Neurochemical Research (1) t91-200 (t976)
S T I M U L A T I O N OF THIAMINE TRIPHOSPHATE METABOLISM IN N E R V E BY C Y C L I C GUANOSINE 3',5'-MONOPHOSPHATE SVEN G. ELIASSON AND JOANNE D. SCARPELLINI Department of Neurology and Neurological Surgery (Neurology) Washington University School of Medicine St, Louis, Missouri 63110
Accepted February 9, 1976
This paper reports that addition of c G M P results in an increase of the amount of 32p~ incorporated into thiamine triphosphate in nerve roots and sympathetic trunks. The effect is present both at rest and during electrical stimulation. Other nucleotides were less effective. Theophylline increased the incorporation, possibly because of phosphodiesterase inhibition. A blocking effect of atropine was noted in sympathetic trunk preparations that contain ganglion cells.
INTRODUCTION Acetylcholine, tetrodotoxin, and electrical stimulation initiate release of thiamine from nerve and cord preparations, and thiamine reverses the ultraviolet light-induced block of nerve impulses (1-3). Simultaneously with the thiamine release, a decrease in thiamine polyphosphates was noted by Gurtner (4) in 4 out of 14 rats. Berman and Fishman (5) were able to show only minor changes in the distribution of thiamine and its phosphate esters on electrical stimulation or K + depolarization of rat cerebral cortex. Rapid turnover of the thiamine triphosphate pool did take place when 32Pi was used as precursor. Also associated with application of cholinergic stimuli, electrical stimuli, and seizures are increases in cyclic guanosine 3',5'-monophosphate (cGMP) (6,7). We 191 O 1976 Plenum Publishing Corporation, 227 West 17tb Street. New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.
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elected to explore the possibility of interaction between exogenous c GMP and thiamine triphosphate levels and metabolism. T h i s p a p e r r e p o r t s t h a t a d d i t i o n o f c G M P i n c r e a s e s t h e a m o u n t o f ~zp incorporated into thiamine triphosphate (TTP) in nerve tissue at rest and during stimulation. Some of the characteristics of TTP metabolism under different conditions are described.
EXPERIMENTAL
PROCEDURE
Materials. Thiamine triphosphate was obtained as a gift from Sankyo Company, Japan; other thiamine compounds were obtained from Sigma Chemicals. Carrier-free orthophosphoric acid and adenosine-5'-triphosphate-7-~-~P, diammonium salt (sp act 5-30 Ci/mM), were purchased from I C N Pharmaceuticals. Tissue Preparation. In these experiments, adult cats were anesthetized; a thoracotomy and laminectomy were performed, and the dorsal and ventral roots, as well as the thoracic sympathetic trunk, were removed. The nervous tissue was preincubated in Krebs-Ringer bicarbonate buffer, pH 7.4, at 37~ in a Dubnoff shaker for 30 min, after which the various experimental conditions were introduced. Preincubation is required to eliminate spontaneous firing in nerve fibers. The tissue was removed, and quickly frozen with liquid nitrogen. The tissue was weighed frozen, then minced and placed in fresh buffer and, to eliminate excess or newly formed ATP-3zP, incubated with excess glucose and hexokinase. Cold 7% perchloric acid was added for 30 min. The volume of acid was adjusted to give a l:I0 homogenate. All homogenizations were carried out in a glass (TenBroeck) homegenizer. The sample was centrifuged and the supernatant was kept cold until removed and neutralized with the addition of saturated potassium carbonate solution. In control experiments, no losses of TTP were noted. Determination of Thiamine Phosphates. The method used for quantization is a slight modification of that given by Itokawa and Cooper (8). After neutralization, the homogenate was subjected to electrophoresis in a Beckman microzone system. The membranes were wetted (not soaked) with 0.05 M acetate buffer at pH 3.8. Sartorius cellulose acetate membranes were used. Strips were spotted with tissue extracts and standards, and electrophoresis was carried out at 8 mA constant current for 9 min. The membrane was then dried in warm air without being removed from the suspension bridge. After the membrane was dried, it was sprayed with a mixture of 30 ml water, 0.6 ml 2% potassium ferricyanide, and 7 ml 15% sodium hydroxide. Spots were visualized with 360 nm U V light, cut out, and placed in fluorometer tubes along with sections of the membrane from blank areas. To the tubes were added 1 ml potassium ferricyanide spray reagent; 289 minutes later, 10/xl 30% hydrogen peroxide were added, and the tubes were gently shaken. The tubes were read 30 min later, using primary filters at 365 nm and secondary filters of 430 nm maximum transmission. All reagents have to be kept fresh and at room temperature for an hour before being read. Tubes must be sized and matched and calibrated with quinine solution. Sensitivity of assay 0.3 rig. Separation of TTP, T D P , TMP, and thiamine is accomplished more rapidly on the Sartorius membrane; the distances between the compounds are the same as documented by Itokawa and Cooper (8). Labeling of Thiamine Phosphates and ATP Analysis. Labeled thiamine phosphates were counted in 10 ml Bray's solution (9) after separation. Several experiments were done to
cGMP AND THIAMINE TRIPHOSPHATE
193
exclude contamination with residual labeled 32pi, The inorganic phosphate travels much faster than any of the assayed compounds and does not contaminate the phosphates. The ATP-T-a2P stays at the origin; after treatment with glucose-hexokinase, some ADP-3~P, which is probably a contaminant, can be located between TDP and TMP. Quench corrections were applied, using external standardization. Measurement of ATP was done according to the method by Lowry and Passonnean (10); the quantity obtained ranged from 1.2 to 3.0 tzmol/g dry weight. The amount of tracer added was equal to or less than 0.02% of the endogenous amount. Stimulation Experiments. Preparations were monitored for action potentials with suction electrodes connected to a Tektronix oscilloscope No. 565. For stimulation, a Grass $8 stimulator was used. Frequency and duration of the impulses are given in connection with the individual experiments. Resting and stimulated preparations were incubated together.
