The action of thiamine and its di- and triphosphates on the slow exponential decline of the ionic currents in the node of Ranvier

Brain Research, 89 (1975) 287-302 ,c~ Elsewer SclenUfic P u b h s h m g Company, Amsterdam - Plinted m The Netherlands

287

T H E A C T I O N OF T H I A M I N E A N D ITS DI- A N D T R I P H O S P H A T E S ON T H E SLOW E X P O N E N T I A L D E C L I N E OF T H E I O N I C C U R R E N T S IN T H E N O D E OF R A N V I E R

J M. FOX AND W D U P P E L

I. Physlologtsches Institut und lnsntut fur Ph)'stologtsche Chemw der Umversltdt de~ Saarlande~, 665 Homburg/Saar (G.F.R ) (Accepted December 15th, 1974)

SUMMARY

Sodium and potassium currents in the node of Ranwer decrease exponenUally with time during long lasting voltage clamp experiments. This dechne is strongly dependent on temperature (Q10 approximately 3). Thiamine and, parucularly, its dland triphosphoric acid esters are shown to prevent this exponential decline of the ~omc currents. Thiamine acts from the outside and from the inside of the nodal membrane, but more potently from the inside. Thiamine diphosphate prevents the exponential dechne of the iomc currents only when applied internally. Thiamine m p h o s p h a t e , the most effective thiamine derivaUve was tested from the inside onl) Bacterial th~aminases apphed externally were not effecUve, presumably because they do not permeate the nodal membrane. Tetrodotoxm, that has been shown by other investigators to induce a release of thiamine from nerve membranes, does not alter the action of thiamine on the exponential decline of currents and vice versa. It ~s concluded that: (1) thiamine dlphosphate or thiamine trlphosphate are the active thiamine compounds in nerve membranes; (2) the site of action is located at the internal surface of the membrane: (3) the reduction of the thiamine concentration m the membrane or in the axoplasm could cause the exponential decline of currents; (4) the release of thiamine from nerve membranes induced by tetrodotoxln is interpreted as a side effect not even related to the mechanism by which tetrodotoxm blocks the sodium channels; (5) thiamine polyphosphates appear to stablhse the intrinsic electric field strength of the nodal membrane in the resting state. Therefore, as a working hypoth-

288 esl~, ~t is suggested that the thiamine der~vatwes control the number of functmnmg lOmC channels by stabfllsmg the denstty of negative surface charges at the tuner ,ide of the nerve membrane.

INTRODUCTION

It ~s generally agreed that the ionic currents in nerve fibres spontaneouslly decrease during an experiment. This 'run-down' of the currents has been attributed to a deficiency of the energy sources in an ~solated preparation. In a recent paper ~', however, it was shown for the node of Ranvier that this decrease of lomc currents depends strongly on the potential at which the nodal membrane is held m ~oltageclamp. It was suggested that this decline of the currents was caused by slow changes m the elecmc field across the membrane due to variations in the density of surtace charges l-' In a communication to this journal 10 it was demonstrated that thmmme does not restore the nodal sodmm currents ~rreverslbly blocked by ultrawolet radmtion. During these prewous experiments a beneficial effect of thiamine reducing the "rundown' of the sodmm and potassium currents was observed. Thiamine and ~ts phosphoric amd esters are known to play an important role m various functmns of nervous tissue~7,25,as, 36,45, though the mechanisms remain obscure. There are two interpretations (1) In several steps of the carbohydrate metabolism thiamine dlphosphate participates as coenzyme. Since the energy for the nervous tissue originates mainly in the metabohsm of carbohydrates, thiamine particularly is essential for excitationS5,27,45 (2) Thiamine or its phosphate esters may be directly linked to the exc~tatmn process. A number of observations support this view, but the mechanism of th~s special actmn of thiamine has not yet been clarified 3,<17,e~,zl,z6 We present here further results on the problems of long-term decline of the runic currents, and report observations which clearly show that thiamine and ~ts d> and tr~phosphate esters apphed intracellularly prevent this deterioration of the mnlc currents m the node of Ranvler. METHODS

Measurement o f currents Motor and sensory fibres isolated from the sciatic nerve of the frog Rana esculenta were investigated under current- and voltage-clamp conditions using the technique developed by Nonner zg. At the beginning of each experiment the Ringer's solution in the side pools (pools C and E) was replaced by isotonic KC1 solution or by an artlficml axoplasm (see below). Then the internodes on either s~de of the node under investigation were cut near the neighbourmg nodes (m pools C and E).

