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Fractionation by zonal centrifugation of brain of normal rats and rats treated with morphine
Pergamon Press
Life Sciences Vol. 11, Part I, pp . 365-373, 1972 . Printed in Great Britain
FRACTIONATION BY ZONAL CENTRIFUGATION OF BRAIN OF NORMAL RATS AND RATS TREATED WITH MORPHINE Richard M . Van Frank and Irving S . Johnson The Lilly Research Laboratories, Eli Lilly and Company Indianapolis, Indiana 46206 (Received 21 December 1971 ; in final form 6 March 1972)
The brains of normal or treated rats were homogenized by The homogenate was explosive nitrogen decompression . fractionated using a sucrose-Ficoll gradient in a B-XXIX zonal centrifuge rotor . Three characteristic peaks of subcellular particulate were routinely obtained from normal rat brains . Brains from rats treated with morphine routinely contained only two of the particulate peaks . The location of peak activities of acetylcholinesterase and acid p'hosphatase were shifted in morphinetreated animals . Introduction Recent reports indicate that morphine is taken up in vitro by various organellae of neural origin (1), or is found associated with synaptosomal (nerve ending) fractions of brain after intra
In contrast, Mule et al . - (3) reported in cisternal injection (2) . an earlier study that morphine was primarily found in the microsomal supernatant or soluble fraction and was not found in significant quantities in the synaptosomal fractions of brain from
either normal or tolerant guinea pigs . The experiments described in this paper were undertaken to investigate the effect of morphine on rat brain at the subcellular level . To do this we have developed a fractionation procedure which permits rapid examination of the total spectrum of particles present in brain homogenates . While this procedure is less elegant than those of Whittaker (4)
or DeRobertis (5), it has the marked advantage that the inevitable loss of enzymes and destruction or modification of organellae that occur during repeated washings and differential centrifugation are considerably reduced .
Methods Preparation of Homogenate . Male Sprague-Dawley rats weighing from 200-250 gm were exsanguinated by incising the cervical blood
365
366
Fractionation of Brain by Centrifugation
Vol. 11, No. 8
vessels .
The skull was rapidly opened and the whole brain or cereberal hemispheres and midbrain (Tel-, Di-, and Mes-, encephalon were removed, weighed, and placed in cold 0 .25M sucrose buffered with 0 .05M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7 .1 . The weight of individual brains ranged from 1 .7-2 .0 gm and the cereberal hemispheres and midbrain from 1 .3-1 .45 gm . Two brains were used for each experiment . Brains were washed once in buffered sucrose, cut into quarters, and washed again . The tissue was then placed in a syringe and forced into a second syringe through a Luer connector . Tissue was forced from syringe to syringe ten times .
Five ml of 0 .25M buffered sucrose were added, and the process was repeated fifteen times . The tissue was then forced back and forth through a 30 mesh stainless steel screen held in a 25 mm Millipore filter holder, eleven times . The screen was washed with 5 ml of buffered sucrose, and the same process was repeated using a 60 mesh screen, seventeen times . The 60 mesh screen was washed with 10 ml of buffered sucrose, and the tissue suspension was diluted to 40 ml with buffered sucrose . Twentyfive ~1 of Antifoam A concentrate (Sigma Chemical Company) were added,
and the suspension was placed in a plastic beaker in a Parr Cell Disruption Bomb (Parr Instrument Company) . In the experiments where cerebral hemispheres and midbrain were used, the Antifoam was added after the homogenate had been removed from the bomb . Using nitrogen, the pressure was slowly increased to 750-800 psi . The previously cooled bomb was maintained at 4 ° C and the tissue suspension was magnetically stirred . After 30 minutes the pressure was rapidly released from the bomb . It should be noted that the tissue suspension was not expelled from the bomb through the de-
livery tube but remained in the bomb as the pressure was released, thus eliminating the shear that occurs when the suspension is forced through the control valve under pressure . The homogenate was diluted to 100 ml with 0 .25M buffered sucrose and thoroughly mixed .
