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Effect of cerebral stimulation on biosynthesis of nucleotides and RNA in brain slices in vitro
285
BIOCHIMICA ET BIOPHYSICA ACTA BBA 96223
E F F E C T OF C E R E B R A L STIMULATION ON B I O S Y N T H E S I S OF N U C L E O T I D E S AND RNA IN B R A I N SLICES I N V I T R O CAROL PRIVES* AND J. H. QUASTEL
Neurochemistry Section, Kinsmen Laboratories, The University of British Columbia, Vancouver 8, B.C. (Canada) (Received February 24th, I969)
SUMMARY
I. Application of electrical impulses to rat brain-cortex slices, incubated aerobically at 37 °, diminishes the rates of conversion of 14C-labelled uridine into uridine polyphosphates and RNA to less than half the control values. The effect is blocked b y the presence of 5 #M tetrodotoxin. 2. Increase of the NaC1 concentration of the incubation medium from 128 to 178 mM and 228 mM greatly decreases the rate of incorporation of l~C-labelled uridine into uridine polyphosphates and RNA in the brain slices. Omission of NaC1 from the incubation medium also diminishes the rates of biosynthesis of uridine nucleotides and RNA. 3. Increase of the KC1 concentration of the incubation medium from 5 to 15 mM results in an increase of the rate of incorporation of 14C-labelled uridine into RNA by approx. 5o %. Further increase of the KC1 concentration to 50 and IOO mM results in a diminished rate of RNA biosynthesis. 4. Addition of I mM acetylcholine (in presence of eserine) to the incubation medium results in an increased rate of incorporation of 14C-labelled uridine into RNA in brain slices. The effect is dependent on the presence of Na + and K +. 5. The rate of conversion of uridine to uridine polyphosphates in presence of ATP, in a dialysed rat brain extract, is diminished by increasing the concentrations of NaC1, or KC1, to IOO mM or more. 6. Possible explanations for these effects, and their significance, are discussed.
INTRODUCTION
In view of the possibility that memory, such as that acquired in learning, is represented by some permanent change in the brain and that this change is brought about as a result of chemical activity in the brain cell, we have carried out experiments to observe whether various stimulating agents will affect nucleotide and RNA biosynthesis in isolated brain. Evidence has already accumulated to indicate that brain excitation, or electrical stimulation in vitro, will affect protein biosynthesis in the brain. SHAPOT1, for example, has demonstrated that the rate of incorporation of * Present address: Department of Molecular Biology, Albert Einstein College of Medicine, 13oo Morris Park Avenue, Bronx, N.Y. 10461 , U.S.A.
Biochim. Biophys. Acta, I82 (I969) 285-294
~86
c. PRIVES, J. H. QUASTEL
[35S]methionine into proteins in the brains of rats, after excitation, is lower than that found in control animals and attributes this to diminished cerebral protein synthesis. ORREGO AND LIPMAI~Nz have concluded that electrical stimulation of brain slices results in inhibition of protein synthesis, as judged b y the diminished rate of incorporation of [14Clvaline into the brain proteins. There are indications also that nucleic acid metabolism in the brain is affected by stimulation; thus GEIGER et al. 3 have found that stimulation for 3 ° sec changes the composition of RNA in tile brain cortex. Marked motor activity causes an increase in the RNA concentration in the motor root cells, whilst sensory stimulation causes changes in RNA concentratiol~ in the vestibular ganglion cells which depend on the intensity of the stimulation 4. Work carried out independently, and at about the same time, by the present authors 5 and by ORREGO6 has shown that electrical stimulation of rat brain cortex i n vitro brings about at least 4 ° % inhibition of RNA biosynthesis, as judged by the rate of incorporation of radioactive uridine into RNA. ORREGO6 has further shown that this m a y be due in part to impaired nucleotide phosphorylation. The following report describes the effects of electrical stimulation, and of changes in the cation composition of the incubation medium, and of acetylcholine, on the rates of incorporation of labelled uridine into uridine nucleotides and into RNA in rat brain cortex slices.
METHODS AND MATERIALS
We have used adult female hooded rats whose brains were quickly removed after stunning and decapitation, and slices (about 0.3-0. 4 m m thick) were prepared from the cerebral hemispheres using a Stadie-Riggs microtome. One dorsal and one lateral slice, weighing a total of 50-75 mg wet wt., were transferred to chilled Warburg manometric vessels each containing 3 ml Krebs-Ringer phosphate medium and IO mM glucose. Radioactive uridine was added from the side arm of the vessel and all other additions were present in the main compartment from the start of the experiment. In experiments with a Na+-free medium the NaC1 was replaced b y sucrose or choline chloride to maintain isotonicity and Tris buffer (IO raM, p H 7-4) was used instead of phosphate buffer. The vessels were equilibrated at 37 ° for IO rain in an atmosphere of 02 prior to tipping in the labelled compound from the side arm and incubation was then carried out for 30 min. Aftei incubation the Slices were remove,d, washed twice with ice-cold Krebs-Ringer medium to remove contaminating radioactivity and homogenized at o ° in 5 % trichloracetic acid. The homogenate was kept at o ° for i h and centrifuged at 600 × g for IO min. The supernatant was analysed for phosphorylated and free uridine after removal of the trichloracetic acid by successive washings with ether. An aliquot (200/A) of the solution was chromatographed on W h a t m a n No. 3 paper, using, as descending solvent, ethanol-ammonium acetate (I M) (pH 7.4) (7:3.5, v/v) for a period of 6 h, which brings about separation of uridine ( + uracil), uridine monophosphate and the combined uridine polyphosphates which are not effectively separated and were therefore estimated together. It should be noted that uridine and uracil are also not separated by this technique, so that spots containing uridine m a y contain uracil derived from the uridine. The resulting spots containing uridine ( + uracil), uridylic acid (UMP) and uridine polyphosphates Biochim. Biophys. Acta, 182 (1969) 285-294
RNA
287
SYNTHESIS IN BRAIN SLICES
( U D P + U T P ) were cut out of the paper in equal sections and their radioactivities were estimated in the liquid scintillation counter after correction for the small quenching effect of the filter paper. The residue, from the centrifuged homogenate, was washed twice with 5 % trichloracetic acid to remove acid-soluble radioactivity and then once with 95 % ethanol and once with a 95 % ethanol-diethyl ether mixture (3 : I, v/v) to remove lipids. The washed residue was dissolved in 2 ml 0.2 M K O H and incubated at 37 ° for 18 h (ref. 7). 0.05 ml 7 ° % HC1Q was added, precipitating the DNA and proteins and leaving the hydrolysed RNA in the supernatant. The radioactivity of the hydrolysed RNA was subsequently determined in the liquid scintillation counter. Electrical stimulation of slices of braiu cortex was carried out in vessels with silver grid electrodes, similar to those used by WALLGRENAND KULONENs, with electric impulses from the alternating frequency generator of the Teratron type described in detail b y WALLGREN9. The impulses had a pulse frequency of IOO cycles/sec with I-msec duration, the peak potential being 4 V. Extracts of cerebral cortex were prepared b y homogenizing the excised cerebral cortex in 12 vol. 0.02 M Tris-HC1 (pH 7.4) in a Potter-Elvehjem glass homogenizer with a fitted teflon pestle. The homogenate was centrifuged at 17 ooo ×g for I h in a Sorvall high-speed centrifuge, and the supernatant was dialysed for 18 h against 0.02 M Tris-HC1 (pH 7.4)-
RESULTS
E]]ects o/electrical stimulation on cerebral incorporation o] uridine into nucleotides and RNA Results given in Table I show that application of electrical impulses has a marked diminishing effect on the rate of conversion, in rat brain cortex slices, of uridine into uridine polyphosphates as well as into RNA. There is little or no effect on the rate of formation of uridine monophosphate and the concentration of free labelled uridine ( + uracil) in the slice rises due to the diminished rate of formation of U D P + UTP. These results confirm those of ORREGO6. They also show that, although the concentration of total labelled uridine ( + uracil), uridine phosphates and RNA in the tissue after 3o min incubation is but little greater than the concentration of
TABLE I EFFECTS OF ELECTRICAL CLEOTIDES IN RAT BRAIN
STIMULATION ON CORTEX SLICES
[2-14C]URIDINE
INCORPORATION
INTO
RNA
AND
NU-
Initial [2-14C]uridine concn.: 5-9/zM; 1. 5 • lO 6 c o u n t s / m i n per vessel. Results expressed as m e a n s of four d e t e r m i n a t i o n s ± S.D.
