Interaction of Local Anesthetics with the Transport System of Glucose in Human Erythrocytes L. LACKO, B. WITTKE AND I. LACKO Gustav-Embden-Zentrum der biotogischen Chernie, Abteilung fur physikalische Biochemie, Theodor-Stern-Kai 7, 0-6000FrankfurUMain 70, Germany
ABSTRACT Local anesthetics inhibit the exchange transport of glucose in human erythrocytes. All compounds tested showed a competitive inhibition except lidocaine and baycaine causing a non-competitive one. Moreover the transport system can bind two inhibitor molecules to one transport site as described for tetracaine and oxybuprocaine. bidest ad 1,000 ml). After the removal of the plasma by centrifugation, the erythrocytes were preloaded with 200 mM glucose by four washings in isotonic NaCl solution containing 200 mM glucose. One hundred and fifty microliters of these preloaded cells were incubated a t 20°C in 10 ml medium consisting of isotonic phosphate buffer pH 7.5 with different concentrations of C14-glucoseand local anesthetics as indicated in the various experiments. After five seconds the incubation was stopped by pouring the suspension into 80 ml ice cold stopping solution (2 mM HgCl,, 310 mM NaC1, 1.25 mM KJ). After centrifugation and washing the inside of the tube with the stopping solution at O"C, the erythrocytes were hemolysed in aqua bidest and the proteins precipitated according to Somogyi ('45). The radioactivity of the C14-glucosewas determined by liquid scintillation counting. For details of the experimental procedure see Lacko et al. ("72). Commercial preparations of the following local anesthetics were used in the experiments: procaine HC1 (Merck, Darmstadt), brufacaine HC1 (Doctor Christian Brunnengraber, Liibeck), dibucaine HC1 syn. cinchocaine (Ciba-Geigy, Wehr/Baden), mepivacaine HCl (ICN Pharmaceuticals GmbH and Co., Eschwege), butacaine-hemisulfate (Sigma, Miinchen), oxybuprocaine HCl (Wander GmbH Frankfurt/Main), baycaine HCl syn. tolycaine (Bayer, Leverkusen) , lidocaine MATERIAL AND METHODS HC1 (Welding and Co., Hamburg) and proHCl, tetracaine HC1, hostaHuman blood of healthy donors was col- cainamide lected in ACD solution (11 gm sodium citrate, caine-phosphate syn. butacetoluid, ultracaine Received Nov. 1, '76. Accepted Jan. 11. '77. 35 gm glucose, 4 gm citric acid with aqua
Local anesthetics are chemical compounds which produce a wide variety of effects on biological processes. First of all, the anesthetic action on excitable membranes by blocking the sodium current in nerve (Ritchie and Greengard, '66) should be mentioned. Additional effects include the induction of membrane expansion (Seeman, '70), the modification of osmotic fragility of erythrocytes (Roth and Seeman, '71), the inhibition of firefly luminiscence (Ueda et al., '76), the effect on phospholipases (Kunze et al., '76) etc. (Browning and Nelson, '76; Poste et al., '75). Some of these effects are tightly linked to the function of cell membranes. Although much attention has been focused on the mode of action of local anesthetics (Papahadjopoulos et al., '75; Mercki et al., '75), this problem has not yet been solved. The assumption can be made, however, that the lipids of the cell membranes play a major role in these processes. According to our findings, membrane lipids are also important for the inhibition of glucose transport by alcohols (Lacko et al., '74) and steroids (Lacko et al., '75). So, one can suppose t h a t local anesthetics will also influence the glucose uptake in erythrocytes. Results on this topic would be of interest not only for developing the concept of the glucose transport in erythrocytes but also for the interaction of local anesthetics with cell membranes (Koblin et al., '75).
.
-
.
.
.
J. CELL. PHYSIOL.,92: 257-264.
.
.
-
.