A 10 ml sample was removed and the remaining 90 ml were loaded into the centrifuge .
Zonal Centrifugation . The B-XXIX rotor was used in these experiments (6) . Gradients were produced using the gradient generator developed by Anderson and Rutenberg (7) . All solutions
were buffered with 0 .05M HEPES . An 1100 ml gradient was generated using as the low density solution 0 .25M buffered sucrose and as the high density solution buffered 23~ Ficoll containing 0 .25M
Fractionation of Brain by Centrifugation
Vol. 11, No. 8
367
One-hundred-and-fifty ml of 25~ buffered Ficoll containsucrose . ing 0 .25M sucrose followed by 250 ml of 1 .57M buffered sucrose were used to fill the rotor . The brain suspension was pumped into the center of the rotor followed by 70 ml of 0 .05M HEPES buffer . The rotor was accelerated to 25,000 rpm and centrifugation was
continued for 30 minutes . After the rotor had decelerated to 4,000 rpm, 60~"sucrose was pumped into the edge and 40 ml fractions were collected as the gradient was displaced out from the center of the rotor .
Analyses .
The gradient was monitored by determining the
refractive index (RI) of each fraction .
Optical density (OD) of
each fraction was determined by either scanning from 350 nm to
240 nm or by determining the OD at 280 nm using a Cary Model 15 spectrophotometer .
Acetylcholinesterase (ACHE) activity was de-
termined using the method of Ellman et al . (8) . Acid phosphatase was determined using p-nitrophenylphosphate as the substrate in the presence of 0 .1~ Triton X-100 (9) . the method of Lowry et al . (10) .
Protein was determined by
Results
Fig . 1 shows the distribution of particulate material as measured by OD se° and the distribution of acid phosphatase and ACHE activity from the fractionation of normal rat brain .
Three
OD peaks are present .
The first, fractions 3-5~ coincide with the sample zone and is composed of soluble material with very
low S values such as nucleic acid and ribosomes, and membranous material of very low isopycnic-banding density . This region of the gradient contains material that shows a sharp absorption peak at 260 nm which suggests the presence of nucleic acid and~or ribosomes .
The second (F 10-17) and the third (F 23-25) peaks
are composéd of particulate that migrates at different rates through the gradient or is isopycnically banded at different densities . Fig . 2 shows the distribution of particulate, acid
phosphatase, and ACHE, from the brain of rats that had received
4 mg of morphine sulfate per 100 gm body weight, subcutaneously,
60 min prior to sacrifice .
The first and second peaks appear
as before but the third peak is now absent .
368
,o
É ô
e
0 à
7 e
~O Um N5 O
O
Fractionation of Brain by Centrifugation
4 3 2 1
Vol . 11, No . 8
~s .lo'
3"10' ACHE VMoI/ml
O U Q 100 90 90 70 BO 50 40 30 20 10
2" 10'
1" 10' 800 800 400 2oa
FIG . 1
Fractionation of normal rat brain
FIG . 2 Fractionation of brains from rats receiving 4 mg morphine per 100 gm
Fig . 3 shows the distribution of protein in the gradients of the experiments summarized in Fig . l, 2, and Table 1 . While OD 28° is not proportional to protein concentration in all regions of the gradient, it is a legitimate measure of the presence of
subcellular particulate . It should be noted, however, that at OD aeo of greater than 2, the comparative value of protein concentration versus OD 28° becomes increasingly discrepant .
FIG . 3 Distribution of protein in gradients from brains of normal and morphine-treated rats
Vol. 11, No. $
Fractionation of Brain by Centrifugation
369
Figs . 4 and 5 are the results of separate duplicate experiments using normal rats (Fig . 4) and morphine-treated (4mg~100) rats (Fig . 5) and show the reproducibility of this fractionation technic . The displacement of the peak in Fig . 4 may be due to a slight difference in the shape of the gradient between the two runs . _
9
5 4
C
1
3
5
1
1
11
13
15
11
1!