Conditions
Control Electrical s t i m u l a t i o n
[14C]Uridine incorporated (pmoles/g wet wt. per 30 rain) as Uridine+ uracil
UMP
UDP4-UTP
RNA
192o4-251 246°±167
5284- 73 5294-113
314o4-47 ° 19354-177
5664-48 2344-37
Biochim. Biophys. Acta, 182 (1969) 285-294
288
c. PRIVES, J. H. QUASTEL
labelled uridine in the bathing medium, the concentration of free labelled uridine in the tissue is much less than that in the medium. Thus, there exists a permeability barrier at the brain-cell membrane limiting the rate of diffusion of uridine into the brain cells. TABLE II EFFECT OF ELECTRICAL STIMULATION IN PRESENCE AND ABSENCE OF TETRODOTOXIN DINE INCORPORATION INTO R N A IN RAT BRAIN CORTEX SLICES
ON
[2-14C]URI -
I n i t i a l [2-14C]uridine conch.: 5.9 t,M; 1. 5. lO6 c o u n t s / m i n p e r vessel. T e t r o d o t o x i n w a s p r e s e n t w h e r e i n d i c a t e d a t a final concn, of 5/~M. R e s u l t s e x p r e s s e d as m e a n s of a t l e a s t t h r e e e x p e r i m e n t s 4-S.D.
Conditions
[14C] Uridine incorporated into
RNA
(pmoles/g wet wt. per 30 min) Control Electrical stimulation Tetrodotoxin Electrical stimulation + tetrodotoxin
5664- 48 234 4- 37 588 4-13 5324-54
Further results (Table II) show that addition of the potent neurotoxin, tetrodotoxin, to the bathing medium blocks the effects of electrical impulses on the incorporation of uridine into RNA. I t is already known that during the application of electrical impulses to brain-cortex slices there is an influx of Na + into brain tissue 1°-12 and that the metabolic effects of electrical impulses on isolated brain m a y be blocked b y the addition of small concentrations of tetrodotoxin 1~ which acts by its inhibitory effect on Na ÷ influx during electrical stimulation. The suppressing action of electrical impulses on the formation of uridine polyphosphates and RNA may, therefore, be due to the influx of Na + into the brain cell. This prediction was verified in the following manner. E//ects o / N a + on uridine incorporation into R N A and uridine nucleotides Results given in Table I I I show that increase of Na + concentration in the bathing medium, from the initial level of 128 mequiv/1 in the Krebs-Ringer medium, TABLE III EFFECTS OF VARIOUS CONCENTRATIONS OF ]_x]'aCl ON [2-14C]URIDINE INCORPORATION INTO R N A A N D NUCLEOTIDES IN RAT BRAIN CORTEX SLICES R e s u l t s e x p r e s s e d as m e a n s 4-S.D. I n i t i a l [2-14C]uridine concn.: 5.9 #M; 3 ' IOe c o u n t s / m i n p e r vessel. I n e x p e r i m e n t s w i t h o a n d 2o mM NaC1, 128 m M c hol i ne c h l o r i d e w a s a d d e d to t h e i nc ub a t i o n m e d i u m to m a i n t a i n i s o t o n i c i t y a n d o . i M Tris-HC1 (pH 7.4) w a s us e d as buffer. I n exp e r i m e n t s w i t h 128, 178 a n d 228 mM NaC1, o . o i M s o d i u m p h o s p h a t e buffe r (pH 7-4) w a s used.
NaCl goncn.
o 20 128 178
228
[14C]Uridine incorporated (pmoles/g wet wt. per 3o rain) as Uridine + uracil
UM P
UD P + U T P
RNA
28664-176 19784-117 17294- 13 25884-167 24294-147
3964- 28 91o4-142 4834- 28 49o4- 34 3944- l o 6
19254- 65 36804-42o 45554- 75 24164-114 17514- 75
1484- IO 4434-89 7 7 6 ± 15 3 4 I t 2I 1214- 9
Biochim. Biophys. Acta, 182 (1969) 285-294
RNA
289
SYNTHESIS IN BRAIN SLICES
leads to a diminution of the rate of uridine incorporation into RNA in rat brain cortex slices. A similar phenomenon occurs with the incorporation of uridine into uridine polyphosphates. These results support the conclusion that influx of Na + into the brain cell, bathed in a physiological saline medium, leads to a suppression in the rates of formation of uridine polyphosphates and RNA. The influx of uridine, however, into the brain tissue is not markedly affected by the rise in NaC1 concentration in the bathing medium. Results in Table III, indicate that when Na + are omitted from the incubation medium, these being replaced by choline to maintain isotonicity, there is a greatly diminished rate of phosphorylation of uridine to uridine monophosphate, as well as to the polyphosphates, and the rate of RNA formation is much suppressed. Addition of 20 mM NaC1 to the Na+-free incubation medium brings about a marked increase of the rate of formation of uridine monophosphate, uridine polyphosphates and RNA. The quantity of free labelled uridine ( + uracil) in the tissue is not diminished b y the absence of Na + from the incubation medium, so that the results cannot be explained as due to suppressed influx of uridine into the tissue.
E//ects o/ouabain on uridine incorporation into R N A Ouabain, at small concentrations which diminish the activity of the Na + pump, by its suppressing effect on membrane bound ATPase, inhibits the incorporation of uridine into RNA (Table IV). The inhibitory effect of ouabain is nullified b y increase of K + concentration in the incubation medium, a phenomenon that m a y be indicative of the well known antagonism between ouabain and K +. TABLE IV EFFECTS OF I/~M OUABAIN ON THE RATE OF [2-14C]URIDINE INCORPORATION INTO R N A IN RAT BRAIN CORTEX SLICES I n i t i a l [2-14Cluridine concn.: 5 ~M; 1.2. i o ~ c o u n t s / m i n p e r vessel.
