosein et al. (10) have shown that the partition eoe~cient of material with acetylcholine-like activity in brain extracts is unrelated to pure acetylcholine. Studies with the bromophenol blue complex calorimetric reaction indicate that substances other than acetylcholine are present in the zone of the ~~orna~grarn of brain extracts where the peak a~etylcholine-like activity occurs. In this paper it was shown that when crystalline acetylcholine was added to the eluate of the zone of the chromatogram where the greatest acetylcholine-like activity occurred, this substance could be recovered and identified by micro-chemical analysis. This meant that if the assayed activity were due to this amount of acetylcholine, the method was sensitive enough to recover and identify it without total destruction. Previous work in this laboratory had shown that with this method of analysis normal brain extracts contain a mixture of choline esters and of butyrobetaine CoA esters. From the results described above, it was shown that in narcotized brain extracts the t,otal acetylcholine-like activity was increased over that extractable from normal animals and this activity was mainly due to acetyl-1-carnitine likely as its CoA ester derivative. This conclusion has been supported by Hosein and Smoly (41) who, synthesized acetyl-1-carnityl choline by incubating the aeetyl-1-carnityl CoA from narcotized brain extract with choline and choline acetyltransferase. The result,s described above for narcotized brain extracts could explain why in the earlier work of Macintosh and Oborin (8) and MarIey and Paton (9) “free” acetylcholine was not available for release during narcosis. Hosein and Ara (6) and Hosein and Proulx (14) had shown earlier that chromatographitally the acetylcholine content (Rf 0.1) of narcotized brain extracts is very small and that the increased activity was mainly in the “bound” fraction with R, 0.5-0.7 which contained the b~~tyrobetaine CoA esters.
AND KOH
It is concluded that narcotics cause an a~~u~~lulation of a~etyl-l-~arnit,yl GoA and other related substances in the subcell~ar particles of brain and this accumulation may be due to t,he failure of these materials to be released from these subcellular components. ACKNOWLEDGMENTS This work was supported by the United States Public Health Service Project NB-01756, Licensed Beverage Industries, Inc. (New York), and the Medical Research Council of Canada. REFERENCES 1. TOBIAS, J. M., LIPTON, M. A., AND LEPINAT, A. A., Proc. Exptl. Biol. Med. 61, 51 (1946). 2. RICHTER, D., AND CROSSLAND, J., Am. J. Physiol. 169, 247 (1949). 3. ELLIOTT, K. A. C., SWANK, R., AND HENDERSON, N., Am. J. Physiol. 162,469 (1950). 4. WAJDA, I., Ph.D. Thesis, University
Birmingham,
of
England (1951).
5. CROSSLAND, J.,
AND MERRICK, A. J., J. PhysioE. 125, 56 (1954). 6. HOSEIN, E. A., AND ARA, R., J. Pharmacol. Exptl. Therap. 136, 230 (1962). 7. GIARMAN, N. J., AND PEPEU, G., Brit. J.
Pharmacol. 19, 226 (1962). 8. MACINTOSH, F. C., AND OBORIN, P. E., Abstr. 19th fntern. PhysioE. Congr. p. 580 (1953). 9. MARLEY, E., AND PATON, W. D. M., Brit. J. PharmacoE. 14, 303 (1959). 10. HOSEIN, E. A., RAMBAUT, P., CIIABROL, J. G., AND ORZE~K, A., Arch. Biochem. Biophys. 111, 540 (1965). 11. HOSEIN, E. A., AND PROULX, P., aware 187, 321 (1960). 12. HOSEIN, E. A., PROULX, P., AND ARA, R., Biochem. J. 82, 341 (1962). 13. HOSEIN, E. A., AND ORZECK, A., T&em. J. Neuropharmaeol. 2, 71 (1964). 14. MASLOBA, A. I?., Federation Proc. a4, No. 3,
Part II, T548 (1965). 15. HOSEIN, E. A., AND PROULX, I)., Arch.
Biothem. Biophys. 106, 267 (1964). 16. WHITTAKER, V. P., Biochem. J. 72, 694 (1959). 17. BANISTER, J., WHITTAKER, V. I’., AND WIJESUNDERA, S., J. PhysioZ. 121, 55 (1953). 18. DALE, H. H., AND DUDLEY, H. W., J. Physiol.