!1
23
FRACTION NO .
25
27
2!
]1
3]
2
1
35 37
3
5
7
!
11
U
15
11
i!
!1
27
FRACTION NO .
25
27
29
31
73
35
OD a6° profile of duplicate frac- OD as° profile of duplicate frac tionations of normal rat brains tionations of brains of rats treated with 4 mg morphine~100 gm The data shown in Fig . 6 is the result of fractionation of the cerebral hemispheres and midbrain from normal rats . Fig . 6a is a plot of the OD ss° while 6b is a plot of the results of ACHE and acid phosphatase assays . Percent recovery of enzymes from duplicate fractionations are found in Table 1 . RI
- t . at .~ 3 .ao - 1 .39 ~O
- 1 .38
RI
N
r
1 .37
-1 .36 -1 .35
1300 É
1100
110 100
900
90 80
N à
70
700
50
500
so
0
40
300
30 20
1
]
5
7
9
Il
1]
15
O
1!
tl 23
FRACTION NO .
25
27
29
71
]]
]5
FIG . 6a OD ae° profile of cerebral hemispheres and midbrain of normal rats
1 .33
D A m
100
10 1
7
5
1
!
9
1]
Ni
A
N
21
2] 25
FRACTION NO . .
77
2!
71
77
35
FIG . 6b Profile of ACHE and acid phosphatase from cerebral hemispheres and midbrain of normal rats
3
370
Fractionation of Brain by Centrifugation
Percent Substance
Vol . 11, No . 8
TABLE 1
Recovery
from Gradient Percent Recovered
Fig .
Exp .
Protein
3 3
1 1
Normal Morphine
ACHE
6b
1 2
Normal Normal
7b
1 2
Morphine Morphine
92 103
6b
1 2
Normal Normal
97 112
7b
1
Morphine Morphine
102 g9
Acid Phosphatase
E Activity in Gradient E Activity in Homogenate
Treatment
132 110 85 81
X 100 = Percent Recovered
Fig . 7 is the result of fractionation of cerebral hemispheres and midbrain from rats which received 2 .0 mg morphine sulphate~100 grams subcutaneously, 60 min prior to sacrifice . Fig . 7a is a plot of the OD~B° while 7b is the enzyme assay profile . Enzyme recovery from duplicate fractionations is shown in Table l . Treatment with morphine results in a marked decrease in the third peak along with a decrease in enzyme activity . The decrease in OD 28° and enzyme concentration is not as great as that produced by treatment with 4 mg morphine~100 grams (Figs . 1 and 2) .
1300 110
1100 D
90
90o
E 100 ~ 90
° ~o
~oo
m 50 ar 50
500
a ao Q
30 20 10
300 100 s i
s
n n~s na ananam111s FRACTION NO .
FIG . 7a FIG . 7b OD 2s° profile of cerebral hemi- Profile of ACHE and acid phosphaspheres and midbrain from rats tase from cerebral hemispheres treated with 2' .0 mg~100 morphine and midbrain of morphine-treated rat s
n 2 rn ,_ ô
3
Vol. 11, No . 8
Fractionation of Brain by Centrifugation
371
Table 2 contains assay data on the homogenates used in duplicate experiments . There appears to be a decrease in acid phosphatase activity in the homogenate from rats treated with morphine . The range in ACHE values makes it difficult to conclude that morphine treatment has any effect on ACHE côncentration . TABLE 2
Enzyme Activity in Homogenates Used in Duplicate Fractionations Enzyme ACHE
Acid Phosphatase
Treatment
Activity ~Mol~ml
Fig .
Exp .
