European Journal of Pharmacology, 218 (1992) 59-68 (0 1992 Elsevier Science Publishers B.V. All rights reserved (X)14-2999/92/$05.00

EJP 52540

Mechanisms of propofol action on ion currents in the myelinated axo of Xenopus laevis o

Flavio Veintcmilla, Fredrik Elinder and Peter Arhem The Nobel Institute for Neurophysiology, Karolinska lnstitutet, S-I04 Ol Stockholm. Sweden

Received 23 December 1991. revised MS received 6 March 1992. accepted 15 April 1992

The effect of the intravenous anaesthetic, propofol (2,6-diisopropylphenol), was investigated on frog myelinated axons ul voltage-clamp conditions. The effect, in the concentration range 60/.tM to 10 raM, was a combination of (i) a negative shift ol steady state activation and inactivation curves for both Na + and K + currents (IN~,IK), (ii) a voltage-independent block of but not of I K, and (iii) a slowed time course of 1K activation. The shift was dose-dependent and, at 1 raM, about - 10 mV fol activation and - 1 6 mV for the inactivation curves. The voltage-independent IN, block showed l : l stoichiometry and : reduction at 2.7 raM. The slowed I K activation showed saturation at 1 mM with a doubled time to half steady state value. All effects were only partially reversible and showed a complex time course at application and washing. The shift of pote dependence may be explained by a general effect on the membrane electric field. The findings suggest effects directly on eha proteins as well as on membrane lipids. Propofol; Ion channels; Myelinated nerve fibre; Voltage-clamp experiment: Anaesthetics (intravenous)

1. I n t r o d u c t i o n T h e basic m e c h a n i s m s of g e n e r a l a n a e s t h e s i a a r e still a m a t t e r o f controversy. A n essential p o i n t in the discussion is the q u e s t i o n of the m o l e c u l a r t a r g e t for the g e n e r a l a n a e s t h e t i c . Classical m e m b r a n e lipid t h e o ries ( O v e r t o n , 1901; H a y d o n et al., 1984) have r e c e n t l y b e e n c h a l l e n g e d by t h e o r i e s of d i r e c t p r o t e i n effects ( F r a n k s a n d Lieb, 1982; 1988). P r o p o f o l ( 2 , 6 - d i i s o p r o p y l p h e n o l ; fig. 1), a p h e n o l derivative, is the n e w e s t i n t r a v e n o u s a n a e s t h e t i c a g e n t available for clinical use. Its clinical usefulness is related to its s m o o t h i n d u c t i o n a n d m a i n t e n a n c e o f general a n a e s t h e s i a ( D e G r o o d , 1987). In spite of its i n c r e a s i n g clinical use, little is k n o w n a b o u t the m e c h a nism o f action of p r o p o f o l . It has b e e n p r o p o s e d to affect G A B A ^ a n d glycine r e c e p t o r s ( H a l e s a n d L a m bert, 1991). R e c e n t l y effects on N a ÷ c h a n n e l s in artificial m e m b r a n e s have also b e e n r e p o r t e d ( F r c n k e l a n d U r b a n , 1991). In o r d e r to analyse the effects o f p r o p o f o l on volta g e - d e p e n d e n t Na ÷ a n d K - c h a n n e l s in nerve m e m b r a n c s , we s t u d i c d its effects on v o l t a g e - c l a m p e d

Correspondence to: P. /~rhem, The Nobel Institute for Neurophysiology, Karolinska Institutet, S-104 01 Stockholm, Sweden. Tel. 46.8.728 6903, fax 46.8.349 544.

m y e l i n a t e d axons. T h e analysis r e v e a l e d s o m e princ d i f f e r e n c e s b e t w e e n the effects of p r o p o f o l a n d tt d c s c r i b e d for o t h e r g e n e r a l a n a e s t h e t i c s . F u r t h e r , results suggest b o t h lipids and c h a n n e l protein: t a r g e t s for its m e c h a n i s m of action.