68, 97 (1929). 19. KRIMBERG, R., 2. Physiol. Chem. 48, 412 (1906). 20. KRIMBERG, R., AND WITTANDT, MT., Biochem. 2. 261, 229 (1932). 21. BREGOFF, H. M., ROBERTS, E., AND DELWICHE, C. C., J. Biot. Chem. 206,565 (1953). 22. CARTER, H. E., BHATTACHARYYA, P. K.,
NARCOSIS
AND
ACETYL-L-~AR~ITI~~
K. R., AND FRAENKEL, G., Arch. B&hem. Biophys. 38, 405 (1952). 23. LINNEWEH, W., Hoppe SeyZ. Z. 181, 42 (1929). 24. HOSEIN, E. A., SMART, M., AND HAWKINS, K., Can. J. Biochem. f’hysiol. 38, 837 (1960). 25. HOSEIN, El. A., SMART, M., HAWKINS, K., ROCHON, S., AND STRASBERO, Z., Arch. Biochem. Biophys. 96, 246 (1962). 26. LYNEN, F., Federation hoc. 12, 683 (1953). 27. STADTMAN, E. R., Abstr. Am. Chem. Sot., i%‘%d Meeting 32~ (1952). 28. MAHLER, H. R., WABIL, S. J., AND BOCK, R. M., J. Biol. Chem. 204,453 (1953). 29. TAUBER, H., Proc. Sot. Ezptl. Biol. Med. 3’7, 600 (1937). 30. FISKE, C. A., AND SUBBAROW, Y. J. Biol. Chem. 68, 375 (1925). 31. WEGER, P., Biochem. Z. 287, 424 (1936). 32. DALLEMAONE, M. J., PHILXPPOT, E., BINON, WE~DM~~,
CoA
101
E. L., Proc. World Congr. Anesthesiol. 1, 285 (1955).
IF., AND DUMO~L~N,
33. DEROBERTJS, E., ARNAIZ, G. R., SALGANXCOFF, L., IRALDI, A. P., AND ZIEHER, L. M., J. Neurochem. 10, 225 (1963). 34. SZERB, J., Nature 197, 1016 (1963). 35. PEPEU, G., SCHMIDT, K. F., AND GIARMAN, N. J., Biochem. Pharmacol. 12,385 (1963). 36. MCLENNAN, II., CURRY, L., AND WALKER, R., Biochem. J. 89, 163 (1963). 37. CROSSLAND, J., AND REDFERN, P., Life Sci. 2, 711 (1963). 38, RYALL, R. W., STONE, IV., END WATKINS, J. C., J. Neuro~hem. 11, 621 (1964). 39. CHANT, H. C., AND GADDUM, J. H., J. Physiol.
79, 255 (1933). 40. HOSEIN, E. A., AND KOH, T. Y., Nature 206, 1119 (1965). 41. HOSEIN, E. A., AND SMOLY, J. M., unpublished data.
OF
BIOCHEMISTRY
AND
Acety~cho~ine-hike
Activity
Particles
114, %-lo1
BIOPHYSICS
of A~ety~-L-Carnity~
of Narcotized
E. A. HOSEIN ~epart~~t
(1966)
Brain TEOW
AND
CoA
in Subcellular
Homogenates YAN
KOH
of ~io~h~~~~tr~, McGW ~~~~~r~t~, Montreal, Canada Received September 14, 1965
The acetylcholine-like activity in the subcellular particles from narcotized rat and rabbit brain was assayedwith the frog rectus preparation and found to be more than that extractable from the brains of normal animals. Chromatographic separation of this material showed that the zone identified with the butyrobetaine CoA esters oontained most of the aoetylcholine-like activity. Chemical analysis revealed the presence of mainly aoetyl-I-oarnitine in almost all the extracts prepared from narcotized brains. From other data presented, it was concluded that narcotics cause an acoumulation of acetyl-1-oarnityl CoA and trace amounts of related substances in the brain. This accumulation may have been caused by inhibition of their release from the subcellular particles.