257
258
L. LACKO, B. WI'ITKE AND I. LACKO
HC1 (Farbwerke Hoechst AG, Frankfurt/ Main). The local anesthetics were dissovled in 0.9% NaCl solution in the described concentrations and added to the incubation medium. To determine the Rf-values thin layer chromatography was carried out on glass plates (20 x 20 ern) coated with a 0.25-mm thick layer of silica gel 60 FZs4(Merck, Darmstadt). The 12 compounds dissolved in water were layered onto the plate (100 pg/spot) and developed for 6.5 hours in a solvent consisting of the upper saturated phase of a mixture of nbutanollacetic-acid/water (4:1:5). a
RESULTS
The effect of the different locl anesthetics on the exchange transport of glucose The investigated local anesthetics contain a lipophilic radical, a n intermediate chain and a hydrophilic secondary or tertiary amino group. Their chemical formulas are shown in table 1. The pK value of the amino group on the benzene ring (if present) is about 2.2 (Killian, '73) which implies that this group is practically not protonized in our experimental conditions. The amino group a t the other end of the molecule has pK values in the range of 8.0 to 9.3 (Buchi and Perlia, '71) indicating that the proportions of the protonized to the non protonized group depend on the pH of the medium. of the The relative inhibition (1 - 3) VO
glucose exchange transport has been studied a t different pH values of the medium. With the glucose concentration at 0.033 mM, the local anesthetic concentrations were adjusted to such values to get reasonable relative inhibitions for the studied pH range, that means the stronger inhibitors were used in lower concentrations and vice versa. It can be seen from table 1that the relative inhibitions of all local anesthetics were smaller a t pH 4 than a t higher pH values. This points t o smaller inhibition effects of the protonized form than of the non-protonized one.
the non-inhibited reaction v, was estimated a t various [SI concentrations, too. The kinetic data were plotted in three ways: as a Lineweaver-Burk plot 1or -.?.- respectively "I
versus -,1 as a Dixon plot IS1
is1
1 Vo
-
versus [I], and
VI
as - versus [I], as proposed by CornishVI
Bowden (Cornish-Bowden, '74). By this procedure the types of inhibition and the dissociation constants of all investigated local anesthetics (except tetracaine and oxybuprocaine) could be determined unambiguously. The kinetics and K, values of the latter two were clarified by further kinetic analysis. As an illustration of the three types of plots, the inhibitions by procaine, lidocaine and tetracaine are shown in figures 1-3.I t can be seen that procaine produces a competitive inhibition; lidocaine, a non-competitive inhibition of the glucose transport. Tetracaine (and oxybuprocaine-not shown in the figure) is a competitive inhibitor according to the Lineweaver-Burk plot. The other two plots did not give straight lines but curves bending towards the ordinate a s observed with other inhibitors, too (Lacko e t al., '74, '75). The result of the graphical analysis for all local anesthetics investigated are summarized in table 2. The lipid solubility corresponds neither with the apparent affinity nor with the inhibition kinetics of the local anesthetics t o the transport system (table 2), as judged from the Rf-values of the thin layer chromatography (Buchi e t al., '75).