6b
1 2
Normal Normal
9,300 7,610
7b
1 2
Morphine Morphine
8,272 7,573
6b
1 2
Normal Normal
60,100 60,400
7b
1 2
Morphine Morphine
47,872 54,787
Discussion
The cell homogenization process used in these experiments differs from the one more commonly used, a rotating Teflon pestle in a glass tube, in that a minimal amount of shear is produced by
The tissue is disrupted by passage through a screen with pore diameters considerably greater than neuronal perikarya ; however, nerve processes are undoubtedly broken off in the process . the process.
The breakage of the cells is accomplished by explosive nitrogen decompression . No washing or sedimentation of the homogenate is done prior to zonal centrifugation . With the exception of the
buffered sucrose used for the washing of the whole and quartered brain, all fluid used in the tissue disruption and cell homogenization process is included in the sample that is centrifuged . Approximately 45 min elapsed from the time the brains were removed until the sample was introduced into the tonal centrifuge . We believe that the rapid processing and conservation of sample by the elimination of differential centrifugation should improve the recovery of enzymes and aid in preservation of the more fragile organellae . Data presented in Figs . 1 and 6, and Figs . 2 and 7 show that treatment with morphine has a profound effect on the
372
Fractionation of Brain by Centrifugation
Vol . 11', No . 8
population of subcellular particulate normally found in the third peak .
This could be due to either a decrease in size or density
of a major portion of an organelle or organellae prior to cell disruption, or because of an increase in the fragility of a major portion of subcellular particles resulting in their being broken up more easily by the homogenization process . However,
changes that occurred in enzyme levels indicate that
if breakage occurred, the enzymes were not released in a free form, since the amount of enzymes in the first peak (sample zone) was not significantly increased .
If the organellae are broken, the
enzymes appear to remain bound to membranes . Since ACHE and acid phosphatase are considered marker enzymes for synaptosomes and
lysosomes, respectively, these are t'he most likely organellae affected and may be the predominant particulates in the third peak . To cause the observed change in the third peak this effect would have to be exerted on a major portion of the brain cells or The effect it would not be detected by the methodology described . is not produced in vitro by the addition of morphine to the cell
homogenate . There appears to be little loss of protein or enzymes during gradient centrifugation .
The homogenates are contaminated with
some red blood cells which would sediment through the gradient to the rotor wall . This could account for some of the apparent loss of ACHE activity . The procedure described permits the rapid fractionation of
brain and the measurement of changes both in physical properties of subcellular particulates and in enzyme concentrations which
should facilitate the study of the effects of drugs on t'he brain . We are also studying the effect produced by methadone,
diisopropylfluorophosphate,
and p-chloroamphetamine . The distribution of particulate and enzymes in the gradient is related to the drug used and is not an artifact of the fractionation technique .
These studies will be reported in a subsequent paper . Electronmicroscopic and histochemical studies are also in progress in an effort to elucidate the mechanism behind the effect reported in this paper .
Acknowle dgments The We thank Mr . J . Redmond for 'his technical assistance . B-XXIX rotor was obtained through a cooperative agreement with
Vol . 11, No . 8
Fractionation of Brain by Centrifugation
373
Dr . N . G . Anderson, Director of the MAN program at Oak Ridge National Laboratory .
1.
2. 3. 4. 5. 6. 7. 8. 9. 10 .