was mainly responsible for t,he acetylcholinelike activity extractable from brain. Recently, hfaslova (14), using polarographic techniques, concluded that of the material with acetylcholine-like activity extractable from ganglia, only 20% of this was actually a~etyleholine. Hosein and Ara (6) have shown that on the basis of paper chromatographic separation of brain extracts prepared from rats narcot,ized with pentobarbital or ether, most of the increased a~etyIcholine-like activity in the extracts was present in the zone of the chromatogram (R,9.5-0.7) normally occupied by the butyrobetaine CoA esters. In that investigation it was nob possible to determine whether any particular butyrobetaine Co-4 ester could have been responsible for the increase in ~etylcholine-like activity. Hosein and Proulx (15) have shown that when brain homogenates are prepared in isotonic sucrose and separated by differential centrifugation into their particular subcellular fractions, further separation of the material(s) with acetyl~hoIine-like activity in each fraction was possible. While bio-assay of trichloroacetic acid extracts of each fraction showed that most of the acetylcholinelike activity in the original homogenate was
It is established that during narcosis, the brain content of material with acetylcholinelike activity increases (l-7). Macintosh and Oborin (8) showed that in GUO,increasing the depth of narcosis caused a diminution of the release of free acetylcholine from brain. Studies by Marley and Paton (9) also showed that narcotics inhibited the release of free acetylcholine from the perfused superior cervical ganglion. Since the total acetylcholinelike activity in the brain increases during narcosis, it is likely that this is mainly in the “bound” form Studies by Hbsein et al. (10) indicated that on the basis of results obtained from (i) partition coefficient studies, (ii) bromophenol blue complex calorimetric determination, and (iii) parallel bioassay, the material with acetyIcholine-like activity in “bound” acetylcholine fraction of brain extracts may not be solely acetylcholine. Earlier, Hosein et al. (11-13) had shown that the material with acetylcholine-like activity in brain extracts is composed of a mixture of acetyl, propionyl and butyryl choline esters and a mixture of the CoA ester derivatives of r-butyrobetaine, crotonbetaine, I-carnitine and acetyl1-carnitine. These authors also showed that the mixture of the butyrobetaine CoA esters 94
NARCOSIS
AND ACETYL-L-CARNITINE
present in the crude ~to~hondrial (Pz) fraction in augment with Whit~taker (16), paper ~h~nlat5graphy showed that the greatest percentage activity in this fraction was contributed by the butyrobetaine CoA est’ers (15). A similar distribution of activity was obtained when subcellular particles from narcotized rats were studied (15). The results presented in this paper are an extension of the work of Hosein and Ara (6) and Hosein and Proulx (15) to determine the effects of several types of narcotic drugs on t’he distribution of material(s) with acetylcholine-like activity in sub-cellular particles prepared from brain homogenates. In addit.ion, chemi(~al identi~cation of the mat,erial(s) responsible for t,he increased acetylcholine-like activity observed under these particular experimental conditions was attempted. METHODS Brain homogenates were prepared in 0.32 M sucrose from normal or narcotized rats with or without 10-S 1!4 eserine and separated into the various fractions by centrifugation as described earlier by Whittaker (16) and by Hosein and Proulx (15). The fractions were extracted with 10% trichloroacetic acid, and paper chromatographic separation in the but,anol-water system was performed as previously described by Banister et al. (17) and by Hosein and Proulx (15). Specific bands on the developed chromatograms were eluted by sha,king in methanol and the acetylcholine-like activity was determined hy bioassay
(‘3).
Chemical identification of the materials present in tissue extra.cts was made through formation of their tetrachloroaurate or chloroplatinate salts as previously des,cribed (12, 13, 18). In each of these experiments, at least 50 rat brains were required. I-Carniti~e was extracted from Difeo beef extract as described by Krimberg (19) and this material was acetylated with acetylchloride exactly as described by Krimberg and Wittandt (20). Narcosis was induced in rats by inhalation or by intraperitoneal injection (pentobarbital 65 mg per kilogram; ethanol 15 ml per kilogram, mixture of equal volumes of absolute ethanol and 5% aqueous glucose) to produce anesthesia of 30 minutes duration. Magnesium narcosis was induced in rabbits by the subcutaneous injection of l-2 gm per kilogram magnesium sulfate, as a 25y0 solution.