RESULTS
Incorporation of 32p Into Endogenous TTP Levels of TTP in cat nerve roots and thoracic sympathetic trunk immediately after removal were variable. Incubation of the nerve material in Krebs-Ringer buffer for 30 rain stabilized the level at 10-20 /,g/g dry weight in roots and 5-10 /zg/g dry weight in the sympathetic trunk. These levels remained stable under resting conditions during continued incubation up to 240 rain. Addition of 32Pi to the incubating roots after the preincubation resulted in rapid uptake of 32p into TTP during the first hour (Fig. 1), after which the rate leveled off over the next 3 h. The amount of TTP/g dry weight remains unchanged. When ATP-T-z2P was used, it appeared to a considerable extent to be hydrolyzed and the inorganic phosphate to be taken up by the nerve. Addition of dinitrophenol or arsenate depressed incorporation of 32Pi more than of ATP-T-a2p (Table I) (11).
Effect of cGMP on TTP Content and Radioactivity The endogenous content of c GMP in roots was determined for us by Dr. James Ferrendelli; it ranged from 20-60 pmol/g wet weight. Exposure of the nerve roots to cyclic GMP produced a marked increase in the incorporation of a2Pi into TTP. The TTP content underwent no constant variation. During the first 15 min, the specific activity is 5-10 times that of the control; it remains 2-3 times over the control values for 2h. The effect of c GMP was concentration-dependent. Optimal effects were obtained at concentrations from 0.01 to 1 /,M (Table II), with the TTP specific activity failing toward control levels at higher concentra-
194
ELIASSON
AND SCARPELLINI
22
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4
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2 10
20
30
40 50 60 TIME (Min)
120
FIG. 1. Effect o f c G M P and varying incubation time on the specific activity of thiamine triphosphate in cat n e r v e roots. T h e roots were left in the m e d i u m for 30 min to stop s p o n t a n e o u s firing. P r e c u r s o r was 0.5 ttCi 3~Pi in 1.25 ml K r e b s - R i n g e r bicarbonate with about 100 m g dorsal roots, c G M P was added to a concentration of 1 /xM, and did not fall below 0 . 8 / x M at 2 h. Values represent m e a n s _+sE for each group.
TABLE I EFFECTS OF INHIBITORS OF OXIDATIVE PHOSPHORYLATION ON LABELING OF TTW TTP
Addition N o n e (6) A r s e n a t e , 10-2 M (6) Dinitrophenol, 10.4 M (6)
F r o m 32Pi (10 -2 x dpm//zg) 730---6 36_+4 60_+8
F r o m ATP-7-3zP (10 -3 • dpm//zg) 210_+ 12 130_+21 95 + - 18
a N e r v e roots from cats were r e m o v e d as described in the text. A quantity of 100-300 m g of roots was preincubated in 1-3 ml K r e b s - R i n g e r bicarbonate for 30 rain, with the inhibitors added as indicated above. Either 32Pi or ATP-y-3~P (0.5/xCi) was added, and incubation c o n t i n u e d for 60 min. At the end of the e x p e r i m e n t , the roots were h o m o g e n i z e d and the h o m o g e n a t e analyzed as described in the text. N u m b e r of e x p e r i m e n t s in p a r e n t h e s e s . Values are reported as the m e a n --+SE.
cGMP AND THIAMINE
195
TRIPHOSPHATE
T A B L E II SPECIFIC ACTIVITY OF T T P AT VARIOUS CONCENTRATIONS OF c G M W Added cGMP
(dpm//~g T T P -+ SE) • 10 -3
6uM)
Roots
TST
0 0.001 0.01 0.1 1 2 10
28-+8 62-+5 140-+10 236-+21 420-+18 305-+9 153-+6
9-+4 14-+3 28-+3 35-+5 30-+8 32_+3 19-+4
a Cat n e r v e roots (60-200 mg) and thoracic sympathetic trunk (160-280 mg) were prepared and preincubated to stop s p o n t a n e o u s electrical activity. Carrier-free o r t h o p h o s p h a t e (0.5/xCi) and varying concentrations of c G M P were then added, and incubation continued for 5 min. Homogenization and a n a l y s e s as in m e t h o d section. Values are the m e a n of 8 samples-+sE.
tions. The thoracic sympathetic trunk (TST) preparation behaved similarly.
Effects of Other Nucleotides Cyclic AMP produced much less of a rise in the incorporation of 32Pi after 5 min (Table III), arid none on prolonged incubation. Guanosine-5TABLE III E F F E C T OF DIFFERENT NUCLEOTIDES ON T T P SPECIFIC ACTIVITY IN NERVE ROOTS a
Addition None G-5-MP cGMP cAMP cIMP cUMP None
C o n c e n t r a t i o n (p~M) Time o f e x p o s u r e -1 1 1 1 1 --
5' 5' 5' 5' 5' 5' 30'
T T P [(dpm-+ SE//zg) • 10-3] 28-+9 7-+3 442-+84 94-+9 40+8 61-+14 582-+85
Mean values +SE from 8 to 10 roots from cats. Isolation, preincubation, and addition of 3zp~ as in Table II. Nucleotides added as indicated. After 5 min, the roots are quick-frozen and thiamine triphosphate quantitated and radioactivity determined as described in m e t h o d s .