289 The holding potential was set to approximately resting potential (no negative after potential in current-clamp; h~ = 0.65-0.70 in voltage-clamp). The basehne of current at holding potential was continuously recorded with an ink writer. After an tmtial stabilisatton period of about 15 min the DC-balance of the voltage-clamp system and the holding potential did not require readjustments Afterwards, the drift of the holding current did not exceed 0.2 nA in good experiments, corresponding to approximately 3 mV change in holding potential. (The calibration was achieved by apphcation of a test change of the holding potential.) The experiments were performed on-hne with a processing computer (Honeywell DDP-516) which was used to sample and digltalise the analogue signals from the output of the voltage-clamp amplifier w~th the aid of an analogue to digital converter (Raytheon, resolution 10 bit; aperture time 100 nsec; throughput rate 50 kc/sec; accuracy 0.1 ~ of full range). Adequate sequences of voltage pulses were apphed to the preparation from the computer using a digital to analogue converter (Honeywell, resolution 10 b~t; accuracy j : 0.1 ~/o; setthng time 8 #sec) The experimental programs were written in a special macroassembler language 4° prowdmg the necessary instructions for measurements under real time conditions and for digital data processing. The digltalised ionic currents were corrected for leakage and for offset currents of the recording system (both separately determined at certain intervals) and were stored on digital magnetic tape.

Nomenclature To describe the Ionic currents, the formahsm of Frankenhaeuser and Huxley a3 was used. Potentials are referred to the external solution and are given as V -E - - Er. 'Hyperpolarlsation' and 'depolarisatlon' have their usual meaning, that is (in the V-scale) a negative or a positive membrane potential, respectively. Inward currents, consequently, are negative. For comparison with earher work the iomc currents were cahbrated as current densities as described elsewhere 11. These current values have to be regarded as rough estimates of the true current densities in the nodal membrane. Solutions The Ringer's solution contained I l0 m M NaCl, 2.5 m M KCI, 1 8 m M CaCl2 and 5 m M Trls (hydroxymethyl)-aminomethane-HC1 buffered at p H 7.3 or p H 6.8. The latter value was used for solutions containing thiamine or thiamine diphosphate in order to prevent chemical changes of the molecules at alkaline pH values. The artificial axoplasm solution being in contact with the cut ends of the fibre contained 103 m M KC1, 10 m M NaC1, and 5 m M Na2HPO4-KHzPO4 (1:1) buffer (pH 6.88) Isotomc K C I (also used in contact with the axoplasm at the cut ends) contained 117 m M KCI. Thiamine, thiamine dlphosphate or thiamine triphosphate (2 and 5 m M ) were added to the Ringer's solution or to the artificial axoplasm solutions before adding NaCl. The p H was adjusted to 6 8 with N a O H and then NaCl was added to a final Na + concentration of 110 m M or 15 raM, respectwely.

290

C]lenllca[,~ :: Thiamine and thmmme diphosphate samples were kindly provided by HoffnmnnLa Roche AG, Basel. A sample of thiamine trlphosphate was donated b) Sank)o L t d , Tokyo. Bacterial thmminases were prepared from cultures of Bacillus thiaminolvttcu,s, A T C C 11376 (thlaminase I) and of Bacdlus aneurinolyttcu,s A T C C 12856 (thmmmase 1I) according to the methods given by Wittliff and Airth ~8,49. The cells were removed from the culture and the thiammases were precipitated w~th ammonium sulphate at 2 °C and p H 7 The precipitate was dissolved m distilled water and dmlysed twice against Ringer's solution or against ~sotomc KC1 solutmn during 8 h. After the fir,~t dmlysls the thiammase solutions contained a few m M of Ca 2+ as checked with the md of a Ca '~~-sensmve electrode After the second dialys~s the Ca '-'~ concentration was smaller than 0.01 m M Further purzficatxon of the thmmmases was not attempted The final concentratmn ol protein was determined to be around 8.7 mg/ml (u~mg ~, method described by Got'nail et al.~5). The activity of the enz~me.~ a~, determined b~ the thmchrome assay according to Awth and Foerster t was 6.3 nmole/mg of protein (mean of 5 cultures) Thmnunase 1 catalyses the d e c o m p o s m o n of thmmme by a base exchange reaction involving a nucleophilic displacement on the methylene group of the pyrtmidme moiety. It l~ specltic for thmmme, ThDP, and other derwatJves with the 4amino group of the pyrHmdme moiety intact. It utdlses pyrithlamine but not oxlth~amine 4s. The molecular we@at is about 42,000. Thlammase 11 catalyseb the hydrolysis of thiamine and thiamine derivatives with the side chain intact at the 5-position of the thmzole moiety. It does not h~drolyse T h D P 49. The molecular weight is approximately 100,000. RESULTS