95
CoA RESULTS SECTION
1
EFFECT OF VARIOUS NARCOTICS ON THE ACETYLCHOLINE-LIKE ACTIFITY OF SUBSTANCES IN SUBCELLULAR FRACTIONS OF RAT BRAIN I~OMOGENATES PREPARED IN (i) ESERINIZE~ OR (ii) ~\ToN-ESERINIZED
0.32
ikf
&JCROSE
Narcosis was induced in rats for 30 minut.es before removal of the brains. The sucrose homogenates were separated by centrifugation, and fractions P1, PZ, and Sz were prepared. Each fraction was ext,racted with trichloroa~etic acid and the extract was chromatogranhed in the water-saturated butanol system. The results obbained are shown in Tables I and II. In these experiments, attempts to remove all the sucrose in the extract by ethanol precipitation were not completely successful. This problem was partially resolved by applying the extract on several papers. The variable recoveries of activity in the eluate of the band w&h R, 0.15-0.50 may be attribut,able to this difficulty. It, can be seen that in many fractions, narcosis caused an increase in the eq~v~lent acetyl~holine-like activity extractable from brain homogenates. This increase in activity in the butyrobetaine CoA esters band (Rf 0.5-0.70) was often statistically significant; that in the acetylcholine band (R, 0.05415) because of the low activity, did not permit accurate assay and thus was of doubtful significance. The majority of the acetylcholine-like activity in the homogenates prepared from normal animals was found in the P, fraction as observed earlier by Whittaker (16) and by Hosein and Proulx (15). Narcosis caused an increase of about 75% with homogenates prepared with eserine while those without eserine showed a greater percentage increase. This indicated that in vivo narcosis in some manner facilitated the accumulation of material(s) with acetylcholine activity within these subcellular particles and that these substances were not entirely destroyed by cholinesterases. Similar experiments were performed with rabbits, which are very susceptible to magnesium narcosis. These results are shown in
None (Controls) Ether Pentobarbital Ethanol Nitrous oxide
Treatment
DISTRIBUTION
None (controls) Ether Pentobarbital Ethanol Nitrous oxide
Treatment
DISTRIBUTION
0.02 0.01 0.03 0.02 0.02
ACh R/ 0.05-0.15
ACTIVITY
PI
f zk It f f
0.16 0.13 0.12 0.04 0.12
0.24 0.48 0.39 0.2 0.21
f f f & f
0.14 0.10 0.18 0.03 0.08
0.22 0.16 0.24 0.07 0.11
f f. f f f
TABLE
I
0.11 0.02 0.12 0.05 0.04
0.26 0.22 0.17 0.11 0.15
f f f f f
TABLE
0.02 0.01 0.03 0.03 0.05
FROM RAT BRAIN
II
0.19 0.56 0.50 0.67 0.46
z!z zk f f zk
0.07 0.23 0.21 0.10 0.16
Substances in band with R/ 0.15-0.50
Pa
0.44 0.84 1.13 1.12 0.89
0.12 0.14 0.13 0.23 0.10
ISOLATED
i f f f f
Butyrobetaine CoA esters Rj 0.504.70
0.02 0.02 0.04 0.04 0.06
0.26 0.82 0.39 0.65 0.55
zk f f f f
0.10 0.35 0.22 0.20 0.20
Substances in band with R/ 15.0450
P2
0.95 1.71 1.61 1.71 1.41
f f f f f
0.14 0.16 0.11 0.22 0.12
Butyrobetaine CoA esters R/ 0.5GO.70
sz
RAT
0.01 0.01 0.01 0.04 0.04
ACh Rf 0.05415
assay)
FROM
0.01 0.01 0.01 0.04 0.03
f f f f f
0.09 0.09 0.07 0.35 0.27
f f f f f
0.12 0.25 0.21 0.30 0.29
52
0.01 0.03 0.01 0.03 0.11
f f f f f
0.02 0.01 0.05 0.04 0.07
0.34 0.50 0.48 0.41 0.61
f f f f f
0.04 0.04 0.07 0.02 0.11
Butyrobetaine CoA esters R/ [email protected]
HOMOGENATES
0.02 0.01 0.02 0.05 0.03
Substances in band with Rj0.15-0.50
BRAIN
0.07 0.06 0.07 0.24 0.16
IN
Butyrobetaine CoA esters R, 0.X-0.70
HOMOGENATES
ACh Substances in band RI 0.05-0.15 with RI 0.150.50
in pg ACh-Cl per half rat brain; mean and SD (frog reck
ACh RI 0.05-0.15
activity
0.13 0.12 0.12 0.03 0.04
Butyrobetaine CoA esters RI 0.50-0.70
Equivalent
ISOLATED
in pg ACh-Cl per half rat brain; mean and SD (frog rectus assay)
ACh Rf 0.05-0.15
activity
IN THE P,, Pz, AND SZ FRACTIONS ABSENCE OF ESERINE
OF SUBSTANCES IN THE Pl, Pz, AND SZ FRACTIONS IN THE PRESENCE OF ESERINE
Substances in band with R~0.15-0.50
PI
ACTIVITY
0.20 0.51 0.29 0.17 0.23
Butyrobetaine CoA esters Rj 0.S3-0.70