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monophosphate was without stimulating effect, and addition of cyclic IMP and cyclic U M P at comparable concentration did not significantly increase TTP specific activity.
Electrical Stimulation, cGMP, and TTP Metabolism A decrease in TTP content of electrically stimulated rat nerve was seen occasionally by Gurtner (4), and Itokawa and Cooper (8) proposed that ion transport associated with conduction could be related to a dephosphorylation of TDP or TTP. The uptake of 32p into TTP from nerve roots increased 10-fold with electrical stimulation (Table IV), although the content remained essentially unchanged. The uptake was stimulated to a higher degree by the addition of cGMP. A combination of c GMP addition and electrical stimulation produced no additional increase in uptake. Atropine blocked the stimulating effect of electrical impulses, but did not affect the c GMP-induced increase in phosphate uptake into TTP.
TABLE IV EFFECTS OF c G M P AND ATROPINE ON T T P LABELING IN NERVE TISSUES WITH ELECTRICAL STIMULATION a'b TTP specific activity (dpm x 10-a//xg) Preparation Nerve roots
Thoracic sympathetic trunk
Additions
Rest
Stimulated
None c G M P 1/~M Atropine 0.024 mM c G M P 1 /zM + Atropine 0.024 mM None c G M P 1 /xM Atropine 0.024 mM c G M P 1 /xM + Atropine 0.024 mM
26 431 182
242 465 92
448 9 21 10 41
405 28 41 6 17
a Stimulation parameters: r o o t s : frequency, 20/sec; impulse duration, 0.1 msec; stimulation length, 5 min; T S T : frequency, 2/sec; impulse duration, 0.5 msec; stimulation length, 5 min. b Average of 3 experiments on about 100 mg roots and sympathetic fibers from cat. After preincubation, no spontaneous activity could be recorded from the nerve preparations. One /xCi 32Pi was added, and the nerves were incubated 5 rain. with or without stimulation, in vessels containing 1.25 ml medium.
c G M P AN D T H I A M I N E T R I P H O S P H A T E
197
TABLE V EFFECT OF THEOPHYLLINE ON cGMP-STIMULATED INCORPORATION OF z2Pi INTO NERVE ROOT a'b TTP Theophylline (1 I~M)
c G M P (0.5 IxM)
Control
+
(dprn/Ng TTP) x 10.3 396+22 810-+68
(dpm//zg TTP) x 10-3 17 + 1 163-+ 10
Mean of 8 samples --+SE of cat lumbar nerve roots, isolated and preincubated for 30 rain. A quantity of 125-150 nag roots in 1.25 ml medium. Theophylline and c G M P added as indicated, together with 0.5 /xCi azPi. After 5 min incubation, the preparation is removed, frozen, and analyzed for TTP quantity and radioactivity, as described in the text.
In experiments with TST (Table IV), the stimulation increased TTP specific activity, although to a lesser degree than in nerve roots. The increased incorporation was totally blocked by atropine. The combined effect of c G M P and electrical stimulation was to raise the T T P specific activity 4-fold, but half this activity was blocked by atropine. Thus, there appears to be a greater sensitivity to atropine in a preparation with ganglion cells.
Theophylline Potentiating Effect A concentration of 1 /zM theophylline in the incubation medium increased the incorporation of 32Pi into T T P (Table V) at the same time as it decreased the TTP content. The TTP level was sometimes decreased to the limit of detectability. Addition of cyclic G M P at a level of 0.5 /xM together with the theophylline produced a very high TTP specific activity.
DISCUSSION The role of cyclic G M P in nervous tissue membrane physiology remains uncertain. Application of dibutyryl cyclic G M P produces a depolarization of the resting membrane of postganglionic neurons from the superior cervical ganglion, and high concentrations will also depolarize the cervical vagus (12). Such a depolarization could result in
198
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prolonged negative after potential with repetitive discharge, as is seen in D D T poisoning (13). Doherty and Matsumura (14) presented evidence that in the latter case the effect is mediated via microsomai proteins in the lobster nerve. If there are a number of proteins, the phosphorylation of which is associated with the control of ion permeability, among the sources of phosphate could be thiamine triphosphate, triphosphoinositide, and adenosine triphosphate. Greengard and Kebabian (15) hypothesized that acetylcholine present in ganglion activates muscarinic receptors; this activation is accompanied by an increase in cyclic GMP, which causes a transient depolarization of the neuron. Routtenberg and Ehrlich (16) found a neuronal membrane protein that is dephosphorylated after addition of cyclic GMP. Phosphorylation of smooth muscle membranes was demonstrated earlier by Casnellie and Greengard (17). Thus, it appears that there are mechanisms present that could mediate the transfer of phosphate in any direction from A T P to TTP via membrane proteins. Fox and Duppel (18) have presented some direct observations supporting the role of thiamine triphosphate in peripheral nerve function. Using voltage clamp conditions, they have shown that in the sciatic nerve of the frog, thiamine triphosphate, probably acting on the internal surface of the axonal membrane, will prevent the eventual exponential decline of the ionic current. They have also proposed as a working hypothesis that thiamine triphosphate, and possibly diphosphate, controis the number of functioning ionic channels by stabilizing the density of negative surface charges at the inner side of the membrane. The time course of cyclic G M P action is so much slower than that of action potentials (minutes as opposed to milliseconds) that one is reluctant to assign cyclic GMP, and for that matter TTP, a role in a conductile process. Promising approaches to further elucidation of thiamine triphosphate function are outlined in the work of Iwata et al. (19) on the inhibitory effect of chlorpromazine on soluble thiamine triphosphatase. The observed stimulatory effect of theophylline could be the result of an increase in endogenous c GMP, which, although small in quantity, may be very effective because of its location. Such increase in endogenous c G M P was found by Ferrendelli et al. (20) in cerebellar slices, and can be attributed to the inhibitory effect of theophylline on cyclic nucleotide phosphodiesterase. The vascular and metabolic effects of theophylline and c G M P that Fredholm described (21) do not apply in our in vitro situation, but may well operate in vivo. The theophylline-induced decrease in T T P quantity is puzzling. An anomalous inhibitory effect of theophylline combined with short periods
cGMP AND THIAMINE TRIPHOSPHATE
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o f e l e c t r i c a l s t i m u l a t i o n o n c A M P a c c u m u l a t i o n in c o r t i c a l s l i c e s w a s n o t e d b y K a k i u c h i et al. (22). F u r t h e r e x p e r i m e n t s will r e v e a l w h e t h e r c G M P a n d t h e o p h y l l i n e t o g e t h e r c a u s e s p o n t a n e o u s firing o f t h e n e r v e and accompanying depletion of stored TTP. I n t h e T S T p r e p a r a t i o n , a n d to a l e s s e r e x t e n t in t h e r o o t p r e p a r a t i o n , t h e p r e s e n c e o f g a n g l i o n cells m a y e x p l a i n t h e a t r o p i n e s e n s i t i v i t y , b e c a u s e it is r e c o g n i z e d t h a t c G M P m a y m e d i a t e t h e p o s t g a n g l i o n i c a c t i v a t i o n at m u s c a r i n i c c h o l i n e r g i c s y n a p s e s (23).