Effects of thiamine and tt.~ di- and triphosphoric acid esters The continuous decrease of the sodium and potassium currents through the nodal membrane can be described by an exponential function with time (Fig I). In additlom the continuous decrease of the steady state sodmm inactivation hoo (V = 0) observed during long lasting experiments follows also an exponentml time course (Fig. 1). The sodium mactivaUon h~ (V -- 0) was determined as the ratio of the sodium current measured w~th a test pulse of 60 mV starting from V = 0 to the current measured with the same test pulse preceded by a condit~omng pulse of 50 msec duration and - - 4 0 mV amphtude, which removed sodium inactlvatmn. The slow decrease m h~ (V ::- 0) ~s due to a permanent shift of the h~ (V) curve along the voltage axis m the negative direction m spite of a constant holding potential 12 Fig. 2 dlustrates the effect of Internal apphcation of thiamine on the exponentml decrease of INa and IK. The solution in the side pools was replaced by another of the * Abbreviations. ThMP thiamine monophosphate, ThDP thiamine triphosphate, TTX tetrodotoxm

thiamine dlphosphate. ThTP

291

[K

[mA/cm

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2O 0

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-~

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T

o__

[m,n

]

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-60 -80 INO [mA/cm

2]

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±

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"--¢,. . . . . . T

T

0.2 C)

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, 20

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i h-0

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, 60

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8J0

, T [m,.]

Fig 1 Exponential decrease with time of the nodal sodium and potassium currents, IN~ and I~, and of the steady state sodium inactivation h~ (V -- 0) possibly related to the density of internal membrane surface charges 12. The experimental points represent the mean values of 5 experiments (3 motor and 2 sensory nerve fibres). The current values were normahsed to the starting points (INa --105 mA/sq cm and IK -- 52 mA/sq.cm) before averaging; the hoo-values were directly averaged The vertical bars indicate the standard deviation from the mean (no vertical bar indicates that the standard deviation is smaller than the symbol) An exponential function of the type I 10" exp (--t/O) was fitted to the data (dashedhnes). O(1N~) 596min, O(IK) -- 696 mm, 6) (h~) 73 8 rain. Test pulses 60 mV and 120 mV for IN,~and 1K, respectively, condltmnlng prepulse. - 4 0 mV, 50 msec All voltages refer to V -- 0 (see Methods). Temperature 14 C -

-

same c o m p o s i t i o n b u t c o n t a i n i n g a d d i t i o n a l l y 2 m M thiamine, which thus could diffuse f r o m b o t h sides a l o n g the a x o p l a s m t o w a r d s the test n o d e ~9 The u p p e r p a r t o f the figure shows the p e a k s o d i u m c u r r e n t (h~o -----1) at a test voltage step to V = 60 mV (see M e t h o d s ) a n d the steady state p o t a s s m m c u r r e n t at a test pulse o f V = 120 mV p l o t t e d v e r s u s time. The lower g r a p h represents the time course o f the steady state s o d i u m i n a c t i v a t i o n hoo (V = 0). A n e x p o n e n t i a l decrease with time o f the 3 variables (fitted to the initial parts o f the d a t a ) has been d r a w n for c o m p a r i s o n with the d a t a o b t a i n e d after the a p p l i c a t i o n o f thiamine. In all cases there is a significant Increase o f the time c o n s t a n t o f decline (see T a b l e I). The effect develops m o r e slowly t h a n expected f r o m the diffusion velocity o f t h i a m i n e a l o n g the axoplasm. This might indicate the necessity o f p h o s p h o r y l a t l o n o f the t h i a m i n e (see below). T h i a m i n e can also act from the outside ( F i g 5 a n d T a b l e I), but n o t as effectively as from the inside. The effect o f T h D P on the s o d i u m a n d p o t a s s i u m currents is shown in Fig. 3 The p H o f the R i n g e r ' s s o l u t i o n c o n t a i n i n g T h D P was a d j u s t e d to p H 6.8. F o r this r e a s o n the c o n t r o l p e r i o d before a p p l i c a t i o n o f the substance was also o b t a i n e d at p H 6.8. The change o f p H h a d no noticeable effect. A f t e r the c o n t r o l period, T h D P was externally a p p l i e d d u r i n g 14 rain. Then, T h D P was a p p l i e d internally two times, while the node was superfused with R i n g e r ' s s o l u t i o n o f p H 6.8. External a p p l i c a t i o n

292 [rnA/crn ~

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l

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I

I

I

T

~°°°°°

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[.

[rain ]

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-~0 -6D -80 INo [ rnF:t/c m2 ]

,f ~"~" / '

~ T

-'.',-" mti TNIAMINE

12 rnM THIAMINE

,/ ~2 mM TNIRMINE /

h~Io) . ~ ~ " 0.6" I

I I,,-0

F ~3.73

I 80

I

I 120

I

""['" ' " " - I " 160 T [rain)