2. M a t e r i a l a n d m e t h o d s Large single m y e l i n a t c d axons o b t a i n e d from sciatic nerve of the c l a w e d t o a d Xenopus laet,is used. T h e axons were m o u n t e d in a P e r s p c x recor, c h a m b e r a n d cut at half i n t e r n o d e length on b o t h s ot" the n o d e u n d e r investigation. T h e r e c o r d i n g ch b e r was c o n n e c t e d via salt b r i d g e s to the v o l t a g e - c l a p p a r a t u s . P r o c e d u r e s for b a l a n c i n g the f e e d b a c k plifiers w e r e essentially the s a m e as d e s c r i b e d by D(

H o H3e~

l

H--C'~'~I H~e~

~ ell3

C--H ~CH 3

Fig. 1. Chemical structure of propofol (2.6-diisopropylpheno]

6()

o

and Frankenhaeuscr (1958), and A r h e m el al. (1973). A TL-I D M A interface (Labmaster, USA) and the p C L A M P software (Axon instruments, U S A ) w c r e uscd for pulse generation and sampling. The pulse rate was usually 1 or 0.5 Hz, unless otherwise stated, and the sampling interval was between 10 and 2(10/zs. In order to obtain good feedback control and recording situations all experiments were performed at a relatively low temperature (10°C). Since only relative current values were essential in the present invcstigation and since the nodal area and axoplasmic rcsistance were

not measured in the experiments, no attempt was n to calibrate the current in absolute values (sec D( and Frankenhaeuscr, 1958). Test solutions were applied externally at the r under investigation. The control Ringer solution rained (in mM): NaCI 115.5, KCI 2.5, CaCI 2 2.0, Tris buffer 5.0 (pH adjusted to 7.2). The solution 1 in the end pools contained (in raM): KCI 120.0, Tris-buffer 5.(1 (pH 7.2). Test solutions of pror (concentration range: 6(I # M - 1 0 mM) were obta by adding appropriate amounts of an aqueous cl

Control ......

-

-~.---- "--

-

mv + 62 my + 82

+42 my , ,, ,

,,

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m,

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,

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mv

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1 haM Propofol

+ 42 mv 4-22 mv

;;2m;

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20

Time (ms) Fig. 2. E f f e c t s o f p r o p o f o l o n c u r r e n t s a s s o c i a t e d with r e c t a n g u l a r p o t e n t i a l s t e p s as i n d i c a t e d . H o l d i n g p o t e n t i a l ( U H) - 9 8 inV. (a) Contr~ 1 m M p r o p o f o l . (c) P e a k c u r r e n t a n d s t e a d y s t a t e c u r r e n t v e r s u s p o t e n t i a l for a x o n in c o n t r o l ( o ) a n d 1 m M p r o p o f o l (t.3). S t e a d y s t a t e cu m e a s u r e d a f t e r 100 ms. T h e s h i f t e d r e v e r s a l p o t e n t i a l o f I ~ , w a s not s e e n r e g u l a r l y .

sion (1% propofol w / v in 10% w / v soya bean oil, 1.2% w / v egg phosphatide and 2.25% w / v glycerol; ICI Pharma AB, Gothenburg) to the Ringer solution. In spite of the extreme hydrophobicity of propofol (oct a n o l / w a t e r coefficient log P = 3.7; Scues and PrysRoberts, 1989), the graded response curves obtained indicated that the solutions were non-saturated. As described below, reversibility was only partial and the time course of effect and recovery was complex. Although thc results from the different expertmerits were qualitatively consistent, we selected for the quantitative analysis three good experiments in which (i) three or more concentrations of propofol were used on the same fibre, (it) the test solution was applied in order of increasing concentration and (iii) the test and Ringer solution were applied three or more consecutive times in order to obtain steady state values,

the higher potential range ( I N ; , by 40% and I K 20%). The leakage current was unaffected. 3.1. E f f e c t s on N a + current

In order to obtain quantitative values for the sh peak Na + permeability versus potential (peak P~, curves were calculated from the constant-field ec tion (Goldman, 1943; Hodgkin and Katz, 1949; Dc and Frankenhacuser, 1959). Figure 3a shows the : mcability curves for the fibre in fig. 2. l)ropofol, 1 r shifted the normalized curve - 8 mV without chanl the slope of the curve. The concentration dependc of the shift is shown in fig. 4 where values ((~ squares) from three fibres and five concentrations collected. The (absolute) value of the shift incre~ with concentration. The concentration depend¢ could be described by the equation: ..IU = A In(Be + 1)