ACKNOWLEDGMENTS W e t h a n k M s . G e n e v a B a n k s f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . Dr. J a m e s F e r r e n d e l l i d e t e r m i n e d t h e l e v e l s o f c G M P in r o o t a n d s y m p a t h e t i c n e r v e t i s s u e . T h i s p r o j e c t w a s s u p p o r t e d b y N I N D S G r a n t 10019.
REFERENCES 1. COOPER,J.R., and PINCUS, J.H. (1967) The role of thiamine in nerve conduction. Ciba Found. Study Group 28, 112-121. 2. ITOKAWA,Y., and COOPER, J.R. (1970) Ion movements and thiamine. II. The release of the vitamin from membrane fragments. Biochim. Biophys. Acta 196, 274-284. 3. EICHENBAUM, J.W., and COOPER, J.R. (1971) Restoration by thiamine of the action potential in ultraviolet irradiated nerve. Brain Res. 32,258-260. 4. GURTNER, H.P. VON. (1961) Aneurin und Nervenerregung Versuche mit z~S-markiertern Aneurin und Aneurinan-timetaboliten. Heir. Physiol. Acta Suppl. XI, 1-47. 5. BEaMAN, K., and FISHMAN,R.A. (1975) Thiamine phosphate metabolism mad possible coenzyme-independent function of thiamine in brain. J. Neurochem. 24,457--465. 6. FERRENDELLI,J.A., STEINER, A.L., McDOUGAL, D.B., JR., and KIPNIS, D.M. (1970) Effect of oxotermorine and atropine on CNS cyclic nucleotides. Biochem. Biophys. Res. Commun. 41, 1061-1067. 7. LUST, W.D., and PASSONNEAU, J.V. (1973) Infuence of certain drugs on cyclic nucleotide levels in mouse brain following electro-convulsive shock. Abstr. Am. Soc. Neurochem. 4, 115. 8. ITOKAWA,Y., and COOPER, J.R. (1970) Electrophoretic separation and fluorometric determination of thiamine and its phosphate esters. Methods Enzymol. 18A, 91, 92. 9. BRAY, G.A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1,279-285. 10. LOWRY, O.H., and PASSONNEAU,J.V. (1972) In A Flexible System of Enzymatic Analysis, Academic Press, New York, pp. 151-155. 11. ELIASSON,S.G., BANKS, G., and SCARPELLINI,J.D. Changes in thiamine phosphates and phosphoinositides during electrical stimulation of peripheral nerves (submitted for publication). 12. MCAFEE, D.A., and GREENGARD,P. (1972) Adenosine 3',5'-monophosphate: electrophysiological evidence for a role in synaptic transmission. Science 178, 310--312.
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13. VAN DEN BERCKEN, J. (1972) The effect of DDT and Dieldrin on myelinated nerve fibres. Eur. J. Pharmacol. 20,205-214. 14. ~)OHERTY,J.D., and MATSUMURA,F. (1974) DDT effect on 3~p incorporation from ylabeled ATP into proteins from lobster nerves. J. Neurochem. 22,765-772. 15. GREENGARD, P., and KEBABIAN, J.W. (1974) Role of cyclic AMP in synaptic transmission in the mammalian peripheral nervous system. Fed. Proc. Fed. Am. Soc. Exp. Biol. 33, 1059-1067. 16. ROUTTENBERG,A., and ErlRLICH, Y.H. (1975) Endogenous phosphorylation of four cerebral cortical membrane proteins: role of cyclic nucleotides-, ATP and divalent cations. Brain Res. 92,415-430. 17. CASNELLIE,J,E., and GREENGARD, P. (1974) Guanosine 3',5'-cyclic monophosphatedependent phosphorylation of endogenous substrate proteins in membranes of mammalian smooth muscle. Proc. Natl. Acad. Sci. U.S.A. 71, 1891-1895. 18. Fox, J.M., and DUPPEL, W. (1975) The action of thiamine and its di- and triphosphates on the slow exponential decline of the ionic currents in the node of Ranvier. Brain Res. 89,287-302. 19. IWATA, H., BABA, A., MATSUDA,T., and TERASHITA,Z. (1975) Properties of thiamine di- and triphosphates in rat brain microsomes: effects of chlorpromazine. J. Neurochem. 24, 1209-1213. 20. FERRENDELLI, J.A., KINSCHERF, D.A., and CHANG, M.M. (1973) Regulation of levels of guanosine cyclic 3',5'-monophosphate in the central nervous system: effects of depolarizing agents. Mol. Pharmacol. 9, 445-454. 21. FREI~HOLM, B.B. (1974) Vascular and metabolic effects of theophylline, dibutyryl cyclic AMP and dibutyryl cyclic GMP in canine subcutaneous adipose tissue in situ. Acta Physiol. Scand. 90, 226-236. 22. KAKIUCHI, S., RALL, R.W., and MCILWAIN, H. (1969) The effect of electrical stimulation upon the accumulation of adenosine 3',5'-phosphate in isolated cerebral tissue. J. Neurochem. 16, 485-491. 23. WEIGHT, F.F., PETZOLD, G., and GREENGARD, P. (1974) Guanosine 3',5'-monophosphate in sympathetic ganglia: increase associated with synaptic transmission. Science 186, 942-944.