Fig. 2. Effect of thiamine (internally applied) o n the exponential decline o f the ionic currents o f the n o d e of Ranvier. T h i a m i n e was applied 3 t i m e s internally. Symbols: [] -- steady state p o t a s s i u m current at V = 120 m V ; + = peak s o d i u m current at V -- 60 m V , preceded by a prepulse of - - 4 0 m V a m p l i t u d e a n d 50 msec d u r a t i o n to r e m o v e s o d i u m inactivation ( h ~ -- 1); × -- steady state sod i u m inactivation determined at zero h o l d i n g potential h ~ (V - 0); d a s h e d lines -- expected e x p o n e n tial decrease of the 3 variables as fitted f r o m the data before application of the vitamine (solid lines). T e m p e r a t u r e 14 °C. T i m e constants: before application of thiamine (solid a n d d a s h e d curves): O (IN~) -- 49.5 min, @ (IK) = 104.3 min, O (h~) = 52.2 m i n ; after application (experimental points): O (IN~) = 214 min, O (Iic) -- 231 min, O (h~) = 420 min. T h e decline o f currents is considerably decreased by the action o f thiamine.

of T h D P had no significant effect. Internal application had a similar but stronger effect than that observed with thiamine. Normally an initial increase particularly of the sodium current was seen. It could not be clarified whether this change was due to a small variation of the electric field across the membrane 12. The holding potential remained stable. Fig. 4 shows the effect of T h T P on the sodium and potassium currents and on boo (V = 0). A large increase of the time constants typical of the exponential decline of currents was observed (compare the curves fitted to the data of the initial period before T h T P was added with the experimental points). The effect of T h T P is still stronger than that observed with ThDP. Again an initial increase of the sodium current is observed similar to that seen with ThDP. Table I summarises the results obtained in a series of experiments like the ones illustrated in Figs. 2, 3, and 4. The effect of each substance is measured as the ratio of the time constants of exponential decline of currents determined after and before application of the agent. The time constants O were evaluated by fitting the function I = I0 exp (--t/O) to the experimental points (see Figs. 3 and 5) in case of external application or control superfusion. In the case of internal application, since the inter-

293

IK

[mR/cm 2]

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I

x~

[mm]

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[mR/cm ~]

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2 mHThDP [INT]

Fig. 3. Effect of ThDP on the exponennal dechne of the Ionic currents m the node of Ranwer Symbols as m Fig 2 After reducUon of the p l l to the ac~dlty of the ThDP solunon, ThDP was first apphed externally (E×T) and then twice internally ([NT). The external apphcahon exhibats no sJgmficant effect, whereas the internal apphcatlon increases the time constants considerably Tameconstants before and during external apphcatlon (dashed curve) O ([Na) = 63 I ram, @(Ix) 47 5 ram, alter internal apphcanon (dotted curve): (9 (I~a) = 516 mm, O (Ix) = 300 mm Temperature 14 'C

0

IK [ ml~l/cm:2

0

,

] -

a

o.m_jjl

F 7.?ho

o

a

o

a

o

u

q

o

~'.-'.t. . . . . . . . . . . . . . . . . . . . . . . . . .

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o

....

t3

q

u

u

......................... LiLT

20" I

I

I

20

-20

I

I

I

bO

I

60

I

T [mln ]

-60 [Na [mR/cm2] h~(o)

÷÷÷'*'~*~

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72

f

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~2

mN ThTP

x~

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I

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T [m,n I

Fig, 4. Effect of T h T P o n the e x p o n e n n a l dechne of the lomc currents o f the node of Ranvler. Symbols as m F]g 2. T h T P was applied twice f r o m the insade. T h e dashed curves represent the expected e x p o n e n n a l decrease o f INa, 1i{ a n d hoo (V - 0), as fitted f r o m the d a t a before applacanon of the substance. T h e dotted curves represent the reduced exponential decrease due to the action of T h T P Tame c o n s t a n t s : before application of T h T P " O (Ir~) = 47 8 ram, (9 (hO -- 48.5 man, G) (hoo) 31.6 r a m ; after application of T h T P : (9 (I.~) = 615 m m , 6) (Ix) -- 1199 man, (9 (ho~) 449 m m T e m p e r a t u r e 14 °C

294 TABLE 1 D E C E L E R A T I O N ()1~ T H [ f X P ( ) N [ N 11AL I)I~CI_IN[ Of- f i l E N O D A L [ O N t (

f U R R E N T S BY 7 H I A M I N F ,

ThDP

~\t)

T h T P , ALt 2 MM T h e time c o n s t a n t s ol decrease o f ~onlc currents, O, before a n d after apphcat~on of the agent were d e t e r m i n e d as described in the text. As a m e a s u r e m e n t of deceleration of the exponentml dechnc S D.). For control the artlficml axoplasm ~oluthe ratio 6) i,,rt,,,~/O ~ao,,,~ was evaluated (mean tlon was replaced by anothel sample of the s a m e c o m p o s i t i o n

Agent

Apphcd

¢9 ,,tt,~rl,'(~)qe~,/ore~ J: S D I\,,

Control Thiamine ThDP ThTP*

Internally Externally Internally Externally Internally Internally

I 18 261 3 16 1.05 60 122

010 , 027 ,~ 0.86 j 002 i 064 59

No. oj

h, r V

O)