Equivalent
OF SUBSTANCES
Substances in band with Rf 0.15-0.50
OF ACh-LIKE
0.01 0.01 0.03 0.04 0.01
ACh RfO.OS-0.15
OF ACh-Lrm
5
s
g g
NARCOSIS
AND
A~ETYL-L-CAR,NITI~E TABLE
97
CoA
III
SUBCELLKLAR DISTRIBUTION OF SUBSTANCES WITH ACh-LIKE ACTIVITY ISOLATED IN ESERINIZFJD 0.32 ICI SUCROSE
IN RABBIT
BRAIN
-
IiOlllXilS
Magnesiumnarcosis
Probable substances
. R, 0.05-0.15 (ACh) Substances in band with Rf 0.15.-0.50 Ilf 0.50-0.70 (butyrobetaine CoA esters) Total a Equivalent
activity
Pz
Pl
ss
0.02” 0.06*0.04
0.02 0.08f0.02
O.l.3*0.04
0.?11.0.07/0.27*0.02
0.21~0.05~0.81~0.07 in pg ACh-Cl
0.02 0.10rt0.03
P-2
PI
0.03 0.05 o.07zko.05jo.15io.03
sz 0.05 0.03zk0.01
O.ZO*O.Ol
0.39f0.02~0.30~0.03
per gm; mean and SD (frog rectus
assay).
The various compounds of eluates of the latter band were separated by fractional crystallization and identified by their melting points and mixed melting points (12, 13). Extracts from normal untreated control rats contained choline and acetylcholine at at Rf 0.25-0.50 Rj 0.05-0.X; butyrylcholine and a mixt.ure of ~-buty~betaine, crotonbeSECTION 2 taine, 1-~arnitine and aeetyl-l-ca~itine at CHEMICAL II)E~TI~~~TION OF SUBSTA~VCE~ RI 0.5-0.7, as described earlier by Hosein et aE. (12, 13). Similar extracts from t,he narWITH ACEXTLCHOLINE-LIKE ACTIVITY cotized rats contained acetyl-1-carnitine IN NARCOTIZED RAT BRAIN (122-126’) at both R, 0.05-0.15 (small (a) Isolation and Identification of Quatenury amount) and R, 0.5-0.7 (large amount). In Ammonium Compounds addition, in this latter zone there was a small In the foregoing experiments, it was con- amount, of I-carnitine (148-150’). In these firmed that narcosis caused an accumulation experiments, it was not possible to weigh of material(s) with acetylcholine-like activthe crystals formed from the material in each ity in rat and rabbit brain. Chromatographeluate as the amount was small. ically, it appeared likely that. these subTo test the efficiency of the method to restances could be identified with the CoA cover and identify added a~etyl~hol~e chloesters of the butyrobetaines. It was therefore ride, an aliquot of an eluate from narcotized necessary to determine chemically the iden- brain extract (Ri 0.5-0.7) was assayed for tity of the material(s) in the eluates of the acetylcholine-like activity with the frog recband of the chromatogram (Rf 0.5-0.7) tus preparation and the remainder was diwhich possessed t,he highest acetylcholinevided into two equal parts. A 200 pg amount like activity. of pure acetylcholine chloride equivalent to (i) Forma,tion of tetrachloroaurate derivathe contained acetylcholine-like activity was tives. One hundred rats were narcotized with added to one part and both samples were pentobarbital for 30 minutes and their exthen converted to their tetrachloroaurate cised brains were extracted with 10 % TCA. derivatives. The added acetylcholine (162After Reinecke salt precipitation at pH 8.5 166”) was recovered and couid be identified (21), the residue was chromatography in the butanol-wat,er system as previously de- with a~etyl-l-carnitine in the mixture whiIe alone was found in the scribed (12, 13). The substances in the elu- acetyl-1-~arnitine control sample. ates of the bands with Rf 0.050.15; 0.15 (ii) Formation of ch~or~l~~nate derivatives. 0.25; 0.25-0.50; and 0.50-0.70 were conOne hnndred rats were narcotized with penverted to their tetrachloroaurate derivatives. Table III. Again, a similar pattern of distribution of act’ivity is obtained as described above with rats when other narcotics were used. The increased activity which was statistically significant (p < 0.05) was again accounted for by the butyrobetaine CoA esters in the PZ fraction.
HOSEIN AND KOH
98
