Journal of Nrurochrmirrrp. 1977 Vol 29. pp 77 81 Pergamon

Press. Printed

in

Great Britain

REDISTRIBUTION OF ADENINE DERIVATIVES AMONG SUBCELLULAR FRACTIONS FROM GUINEA PIG NEOCORTICAL TISSUES, ON INCUBATION IN VITRO C. BARBERIS'and H. MCILWAIN Department of Biochemistry, Institute of Psychiatry (British Postgraduate Medical Federation, University of London), De Crespigny Park, London, SE5 SAF, UK (Received 19 November 1976. Accepred 20 January 1977)

Abstract-After the brief in virro exposure of guinea-pig neocortical tissue to CL4C]adenine,synaptosoma1 fractions prepared from the incubated tissue contained about 6% of its retained I4C. On continued incubation and superfusion with or without stimulation, the synaptosomal proportion of the I4C increased, while the protein and K content of the fraction underwent smaller changes only. Colchicine, 0.5mw, diminished the synaptosomal enrichment in ["Cladenine derivatives and also in some cases increased the I4C emuent from tissues to superfusates. Colchicine also diminished the uptake of adenosine, but not of adenine, to the neocortical tissues. It is concluded that nerve terminal regions receive adenine derivatives from other tissue components as part of their normal metabolism, and that much of this can arrive by extracellular fluids; transport cytoplasmically is not excluded.

~

SYNAPTOSOME preparations from guinea-pig neo- lar space have been shown in parts of the brain in cortex were found on incubation to lose up to 1.50,: v i m (SCHUBERTet al., 1976) by histochemical of their acid-soluble adenine derivatives per minute methods. Subcellular fractionation could give comple& MCILWAIN,1976). For this loss to be mentary information. and we have measured redistri(BARBERIS a normal occurrence rather than a peculiarity of the bution after imposing various conditions in the prespreparation used, compensatory processes supplying ence and absence of colchicine, which inhibits cyto1976; OCHS,1975a, b ; WILadenine derivatives to the terminals must normally plasmic flow (LUBINSKA, be in progress. Cerebral tissues are capable of de n o w sob. 1975). synthesis of purine derivatives (see MCILWAIN& BACHELARD, 1971) but rates are relatively low. Supply MATERIALS AND METHODS from other parts of the tissue appears more likely, and we have now sought to demonstrate such proPreparation and labelling of tissue slices. Guinea-pig neocesses during the incubation of tissue slices. It cortical tissues were prepared promptly, mounted in quickappeared feasible to d o this by using isotopically- transfer holders, incubated and superfused as described by (19720) and MCILWAIN (1975). Eight labelled adenine as a precursor, for it has been con- PULL& MCILWAIN cluded that the adenine is preferentially taken up to tissue samples. 0.35 m m thick, from 2 animals were nora limited region only of the tissue, probably to neur- mally prepared together and incubated in separate tissueholding electrodes in one experiment. Each holder was onal cell bodies (DALY,1972; MCILWAIN, 1974). placed in an incubation vessel which contained 5ml of We now report the redistribution of [I4C]adenine incubation medium, at 37°C. After preincubation for derivatives on continued incubation of isolated cere- 20min and unless specified otherwise, 1 pCi of [8-I4C]bral tissues, performed in a superfusion system in adenine (58 mCi/mmol), was added to each beaker, mixed order to minimize their extracellular accumulation. by shaking and tissues further incubated for 3 or 1Omin. Orthodox methods of subcellular fractionation are Superfusion at approx. 3.5 ml/min with medium lacking applicable to tissues after their in uitro incubation adenine was then commenced for periods described in indi(WOLFE& MCILWAIN,1961; KURODA& MCILWAIN, vidual experiments. The incubation medium was a bicarbonate-glucose 1973). but to guard against misinterpretations which might follow from change in centrifugal deposition saline and contained 120 mM-NaC1, 3.5 mw-KCI, 1.25 mMof fractions secondary to the continued incubation, KH,P04, 1.2m~-MgSO,,0.75mM-CaC12,25 mM-NaHCO,, IOmht-glucose and was equilibrated with a gas mixture the protein content of the separated fractions has also of 02.95% and CO,, 5%. Added compounds, and electribeen determined. We consider also the routes by cal stimulation (with alternating exponential pulses of peak which redistribution may take place. Movement of potential IOV; Q4ms time-constant and frequency 20 or adenine derivatives cytoplasmically and by extracellu- 50Hz)were applied to specified tissues at chosen times. Subcellular fracfionation of labelled rissues. All the procedures were carried out at 0 to 4°C. as described by I Present address: Laboratoire de Physiologie Gcnerale, KURODA& MCILWAIN (1973) and BARBERIS & MCILWAIN Universite de Nancy 1- -54037, France. ~~