099 + 2.96A 3 84 : 070 71 66

0.10 1.34 1.10 010 1.94 38

1t, IOl ~ 0 0 8 172±035 8.15 :i 0 4 8 09 =0.18 69 L 210 100 ! 7 3

~' Y p e r l m ¢ ' / / I

4 3 3 2 4 3

* In one experiment a 6-h-old T h T P solution was used which caused a rather low increase in the time c o n s t a n t s c o m p a r e d to the other two expernments performed w~th fresh solution

nal concentratton of the agent at the test node could not be kept constant, the experimental points did not follow an exponential t~me course. So, a lower limit of O was estimated assuming that the current values at the end of an experiment were reached by an exponential dechne starting at the moment of the apphcat~on of the substance (see Figs 2 and 4). For control the internal application was simulated by replacing the solution m the side pools by another sample of ldentzcal composmon. Thts manipulation did not significantly alter the exponential dechne of the ionic currents. External apphcatnon of thiamine slowed the process of exponenttal dechne 2 or 3 times, internal apphcatmn more than 3 times. Internal application of T h D P and ThTP caused an increase of the time constant by a factor of about 6 and of more than 10, respectwely.

Apparent lack of e//eet oJ bacterial th&minases With the aid of bacterial thiaminases two aspects of the action of thiamine in nervous tissue can be stud~ed : ( 1) with their different substrate specifity (see Methods) it may be possible to identify the acttve substance (thiamine or one of its phosphoric acid esters); (2) the enzymatic degradation of thiamine derivatives m the nerve membrane might either mimic the exponentml decline of the nonlc currents on a shorter time scale or may reveal additional effects, indicating that thiamine takes part m several different mechanisms related to excitation. Bacterial thiaminase l from 4 different cultures was apphed externally (7 experiments) and internally (2 experiments) to the nodal membrane, and bacterial thiammase II from one culture was applied externally (2 experiments), but in all cases without s~gmficant effect on the exponential decline of the ionic currents. One explanation of this negative result could be found in the rather low actl~t~

295 TABLE 11 EFFECT OF TEMPERATURE ON THE EXPONENTIAL DECLINE OF THE IONIC CURRENTS

The time constants of current decrease, (-), were determined by fitting the function 1 lo exp (--t/(~)) to the data points of the initial periods of the experiments (before apphcatlon of an agent) Mean i S.D

(-)(145'C)(mtn) (-)(65 'C)(mln) Qio

L.\',

h c~ ( V

O)

59 -- 5 197 ~ 55 45 k 1 5

63 11 141 ~ 2 3 2.7 i_ 09

In

No of experiments

64 ~ 6 174 ±41 3.5z 1 3

9 4

o f the applied enzyme preparations. However, thc apparent lack of an effect o f the thlaminases was not surprising after the finding that T h D P was only effective from the inside. The high molecular weight o f the enzymes practically excludes their penetration t h r o u g h the nodal m e m b r a n e A diffusion along the axoplasm would be too slow, so that the enzyme concentration built up the inner side o f the nodal m e m b r a n e would be lower than a minimum required level. Effect o f temperature Low temperature increases the t~me constants o f the exponential decline conslderably. This is shown in Table II. The time constants of current decrease were evaluated by fitting an exponential function to the experimental points o f 9 experiments at 14.5 ' C and o f 4 experiments at 6.5 °C F r o m the mean values o f the time constants the Q10 of the rate o f exponential decline o f currents was found to be in the order o f 3 EfJect o f T T X plus thiamine ltokawa and C o o p e r zl reported that T T X induces a release o f thiamine from nerve membranes. In case of a direct relationship between the function o f T T X to block sodium channels and its ability to release thmmine, an excess o f thiamine should reduce or even inhibit the T T X effect. T h o u g h this was not probable it was investigated in experiments of the type illustrated in Fig. 5. A low concentration of T T X (6 n M ) was chosen which blocked approximately two-thirds of the sodium channels and thus easily allowed the determination o f changes in the remaining sodium current After the partml inhibition of the sodium current was complete the external T T X - R i n g e r ' s solution was replaced by another containing additionally 2 m M thiamine There was no reduction o f the blocking effect o f TTX. Nevertheless, thiamine clearly reduced the exponential decline of the sodium current as usual. The time constant o f current decrease before apphcation o f TTX, (01, was calculated as above (sohd line, labelled O1) The dashed curve (labelled O1) drawn as a continuation of the solid curve represents the time course o f the sodium current expected without the application o f T T X (see Fig. 1). Since T T X does not slgmficantly change the exponential decline o f the sodium current as evidenced by the results o f 3 separate

296 20

~0

I

I

T [rnin ]

60

I

I

I

I

I

I

6 nM TTX

-20.

O1

I ........................