Conditions
Uridine incorporated into R N A (pmoles/g wet wt. tissue)
5 15 5 15
41o4-34 560+ 5 28o+ 9 4 9 7 i 16
mM mM mM mM
KC1 KC1 KCl+ouabain KCl+oaubain
EHects o] K + on the rate o[ incorporation o[ uridine into R N A in rat brain cortex slices Turning to the effects of increased concentrations of K + on the rate of RNA biosynthesis, as determined b y uridine incorporation, we have found 18 that an increase of KC1 concentration, from 5 to 15 mM, in the incubation medium increases the rate of RNA formation Typical results are shown in Fig. I. The presence of 5 mequiv/l K + nearly doubles the rate of incorporation of labelled uridine into RNA found in the absence of added K+, whilst the respiratory rate increases from 9.9/~1 03 consumed per mg dry wt. brain tissue to II.O. Addition of K+ to 15 mequiv]l brings about a further increase in the rate of labelled uridine incorporation into RNA, but a still greater Biochim. Biophys. Acta, 182 (I969) 285-294
290
c. PRIVES, J. H. QUASTEL
P,, \
/
O
En <
'~000
\x
iI
I
~z
\k \\
'\
~-~ "O •
5
I
I
15 .50 KCt conch. (raM)
I
100
Fig. I. E f f e c t s of v a r y i n g c o n c e n t r a t i o n s of KC1, in t h e presence a n d a b s e n c e of i m M acetylcholine ( + i m M eserine) o n t h e r a t e of E2-14C]uridine i n c o r p o r a t i o n into R N A in r a t brainc o r t e x slices. Initial c o n c e n t r a t i o n of uridine = 5.9 #M. Q , no acetylcholine; O , w i t h acetylcholine.
increase to 5 ° or IOO mequiv/1 brings about a fall in the rate of incorporation, although the respiratory rate increases to the value of 18.3/,1 O 3 consumed per mg dry wt. brain tissue per h. The fact that a concentration of K+ appreciably higher than 15 mequiv/1 brings about a fall in the rate of incorporation of uridine into RNA m a y be correlated with the well known fact that, with a marked increase of K + in the incubation medium, there is a fall in the brain cell level of ATP 14-17, a phenomenon associated with the increased activity of the membrane-bound (Na+-K+)-sensitive ATPase. E[]ects o] N a + and K + on uridine phosphorylation in a dialysed rat brain extract The rates of uridine phosphorylation b y a dialysed brain extract obtained from 5 ° mg wet wt. brain tissue, in the presence of ATP, are far greater than those obtained by an equal weight of rat brain cortex slices incubated under optimal respiratory conditions. Results given in Table V show that the total quantity of phosphorylated uridine formed from 7.5 nmoles uridine in 30 rain approximates to 3 nmoles, whereas the total formed, in 50 mg wet wt. brain slices, from 17. 7 nmoles uridine in the same time does not exceed 0.25 nmoles (Table III). Nevertheless, the concentration of total phosphorylated uridine found in a brain extract, equivalent to 50 mg wet wt. tissue, amounts at the most to 3.6 ~M (Table V), whereas the concentration of total phosphorylated uridine in the brain slice m a y amount to 6.3 #M (assuming that the rat brain slice contains 80 % water) (Table I I I ) . These results are presumably largely due to the permeability barrier, imposed by the brain cell membrane, to uridine, which limits the amount of uridine to be metabolised and to the higher concentrations of ATP in the brain cell (about 2.2 raM) than that used with the brain extract (I mM). The marked diminution of the rate of uridine phosphorylation, due to absence of Na + from the incubation medium, in brain cortex slices is not evident with a dialysed rat brain extract in the presence of ATP (Table V). In such an extract, increase of the concentration of Na + to ioo mequiv/1 leads to a diminished rate of formation of uridine polyphosphates, whilst the rate of uridine monophosphate formation is Biochim. Biophys. Acta, 182 (1969) 285-294
RNA
291
SYNTHESIS IN BRAIN SLICES
TABLE V PHOSPHORYLATION
OF [2-14C]URIDINE
IN A DIALYSED
EXTRACT
FROM RAT BRAIN
CORTEX
IN THE
PRESENCE OF VARYING CONCENTRATIONS OF NaC1 OR OF KC1. Results expressed as m e a n s ~:S.D. Initial [2-14C]uridine concn.: 5.1/~M; 3.5" lOS c o u n t s / m i n per vessel. E a c h vessel contained i mM K , A T P in the presence of v a r y i n g concentrations of NaCl, or I mM N a , A T P in the presence of v a r y i n g concentrations of KC1, 5 mM MgCI v o.o2 M T r i s HC1 (pH 7.4) and 0. 5 ml e x t r a c t (corresponding to 50 m g w e t wt. brain tissue), tile final volume being 1.5 ml. I n c u b a t i o n w a s carried o u t at 37 ° for 3 ° rain, after which the reaction w a s s t o p p e d b y the addition of ioo % trichloroacetic acid to a final concn, of 5 % trichloroacetic acid. The t u b e s were k e p t at o ° for i h and t h e n centrifuged for io m i n at m a x i m u m speed in the clinical centrifuge. The s u p e r n a t a n t s were r e m o v e d and s h a k e n 3 times w i t h 2 vol. ether to r e m o v e trichloroacetic acid and a 20o-/11 aliquot was c h r o m a t o g r a p h e d .
Conditions
?*C]Uridine (pmoles]ml per 30 rain) as
Uridine + uracil
UMP
UDP + UTP
NaCl (M)
o 5 15 ioo 200
I36O~:33 1297£55 1317±17 15824-74 16534-20
7 7 8 i 12 743~ 9 795-4- IO 1 2 o 3 + 19 1581+1o 5
285o4- 83 28o8+ 7 27234- 59 1763-4- 9o i312ii38
KCl (M)
o 5 15 IOO 200
12374-68 13174-16 I229i42 16994-28 19974- 4
691-4- 2 706-4- 9 6534- 14. 1o354- 42 1 4 5 4-4 14
2678+119 27354- 65 26614- 99 2o554- 58 11004- io
undiminished or possibly increased. Evidently, the effect of increasing the Na+ concentration of the brain cell is to bring about a suppression of the phosphorylation of uridylic acid and, thereby, a suppression of the rate of incorporation of uridine into RNA. The effects of K + on uridine phosphorylation by a dialysed rat brain extract in the presence of A T P resemble those due to Na + (Table V). High concentrations of K + in the extract lead to diminished rates of uridylic acid phosphorylation. The rate of formation of uridylic acid is undiminished or, possibly, accelerated.
E]/ects o] acetylcholine on uridine phosphorylation and incorporation into R N A In view of the neurophysiological effects of acetylcholine on brain cell permeability TM, we have carried out experiments to observe its effects on uridine phosphorylation and conversion to RNA. The addition of acetylcholine (I mM), in the presence of eserine, brings about an increased rate of incorporation of labelled uridine into RNA of rat brain cortex slices incubated in a glucose-Ringer medium, the increase being largest in the presence of 15 mM KC1. Results are shown in Fig. I where it is evident that acetylcholine is least active in the absence of K + from the incubation medium. The combination of 15 mM KC1 and acetylcholine brings about a doubling of the rate of uridine incorporation into RNA found in the normal Ringer medium. Acetylcholine is without effect in the absence of eserine. Eserine, alone, at the concentration used (I mM) brings about a small (about 20 %) acceleration of the rate of uridine incorporation. Carbamylcholine (in the absence of eserine) is equally as effective as acetylcholine (in the presence of eserine) in bringing about a stimulation of RNA formation. Biochim. Biophys. Acta, 182 (1969) 285-294
292
C. PRIVES, J. H. QUASTEL
Acetylcholine is without effect on uridine phosphorylation in brain extracts and its stimulatory effect in brain slices is greatly diminished in the absence of Na +. To obtain information about the effect of acetylcholine under conditions where RNA synthesis is blocked, experiments were carried out to observe the effects of acetylcholine (in the presence of eserine) on cerebral uridine phosphorylation in the presence of actinomycin D which was added to the incubation medium at a concentration of 3 ~,g/ml. Results are shown in Table VI. TABLE VI EFFECTS OF VARIOUS CONCENTRATIONS OF I{CI-[-ACETYLCHOLINE (q-ESERINE) ON E2-14C]uRIDINE INCORPORATION INTO NUCLEOTIDES AND R N A IN THE PRESENCE OF ACTINOMYCIN D IN RAT BRAINCORTEX SLICES
[2-14ClUridine i n p u t : 3 ' lO6 c o u n t s / m i n per vessel; 12/zM. Acetylcholine w a s p r e s e n t where specified at a concentration of i mM in the presence of eserine: i raM. Actinomycin D was p r e s e n t at a concentration of 3 / , g / m l . Results are m e a n s of two determinations.