References and A . LAJTHA, Brain Research 24 :534-538 (1970) " S . NAVON J . T . SCRAFANI, N . WILLIAMS and D . H . CLOUET, The Pharma colo~ist 12 2 :175 (1970) . S . J . MULE, C . M . REDMAN and J . W . FLESHER, J . Pharmacol . Exp . Therap . 15 7 2) :459-471 (1967) " V . P . WHITTAKER, Handbook of Neurochemistr 2_ :327-324 Plenum 19 9 . Press, New York, E . De ROBERTIS, E . RODRIGUEZ and G . De LORES ARNAIZ, Handbook 1969 . of Neurochemistry 2 ;365-392 " Plenum Press, New York, N . G . ANDERSON, C . E . NUNLEY and C . T . RANKIN, JR ., Anal . Biochem . 31 :255-271 (1969) " N . G . ANDERSON and E . RUTENBERG, Anal . Biochem . _21 :259-265 (1967) " G . L . ELLMAN, K . D . COURTNEY, V . ANDRES, JR . and R . M . FEATHERSTONE, Biochem . Pharmacol . 7 :88-95 (1961) K . LINHARDT and K . WALTER, Methods of Enz matit Anal sis 19 5 p " 779-785 " Academic Press, New York, N . Y . 0 . H . LOWRY, N . J . ROSEBROUGH, A . L . FARR, and R . J . RANDALL, J . Biol . Chem . 193 :265-275 (1951) "
Life Sciences Vol. 11, Part I, pp . 365-373, 1972 . Printed in Great Britain
FRACTIONATION BY ZONAL CENTRIFUGATION OF BRAIN OF NORMAL RATS AND RATS TREATED WITH MORPHINE Richard M . Van Frank and Irving S . Johnson The Lilly Research Laboratories, Eli Lilly and Company Indianapolis, Indiana 46206 (Received 21 December 1971 ; in final form 6 March 1972)
The brains of normal or treated rats were homogenized by The homogenate was explosive nitrogen decompression . fractionated using a sucrose-Ficoll gradient in a B-XXIX zonal centrifuge rotor . Three characteristic peaks of subcellular particulate were routinely obtained from normal rat brains . Brains from rats treated with morphine routinely contained only two of the particulate peaks . The location of peak activities of acetylcholinesterase and acid p'hosphatase were shifted in morphinetreated animals . Introduction Recent reports indicate that morphine is taken up in vitro by various organellae of neural origin (1), or is found associated with synaptosomal (nerve ending) fractions of brain after intra
In contrast, Mule et al . - (3) reported in cisternal injection (2) . an earlier study that morphine was primarily found in the microsomal supernatant or soluble fraction and was not found in significant quantities in the synaptosomal fractions of brain from
either normal or tolerant guinea pigs . The experiments described in this paper were undertaken to investigate the effect of morphine on rat brain at the subcellular level . To do this we have developed a fractionation procedure which permits rapid examination of the total spectrum of particles present in brain homogenates . While this procedure is less elegant than those of Whittaker (4)
or DeRobertis (5), it has the marked advantage that the inevitable loss of enzymes and destruction or modification of organellae that occur during repeated washings and differential centrifugation are considerably reduced .
Methods Preparation of Homogenate . Male Sprague-Dawley rats weighing from 200-250 gm were exsanguinated by incising the cervical blood
365
366
Fractionation of Brain by Centrifugation
Vol. 11, No. 8
vessels .
The skull was rapidly opened and the whole brain or cereberal hemispheres and midbrain (Tel-, Di-, and Mes-, encephalon were removed, weighed, and placed in cold 0 .25M sucrose buffered with 0 .05M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7 .1 . The weight of individual brains ranged from 1 .7-2 .0 gm and the cereberal hemispheres and midbrain from 1 .3-1 .45 gm . Two brains were used for each experiment . Brains were washed once in buffered sucrose, cut into quarters, and washed again . The tissue was then placed in a syringe and forced into a second syringe through a Luer connector . Tissue was forced from syringe to syringe ten times .