Y +2 mM T N I R M I N E

-~0-

......... ::: ............................... ~

-60, ]No [mR/cm

2]

...................... "',

J

"

I RINGER'S ~/

F 1~.73

Fig. 5. Effect of the c o m b i n e d action of T T X (6 n M ) a n d t h i a m i n e (2 m M ) o n the s o d i u m current, I ~ . Solid curve (labelled O1) = time course of the s o d i u m current as fitted f r o m t h e data before application of tetrodotoxin. D a s h e d curves (labelled Oi) are constructed u s i n g O1 a n d represent the expected time course of IN~ with a n d without the poison present. Solid curve (labelled OD -- time c o u r s e o f INa during the action of T T X plus t h i a m i n e as fitted f r o m the data. D o t t e d curve (labelled 02) = expected curve in case of t h i a m i n e being present solely, as constructed u s i n g 02. N o interference o f the action o f both substances. O1 -- 72 rain, 02 -- 244 rain. T e m p e r a t u r e 14 °C.

experiments, the dashed line starting from the partly blocked current values represents the expected sodium current with TTX present but without thiamine. However, the time constant of the exponential decline is increased (solid line labelled 02), and the time course of the sodium current after wash out of the poison can be satisfactorily described by this increased time constant, 02, due to the action of thiamine (dotted curve). Thus, the effect of thiamine does not depend on the presence of TTX. On the other hand, an excess of thiamine does not alter the blocking ability of the poison. Another argument for an independence of the two TTX effects can be drawn from the observation that thiamine acts on the potassium current as well (see Fig. 2), whereas TTX, as a general experience, has no effect on the potassium current. DISCUSSION

Considerable knowledge on the function of thiamine and its phosphoric acid esters in nerves has been accumulated, but the biochemical mechanisms are far from being clarified. The results presented in this paper suggest that thiamine and/or its energy rich phosphates are important for keeping the ionic channels functioning. These findings have to be discussed with respect to the following points. (1) The di- and triphosphoric acid esters of thiamine are likely to be the functional compounds rather than thiamine. (2) Thiamine or its phosphates may exert their function on excitability apart from the coenzyme function in oxidative decarboxylation. (3) The 'thiamine receptor' seems to be located on the inner side of the nerve membrane. The relative distribution of thiamine and its phosphate esters ThMP, ThDP and ThTP is essentially equal in most mammalian cells6, 42: thiamine 6 ~ , T h M P 14 ~ , ThDP 72 ~ , and ThTP 8 ~. No conclusion, therefore, can be drawn with respect to a special role of these compounds in the excitation process from their relative distribu-

297 tion in nervous tissue. Most of the thiamine (about 72 ~ ) is found in the dlphosphorylated form, and the main function of ThDP, namely the coenzyme part in oxidative decarboxylatlon, is well documented. The role of T h T P is essentially unknown. However, the early observation of M m z z3 and subsequent investigations of yon Muralt and collaborators 3z,z4,35,37,44,5° suggested that thiamine was 'liberated' from peripheral nerves by electrical st,mulation. The process o f ' l i b e r a t m n ' was shown by Gurtner 17 to be a release of thiamine or T h M P from T h D P and ThTP by a dephosphorylation obviously being connected ,n some unknown manner with excitat,on. These results obtained with peripheral nerves of rats were confirmed by Cooper et al. 7 using a spinal cord preparation from frogs. Additional information was provided by Itokawa and Cooper ~'2,'z showing that neuroactive agents, such as acetylchohne, tetrodotoxm, ouabain, 5-hydroxytryptamine and lyserglc acid diethylamlde were able to release thiamine by apparently the same process, namely a dephosphorylation of T h D P and ThTP. The total amount of thiamine (including the phosphate esters) was not reduced (if not washed out) from the preparation, as was shown by Gurtner 17 and as also was stated by ltokawa and Cooper 2~'. So, the 'release' of thiamine was the result of the shift in the relative distr,but~on of the phosphoric acid esters by the dephosphorylat,on. Further investigations22, ~z evidenced that the release of thiamine by neuroact,ve drugs did not occur similarly m all thiamine containing cell fractions of nervous tissue, but was entirely restricted to the membrane fraction. This was found In preparations of brain, spinal cord, and peripheral nerve from rats and bullfrogs as well. A complete set of enzyme systems catalysing the lnterconversion of all 4 compounds has been detected, isolated and purified from nervous tissue T h T P is hydrolysed by a specific ThTPase which was detected in nervous tissue by Greiling and K~esow t6 and wh,ch was simultaneously characterised by Hashitanl and Cooper is and Barchi and Braun3; the stoichiometry was shown to be: ThTP