S T I M U L A T I O N OF THIAMINE TRIPHOSPHATE METABOLISM IN N E R V E BY C Y C L I C GUANOSINE 3',5'-MONOPHOSPHATE SVEN G. ELIASSON AND JOANNE D. SCARPELLINI Department of Neurology and Neurological Surgery (Neurology) Washington University School of Medicine St, Louis, Missouri 63110
Accepted February 9, 1976
This paper reports that addition of c G M P results in an increase of the amount of 32p~ incorporated into thiamine triphosphate in nerve roots and sympathetic trunks. The effect is present both at rest and during electrical stimulation. Other nucleotides were less effective. Theophylline increased the incorporation, possibly because of phosphodiesterase inhibition. A blocking effect of atropine was noted in sympathetic trunk preparations that contain ganglion cells.
INTRODUCTION Acetylcholine, tetrodotoxin, and electrical stimulation initiate release of thiamine from nerve and cord preparations, and thiamine reverses the ultraviolet light-induced block of nerve impulses (1-3). Simultaneously with the thiamine release, a decrease in thiamine polyphosphates was noted by Gurtner (4) in 4 out of 14 rats. Berman and Fishman (5) were able to show only minor changes in the distribution of thiamine and its phosphate esters on electrical stimulation or K + depolarization of rat cerebral cortex. Rapid turnover of the thiamine triphosphate pool did take place when 32Pi was used as precursor. Also associated with application of cholinergic stimuli, electrical stimuli, and seizures are increases in cyclic guanosine 3',5'-monophosphate (cGMP) (6,7). We 191 O 1976 Plenum Publishing Corporation, 227 West 17tb Street. New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.
192
ELIASSON AND SCARPELLINI
elected to explore the possibility of interaction between exogenous c GMP and thiamine triphosphate levels and metabolism. T h i s p a p e r r e p o r t s t h a t a d d i t i o n o f c G M P i n c r e a s e s t h e a m o u n t o f ~zp incorporated into thiamine triphosphate (TTP) in nerve tissue at rest and during stimulation. Some of the characteristics of TTP metabolism under different conditions are described.
EXPERIMENTAL
PROCEDURE
Materials. Thiamine triphosphate was obtained as a gift from Sankyo Company, Japan; other thiamine compounds were obtained from Sigma Chemicals. Carrier-free orthophosphoric acid and adenosine-5'-triphosphate-7-~-~P, diammonium salt (sp act 5-30 Ci/mM), were purchased from I C N Pharmaceuticals. Tissue Preparation. In these experiments, adult cats were anesthetized; a thoracotomy and laminectomy were performed, and the dorsal and ventral roots, as well as the thoracic sympathetic trunk, were removed. The nervous tissue was preincubated in Krebs-Ringer bicarbonate buffer, pH 7.4, at 37~ in a Dubnoff shaker for 30 min, after which the various experimental conditions were introduced. Preincubation is required to eliminate spontaneous firing in nerve fibers. The tissue was removed, and quickly frozen with liquid nitrogen. The tissue was weighed frozen, then minced and placed in fresh buffer and, to eliminate excess or newly formed ATP-3zP, incubated with excess glucose and hexokinase. Cold 7% perchloric acid was added for 30 min. The volume of acid was adjusted to give a l:I0 homogenate. All homogenizations were carried out in a glass (TenBroeck) homegenizer. The sample was centrifuged and the supernatant was kept cold until removed and neutralized with the addition of saturated potassium carbonate solution. In control experiments, no losses of TTP were noted. Determination of Thiamine Phosphates. The method used for quantization is a slight modification of that given by Itokawa and Cooper (8). After neutralization, the homogenate was subjected to electrophoresis in a Beckman microzone system. The membranes were wetted (not soaked) with 0.05 M acetate buffer at pH 3.8. Sartorius cellulose acetate membranes were used. Strips were spotted with tissue extracts and standards, and electrophoresis was carried out at 8 mA constant current for 9 min. The membrane was then dried in warm air without being removed from the suspension bridge. After the membrane was dried, it was sprayed with a mixture of 30 ml water, 0.6 ml 2% potassium ferricyanide, and 7 ml 15% sodium hydroxide. Spots were visualized with 360 nm U V light, cut out, and placed in fluorometer tubes along with sections of the membrane from blank areas. To the tubes were added 1 ml potassium ferricyanide spray reagent; 289 minutes later, 10/xl 30% hydrogen peroxide were added, and the tubes were gently shaken. The tubes were read 30 min later, using primary filters at 365 nm and secondary filters of 430 nm maximum transmission. All reagents have to be kept fresh and at room temperature for an hour before being read. Tubes must be sized and matched and calibrated with quinine solution. Sensitivity of assay 0.3 rig. Separation of TTP, T D P , TMP, and thiamine is accomplished more rapidly on the Sartorius membrane; the distances between the compounds are the same as documented by Itokawa and Cooper (8). Labeling of Thiamine Phosphates and ATP Analysis. Labeled thiamine phosphates were counted in 10 ml Bray's solution (9) after separation. Several experiments were done to
cGMP AND THIAMINE TRIPHOSPHATE
193
exclude contamination with residual labeled 32pi, The inorganic phosphate travels much faster than any of the assayed compounds and does not contaminate the phosphates. The ATP-T-a2P stays at the origin; after treatment with glucose-hexokinase, some ADP-3~P, which is probably a contaminant, can be located between TDP and TMP. Quench corrections were applied, using external standardization. Measurement of ATP was done according to the method by Lowry and Passonnean (10); the quantity obtained ranged from 1.2 to 3.0 tzmol/g dry weight. The amount of tracer added was equal to or less than 0.02% of the endogenous amount. Stimulation Experiments. Preparations were monitored for action potentials with suction electrodes connected to a Tektronix oscilloscope No. 565. For stimulation, a Grass $8 stimulator was used. Frequency and duration of the impulses are given in connection with the individual experiments. Resting and stimulated preparations were incubated together.