77 v.c. 2911 --I

78

C. BARBERISand H. MC~LWAIN

(1976).After incubation the slices were released from the RESULTS holders into chilled 0.32 M-sucrose solution, removed from this after 10s with a bent platinum wire, and transferred Redistribution on continued incubation. Observato a Perspex- glass homogenizer containing 0.32 M-SUCIOSe tions of tissue constituents followed a period of preinwith 0.5 mM-EDTA at pH 7.4; in specified instances the cubation which allowed stable conditions to be resucrose contained also, 5 m~-3-isobutyl-l-methylxanthine. established in the tissues after excision and slicing (see Primary fractions of the homogenate were prepared by the following sequence. modified to a small extent only from MCILWAIN,1975). Towards the end of this period the [''Cladenine was added, some minutes allowed for the procedure of WHITTAKER er al. (1964) and carried out with a Beckman L265 ultracentrifuge. The 'nuclear' frac- incorporation, and the excess I4C removed by supertion PI was prepared by centrifuging at lo00 g for 10 min fusion. It was found that 4-10min sufficed for reand was washed except when specified otherwise in the moval; these times represented the shortest startingTables. Fraction S, was separated at 11.ooO g for 20 min point for observing possible redistribution. From 4 and the deposited material fractionated using sucrose gra- or 10, to 20 or 90min were chosen as periods for dients, that between 0.8 and 1.2 M-sucrose being collected continued incubation with superfusion, during which as the synaptosomal fraction. It is recognized that this frac- redistribution might be seen. It was found that during tion includes some resealed membranes from sources other these periods the tissues lost a small proportion of than nerve terminals (COTMAN ef a/., 1970; MARCHBANKS, 1975). Pellets were suspended in appropriate volumes of their protein and 14C (Table 1) while that remaining underwent redistribution (Table 2). 0.32 M-sucrose and portions used for assay. Redistribution in favour of the synaptosomal fracFurther froctionution of crude mitochondria/ Jracfion. The crude mitochondria1 fraction was suspended in 1 ml of the tion was first demonstrated by experiments 1 to 3 0.32 wsucrose. 0.5 mM-EDTA Tris solution, and was of Table 2. These showed its occurrence in experilayered into a discontinuous density gradient consisting ments which differed in period of exposure to adenine, of 0.3 ml of 1.4 M-SUCTOSe. 2 ml of 1.2 M-sucrose and 2 ml and in the manner of synaptosome preparation. Colof 0.8~-sucrose.This was centrifuged at 105.o00g for lectively, the three experiments showed the synapto6Omin in a SW50 head of the Beckman ultracentrifuge. some fractions to gain a variable 1804 12% of proBands were separated using bent-tipped Pasteur pipettes tein and a larger and more consistent 377; f 4% of with suction. ['4C]adenine derivatives (mean and S.D. of the 3 Deposition o f r he synapfosom[I/fracrionas beds and supervalues quoted; difference of mean from unity in oneJusion. The procedures used for deposition were based on those of DE BELLEROCHE & BRADFORD(1972). carried out tailed t test, 0.025 < P < 0.05 and P < 0.0005 reas described by KURODA& M C ~ L W A I(1974) N and BAR- spectively). Gain in adenine derivatives of the same BERS & M C ~ L W A (1976). I N After periods of incubation in magnitude was also obtained in tissues which had and superfusion with the glucose bicarbonate saline as been electrically stimulated in the presence or absence noted in Table 2, the beds were plunged into loo/, (w/v) of histamine (experiments 4 to 6 of Table 2), conditrichloracetic acid for extraction. tions which were chosen to alter the tissue adenine Dererminarion of [ 14C]ridenine deriuafices. A sample of nucleotides (see KAKIUCHIet a!., 1969). Mean values a subcellular fraction was added to an equal volume of from these three experiments were: synaptosomal ice-cold 200: (wjv) trichloroacetic acid, made up to at least 1 ml with 1014 trichloroacetic acid in a homogenizing tube enrichment in adenine derivatives, 41 _+ 4%; in proand homogenized. After centrifugation. the supernatant tein, 14 le"/, (given _+KO.; difference of mean from was decanted. the residue treated with 0.5 ml of lo:/, (wjv) unity in one-tailed t-test, P < 0.005 and >0.1, retrichloroacetic acid. again centrifuged and the supernatants spectively). were combined. Portions (0.1 0.2 ml) of the trichloroacetic Distribution of protein and [ I4C]adenine derivaextract were added to vials and their volume was adjusted tives were determined also among other subcellular to 1 ml hy adding water. A toluene: Triton X-100 (2:l. fractions in the same experiments, by calculating v/v) scintillation fluid (10 ml) that contained 2.5-diphenyl- ratios corresponding to B / A of Table 2. These did oxazole (OSY,, w/v) and 1,4-bis-(4-methyl-5-phenyloxazol-not show redistribution comparable to that found in 2-yl) benzene (0..1?,,, wjv) was then added. Counting was the synaptosomal fraction. In particular, the propor(19724. carried out as described by PULL& MC~LWAIN Determination of potassium was as desaibed by SWANSOWtinns found in the nuclear fractions as a yd of those & MC~LWAIN (1965). The protein content of the trichloro- in the homogenates were for protein 109 14 and acetic acid precipitates was determined after addition of for I4C, 109 & 5 (mean f S.D.for 3 values; differences M-NaOH by the method of MILLER(1959) using bovine of mean from unity by one-tailed t-test: P > 0.15, and serum albumin as standard. Lactic acid in superfusion between 0.0125 and 0.025 respectively). fluids was determined by an automated fluorometric Exchange with Juid; actions of colchicine. Colchimethod with lactate dehydrogenasc as described by PLJIL cine at 0.5 mM diminished the CL4C]adenine deriva& MCII.WAIK (1972~). tives found in the synaptosome fraction after conMafericils.[8-14C]Adeninewas obtained from the Radio- tinued tissue-incubation (Table 3, exp. 1). A possible chemical Centre, Amersham Bucks., U.K. Colchicine was basis for this action was tested by the other experipurchased from Sigma Chemical Co. (St Louis. MO) 3-isobutyl-I-methyl-xanthine was given by Reecham Pharma- ments of Table 3. Formation of lactic acid, which ceuticals (Research Division). Brockham Park, Betchworth. is modified in cerebral tissues by some deleterious Surrey. U.K.. and was also obtained from Aldrich Chemi- conditions, was unaffected by colchicine. Nor did colchicine displace ['4C]adenine dcrivatives from a cal Co.. Milwaukee, W1. USA.