Five ml of 0 .25M buffered sucrose were added, and the process was repeated fifteen times . The tissue was then forced back and forth through a 30 mesh stainless steel screen held in a 25 mm Millipore filter holder, eleven times . The screen was washed with 5 ml of buffered sucrose, and the same process was repeated using a 60 mesh screen, seventeen times . The 60 mesh screen was washed with 10 ml of buffered sucrose, and the tissue suspension was diluted to 40 ml with buffered sucrose . Twentyfive ~1 of Antifoam A concentrate (Sigma Chemical Company) were added,
and the suspension was placed in a plastic beaker in a Parr Cell Disruption Bomb (Parr Instrument Company) . In the experiments where cerebral hemispheres and midbrain were used, the Antifoam was added after the homogenate had been removed from the bomb . Using nitrogen, the pressure was slowly increased to 750-800 psi . The previously cooled bomb was maintained at 4 ° C and the tissue suspension was magnetically stirred . After 30 minutes the pressure was rapidly released from the bomb . It should be noted that the tissue suspension was not expelled from the bomb through the de-
livery tube but remained in the bomb as the pressure was released, thus eliminating the shear that occurs when the suspension is forced through the control valve under pressure . The homogenate was diluted to 100 ml with 0 .25M buffered sucrose and thoroughly mixed .
A 10 ml sample was removed and the remaining 90 ml were loaded into the centrifuge .
Zonal Centrifugation . The B-XXIX rotor was used in these experiments (6) . Gradients were produced using the gradient generator developed by Anderson and Rutenberg (7) . All solutions
were buffered with 0 .05M HEPES . An 1100 ml gradient was generated using as the low density solution 0 .25M buffered sucrose and as the high density solution buffered 23~ Ficoll containing 0 .25M
Fractionation of Brain by Centrifugation
Vol. 11, No. 8
367
One-hundred-and-fifty ml of 25~ buffered Ficoll containsucrose . ing 0 .25M sucrose followed by 250 ml of 1 .57M buffered sucrose were used to fill the rotor . The brain suspension was pumped into the center of the rotor followed by 70 ml of 0 .05M HEPES buffer . The rotor was accelerated to 25,000 rpm and centrifugation was
continued for 30 minutes . After the rotor had decelerated to 4,000 rpm, 60~"sucrose was pumped into the edge and 40 ml fractions were collected as the gradient was displaced out from the center of the rotor .
Analyses .
The gradient was monitored by determining the
refractive index (RI) of each fraction .
Optical density (OD) of
each fraction was determined by either scanning from 350 nm to
240 nm or by determining the OD at 280 nm using a Cary Model 15 spectrophotometer .
Acetylcholinesterase (ACHE) activity was de-
termined using the method of Ellman et al . (8) . Acid phosphatase was determined using p-nitrophenylphosphate as the substrate in the presence of 0 .1~ Triton X-100 (9) . the method of Lowry et al . (10) .
Protein was determined by
Results
Fig . 1 shows the distribution of particulate material as measured by OD se° and the distribution of acid phosphatase and ACHE activity from the fractionation of normal rat brain .
Three
OD peaks are present .
The first, fractions 3-5~ coincide with the sample zone and is composed of soluble material with very
low S values such as nucleic acid and ribosomes, and membranous material of very low isopycnic-banding density . This region of the gradient contains material that shows a sharp absorption peak at 260 nm which suggests the presence of nucleic acid and~or ribosomes .
The second (F 10-17) and the third (F 23-25) peaks
are composéd of particulate that migrates at different rates through the gradient or is isopycnically banded at different densities . Fig . 2 shows the distribution of particulate, acid
phosphatase, and ACHE, from the brain of rats that had received
4 mg of morphine sulfate per 100 gm body weight, subcutaneously,
60 min prior to sacrifice .
The first and second peaks appear
as before but the third peak is now absent .
368
,o
É ô
e
0 à
7 e
~O Um N5 O
O
Fractionation of Brain by Centrifugation
4 3 2 1
Vol . 11, No . 8
~s .lo'
3"10' ACHE VMoI/ml
O U Q 100 90 90 70 BO 50 40 30 20 10
2" 10'
1" 10' 800 800 400 2oa
FIG . 1
Fractionation of normal rat brain
FIG . 2 Fractionation of brains from rats receiving 4 mg morphine per 100 gm
Fig . 3 shows the distribution of protein in the gradients of the experiments summarized in Fig . l, 2, and Table 1 . While OD 28° is not proportional to protein concentration in all regions of the gradient, it is a legitimate measure of the presence of
subcellular particulate . It should be noted, however, that at OD aeo of greater than 2, the comparative value of protein concentration versus OD 28° becomes increasingly discrepant .