RESULTS
Incorporation of 32p Into Endogenous TTP Levels of TTP in cat nerve roots and thoracic sympathetic trunk immediately after removal were variable. Incubation of the nerve material in Krebs-Ringer buffer for 30 rain stabilized the level at 10-20 /,g/g dry weight in roots and 5-10 /zg/g dry weight in the sympathetic trunk. These levels remained stable under resting conditions during continued incubation up to 240 rain. Addition of 32Pi to the incubating roots after the preincubation resulted in rapid uptake of 32p into TTP during the first hour (Fig. 1), after which the rate leveled off over the next 3 h. The amount of TTP/g dry weight remains unchanged. When ATP-T-z2P was used, it appeared to a considerable extent to be hydrolyzed and the inorganic phosphate to be taken up by the nerve. Addition of dinitrophenol or arsenate depressed incorporation of 32Pi more than of ATP-T-a2p (Table I) (11).
Effect of cGMP on TTP Content and Radioactivity The endogenous content of c GMP in roots was determined for us by Dr. James Ferrendelli; it ranged from 20-60 pmol/g wet weight. Exposure of the nerve roots to cyclic GMP produced a marked increase in the incorporation of a2Pi into TTP. The TTP content underwent no constant variation. During the first 15 min, the specific activity is 5-10 times that of the control; it remains 2-3 times over the control values for 2h. The effect of c GMP was concentration-dependent. Optimal effects were obtained at concentrations from 0.01 to 1 /,M (Table II), with the TTP specific activity failing toward control levels at higher concentra-
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ELIASSON
AND SCARPELLINI
22
)
-~ 2O
~16 ~
'~ 14i I
4
,}/' /1 /
2 10
20
30
40 50 60 TIME (Min)
120
FIG. 1. Effect o f c G M P and varying incubation time on the specific activity of thiamine triphosphate in cat n e r v e roots. T h e roots were left in the m e d i u m for 30 min to stop s p o n t a n e o u s firing. P r e c u r s o r was 0.5 ttCi 3~Pi in 1.25 ml K r e b s - R i n g e r bicarbonate with about 100 m g dorsal roots, c G M P was added to a concentration of 1 /xM, and did not fall below 0 . 8 / x M at 2 h. Values represent m e a n s _+sE for each group.
TABLE I EFFECTS OF INHIBITORS OF OXIDATIVE PHOSPHORYLATION ON LABELING OF TTW TTP
Addition N o n e (6) A r s e n a t e , 10-2 M (6) Dinitrophenol, 10.4 M (6)
F r o m 32Pi (10 -2 x dpm//zg) 730---6 36_+4 60_+8
F r o m ATP-7-3zP (10 -3 • dpm//zg) 210_+ 12 130_+21 95 + - 18
a N e r v e roots from cats were r e m o v e d as described in the text. A quantity of 100-300 m g of roots was preincubated in 1-3 ml K r e b s - R i n g e r bicarbonate for 30 rain, with the inhibitors added as indicated above. Either 32Pi or ATP-y-3~P (0.5/xCi) was added, and incubation c o n t i n u e d for 60 min. At the end of the e x p e r i m e n t , the roots were h o m o g e n i z e d and the h o m o g e n a t e analyzed as described in the text. N u m b e r of e x p e r i m e n t s in p a r e n t h e s e s . Values are reported as the m e a n --+SE.
cGMP AND THIAMINE
195
TRIPHOSPHATE
T A B L E II SPECIFIC ACTIVITY OF T T P AT VARIOUS CONCENTRATIONS OF c G M W Added cGMP
(dpm//~g T T P -+ SE) • 10 -3
6uM)
Roots
TST
0 0.001 0.01 0.1 1 2 10
28-+8 62-+5 140-+10 236-+21 420-+18 305-+9 153-+6
9-+4 14-+3 28-+3 35-+5 30-+8 32_+3 19-+4
a Cat n e r v e roots (60-200 mg) and thoracic sympathetic trunk (160-280 mg) were prepared and preincubated to stop s p o n t a n e o u s electrical activity. Carrier-free o r t h o p h o s p h a t e (0.5/xCi) and varying concentrations of c G M P were then added, and incubation continued for 5 min. Homogenization and a n a l y s e s as in m e t h o d section. Values are the m e a n of 8 samples-+sE.
tions. The thoracic sympathetic trunk (TST) preparation behaved similarly.