ThTPase, Mg z+ ~ ~ T h D P 4- P~

Actually two different ThTPases seem to exist in nervous tissue, one with an activity peak around p H 6.5, which is membrane associated 3 and another with an activity peak around p H 9.518. The major part of the 'alkaline' ThTPase (about 6 0 ~ of the total activity) is occurring m the soluble fraction rather than in the membrane (about 7 ~o) or mitochondrial (about 3 ~ ) fraction, so hi vivo it must be located in the axoplasm. TTX, ouabain, acetylchohne, tripropyltln, pyrith,amine, oxythiamlne, and Na + as well as K + had no effect on the enzyme activity s,ls. T h D P is similarly hydrolysed in nervous tissue by a ThDPase, which was evidenced by Barchi and Braun a to be most probably identical with a non-specific nucleoside diphosphatase (EC 3.6.1.6 nucleosldediphosphate phosphohydrolase): ThDP

\

ThDPase, Mg 2+ ~ T h M P 4- P~

This enzyme is completely unaffected by ATP 4. As in the case of the ThTPase neuroactive agents did not significantly alter the activity of the ThDPase in vitro 4.

298 The reverse pathway, the synthesis of the phosphoric acid esters b~ ,~ pho~phorylation of thmmme has been demonstrated to occur m nervou~s tissue m ~t~, by Gurtner t7 using :~S-labelled thiamine in rats and frogs. An en/\.me sy,tem thal catalyses the phosphorylatlon of thmimne to ThMP was shown to cxl~t m the ~pmal cord of swine by Eckert and M6bus ~ Johnson and Gubler '-'4 ~solated and purJhed a thiamine dlphosphokmase (ATP'thmmine dlphosphotransferase, EC 2 2. l I) from rat brain which catalyses the resynthesis of ThDP from thiamine" Mg ~', Th-dlphosphokmase Thiamine ~t ATP

-

ThDP-t AMP

The interesting point of this reaction i~ that the phosphorylatlon takes place without the intermediate ThMP. The locahsatlon of the enzyme within the cell was not reported. Eckert and M/)bus :}also detected a thiamine d~phosphate phosphotranslerase system that catalyses the resynthesl~ of ThTP according to" P-transferase T h D P + ATP

~ ThTP t- A D P

This enzyme system was isolated from rat brain and characterised by ltokawa and Cooper 20. From all knowledge accumulated so far it is reasonable to conclude that T h D P and/or ThTP are the functional compounds m nervous tissue rather than ThMP or thiamine ~tself. Thts conclusion ~s in agreement with the results presented in this paper showing that ThTP and T h D P are far more effective m reducing the exponential dechne of the somc currents than thiamine. However, ~t cannot be dec~ded whether or not the thmmme compounds take part in the excitation process b? a special function apart from the well-known coenzyme role m carbohydrate metabolism. ThTP cannot serve as a substitute for ThDP as a coenzyme s,z6,~3. Therefore, serving as a coenzyme, ThTP has to be dephosphorylated. On the other hand, ~t is hard to enwsage that ThTP would be used for nothing else than a store for ThDP, since ThDP can be synthesised from thmmme. The cychc dephosphorylation and rephosphorylation of ThTP ~uggested by |tokawa and Cooper 22 to be assocmted with the excitation process would principally be possible, since all necessary enzymes exist m nervous tissue. On the other hand, none of these enzymes ~s sensitive to one of those neuroactive agents that have been stated to cause a release of thiamine from nerve membranes by exactly this mechanism 2~,23. This fact, however, might indicate that the dephosphorylation and rephosphorylatlon process might not directly be coupled to the excitation mechamsm. Our observation, that the action of TTX and of thiamine do not interfere, supports this view. If there were a direct relation between the blocking action and the release of thiamine, thiamine should not be able to reduce the exponential decline of the lOmC currents during the presence of TTX, or the blocking effect had to be reduced as well. Furthermore, the thiamine deficiency is acting on both, the sodium and potassium channels, whereas TTX is specific for the sodium channels only. Other neuroactlve agents that release thiamine (such as ouabain) do not exhibit any direct effect on excitation. An explanatmn of the thiamine releasing effect of neuro-