tobarbital, the brains were extracted with TCA, and Reinecke salt precipitation was carried out at# pH 8.5. Both the chloroplatinate (18) and tetrachloroa~ate derivatives were prepared from the Reinecke salt precipitable material. Acetyl-I-carnitine was identified by the melting point of its tetrachloroa~ate (122-126’) and of its chloroplatinate (185”). These values compare favorably with 128” and 187“ described by Krimberg and Wittandt (20) and were confirmed in prelill~inary experilnent,s with synthetic acetyl-1-carnitine. The tetrachloroaurate derivative of acetyl1-carnitine isolated from brain extract was di~c~tly soluble in water, insoluble in ether and soluble in alcohol. The ~hloroplat,inate derivative also was difficultly soluble in water (20). (iii) F~~~~o~ of cro~onbe~~~ne.To confirm further t,he identity of acetyl-l-carnitine in the extract, Reinecke salt precipitable material from the extract prepared from one hundred narcotized rats was heated with concent~ted sulphuric acid to destroy the ester linkage and dehydrate the carnitine to crotonbetaine (22). The product of the reaction was converted to its tekachloroaurate derivative, which mehed at 193-195”. Using this react,ion, Carter et cd. (22) obtained 192-200” for crotonbetaine. These values are lower than those for pure crotonbetaine t,etrachloroa~ate (212-214°) reported by Linneweh (23). This was likely due to the fact that after treatment with the sulphuric acid the crotonbetaine formed was not isolated before father treatment was carried out. (b) IdentiJcation
of Acyl CoA Estem
The results from the experin~ents described above indicated that in extracts prepared from narcotized rat brain, acetyl-lcarnitine and I-narnitine were found on the chro~~a~gran~. These substances were found pre~omin~tly in the band w&h Rf 0.5-0.7 which earlier work (12, 13) showed to contain the CoA esters of the butyrobetaines. These quaternary ammonium compounds identified in the narcotized brain extracts originally were precipitated as reineckates at pH 8.5. Bregoff et al. (21) have shown that at this pH, betaine esters, choline and ace-
tylcholine are precipitable as reineckntes while at pH 1.2 the unesterified betaines are precipitable. Since the mat,erial(s) identified above were obt.ained by precipitation at pH 8.5, it is likely that they were betaine esters rather than unesterified betaines. This was further supported by chromatographie data. Acetyl-1-~arnit,ine and I-carnitir~e have RI 0.05-0.15 in the butanol-water system. In the experiments described above, these substances were identified in eluates of the band with R.f 0.5-0.7. ~st.erifi~ation of gammabutyrobetaine either with an alkyl radicle (24) or with CoA-SH (25) increases it’s mobility in this chromatographic system. Since Hosein et al. (12, 13) had earlier identified the CoA est,ers of several butyrobetaines in this band of the chromatogram, similar experiments were performed on extracts from the brains of narcotized rats. Lynen (26), Stadtman and Soadtman (27), and Mahler et al. (28) have shown that COA esters can be identified by measuring the difference in optical density at 232 and 260 rnp before and after alkaline hydrolysis. When this experiment was performed on an eluate of the band with Rf 0.5-0.7 from narcotized brain extract a difference in optical density was observed at 232 but not at, 260; indicative of thiol ester destruction (26-28). These result’s were similar to those described earIier by Hosein and Proulx (15). It now appeared likely t~hat acetyl-I-carnitine and 1-carnitine were present in the eluate as CoA esters. To check this point further, the mother liquor after tetrachloroa~ate forl~ation of acetyl-1-carnitine and 1-carnitine (Section 2, a, i) was treated with HZS to remove excess gold and the clear filtrate was further analysed. Ribose (29), adenine (26) and phosphate (30) were found to be present. These substances are components of several nucleotides including CoA-SH. Since all such nucIeotides have little or no mobilit,y in the butanol-water system, it is likely that their presence in this band of the chromatogram with R, 0.5--Q.7 was in conjugation with other material. These results are similar to those described earlier by Hosein et al. (12, 13, 25) for other butyrobetaine CoA esters and suggest that the mat,eriaI with acetylcholine-like activity