3. Results Figure 2a,b shows the effect of 1 mM propofol on the currents associated with rectangular potential steps in increments of 20 mV in a voltage-clamp experiment. It is evident that both N a ' and K + current (IN~, I K) were reduced, and that the l K activation time course was slowed. Figure 2c shows the current versus potential ( I - U ) curves for peak I s , and steady state I~; for the same axon. As seen the I N , - U and I ~ - U curves were shifted in a negative direction along the potential axis (about 10 mV). and 1N;, and i K were reduced in

where ,~U is the shift, c the concentration (in mM propofol, and A and B are constants with values mV and 25 mM- ~ respectively (see Discussion for possible meaning of the equation). Part of the I ~,, reduction was due to effects on steady state inactivation. This was studied by mea ing peak IN~ at a test step to - 10 mV after prept of different amplitudes and a duration of 1 s (p frequency 0.5 Hz). Figure 3b shows the non-normal peak l~;,-U~,p relation for four concentration,, propofol (60 p,M to 10 raM) on one fibre (open bols). The effect could be described as a combina

c

-~

eakage

-1

Potential (mV) Fig. 2 (continued).

62 o f t w o e f f e c t s : (i) a s h i f t o n t h e c u r v e in a n e g a t i v e d i r e c t i o n w i t h o u t c h a n g e in t h e n o r m a l i z e d s l o p e o f t h e

potcntial curves for the control solution and 1 r p r o p o f o l , r e s p e c t i v e l y , f o r t h e f i b r e o f fig. 2. T h e cu

curve, and (ii)a potential-independent

w a s s h i f t e d a b o u t 6 m V in a n e g a t i v e d i r e c t i o n . "

current.

The

reduction of thc

(absolute) value of the

shift i n c r e a s e d

t i m e c o n s t a n t w a s d e t e r m i n e d f r o m t h e e a r l y phas~

w i t h c o n c e n t r a t i o n : at 1 m M it w a s a b o u t - 16 m V , a n d at 5 m M a b o u t - 2 5 m V . F i g u r e 4 s h o w s v a l u c s f o r

s e m i - l o g a r i t h m i c p l o t s o f t h e a b s o l u t e v a l u e o f IN., t h e p o t e n t i a l r a n g e s t u d i e d t h e c o n t r i b u t i o n o f 1K

t w o f i b r e s a n d five c o n c e n t r a t i o n s ( s o l i d s q u a r e s ) . T h e

the

l o w e r c u r v e s h o w s t h e s o l u t i o n o f Eq. (1) f o r A = - 5

disregarded

m V a n d B = 25 m M - ~

Frankenhacuser,

The

time course of the

IN.

inactivation was also

time

constant

measured

(Dodge

is v e r y s m a l l

and

and

Frankenhaeuser,

' 1~

1962).

T h e v o l t a g e - i n d e p e n d e n t r e d u c t i o n c o u l d b e stud

a f f e c t e d . T h i s w a s m o s t e a s i l y s e e n at t h e l o w e r p o t c n -

in i s o l a t i o n at v e r y n e g a t i v e p r e p o t e n t i a l s . F i g u r e

tial s t e p s . F i g u r e 3d s h o w s t h e t i m e c o n s t a n t v e r s u s

shows the

effect of propofol

at

-140

.

.

mV

for

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1.0 -

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0.5-

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n

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.

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+25

-140

Potential (mV)

.

.

.

-120

. -100

. -80

-60

Potential (mV)

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d

6

1.0-

5

,0.5 -

3

c

J

2 10C o n c e n t r a t i o n (mM)

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Potential (mV)

Fig. 3. Effects o f propofol on the Na ~ system. (a) Normalized peak Na ~ permeability (PN,) versus potential in control (c.:) and 1 m M pro] ( [ J ) solutions. LII~ = - 9 8 mV. ('ontinous lines solutions o f : P (or I ) = 1/[1 + e x p ( ( U i j 2 - U ) / k ) J Eq. (3), where P is peak permeabilit

test-step potential, LJt .~ potential at 5(F'~ of maximum peak P,~,, and k slope value. U~/. = - 3 2 mV, and - 4 0 mY: k ~ 8 mV Non-normalized steady state I y,, inactivation versus prepulse potential. Measured from peak I y,, at a test step to - It) mV from prepulses ¢ duration (open symbols). Control (O). 0.06 (12). 0.6 (

Mechanisms of propofol action on ion currents in the myelinated axon of Xenopus laevis.

The effect of the intravenous anaesthetic, propofol (2,6-diisopropylphenol), was investigated on frog myelinated axons under voltage-clamp conditions...
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