Effects of Other Nucleotides Cyclic AMP produced much less of a rise in the incorporation of 32Pi after 5 min (Table III), arid none on prolonged incubation. Guanosine-5TABLE III E F F E C T OF DIFFERENT NUCLEOTIDES ON T T P SPECIFIC ACTIVITY IN NERVE ROOTS a
Addition None G-5-MP cGMP cAMP cIMP cUMP None
C o n c e n t r a t i o n (p~M) Time o f e x p o s u r e -1 1 1 1 1 --
5' 5' 5' 5' 5' 5' 30'
T T P [(dpm-+ SE//zg) • 10-3] 28-+9 7-+3 442-+84 94-+9 40+8 61-+14 582-+85
Mean values +SE from 8 to 10 roots from cats. Isolation, preincubation, and addition of 3zp~ as in Table II. Nucleotides added as indicated. After 5 min, the roots are quick-frozen and thiamine triphosphate quantitated and radioactivity determined as described in m e t h o d s .
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ELIASSON AND SCARPELLINI
monophosphate was without stimulating effect, and addition of cyclic IMP and cyclic U M P at comparable concentration did not significantly increase TTP specific activity.
Electrical Stimulation, cGMP, and TTP Metabolism A decrease in TTP content of electrically stimulated rat nerve was seen occasionally by Gurtner (4), and Itokawa and Cooper (8) proposed that ion transport associated with conduction could be related to a dephosphorylation of TDP or TTP. The uptake of 32p into TTP from nerve roots increased 10-fold with electrical stimulation (Table IV), although the content remained essentially unchanged. The uptake was stimulated to a higher degree by the addition of cGMP. A combination of c GMP addition and electrical stimulation produced no additional increase in uptake. Atropine blocked the stimulating effect of electrical impulses, but did not affect the c GMP-induced increase in phosphate uptake into TTP.
TABLE IV EFFECTS OF c G M P AND ATROPINE ON T T P LABELING IN NERVE TISSUES WITH ELECTRICAL STIMULATION a'b TTP specific activity (dpm x 10-a//xg) Preparation Nerve roots
Thoracic sympathetic trunk
Additions
Rest
Stimulated
None c G M P 1/~M Atropine 0.024 mM c G M P 1 /zM + Atropine 0.024 mM None c G M P 1 /xM Atropine 0.024 mM c G M P 1 /xM + Atropine 0.024 mM
26 431 182
242 465 92
448 9 21 10 41
405 28 41 6 17
a Stimulation parameters: r o o t s : frequency, 20/sec; impulse duration, 0.1 msec; stimulation length, 5 min; T S T : frequency, 2/sec; impulse duration, 0.5 msec; stimulation length, 5 min. b Average of 3 experiments on about 100 mg roots and sympathetic fibers from cat. After preincubation, no spontaneous activity could be recorded from the nerve preparations. One /xCi 32Pi was added, and the nerves were incubated 5 rain. with or without stimulation, in vessels containing 1.25 ml medium.
c G M P AN D T H I A M I N E T R I P H O S P H A T E
197
TABLE V EFFECT OF THEOPHYLLINE ON cGMP-STIMULATED INCORPORATION OF z2Pi INTO NERVE ROOT a'b TTP Theophylline (1 I~M)
c G M P (0.5 IxM)
Control
+
(dprn/Ng TTP) x 10.3 396+22 810-+68
(dpm//zg TTP) x 10-3 17 + 1 163-+ 10
Mean of 8 samples --+SE of cat lumbar nerve roots, isolated and preincubated for 30 rain. A quantity of 125-150 nag roots in 1.25 ml medium. Theophylline and c G M P added as indicated, together with 0.5 /xCi azPi. After 5 min incubation, the preparation is removed, frozen, and analyzed for TTP quantity and radioactivity, as described in the text.
In experiments with TST (Table IV), the stimulation increased TTP specific activity, although to a lesser degree than in nerve roots. The increased incorporation was totally blocked by atropine. The combined effect of c G M P and electrical stimulation was to raise the T T P specific activity 4-fold, but half this activity was blocked by atropine. Thus, there appears to be a greater sensitivity to atropine in a preparation with ganglion cells.
Theophylline Potentiating Effect A concentration of 1 /zM theophylline in the incubation medium increased the incorporation of 32Pi into T T P (Table V) at the same time as it decreased the TTP content. The TTP level was sometimes decreased to the limit of detectability. Addition of cyclic G M P at a level of 0.5 /xM together with the theophylline produced a very high TTP specific activity.