Kinetics of the inhibition by tetracaine and oxy buprocaine We wanted to test whether the bending of the curves (fig. 3) is in agreement with the assumption that two molecules of the inhibitor [I] are bound to one transport site [Cl. In this case, for competitive inhibition the following is valid: IS1 "I = Vm,, iil [I]' ' (1) ISI+& (1 + -+ -1
Kinetics of the inhibition ofglucose exchange K, (2) To characterize the inhibition by local anes- where Ki = 1c1 . [I1 qi = lCIl . I11 ICIl ' ICIIl ' thetics, the relation between the uptake velocity vi and glucose concentration [Sl a t Transforming this relationship a t two concenconstant local anesthetic concentration [I1 trations [I,] and [I,] of a (given) inhibitor and between v, and [I1 a t constant [SI was (similarly as was done for non-competitive studied; the velocity of the glucose uptake of inhibition, Lacko et al., '75) and plotting ,
~
~
259
INHIBITION OF GLUCOSE TRANSPORT BY LOCAL ANESTHETICS TABLE 1
Inhibition ofglucose exchange transport by diferent local anesthetics S t r u c t u r a l .oea1.nesthe t ic s
Aromatic residue
I
oncenration
F o r m u l a s
Intermediate chain
1
Amino group
(mM) . . -
45.0
36.0
'rocainarnide
Relative Inhibition In 9
40
PH
PH 75'
PH 9.0
55
59
2s
45
54
1
46
52
7
43
az
11
15.0
lepivacaine
12.0
'rocaine
.idocaine
8.0
9
40
64
Hostacaine
6.0
1
48
80
Jltracaine
4.5
0
42
60
Iletracaine
1.5
0
41
81
3.0
3
43
65
1.2
4
40
78
1.6
5
42
80
35
44
,CH,-
Baycaine
CH,
kybuprocaine
\
c y- cH,
PIltacaine
Xbucaine
C 0 - N H - C ;H CHa-
t, calculated from Ki, except tetracaine and oxybuprocaine.
CH;
CH,
0.35
5
260
L. LACKO, B. WITTKE AND I. LACKO
A
-viS
-1
1
V
6
Vi
5-
-v1
c
5-
A
-1
B
vi
5-
100
-S
C
vi
-
5-
:k -
rl "i
3
2
1
w
@
&-tMGLucose)-4
12
2P
I (mM Tetracaine)
w
1,2 2,0 I ( m M Tetracaine)
Figs. 1-2-3 Kinetics of procaine, lidocaine and tetracaine-inhibition of the glucose transport. Erythrocytes were preloaded with 200 mM glucose. The incubation was carried out for five seconds a t 2 0 T in phosphate buffer (pH 7.5) a t various concentrations of glucose and inhibitors, as described in A,B,C. A Double reciprocal plot of glucose uptake. The incubation medium contained five different C "-glucose concentrations between 0.5 and 2.5 mM with ( 0 )and without (0) the inhibitor. The inhibitor concentrations were: figure 1: 12 mM procaine; figure 2: 8 mM lidocaine and figure 3: 1.2 mM tetracaine. The reciprocal value of t h e uptake velocity, sec. mM I , of the glucose is plotted against the reciprocal value of the glucose concentration in the incubation medium. B The dependence of glucose exchange flux on the inhibitor concentrations [I].The incubation medium contained the following glucose and inhibitor concentrations: in figure 1: W 1 mM glucose, A 3 mM glucose and t h e procaine concentrations between 5 and 20 mM; in figure 2: 0.033 mM glucose, A 0.066 mM glucose and lidocaine concentrations between 3 and 15 mM; in figure 3: A 0.5 mM glucose, 1.0 mM glucose, 0 2.0 mM glucose and the tetracaine concentrations between 0.4 and 2.0 mM. The reciprocal value of the uptake velocity, sec. mM-', of glucose is plotted against the inhibitor concentration 111. C The reciprocal value of the relative flux (@-I against the inhibitor concentration [I]. Values and symVi
bols for figures 1 and 3 as in B and for figure 2: A 0.066mM,
+ 1.0 mM,
3 mM glucose.