*

*

Redistribution of neocortical adenine derivatives

TABLE 1.

CONSTITIJENTS OF TISSUFS INCUBATED FOR

79

10-90 mill

Period of superfusion (min) Constituent; units

Measured in

Protein mg/100 mg tissue Protein of total recovered Protein 0; of that in mitochondrial I4C from adenine nCi/lOO mg protein I4C from adenine n o of total recovered 14C from adenine 'b of that in mitochondrial fraction K'. ltequiv./100 mg protein

K + , pequiv./100 mg protein

10

17

Primary mitochondrial fraction Synaptosome fraction

8.08 f 0.16 (4) 40.8 f 2.9 (3) 47.6 (2) 659 (2) 25.6 f 8.7 (3) 39.7 (2)

8.34 It 1.45 (7) 42.6 k 6.0 (3) 53.9 0.6 (3) 476 f 118 (5) 27.8 10.6 (3) 43.3 & 6.8 (3)

Total tissue homogenate Synaptosome fraction

85.4 f 7.6 (3) 36.8 0.3 (3)

Total tissue homogenate Primary mitochondrial fraction Synaptosome fraction Total tissue

+

90

1.72

0.62 (6) 40.8 f 4.1 (4) 49.2 f 3.4 (3) 530 f 93 (3) 27.7 7.6 (4) 42.5 5.6 (3)

+

91.3 f 12.1 (3) 40.0 5 4.4 (3)

The tissues were incubated with [14C]adenine for 3min and then superfused for a period of time quoted in the table. They were homogenized and subcellular fractions were prepared and analysed as described in the experimental section. Values quoted were derived from 17 experiments, each using 8 tissue samples obtained from 2 guinea pigs. Data are given as mean values f S.D. for the number of determinations quoted in brackets when three or more experiments were run. synaptosome preparation, or diminish the uptake of [I4C]adenine t o tissue slices. However, when slices previously exposed to [ lAC]adenine were superfused with colchicine. more I4C appeared in the effluent (Table 3, exp. 2). This difference was not evident when the tissues were stimulated electrically. but colchicine

TABLE 2.

proved to diminish the uptake of adenosine to the tissue (Table 3. exp. 8). DISCUSSION

The synaptosomal fraction initially contained a relatively small proportion of the ["Cladenine deri-

REUISTRIBUTION OF TISSUE CONSTITUENTS DURING SUPERFUSION

Proportion (%) of constituent in synaptosomes after superfusion for Experiment; conditions 1. Adenine preincubation.