NARCOSIS
AND ACETYL-I,-CAI-LNITINE
in the band of the chromatogram with RI 0.5-0.7 was acet’yl-1-carnityl CoA and lcarnityl CoA. Since acetyl-1-carnitine rather than 1-carnitine possesses acetyl~holine-like activity (31,32), it would appear that acetylI-carnityl CoA is mainly responsible for the acetylcholine-like activity in narcotized rat brain. DISCUSSION
The increased acetylcholine-like activity in the narcotized brain could be due to the non-utilization of t,he transmitter substance for nervous activit,y. Indeed, in most of the instances studied, the animals were invariably fully anest,hetized prior to killing. Nonutilization of the transmitter substances may be attribut’able t’o failure of release of these materials from subcellular particles. The I’2 fraction which Whitt,aker (15) and others (33) have shown to be a crude mitochondrial contains t,he subfraction with fraction, pinched-off nerve endings and the synaptic vesicles. S&e these subfractions in turn contain most of the aeetyl~holine~like activity (15,16) it appears that the narcotic agent may in some manner affect the release of these substances from the subcellular particles. The acet,ylcholine-like activity of the material in these particles has been identified exclusively with acetylcholine (16). However, failure to identify a~etylcholine itself with more than 20% of the acetylcholinelike activity in ganglia (14) suggests that the bulk of the acetylcholine-like activity was due to some other material. Hosein et al. (12, 13) have shown by microchemical analyses of Reinecke salt precipitable material from extra&s of large numbers of fresh rat brain, that the acetyl~holine-like activity in such extracts comprises a mixture of choline esters and butyrobet8aine CoA esters. Several investigators (34-37) have implied that they have repeated these experiments. Examination of t,heir data reveals that they did not, attempt the complete procedure as described by Hosein er’ al. (12) but rather they merely performed ~hrol~~atographic separation in butanol-wat,er of tri~hloroaceti~ acid extracts prepared from a small number of rat brains. Using the ehromatographic system originally described by Banister et al. (17) for the
CoA
99
separation of choline esters in ox spleen as applied by Hosein et! al. (12) for trichloroacetic acid extracts of brain, Szerb (34) obtained peak a~etyl~holine-like activity in a brain extract in the band with R, 0.05-0.15; Pepeu et al. (35) between 0.2 and 0.6, and McLennan et al. (36) in two zones, 0.1 and 0.6. Crossland and Redfern (37), on the other hand, could fmd no activit,y below 0.5, but found peak activity between 0.5 and 0.9. l’epeu et al. (35) also performed chromatographic separation of Reinecke salt precipitable material and found peak activity at R, 0.12-0.36. These authors (35) and Crossland and Redfern (37) added acetylcholine chloride to their extracts and found that the added acetylcholine had Rf 0.12-0.36 (35) and 0.5-0.9 (37), respectively, rather than 0.05-0.15 for pure acetylcholine (12, 17). Despite the wide discrepancy between the Rf of pure ~cetylcholi~e and those in the various experiments, these authors concluded that the active material present was solely acetylcholine. This discrepancy between t#he Rf of pure a~etyleholi~e and that added to brain extracts was not observed by Hosein et al. (lo), who obtained Rf 0.05-0.15 for acetylcholine chloride in both systems. Richter and Crossland (2), Szerb (34), McLennan et aE. (36), Crossland and Redfern (37), and Ryall et al. (38) have used the method of parallel bio-assay to support the identit.y of the acetylcholine-like activity in brain extracts with acetylcholine. In all these instances, these