FIG . 3 Distribution of protein in gradients from brains of normal and morphine-treated rats
Vol. 11, No. $
Fractionation of Brain by Centrifugation
369
Figs . 4 and 5 are the results of separate duplicate experiments using normal rats (Fig . 4) and morphine-treated (4mg~100) rats (Fig . 5) and show the reproducibility of this fractionation technic . The displacement of the peak in Fig . 4 may be due to a slight difference in the shape of the gradient between the two runs . _
9
5 4
C
1
3
5
1
1
11
13
15
11
1!
!1
23
FRACTION NO .
25
27
2!
]1
3]
2
1
35 37
3
5
7
!
11
U
15
11
i!
!1
27
FRACTION NO .
25
27
29
31
73
35
OD a6° profile of duplicate frac- OD as° profile of duplicate frac tionations of normal rat brains tionations of brains of rats treated with 4 mg morphine~100 gm The data shown in Fig . 6 is the result of fractionation of the cerebral hemispheres and midbrain from normal rats . Fig . 6a is a plot of the OD ss° while 6b is a plot of the results of ACHE and acid phosphatase assays . Percent recovery of enzymes from duplicate fractionations are found in Table 1 . RI
- t . at .~ 3 .ao - 1 .39 ~O
- 1 .38
RI
N
r
1 .37
-1 .36 -1 .35
1300 É
1100
110 100
900
90 80
N à
70
700
50
500
so
0
40
300
30 20
1
]
5
7
9
Il
1]
15
O
1!
tl 23
FRACTION NO .
25
27
29
71
]]
]5
FIG . 6a OD ae° profile of cerebral hemispheres and midbrain of normal rats
1 .33
D A m
100
10 1
7
5
1
!
9
1]
Ni
A
N
21
2] 25
FRACTION NO . .
77
2!
71
77
35
FIG . 6b Profile of ACHE and acid phosphatase from cerebral hemispheres and midbrain of normal rats
3
370
Fractionation of Brain by Centrifugation
Percent Substance
Vol . 11, No . 8
TABLE 1
Recovery
from Gradient Percent Recovered
Fig .
Exp .
Protein
3 3
1 1
Normal Morphine
ACHE
6b
1 2
Normal Normal
7b
1 2
Morphine Morphine
92 103
6b
1 2
Normal Normal
97 112
7b
1
Morphine Morphine
102 g9
Acid Phosphatase
E Activity in Gradient E Activity in Homogenate
Treatment
132 110 85 81
X 100 = Percent Recovered
Fig . 7 is the result of fractionation of cerebral hemispheres and midbrain from rats which received 2 .0 mg morphine sulphate~100 grams subcutaneously, 60 min prior to sacrifice . Fig . 7a is a plot of the OD~B° while 7b is the enzyme assay profile . Enzyme recovery from duplicate fractionations is shown in Table l . Treatment with morphine results in a marked decrease in the third peak along with a decrease in enzyme activity . The decrease in OD 28° and enzyme concentration is not as great as that produced by treatment with 4 mg morphine~100 grams (Figs . 1 and 2) .
1300 110
1100 D
90
90o
E 100 ~ 90
° ~o
~oo
m 50 ar 50
500
a ao Q
30 20 10
300 100 s i
s
n n~s na ananam111s FRACTION NO .