IOmin 2. Adenine preincubation, 3 min 3. As 2; nuclear fraction not washed 4. As I ; with histamine and electrical stimulation 5. As 2; with electrical stimulation at 20 Hz 6. 7. As 2; with histamine and electrical stimulation; nuclear fraction not washed

Measured

A = 10min

B = 90min

Protein I4C Protein

19.18 4.76 19.86 10.17 6.84 3.45 11.84 3.30 A = 4min 17.50 9.40 9.61 5.20

23.33 6.45 20.66 14.48 8.76 4.61 11.18 4.67 B = l7min 21.18 12.91 10.73 7.42

l

4c

Protein 14C Protein

4c

Protein I4C Protein 14C

'

Synaptosomal enrichment between time A and B (ratio B / A ) 1.21 1.35 1.04 1.42 1.28 1.34 0.94 1.42 1.21 1.37 1.11 I .43

After 30 min preincubation. neocortical tissues were incubated with ['4C]adenine for the times indicated in the first column, and then superfused with glucose -bicarbonate saline for times stated under A and B; they were then removed and homogenized. In experiment 4. they were superfused with glucose-bicarbonate saline containing 0.1 mM-histamine. After IOmin superfusion, they were stimulated for 6min while the superfusion was continued; the slices A were then quickly homogenized while slices B were superfused for a further 74 min before being homogenized. In experiments 5 to 7, the slices were preincubated and superfused with glucose-bicarbonate saline containing 2.6 mM-CaCI2 and histamine, 0.1 m M (6, 7). After incubation with [14C]adenine for 3 min, they were superfused and stimulated for 4min. In A the slices were then homogenized and in B they were superfused for a further 13min. Subcellular fractions were prepared as described in the text except for experiments 3, 6 and 7 in which the nuclear fraction was not washed. In experiments 5, 6 and 7, they were prepared in a sucrose solution containing 5 mw-3-isobutyl-l -methylxanthine. Values are quoted from individual experiments, each using 4 slices.

80

C. BARBERISand 11. MCII.WAIN 1‘ARLh

3.

TISSUE METABOLISM W1’111A N D WITHOLT ADDED COLCIllClVll ~~_______ ~ ~ _ _ _ _ _

Colchicine (mM) Preparation ; Expcrimcnt ; property examined 1. Subcellular distribution of ’ “ C adenine

derivativcs aftcr superfusion and

stimulation 2. Output of I 4 c on superfusion. adenine-frcc medium, tinstimulated 3. output o f 1 4 ( . on superfusion. adenine-free medium. stimulated 4. Output of lactate during experiments 2 5. Output of laclate during experiments 3 6. lJptake of “ C adenine on incubation with 0.2 /iCi:ml for IOmin 7. Content of “C (taken up as “C adenine in absence of colcliicinc)after superfusion and stimulation 8. Uptake of I4C adenosine on incubation with 0.1 pCi;ml for IOmin

units

0

0.5

”’, of tissue content

4.4x fO.15

3.53 k0.14

in synaptosomes Neocortex, slice; of contentimin Neocortex, slice: of contentimin pnoles/g. h pmoles/g. h Neocortex. slice; nCi/g Synaptosonie bed; nCi/mg protein

0.046 f0.009 0.34Y f0.05 I 14.6 f 5.9 122 f 25 2875 k281 2.59 f0.26

Neocortex, slice;

‘I,,

‘lo

Neocortex, slice; nCi/g

0.349

1045 + I66

0.073 f 0.010

0.341

k 0.048 12.7 f 3.4 124 27 3043 f 328 2.63 - 0.20

+

716 f 77

Data are quoted as mean f S.E.M. or 4 values. Experiments I were carried out as those of Exp. 4R, Table 2, except that the period of incubation after stimulation was 20 min and the total time of exposure to colchicine, 40 min. In output experiments 2 and 3. tissues were labelled by incubating with [l*C]adenine for 30min. and superfusion fluids collected for analysis before and during a period of stimulation at 20 Hz. Uptake experiments 6 and 8 were carried out as described in the experimental section. with superfusion for 60min before sampling. The synaptosome beds 7 were preincubated for 20min. exposed to ‘*C adenine at O.ZptCi/ml for 3min. superfused for 6min with stirnulation at 50 Hz and subsequently for 10 min without stimulation; they were then extracted for determination of ‘“C. votives retained hy the tissue: averaging 6.1?,

Redistribution of adenine derivatives among subcellular fractions from guinea pig neocortical tissues, on incubation in vitro.

Journal of Nrurochrmirrrp. 1977 Vol 29. pp 77 81 Pergamon Press. Printed in Great Britain REDISTRIBUTION OF ADENINE DERIVATIVES AMONG SUBCELLULAR...
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