Conditions
5 mM 5 mM 15 mM 15 mM 5omM 5omM
KC1 KCl+acetylcholine KC1 KCl+acetylcholine 1KC1 KCl+acetylcholine
[14C]Uridine incorporated (pmoles/g wet wt./3o rain) as Uridine+uracil
UMP
UDP+ UTP
RNA
4920-[-342 438o:~ 23 4145-[" 47 404032217 418o-[,247 384o324oo
78832 29 97532 71 9o6~z 26 lO65-[" 59 52832125 1135-[,112
56IOd~ 194 645o-['4Ol 562o~423 6360-[- 71 525032323 6750-[-754
I78"32 7 187 ± 3 182 32 i 195 -[,18 154 :~IO 167 ± 8
" Control figure for R N A in the absence of actinomycin D = lO6O pmoles/g wet wt./3o rain.
It will be seen that the rate of uridine incorporation into RNA falls from a normal value of 1.o6 nmoles/g per 30 rain to o.178 nmoles/g per 30 rain. The greatly diminished rat of RNA formation is not affected, within experimental error, b y the presence of acetylcholine at the various concentrations of K + investigated. However, a significant increase in the rate of phosphorylation of uridine is brought about by the addition of acetylcholine (in the presence of eserine) at all concentrations of K+ investigated. The rise is largest in the presence of 50 mM KC1 where the rate of incorporation of labelled uridine (12/~M) into UMP rises from 528+125 to I I 3 5 d z I I 2 pmoles and into combined U D P + U T P from 525od~323 to 675odz754 pmoles/g wet wt. tissue in an incubation time of 30 rain. Preliminary experiments have now shown that the effect of acetylcholine m a y be due to its dinlinution or removal of the timelag observed 19 during the course of uridine incorporation into RNA. Acetylcholine does not affect the diminished rate of RNA biosynthesis brought about by electrical impulses. DISCUSSION
The results reported in this communication show that the suppressing effect of electrical impulses on the formation of uridine polyphosphates and RNA in brain cortex slices, incubated aerobically in a Krebs-Ringer-glucose medium, is probably due to the influx of Na + into the brain cells. Increase of Na+ concentration in the incubation medium is itself sufficient to diminish the rates of incorporation of labelled Biochim. Biophys. Acta, 182 (1969) 285-294
RNA SYNTHESIS IN BRAIN SLICES
293
uridine into uridine polyphosphates and RNA. The influx of labelled uridine into the brain cell is not suppressed. When Na + are entirely omitted from the incubation medium there also follows a diminished rate of uridine phosphorylation and of RNA formation, there being no diminution of influx of uridine into the brain tissue. Moreover, the presence of ouabain (I/zM) in the normal incubation medium suppresses the rate of uridine incorporation into RNA. I t would appear, therefore, that the process of uridine conversion to RNA in brain cortex slices depends to some extent on the operation of the Na + pump. As is well known, suppression of the activity of the Na + p u m p leads to leakage of K ÷ from the brain cell. Our experiments (Fig. I) show that omission of K ÷ from the incubation medium leads to a diminished rate of incorporation of uridine into RNA. The results also indicate that increase of the K + concentration in the incubation medium to 15 mequiv/1 leads to a marked increase of the rate of incorporation of uridine into RNA over that found in the normal incubation medium. The presence of relatively high concentrations of K + ( > 50 mequiv/1) brings about, on the other hand, a decreased rate of uridine incorporation into RNA. The effects of changing the K ÷ content of the incubation medium m a y be explained by the occurrence of the following phenomena: (a) a stimulation of the rate of uridine incorporation directly dependent on the K ÷ content of the brain cell; (b) an inhibition partly due to the fall in the cell level of ATP, dependent on the K + concentration of the incubation medium and partly due to the inhibitory effect of a high KCI concentration on the rate of formation of uridine polyphosphates (Table V). Studies of the activities of dialysed brain extracts, in the presence of A T P and Mg *+, show that increase of the concentration of Na + to IOO mequiv/1 leads to a suppression of the rate of phosphorylation of uridylic acid and presumably, therefore, to a suppression of the rate of incorporation into RNA in the intact cell. This result makes it possible to understand the effects, on RNA biosynthesis, of electrical stimulation, or of ouabain, either of which causes an appreciable increase in the brain cell concentration of Na ÷. Studies with brain extracts, moreover, show that the stimulatory effects of K +, on uridine phosphorylation and conversion to RNA in brain slices, are not due to a direct action of the ions on the process of uridine phosphorylation in the presence of ATP. Conceivably the stimulatory action of K ÷, seen in Fig. I, is partly due to increased retention of K + within the brain cell, leading to an increased rate of formation of pyruvate and of ATP (by their well known accelerating effect on the activity of pyruvate phosphokinase). I t is known that in the absence of K ÷ there is a diminished brain cell level of ATP TM. Possibly there m a y be a direct accelerating effect of K + on RNA biosynthesis. BARONDESTM has shown, in this connection, that the rate of incorporation of [2-14C]CTP into RNA in rat brain preparations is accelerated by the addition of high concentrations of KC1, that are, however, considerably higher than those we have employed. The presence of acetylcholine (I mM), in the presence of eserine, brings about an acceleration of the rate of uridine incorporation into RNA and on the rate of uridine phosphorylation in brain slices so long as Na + and K + are present in the incubation medium. I t does not affect uridine phosphorylation in brain extracts. Its precise mode of action is, at present, unknown. Biochim. Biophys. Acta, 182 (1969) 285-294
294
C. PRIVES, J. H. QUASTEL
ACKNOWLEDGEMENT
We gratefully acknowledge financial support for this investigation from the Medical Research Council of Canada.