299 active drugs could possibly be found in the observation of Galzigna la that thiamine forms complexes with most of those agents (acetylcholine, norepinephrine, 5-hydroxytryptamlne). After dephosphorylation of the thiamine phosphates, thiamine would be bound to the neuroactive substances and washed out so that a rephosphorylatlon would be prevented. In fact, Heller and Hesse 19 observed that thiamine is released and washed out from isolated sciatic nerves of rats which are incubated in a phosphate-buffered glucose-free medium. The diffusible thiamine was stated to be derived from thiamine phosphate compounds. A further observation seems to be important, the loss of thiamine is prevented by the presence of thiamine or glucose m the incubation medium, which obviously stablhse the phosphorylated thiamine compounds. The thiamine depletion of the nerve is associated with an activation of the respiratory rate of the nerve. The high temperature dependence (Q10 approximately 3) of the exponential decline of the nodal ionic currents presented in this paper suggests the involvement of a metabolic process. Thus, the reduction of the exponential dechne by the action of thiamine phosphate esters can be thought to occur in 3 ways: the first possibility is that liberated thiamine is washed out from the preparation ~9 during the experiment and is no longer available for resynthesis. The dephosphorylation and therefore the liberation and wash out are faster at high than at low temperatures The second possibility would be that there is a deteriorating metabolic process which is stopped or counterbalanced by the action of the thiamine compounds, as long as they are sufficiently available. The temperature dependence of the exponential dechne of currents would then be due to the deteriorating process and the time constant of the inactivation effect would depend on the amount of thiamine present in the preparation. A third possibility would be an interruption of the rephosphorylatlon of thiamine by either the lack of energy (ATP) or the occurrence of a poisoning metabohte. A few observations seem not to show a dependence of the effect on ATP. A decision between the 3 possibihtles cannot be made from the experiments. Nevertheless, in either case, a direct involvement of thiamine or its phosphoric acid esters in the excitation mechanism itself is not required (but cannot be excluded) The site of the thiamine action is apparently located at the inner side of the membrane, since it was demonstrated in this paper that thiamine IS more effective from the inside than from the outside and that ThDP exhibits its effect from the inside only. Additional proof was obtained from the application of the bacterial thiamlnases which did not exert any effect from the outside and will certainly not permeate the membrane because of their high molecular weight (see Methods). This conclusion is in agreement with the finding of Armett and Cooper ~-that oxythiamine, which exhibits no function as a thiamine antagonist in nervous tissue, does not enter the cells, whereas pyrithiamine, an effective antagonist, permeates the membrane. There is one opposing statement by yon Muralt a6 that ThDP permeates the nerve membrane, but neither a p r o o f nor a reference ~s g~ven in his paper Another discrepancy seems to arise from the observations that 'thlaminases" externally applied to isolated nerve preparations show effects antagonistic to thiamine "s.al,3s,41. But both materials turned out not to be real enzymes. The active substance from fern

300 extract ,s most hkely identical with the 3,4-dihydroxycmnamic acid molecule with the molecular weight of 180 "~,46, and the active materml from the carp intestine ~va~ shown to be a hemin-Cl hke molecule of a molecular weight of around 651),*°,~7 These substances are hkely to penetrate through the membrane and ~t Js therelore not surprising that they can exhibit a thmmine antagomstic function m contrasl to the enzymes thiaminases ! and I1 with their high molecular weight CONCLUSION The exponentml dechne of the nodal ~onic currents was hypotheslsed to be connected to the density of the surface charges on the tuner side of the membrane~L Thiamine, most probably m its phosphorylated form, reduces this exponential decline by preventing the further shift of the h a (V) curve, t.e., by stabdismg the electric field strength in the nodal membrane m the resting state. Thiamine exerts this specml function presumably apart from its coenzyme role, but being not directly revolved in the excitation process itself As a working hypothesis for further investigations ~t is tentatwely suggested that the specific role of thmmme in excitation, which was postulated long ago, is to control the number of functioning ionic channels by stabihsmg the density of negative surface charges presumably at the tuner side of the nerve membrane. This mechamsm may be considered as an ad&tional fine regulative for maintaining excitabdity apart from the regulation of the membrane potential by the ~onic concentrations NOTE ADDED IN PROOF The question whether thiamine plays an essential role in the permeabihty changes underlying sodium activation during the action potential was recently reinvestigated by Goldberg* using thiamine antlmetabolites of the pyrithiamine class and fern extracts on lobster and squid giant axons. It was shown that thiamine anttmetabolites do not interact exclusively with the sodium channel and it was concluded that 'even ~f thiamine plays a role in conduction, it does not confer excitabdity on a membrane.' This is on the hne of the conclusion from our experimental results. ACKNOWLEDGEMENTS We gratefully acknowledge the continuous support of our work by Prof. Dr. R. St/impfli and Prof. Dr. V. Ullrich. We are indebted to Prof. Dr. K. Kanig, Ass. Prof. Dr. W. Nonner and Prof. Dr. E. Rojas for their valuable suggestions and their criticism of the manuscript. We appreciate the helpful discussions with Prof. Dr. H. J. Haas and Dr. B. Neumcke. We are grateful to Mrs. H. Gartenhof for her technical assistance and to Mrs G. Mtiller and Mr. O. Schneider for their help with * GOLDBERG, D. J., Thiamme New Haven, Corm, 1974.

m nerve ttssue

A cellular approach,

Ph.D. Thesis, Yale University

301 t h e e v a l u a t t o n o f t h e d a t a . L a s t b u t n o t l e a s t , w e w~sh t o t h a n k M r s . M . S i n n w e l l for her continuous efforts with this research project. Supported

by Deutsche Forschungsgemeinschaft,

Sonderforschungsbereich

38

' Membranforschung'.

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302 24 JOHNSON, L R., AND ( J ~ L ~ l { ,

25

26 27 28

29

30 31 32 33 34 35 36 37 38 39 40

41 42 43

44 45 46 47 48

49

50

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