FIG . 7a FIG . 7b OD 2s° profile of cerebral hemi- Profile of ACHE and acid phosphaspheres and midbrain from rats tase from cerebral hemispheres treated with 2' .0 mg~100 morphine and midbrain of morphine-treated rat s
n 2 rn ,_ ô
3
Vol. 11, No . 8
Fractionation of Brain by Centrifugation
371
Table 2 contains assay data on the homogenates used in duplicate experiments . There appears to be a decrease in acid phosphatase activity in the homogenate from rats treated with morphine . The range in ACHE values makes it difficult to conclude that morphine treatment has any effect on ACHE côncentration . TABLE 2
Enzyme Activity in Homogenates Used in Duplicate Fractionations Enzyme ACHE
Acid Phosphatase
Treatment
Activity ~Mol~ml
Fig .
Exp .
6b
1 2
Normal Normal
9,300 7,610
7b
1 2
Morphine Morphine
8,272 7,573
6b
1 2
Normal Normal
60,100 60,400
7b
1 2
Morphine Morphine
47,872 54,787
Discussion
The cell homogenization process used in these experiments differs from the one more commonly used, a rotating Teflon pestle in a glass tube, in that a minimal amount of shear is produced by
The tissue is disrupted by passage through a screen with pore diameters considerably greater than neuronal perikarya ; however, nerve processes are undoubtedly broken off in the process . the process.
The breakage of the cells is accomplished by explosive nitrogen decompression . No washing or sedimentation of the homogenate is done prior to zonal centrifugation . With the exception of the
buffered sucrose used for the washing of the whole and quartered brain, all fluid used in the tissue disruption and cell homogenization process is included in the sample that is centrifuged . Approximately 45 min elapsed from the time the brains were removed until the sample was introduced into the tonal centrifuge . We believe that the rapid processing and conservation of sample by the elimination of differential centrifugation should improve the recovery of enzymes and aid in preservation of the more fragile organellae . Data presented in Figs . 1 and 6, and Figs . 2 and 7 show that treatment with morphine has a profound effect on the
372
Fractionation of Brain by Centrifugation
Vol . 11', No . 8
population of subcellular particulate normally found in the third peak .
This could be due to either a decrease in size or density
of a major portion of an organelle or organellae prior to cell disruption, or because of an increase in the fragility of a major portion of subcellular particles resulting in their being broken up more easily by the homogenization process . However,
changes that occurred in enzyme levels indicate that
if breakage occurred, the enzymes were not released in a free form, since the amount of enzymes in the first peak (sample zone) was not significantly increased .
If the organellae are broken, the
enzymes appear to remain bound to membranes . Since ACHE and acid phosphatase are considered marker enzymes for synaptosomes and
lysosomes, respectively, these are t'he most likely organellae affected and may be the predominant particulates in the third peak . To cause the observed change in the third peak this effect would have to be exerted on a major portion of the brain cells or The effect it would not be detected by the methodology described . is not produced in vitro by the addition of morphine to the cell
homogenate . There appears to be little loss of protein or enzymes during gradient centrifugation .
The homogenates are contaminated with
some red blood cells which would sediment through the gradient to the rotor wall . This could account for some of the apparent loss of ACHE activity . The procedure described permits the rapid fractionation of
brain and the measurement of changes both in physical properties of subcellular particulates and in enzyme concentrations which
should facilitate the study of the effects of drugs on t'he brain . We are also studying the effect produced by methadone,
diisopropylfluorophosphate,
and p-chloroamphetamine . The distribution of particulate and enzymes in the gradient is related to the drug used and is not an artifact of the fractionation technique .
These studies will be reported in a subsequent paper . Electronmicroscopic and histochemical studies are also in progress in an effort to elucidate the mechanism behind the effect reported in this paper .
Acknowle dgments The We thank Mr . J . Redmond for 'his technical assistance . B-XXIX rotor was obtained through a cooperative agreement with
Vol . 11, No . 8
Fractionation of Brain by Centrifugation
373
Dr . N . G . Anderson, Director of the MAN program at Oak Ridge National Laboratory .
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2. 3. 4. 5. 6. 7. 8. 9. 10 .
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