REFERENCES I V. S. SHAPOT, in D. RICHTER, Metabolism of the Nervous System, P e r g a m o n Press, L o n d o n , 1957, p. 257. 2 F. ORREGO AND F. LIPMANN, J. Biol. Chem., 242 (1967) 665. 3 A. GEIGER, S. YAMASAKI AND R. LYONS, Am. J. Physiol., 184 (1956) 239. 4 H. HYDEN, in K. A. C. ELLIOTT, I. H. PAGE AND J. H. QUASTEL, Neurochemistry, T h o m a s , Springfield, 2nd Ed., 1962, p. 331. 5 C. PRIVES AND J. H. QUASTEL, Proc. Can. Federation Biol. Soc., IO (1967) 31. 6 F. ORREGO, J. Neurochem., 14 (1967) 851. 7 G. SCHMIDT AND S. J. THANNHAUSER, J. Biol. Chem., 161 (1945) 83. 8 H. WALLGREN AND E. KULONEN, Biochem. J., 75 (196o) 15o. 9 H. WALLGREN, J. Neurochem., io (1963) 349. io J. C. KEESEY, H. WALLGREN AND H. MclLWAIN, Biochem. J., 95 (1965) 289. t i J. C. KEESEY AND H. WALLGREN, Biochem. J., 95 (1965) 3 ol12 S. L. CHAN AND J. H. QUASTEL, Science, 156 (1967) 1752. 13 J. H. QUASTEL, C. PRIVES AND H. MENON, Abstr. Ist Intern. Meeting Intern. Soc. Neurochem., Strasburg, 1967, p. 174. 14 R. J. ROSSlTER, in D. RICHTER, Metabolism of the Nervous System, P e r g a m o n Press, L o n d o n , 1957, P- 355. 15 Y. TSUKADA AND O. TAKAGUCHI, Nature, 175 (1955) 725 . 16 P. J. HEALD, Biochem. J., 57 (1954) 673. 17 P. N. ABADOM, AND P. G. SCHOLEFIELD, Can. J. Biochem. Physiol., 4 ° (1962) 16o 3. 18 C. O. HEBB AND K. KRNJEVIC, ill K. A. C. ELLIOTT, I. H. PAGE AND J. H. QUASTEL, Neurochemistry, T h o m a s , Springfield, 2nd Ed., 1962, p. 452. 19 S. H. BARONDES, J. Neurochem., i i (1964) 663. 20 G. GUROFF, A. F. HOGANS AND S. UDENFRIEND, J. Neurochem., 15 (1968) 489.
Biochim. Biophys. Acta, 182 (1969) 285-294
BIOCHIMICA ET BIOPHYSICA ACTA BBA 96223
E F F E C T OF C E R E B R A L STIMULATION ON B I O S Y N T H E S I S OF N U C L E O T I D E S AND RNA IN B R A I N SLICES I N V I T R O CAROL PRIVES* AND J. H. QUASTEL
Neurochemistry Section, Kinsmen Laboratories, The University of British Columbia, Vancouver 8, B.C. (Canada) (Received February 24th, I969)
SUMMARY
I. Application of electrical impulses to rat brain-cortex slices, incubated aerobically at 37 °, diminishes the rates of conversion of 14C-labelled uridine into uridine polyphosphates and RNA to less than half the control values. The effect is blocked b y the presence of 5 #M tetrodotoxin. 2. Increase of the NaC1 concentration of the incubation medium from 128 to 178 mM and 228 mM greatly decreases the rate of incorporation of l~C-labelled uridine into uridine polyphosphates and RNA in the brain slices. Omission of NaC1 from the incubation medium also diminishes the rates of biosynthesis of uridine nucleotides and RNA. 3. Increase of the KC1 concentration of the incubation medium from 5 to 15 mM results in an increase of the rate of incorporation of 14C-labelled uridine into RNA by approx. 5o %. Further increase of the KC1 concentration to 50 and IOO mM results in a diminished rate of RNA biosynthesis. 4. Addition of I mM acetylcholine (in presence of eserine) to the incubation medium results in an increased rate of incorporation of 14C-labelled uridine into RNA in brain slices. The effect is dependent on the presence of Na + and K +. 5. The rate of conversion of uridine to uridine polyphosphates in presence of ATP, in a dialysed rat brain extract, is diminished by increasing the concentrations of NaC1, or KC1, to IOO mM or more. 6. Possible explanations for these effects, and their significance, are discussed.
INTRODUCTION
In view of the possibility that memory, such as that acquired in learning, is represented by some permanent change in the brain and that this change is brought about as a result of chemical activity in the brain cell, we have carried out experiments to observe whether various stimulating agents will affect nucleotide and RNA biosynthesis in isolated brain. Evidence has already accumulated to indicate that brain excitation, or electrical stimulation in vitro, will affect protein biosynthesis in the brain. SHAPOT1, for example, has demonstrated that the rate of incorporation of * Present address: Department of Molecular Biology, Albert Einstein College of Medicine, 13oo Morris Park Avenue, Bronx, N.Y. 10461 , U.S.A.
Biochim. Biophys. Acta, I82 (I969) 285-294
~86
c. PRIVES, J. H. QUASTEL
[35S]methionine into proteins in the brains of rats, after excitation, is lower than that found in control animals and attributes this to diminished cerebral protein synthesis. ORREGO AND LIPMAI~Nz have concluded that electrical stimulation of brain slices results in inhibition of protein synthesis, as judged b y the diminished rate of incorporation of [14Clvaline into the brain proteins. There are indications also that nucleic acid metabolism in the brain is affected by stimulation; thus GEIGER et al. 3 have found that stimulation for 3 ° sec changes the composition of RNA in tile brain cortex. Marked motor activity causes an increase in the RNA concentration in the motor root cells, whilst sensory stimulation causes changes in RNA concentratiol~ in the vestibular ganglion cells which depend on the intensity of the stimulation 4. Work carried out independently, and at about the same time, by the present authors 5 and by ORREGO6 has shown that electrical stimulation of rat brain cortex i n vitro brings about at least 4 ° % inhibition of RNA biosynthesis, as judged by the rate of incorporation of radioactive uridine into RNA. ORREGO6 has further shown that this m a y be due in part to impaired nucleotide phosphorylation. The following report describes the effects of electrical stimulation, and of changes in the cation composition of the incubation medium, and of acetylcholine, on the rates of incorporation of labelled uridine into uridine nucleotides and into RNA in rat brain cortex slices.
METHODS AND MATERIALS
We have used adult female hooded rats whose brains were quickly removed after stunning and decapitation, and slices (about 0.3-0. 4 m m thick) were prepared from the cerebral hemispheres using a Stadie-Riggs microtome. One dorsal and one lateral slice, weighing a total of 50-75 mg wet wt., were transferred to chilled Warburg manometric vessels each containing 3 ml Krebs-Ringer phosphate medium and IO mM glucose. Radioactive uridine was added from the side arm of the vessel and all other additions were present in the main compartment from the start of the experiment. In experiments with a Na+-free medium the NaC1 was replaced b y sucrose or choline chloride to maintain isotonicity and Tris buffer (IO raM, p H 7-4) was used instead of phosphate buffer. The vessels were equilibrated at 37 ° for IO rain in an atmosphere of 02 prior to tipping in the labelled compound from the side arm and incubation was then carried out for 30 min. Aftei incubation the Slices were remove,d, washed twice with ice-cold Krebs-Ringer medium to remove contaminating radioactivity and homogenized at o ° in 5 % trichloracetic acid. The homogenate was kept at o ° for i h and centrifuged at 600 × g for IO min. The supernatant was analysed for phosphorylated and free uridine after removal of the trichloracetic acid by successive washings with ether. An aliquot (200/A) of the solution was chromatographed on W h a t m a n No. 3 paper, using, as descending solvent, ethanol-ammonium acetate (I M) (pH 7.4) (7:3.5, v/v) for a period of 6 h, which brings about separation of uridine ( + uracil), uridine monophosphate and the combined uridine polyphosphates which are not effectively separated and were therefore estimated together. It should be noted that uridine and uracil are also not separated by this technique, so that spots containing uridine m a y contain uracil derived from the uridine. The resulting spots containing uridine ( + uracil), uridylic acid (UMP) and uridine polyphosphates Biochim. Biophys. Acta, 182 (1969) 285-294
RNA
287
SYNTHESIS IN BRAIN SLICES
( U D P + U T P ) were cut out of the paper in equal sections and their radioactivities were estimated in the liquid scintillation counter after correction for the small quenching effect of the filter paper. The residue, from the centrifuged homogenate, was washed twice with 5 % trichloracetic acid to remove acid-soluble radioactivity and then once with 95 % ethanol and once with a 95 % ethanol-diethyl ether mixture (3 : I, v/v) to remove lipids. The washed residue was dissolved in 2 ml 0.2 M K O H and incubated at 37 ° for 18 h (ref. 7). 0.05 ml 7 ° % HC1Q was added, precipitating the DNA and proteins and leaving the hydrolysed RNA in the supernatant. The radioactivity of the hydrolysed RNA was subsequently determined in the liquid scintillation counter. Electrical stimulation of slices of braiu cortex was carried out in vessels with silver grid electrodes, similar to those used by WALLGRENAND KULONENs, with electric impulses from the alternating frequency generator of the Teratron type described in detail b y WALLGREN9. The impulses had a pulse frequency of IOO cycles/sec with I-msec duration, the peak potential being 4 V. Extracts of cerebral cortex were prepared b y homogenizing the excised cerebral cortex in 12 vol. 0.02 M Tris-HC1 (pH 7.4) in a Potter-Elvehjem glass homogenizer with a fitted teflon pestle. The homogenate was centrifuged at 17 ooo ×g for I h in a Sorvall high-speed centrifuge, and the supernatant was dialysed for 18 h against 0.02 M Tris-HC1 (pH 7.4)-
RESULTS
E]]ects o/electrical stimulation on cerebral incorporation o] uridine into nucleotides and RNA Results given in Table I show that application of electrical impulses has a marked diminishing effect on the rate of conversion, in rat brain cortex slices, of uridine into uridine polyphosphates as well as into RNA. There is little or no effect on the rate of formation of uridine monophosphate and the concentration of free labelled uridine ( + uracil) in the slice rises due to the diminished rate of formation of U D P + UTP. These results confirm those of ORREGO6. They also show that, although the concentration of total labelled uridine ( + uracil), uridine phosphates and RNA in the tissue after 3o min incubation is but little greater than the concentration of
TABLE I EFFECTS OF ELECTRICAL CLEOTIDES IN RAT BRAIN
STIMULATION ON CORTEX SLICES
[2-14C]URIDINE
INCORPORATION
INTO
RNA
AND
NU-
Initial [2-14C]uridine concn.: 5-9/zM; 1. 5 • lO 6 c o u n t s / m i n per vessel. Results expressed as m e a n s of four d e t e r m i n a t i o n s ± S.D.