DISCUSSION The role of cyclic G M P in nervous tissue membrane physiology remains uncertain. Application of dibutyryl cyclic G M P produces a depolarization of the resting membrane of postganglionic neurons from the superior cervical ganglion, and high concentrations will also depolarize the cervical vagus (12). Such a depolarization could result in
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prolonged negative after potential with repetitive discharge, as is seen in D D T poisoning (13). Doherty and Matsumura (14) presented evidence that in the latter case the effect is mediated via microsomai proteins in the lobster nerve. If there are a number of proteins, the phosphorylation of which is associated with the control of ion permeability, among the sources of phosphate could be thiamine triphosphate, triphosphoinositide, and adenosine triphosphate. Greengard and Kebabian (15) hypothesized that acetylcholine present in ganglion activates muscarinic receptors; this activation is accompanied by an increase in cyclic GMP, which causes a transient depolarization of the neuron. Routtenberg and Ehrlich (16) found a neuronal membrane protein that is dephosphorylated after addition of cyclic GMP. Phosphorylation of smooth muscle membranes was demonstrated earlier by Casnellie and Greengard (17). Thus, it appears that there are mechanisms present that could mediate the transfer of phosphate in any direction from A T P to TTP via membrane proteins. Fox and Duppel (18) have presented some direct observations supporting the role of thiamine triphosphate in peripheral nerve function. Using voltage clamp conditions, they have shown that in the sciatic nerve of the frog, thiamine triphosphate, probably acting on the internal surface of the axonal membrane, will prevent the eventual exponential decline of the ionic current. They have also proposed as a working hypothesis that thiamine triphosphate, and possibly diphosphate, controis the number of functioning ionic channels by stabilizing the density of negative surface charges at the inner side of the membrane. The time course of cyclic G M P action is so much slower than that of action potentials (minutes as opposed to milliseconds) that one is reluctant to assign cyclic GMP, and for that matter TTP, a role in a conductile process. Promising approaches to further elucidation of thiamine triphosphate function are outlined in the work of Iwata et al. (19) on the inhibitory effect of chlorpromazine on soluble thiamine triphosphatase. The observed stimulatory effect of theophylline could be the result of an increase in endogenous c GMP, which, although small in quantity, may be very effective because of its location. Such increase in endogenous c G M P was found by Ferrendelli et al. (20) in cerebellar slices, and can be attributed to the inhibitory effect of theophylline on cyclic nucleotide phosphodiesterase. The vascular and metabolic effects of theophylline and c G M P that Fredholm described (21) do not apply in our in vitro situation, but may well operate in vivo. The theophylline-induced decrease in T T P quantity is puzzling. An anomalous inhibitory effect of theophylline combined with short periods
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199
o f e l e c t r i c a l s t i m u l a t i o n o n c A M P a c c u m u l a t i o n in c o r t i c a l s l i c e s w a s n o t e d b y K a k i u c h i et al. (22). F u r t h e r e x p e r i m e n t s will r e v e a l w h e t h e r c G M P a n d t h e o p h y l l i n e t o g e t h e r c a u s e s p o n t a n e o u s firing o f t h e n e r v e and accompanying depletion of stored TTP. I n t h e T S T p r e p a r a t i o n , a n d to a l e s s e r e x t e n t in t h e r o o t p r e p a r a t i o n , t h e p r e s e n c e o f g a n g l i o n cells m a y e x p l a i n t h e a t r o p i n e s e n s i t i v i t y , b e c a u s e it is r e c o g n i z e d t h a t c G M P m a y m e d i a t e t h e p o s t g a n g l i o n i c a c t i v a t i o n at m u s c a r i n i c c h o l i n e r g i c s y n a p s e s (23).
ACKNOWLEDGMENTS W e t h a n k M s . G e n e v a B a n k s f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . Dr. J a m e s F e r r e n d e l l i d e t e r m i n e d t h e l e v e l s o f c G M P in r o o t a n d s y m p a t h e t i c n e r v e t i s s u e . T h i s p r o j e c t w a s s u p p o r t e d b y N I N D S G r a n t 10019.
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13. VAN DEN BERCKEN, J. (1972) The effect of DDT and Dieldrin on myelinated nerve fibres. Eur. J. Pharmacol. 20,205-214. 14. ~)OHERTY,J.D., and MATSUMURA,F. (1974) DDT effect on 3~p incorporation from ylabeled ATP into proteins from lobster nerves. J. Neurochem. 22,765-772. 15. GREENGARD, P., and KEBABIAN, J.W. (1974) Role of cyclic AMP in synaptic transmission in the mammalian peripheral nervous system. Fed. Proc. Fed. Am. Soc. Exp. Biol. 33, 1059-1067. 16. ROUTTENBERG,A., and ErlRLICH, Y.H. (1975) Endogenous phosphorylation of four cerebral cortical membrane proteins: role of cyclic nucleotides-, ATP and divalent cations. Brain Res. 92,415-430. 17. CASNELLIE,J,E., and GREENGARD, P. (1974) Guanosine 3',5'-cyclic monophosphatedependent phosphorylation of endogenous substrate proteins in membranes of mammalian smooth muscle. Proc. Natl. Acad. Sci. U.S.A. 71, 1891-1895. 18. Fox, J.M., and DUPPEL, W. (1975) The action of thiamine and its di- and triphosphates on the slow exponential decline of the ionic currents in the node of Ranvier. Brain Res. 89,287-302. 19. IWATA, H., BABA, A., MATSUDA,T., and TERASHITA,Z. (1975) Properties of thiamine di- and triphosphates in rat brain microsomes: effects of chlorpromazine. J. Neurochem. 24, 1209-1213. 20. FERRENDELLI, J.A., KINSCHERF, D.A., and CHANG, M.M. (1973) Regulation of levels of guanosine cyclic 3',5'-monophosphate in the central nervous system: effects of depolarizing agents. Mol. Pharmacol. 9, 445-454. 21. FREI~HOLM, B.B. (1974) Vascular and metabolic effects of theophylline, dibutyryl cyclic AMP and dibutyryl cyclic GMP in canine subcutaneous adipose tissue in situ. Acta Physiol. Scand. 90, 226-236. 22. KAKIUCHI, S., RALL, R.W., and MCILWAIN, H. (1969) The effect of electrical stimulation upon the accumulation of adenosine 3',5'-phosphate in isolated cerebral tissue. J. Neurochem. 16, 485-491. 23. WEIGHT, F.F., PETZOLD, G., and GREENGARD, P. (1974) Guanosine 3',5'-monophosphate in sympathetic ganglia: increase associated with synaptic transmission. Science 186, 942-944.