authors assumed that the material with acetylcholine-like activity in the crude brain extract was acetylcholine because of the close correspondence of assay values between several test preparations for the same brain extract (39). This method has never been proven for mixtures of substances with acet,ylcholine-like activity. Hosein and Koh (40) have shown that this method of parallel bio-assay failed to identify acetylcholine in mixtures of materials with acetylcholine-like activity. In other work it was shown that ratios close to unity could be obt#ained with several test preparations when samples prepared without acetylcholine but containiI~g mixtures of non-natural substances with acetylcholine-like activity were tested (10). The reliability of the method of parallel bioassay for t,he identification of
100
HOSEIN
acetylcholine in crude tissue extracts is also of questionable validity. If the methods of chromatographi~ separation and parallel bio-assay fail to provide proof of the identity of the mat#erial(s) with acetylcholine-like activity found in tissue extracts, then other methods must8 be sought. Hosein et al. (10) have shown that the partition eoe~cient of material with acetylcholine-like activity in brain extracts is unrelated to pure acetylcholine. Studies with the bromophenol blue complex calorimetric reaction indicate that substances other than acetylcholine are present in the zone of the ~~orna~grarn of brain extracts where the peak a~etylcholine-like activity occurs. In this paper it was shown that when crystalline acetylcholine was added to the eluate of the zone of the chromatogram where the greatest acetylcholine-like activity occurred, this substance could be recovered and identified by micro-chemical analysis. This meant that if the assayed activity were due to this amount of acetylcholine, the method was sensitive enough to recover and identify it without total destruction. Previous work in this laboratory had shown that with this method of analysis normal brain extracts contain a mixture of choline esters and of butyrobetaine CoA esters. From the results described above, it was shown that in narcotized brain extracts the t,otal acetylcholine-like activity was increased over that extractable from normal animals and this activity was mainly due to acetyl-1-carnitine likely as its CoA ester derivative. This conclusion has been supported by Hosein and Smoly (41) who, synthesized acetyl-1-carnityl choline by incubating the aeetyl-1-carnityl CoA from narcotized brain extract with choline and choline acetyltransferase. The result,s described above for narcotized brain extracts could explain why in the earlier work of Macintosh and Oborin (8) and MarIey and Paton (9) “free” acetylcholine was not available for release during narcosis. Hosein and Ara (6) and Hosein and Proulx (14) had shown earlier that chromatographitally the acetylcholine content (Rf 0.1) of narcotized brain extracts is very small and that the increased activity was mainly in the “bound” fraction with R, 0.5-0.7 which contained the b~~tyrobetaine CoA esters.
AND KOH
It is concluded that narcotics cause an a~~u~~lulation of a~etyl-l-~arnit,yl GoA and other related substances in the subcell~ar particles of brain and this accumulation may be due to t,he failure of these materials to be released from these subcellular components. ACKNOWLEDGMENTS This work was supported by the United States Public Health Service Project NB-01756, Licensed Beverage Industries, Inc. (New York), and the Medical Research Council of Canada. REFERENCES 1. TOBIAS, J. M., LIPTON, M. A., AND LEPINAT, A. A., Proc. Exptl. Biol. Med. 61, 51 (1946). 2. RICHTER, D., AND CROSSLAND, J., Am. J. Physiol. 169, 247 (1949). 3. ELLIOTT, K. A. C., SWANK, R., AND HENDERSON, N., Am. J. Physiol. 162,469 (1950). 4. WAJDA, I., Ph.D. Thesis, University
Birmingham,
of
England (1951).
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