Conditions
Control Electrical s t i m u l a t i o n
[14C]Uridine incorporated (pmoles/g wet wt. per 30 rain) as Uridine+ uracil
UMP
UDP4-UTP
RNA
192o4-251 246°±167
5284- 73 5294-113
314o4-47 ° 19354-177
5664-48 2344-37
Biochim. Biophys. Acta, 182 (1969) 285-294
288
c. PRIVES, J. H. QUASTEL
labelled uridine in the bathing medium, the concentration of free labelled uridine in the tissue is much less than that in the medium. Thus, there exists a permeability barrier at the brain-cell membrane limiting the rate of diffusion of uridine into the brain cells. TABLE II EFFECT OF ELECTRICAL STIMULATION IN PRESENCE AND ABSENCE OF TETRODOTOXIN DINE INCORPORATION INTO R N A IN RAT BRAIN CORTEX SLICES
ON
[2-14C]URI -
I n i t i a l [2-14C]uridine conch.: 5.9 t,M; 1. 5. lO6 c o u n t s / m i n p e r vessel. T e t r o d o t o x i n w a s p r e s e n t w h e r e i n d i c a t e d a t a final concn, of 5/~M. R e s u l t s e x p r e s s e d as m e a n s of a t l e a s t t h r e e e x p e r i m e n t s 4-S.D.
Conditions
[14C] Uridine incorporated into
RNA
(pmoles/g wet wt. per 30 min) Control Electrical stimulation Tetrodotoxin Electrical stimulation + tetrodotoxin
5664- 48 234 4- 37 588 4-13 5324-54
Further results (Table II) show that addition of the potent neurotoxin, tetrodotoxin, to the bathing medium blocks the effects of electrical impulses on the incorporation of uridine into RNA. I t is already known that during the application of electrical impulses to brain-cortex slices there is an influx of Na + into brain tissue 1°-12 and that the metabolic effects of electrical impulses on isolated brain m a y be blocked b y the addition of small concentrations of tetrodotoxin 1~ which acts by its inhibitory effect on Na ÷ influx during electrical stimulation. The suppressing action of electrical impulses on the formation of uridine polyphosphates and RNA may, therefore, be due to the influx of Na + into the brain cell. This prediction was verified in the following manner. E//ects o / N a + on uridine incorporation into R N A and uridine nucleotides Results given in Table I I I show that increase of Na + concentration in the bathing medium, from the initial level of 128 mequiv/1 in the Krebs-Ringer medium, TABLE III EFFECTS OF VARIOUS CONCENTRATIONS OF ]_x]'aCl ON [2-14C]URIDINE INCORPORATION INTO R N A A N D NUCLEOTIDES IN RAT BRAIN CORTEX SLICES R e s u l t s e x p r e s s e d as m e a n s 4-S.D. I n i t i a l [2-14C]uridine concn.: 5.9 #M; 3 ' IOe c o u n t s / m i n p e r vessel. I n e x p e r i m e n t s w i t h o a n d 2o mM NaC1, 128 m M c hol i ne c h l o r i d e w a s a d d e d to t h e i nc ub a t i o n m e d i u m to m a i n t a i n i s o t o n i c i t y a n d o . i M Tris-HC1 (pH 7.4) w a s us e d as buffer. I n exp e r i m e n t s w i t h 128, 178 a n d 228 mM NaC1, o . o i M s o d i u m p h o s p h a t e buffe r (pH 7-4) w a s used.
NaCl goncn.
o 20 128 178
228
[14C]Uridine incorporated (pmoles/g wet wt. per 3o rain) as Uridine + uracil
UM P
UD P + U T P
RNA
28664-176 19784-117 17294- 13 25884-167 24294-147
3964- 28 91o4-142 4834- 28 49o4- 34 3944- l o 6
19254- 65 36804-42o 45554- 75 24164-114 17514- 75
1484- IO 4434-89 7 7 6 ± 15 3 4 I t 2I 1214- 9
Biochim. Biophys. Acta, 182 (1969) 285-294
RNA
289
SYNTHESIS IN BRAIN SLICES
leads to a diminution of the rate of uridine incorporation into RNA in rat brain cortex slices. A similar phenomenon occurs with the incorporation of uridine into uridine polyphosphates. These results support the conclusion that influx of Na + into the brain cell, bathed in a physiological saline medium, leads to a suppression in the rates of formation of uridine polyphosphates and RNA. The influx of uridine, however, into the brain tissue is not markedly affected by the rise in NaC1 concentration in the bathing medium. Results in Table III, indicate that when Na + are omitted from the incubation medium, these being replaced by choline to maintain isotonicity, there is a greatly diminished rate of phosphorylation of uridine to uridine monophosphate, as well as to the polyphosphates, and the rate of RNA formation is much suppressed. Addition of 20 mM NaC1 to the Na+-free incubation medium brings about a marked increase of the rate of formation of uridine monophosphate, uridine polyphosphates and RNA. The quantity of free labelled uridine ( + uracil) in the tissue is not diminished b y the absence of Na + from the incubation medium, so that the results cannot be explained as due to suppressed influx of uridine into the tissue.
E//ects o/ouabain on uridine incorporation into R N A Ouabain, at small concentrations which diminish the activity of the Na + pump, by its suppressing effect on membrane bound ATPase, inhibits the incorporation of uridine into RNA (Table IV). The inhibitory effect of ouabain is nullified b y increase of K + concentration in the incubation medium, a phenomenon that m a y be indicative of the well known antagonism between ouabain and K +. TABLE IV EFFECTS OF I/~M OUABAIN ON THE RATE OF [2-14C]URIDINE INCORPORATION INTO R N A IN RAT BRAIN CORTEX SLICES I n i t i a l [2-14Cluridine concn.: 5 ~M; 1.2. i o ~ c o u n t s / m i n p e r vessel.
Conditions
Uridine incorporated into R N A (pmoles/g wet wt. tissue)
5 15 5 15
41o4-34 560+ 5 28o+ 9 4 9 7 i 16
mM mM mM mM
KC1 KC1 KCl+ouabain KCl+oaubain
EHects o] K + on the rate o[ incorporation o[ uridine into R N A in rat brain cortex slices Turning to the effects of increased concentrations of K + on the rate of RNA biosynthesis, as determined b y uridine incorporation, we have found 18 that an increase of KC1 concentration, from 5 to 15 mM, in the incubation medium increases the rate of RNA formation Typical results are shown in Fig. I. The presence of 5 mequiv/l K + nearly doubles the rate of incorporation of labelled uridine into RNA found in the absence of added K+, whilst the respiratory rate increases from 9.9/~1 03 consumed per mg dry wt. brain tissue to II.O. Addition of K+ to 15 mequiv]l brings about a further increase in the rate of labelled uridine incorporation into RNA, but a still greater Biochim. Biophys. Acta, 182 (I969) 285-294
290
c. PRIVES, J. H. QUASTEL
P,, \
/
O
En <
'~000
\x
iI
I
~z
\k \\
'\
~-~ "O •
5
I
I
15 .50 KCt conch. (raM)
I
100
Fig. I. E f f e c t s of v a r y i n g c o n c e n t r a t i o n s of KC1, in t h e presence a n d a b s e n c e of i m M acetylcholine ( + i m M eserine) o n t h e r a t e of E2-14C]uridine i n c o r p o r a t i o n into R N A in r a t brainc o r t e x slices. Initial c o n c e n t r a t i o n of uridine = 5.9 #M. Q , no acetylcholine; O , w i t h acetylcholine.
increase to 5 ° or IOO mequiv/1 brings about a fall in the rate of incorporation, although the respiratory rate increases to the value of 18.3/,1 O 3 consumed per mg dry wt. brain tissue per h. The fact that a concentration of K+ appreciably higher than 15 mequiv/1 brings about a fall in the rate of incorporation of uridine into RNA m a y be correlated with the well known fact that, with a marked increase of K + in the incubation medium, there is a fall in the brain cell level of ATP 14-17, a phenomenon associated with the increased activity of the membrane-bound (Na+-K+)-sensitive ATPase. E[]ects o] N a + and K + on uridine phosphorylation in a dialysed rat brain extract The rates of uridine phosphorylation b y a dialysed brain extract obtained from 5 ° mg wet wt. brain tissue, in the presence of ATP, are far greater than those obtained by an equal weight of rat brain cortex slices incubated under optimal respiratory conditions. Results given in Table V show that the total quantity of phosphorylated uridine formed from 7.5 nmoles uridine in 30 rain approximates to 3 nmoles, whereas the total formed, in 50 mg wet wt. brain slices, from 17. 7 nmoles uridine in the same time does not exceed 0.25 nmoles (Table III). Nevertheless, the concentration of total phosphorylated uridine found in a brain extract, equivalent to 50 mg wet wt. tissue, amounts at the most to 3.6 ~M (Table V), whereas the concentration of total phosphorylated uridine in the brain slice m a y amount to 6.3 #M (assuming that the rat brain slice contains 80 % water) (Table I I I ) . These results are presumably largely due to the permeability barrier, imposed by the brain cell membrane, to uridine, which limits the amount of uridine to be metabolised and to the higher concentrations of ATP in the brain cell (about 2.2 raM) than that used with the brain extract (I mM). The marked diminution of the rate of uridine phosphorylation, due to absence of Na + from the incubation medium, in brain cortex slices is not evident with a dialysed rat brain extract in the presence of ATP (Table V). In such an extract, increase of the concentration of Na + to ioo mequiv/1 leads to a diminished rate of formation of uridine polyphosphates, whilst the rate of uridine monophosphate formation is Biochim. Biophys. Acta, 182 (1969) 285-294
RNA
291
SYNTHESIS IN BRAIN SLICES
TABLE V PHOSPHORYLATION
OF [2-14C]URIDINE
IN A DIALYSED
EXTRACT
FROM RAT BRAIN
CORTEX
IN THE
PRESENCE OF VARYING CONCENTRATIONS OF NaC1 OR OF KC1. Results expressed as m e a n s ~:S.D. Initial [2-14C]uridine concn.: 5.1/~M; 3.5" lOS c o u n t s / m i n per vessel. E a c h vessel contained i mM K , A T P in the presence of v a r y i n g concentrations of NaCl, or I mM N a , A T P in the presence of v a r y i n g concentrations of KC1, 5 mM MgCI v o.o2 M T r i s HC1 (pH 7.4) and 0. 5 ml e x t r a c t (corresponding to 50 m g w e t wt. brain tissue), tile final volume being 1.5 ml. I n c u b a t i o n w a s carried o u t at 37 ° for 3 ° rain, after which the reaction w a s s t o p p e d b y the addition of ioo % trichloroacetic acid to a final concn, of 5 % trichloroacetic acid. The t u b e s were k e p t at o ° for i h and t h e n centrifuged for io m i n at m a x i m u m speed in the clinical centrifuge. The s u p e r n a t a n t s were r e m o v e d and s h a k e n 3 times w i t h 2 vol. ether to r e m o v e trichloroacetic acid and a 20o-/11 aliquot was c h r o m a t o g r a p h e d .
Conditions
?*C]Uridine (pmoles]ml per 30 rain) as
Uridine + uracil
UMP
UDP + UTP
NaCl (M)
o 5 15 ioo 200
I36O~:33 1297£55 1317±17 15824-74 16534-20
7 7 8 i 12 743~ 9 795-4- IO 1 2 o 3 + 19 1581+1o 5
285o4- 83 28o8+ 7 27234- 59 1763-4- 9o i312ii38
KCl (M)
o 5 15 IOO 200
12374-68 13174-16 I229i42 16994-28 19974- 4
691-4- 2 706-4- 9 6534- 14. 1o354- 42 1 4 5 4-4 14
2678+119 27354- 65 26614- 99 2o554- 58 11004- io
undiminished or possibly increased. Evidently, the effect of increasing the Na+ concentration of the brain cell is to bring about a suppression of the phosphorylation of uridylic acid and, thereby, a suppression of the rate of incorporation of uridine into RNA. The effects of K + on uridine phosphorylation by a dialysed rat brain extract in the presence of A T P resemble those due to Na + (Table V). High concentrations of K + in the extract lead to diminished rates of uridylic acid phosphorylation. The rate of formation of uridylic acid is undiminished or, possibly, accelerated.
E]/ects o] acetylcholine on uridine phosphorylation and incorporation into R N A In view of the neurophysiological effects of acetylcholine on brain cell permeability TM, we have carried out experiments to observe its effects on uridine phosphorylation and conversion to RNA. The addition of acetylcholine (I mM), in the presence of eserine, brings about an increased rate of incorporation of labelled uridine into RNA of rat brain cortex slices incubated in a glucose-Ringer medium, the increase being largest in the presence of 15 mM KC1. Results are shown in Fig. I where it is evident that acetylcholine is least active in the absence of K + from the incubation medium. The combination of 15 mM KC1 and acetylcholine brings about a doubling of the rate of uridine incorporation into RNA found in the normal Ringer medium. Acetylcholine is without effect in the absence of eserine. Eserine, alone, at the concentration used (I mM) brings about a small (about 20 %) acceleration of the rate of uridine incorporation. Carbamylcholine (in the absence of eserine) is equally as effective as acetylcholine (in the presence of eserine) in bringing about a stimulation of RNA formation. Biochim. Biophys. Acta, 182 (1969) 285-294
292
C. PRIVES, J. H. QUASTEL
Acetylcholine is without effect on uridine phosphorylation in brain extracts and its stimulatory effect in brain slices is greatly diminished in the absence of Na +. To obtain information about the effect of acetylcholine under conditions where RNA synthesis is blocked, experiments were carried out to observe the effects of acetylcholine (in the presence of eserine) on cerebral uridine phosphorylation in the presence of actinomycin D which was added to the incubation medium at a concentration of 3 ~,g/ml. Results are shown in Table VI. TABLE VI EFFECTS OF VARIOUS CONCENTRATIONS OF I{CI-[-ACETYLCHOLINE (q-ESERINE) ON E2-14C]uRIDINE INCORPORATION INTO NUCLEOTIDES AND R N A IN THE PRESENCE OF ACTINOMYCIN D IN RAT BRAINCORTEX SLICES
[2-14ClUridine i n p u t : 3 ' lO6 c o u n t s / m i n per vessel; 12/zM. Acetylcholine w a s p r e s e n t where specified at a concentration of i mM in the presence of eserine: i raM. Actinomycin D was p r e s e n t at a concentration of 3 / , g / m l . Results are m e a n s of two determinations.
Conditions
5 mM 5 mM 15 mM 15 mM 5omM 5omM
KC1 KCl+acetylcholine KC1 KCl+acetylcholine 1KC1 KCl+acetylcholine
[14C]Uridine incorporated (pmoles/g wet wt./3o rain) as Uridine+uracil
UMP
UDP+ UTP
RNA
4920-[-342 438o:~ 23 4145-[" 47 404032217 418o-[,247 384o324oo
78832 29 97532 71 9o6~z 26 lO65-[" 59 52832125 1135-[,112
56IOd~ 194 645o-['4Ol 562o~423 6360-[- 71 525032323 6750-[-754
I78"32 7 187 ± 3 182 32 i 195 -[,18 154 :~IO 167 ± 8
" Control figure for R N A in the absence of actinomycin D = lO6O pmoles/g wet wt./3o rain.
It will be seen that the rate of uridine incorporation into RNA falls from a normal value of 1.o6 nmoles/g per 30 rain to o.178 nmoles/g per 30 rain. The greatly diminished rat of RNA formation is not affected, within experimental error, b y the presence of acetylcholine at the various concentrations of K + investigated. However, a significant increase in the rate of phosphorylation of uridine is brought about by the addition of acetylcholine (in the presence of eserine) at all concentrations of K+ investigated. The rise is largest in the presence of 50 mM KC1 where the rate of incorporation of labelled uridine (12/~M) into UMP rises from 528+125 to I I 3 5 d z I I 2 pmoles and into combined U D P + U T P from 525od~323 to 675odz754 pmoles/g wet wt. tissue in an incubation time of 30 rain. Preliminary experiments have now shown that the effect of acetylcholine m a y be due to its dinlinution or removal of the timelag observed 19 during the course of uridine incorporation into RNA. Acetylcholine does not affect the diminished rate of RNA biosynthesis brought about by electrical impulses. DISCUSSION
The results reported in this communication show that the suppressing effect of electrical impulses on the formation of uridine polyphosphates and RNA in brain cortex slices, incubated aerobically in a Krebs-Ringer-glucose medium, is probably due to the influx of Na + into the brain cells. Increase of Na+ concentration in the incubation medium is itself sufficient to diminish the rates of incorporation of labelled Biochim. Biophys. Acta, 182 (1969) 285-294
RNA SYNTHESIS IN BRAIN SLICES
293
uridine into uridine polyphosphates and RNA. The influx of labelled uridine into the brain cell is not suppressed. When Na + are entirely omitted from the incubation medium there also follows a diminished rate of uridine phosphorylation and of RNA formation, there being no diminution of influx of uridine into the brain tissue. Moreover, the presence of ouabain (I/zM) in the normal incubation medium suppresses the rate of uridine incorporation into RNA. I t would appear, therefore, that the process of uridine conversion to RNA in brain cortex slices depends to some extent on the operation of the Na + pump. As is well known, suppression of the activity of the Na + p u m p leads to leakage of K ÷ from the brain cell. Our experiments (Fig. I) show that omission of K ÷ from the incubation medium leads to a diminished rate of incorporation of uridine into RNA. The results also indicate that increase of the K + concentration in the incubation medium to 15 mequiv/1 leads to a marked increase of the rate of incorporation of uridine into RNA over that found in the normal incubation medium. The presence of relatively high concentrations of K + ( > 50 mequiv/1) brings about, on the other hand, a decreased rate of uridine incorporation into RNA. The effects of changing the K ÷ content of the incubation medium m a y be explained by the occurrence of the following phenomena: (a) a stimulation of the rate of uridine incorporation directly dependent on the K ÷ content of the brain cell; (b) an inhibition partly due to the fall in the cell level of ATP, dependent on the K + concentration of the incubation medium and partly due to the inhibitory effect of a high KCI concentration on the rate of formation of uridine polyphosphates (Table V). Studies of the activities of dialysed brain extracts, in the presence of A T P and Mg *+, show that increase of the concentration of Na + to IOO mequiv/1 leads to a suppression of the rate of phosphorylation of uridylic acid and presumably, therefore, to a suppression of the rate of incorporation into RNA in the intact cell. This result makes it possible to understand the effects, on RNA biosynthesis, of electrical stimulation, or of ouabain, either of which causes an appreciable increase in the brain cell concentration of Na ÷. Studies with brain extracts, moreover, show that the stimulatory effects of K +, on uridine phosphorylation and conversion to RNA in brain slices, are not due to a direct action of the ions on the process of uridine phosphorylation in the presence of ATP. Conceivably the stimulatory action of K ÷, seen in Fig. I, is partly due to increased retention of K + within the brain cell, leading to an increased rate of formation of pyruvate and of ATP (by their well known accelerating effect on the activity of pyruvate phosphokinase). I t is known that in the absence of K ÷ there is a diminished brain cell level of ATP TM. Possibly there m a y be a direct accelerating effect of K + on RNA biosynthesis. BARONDESTM has shown, in this connection, that the rate of incorporation of [2-14C]CTP into RNA in rat brain preparations is accelerated by the addition of high concentrations of KC1, that are, however, considerably higher than those we have employed. The presence of acetylcholine (I mM), in the presence of eserine, brings about an acceleration of the rate of uridine incorporation into RNA and on the rate of uridine phosphorylation in brain slices so long as Na + and K + are present in the incubation medium. I t does not affect uridine phosphorylation in brain extracts. Its precise mode of action is, at present, unknown. Biochim. Biophys. Acta, 182 (1969) 285-294
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ACKNOWLEDGEMENT
We gratefully acknowledge financial support for this investigation from the Medical Research Council of Canada.
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Biochim. Biophys. Acta, 182 (1969) 285-294