261
INHIBITION OF GLUCOSE TRANSPORT BY LOCAL ANESTHETICS TABLE 2
The mode of inhibition and Ki values ofglucose uptake from different local anesthetics Local anesthetics bound
Local anesthetic
Type of inhibition
Brufacaine Procainamide Mepivacaine Procaine Lidocaine Hostacaine Ultracaine Tetracaine Baycaine Oxybuprocaine Butacaine Dibucaine
Competitive Competitive Competitive Competitive Non-competit ive Competitive Competitive Competitive Non-competitive Competitive Competitive Competitive
against the sum [I,] + [I,] gives a straight line with the intercept (1 on )the ordinate KI
and the intercept
-K,, on the abscissa;
+ Km ). Km From figure 4, one can see that for both tetracaine and oxybuprocaine the inhibition kinetics correspond to the binding of two molecules to one transport site. The values for tetracaine were K, = 5.6 mM, K,, = 0.9 mM and for oxybuprocaine K, = 2.5 mM and K,, = 1.0 mM. Since for both inhibitors K,, < K, it has to be concluded that the binding of the first inhibitor molecule facilitates the binding of the second one (Lacko et al., '74). (f =- IS1
Combined action of a competitive inhibitor and a non-competitive one We wanted to find out whether the competitive inhibitors tetracaine and oxybuprocaine also exhibit a non-competitive component. We estimated the uptake velocity of glucose with the non-competitive inhibitors lidocaine and baycaine (vJ, with the tested competitive inhibitors tetracaine and oxybuprocaine (v,) and under the combined influence of one of the competitive and one of the non-competitive inhibitors (v,,). The non-inhibited glucose uptake (v,) was also determined. The results were inserted in the formerly derived relationship (Lacko et al., '75): -1 + - 1
Ki (mM)
54.0 45.0 18.0 16.0 12.0 6.6 6.3 5.6 4.0 2.5 2.2 0.33
1 1 1 1 1
1 1 2 1
2 1 1
Kii (mM)
Rpvalues
-
0.25 0.22 0.37 0.30 0.36 0.58 0.51 0.38 0.33 0.39 0.54 0.42
-
0.9
1.0
-
the competitive and the non-competitive inhibitors do not influence each others' binding and action: the tested inhibitor shows only one component. If @ < 1and T = 1, the binding of one type of inhibitor is prevented by the binding of the other one. From table 3 it can be seen that oxybuprocaine in contrast to tetracaine shows a non-competitive component, too. This implies that oxybuprocaine also binds to some
/A
-1,5
@
-0,5
I
I
0,s
I
1,5
4,5
Fig. 4 Graphical evaluation to determine the dissociation constants of tetracaine (line 1) and oxybuprocaine (line 2). The glucose concentrations are A 0.5 mM, 1.0 mM and 0 2.0 mM. I + I, is the sum of different concentrations of one inhibitor. f is a constant, calculated from [" Km ; it is for 0.5 mM glucose 1.2, for 1.0 mM glucose +
vj
3,5
1,+ xi (mM I
Km
Vi
23
1.4 and for 2.0 mM glucose 1.8.
262
L. LACKO. B. WITTKE AND I. LACKO TABLE :I
Combined action ofbaycaine or lidocaine respectively with oxybuprocaine or tetracaine respectively in theglucose exchange transport rt,
v 1.05 1.05 1.03 1.05 1.12 1.15 1.10 1.15
Baycaine
+ oxybuprocaine
0.80 0.82
Lidocaine
+ oxybuprocaine
0.82 0.82
Baycaine
+ tetracaine
Lidocaine
+ tetracaine
1.04 0.99 1.00 0.99
The glucose concentration was 0.033 rnM and the inhibitor concentration: for baycaine 3 mM. for lidocaine 8mM, for oxybuprocaine 12 m M and for tetracaine 1.5 mM
extent t o the non-competitive site and prevents the binding of lidocaine or baycaine. DISCUSSION
Most of the investigated local anesthetics exerted (like the steroids, Lacko et al., '75) a competitive inhibition kinetic; only two out of 12 local anesthetics showed non-competitive ones. The non-competitive inhibitors lidocaine and baycaine are characterized by the following features: they have no amino group on the benzene ring, the intermediate chain is - NH - CO - CH2- and the tertiary aminogroup is - N
ABSTRACT Local anesthetics inhibit the exchange transport of glucose in human erythrocytes. All compounds tested showed a competitive inhibition except lidocaine and baycaine causing a non-competitive one. Moreover the transport system can bind two inhibitor molecules to one transport site as described for tetracaine and oxybuprocaine. bidest ad 1,000 ml). After the removal of the plasma by centrifugation, the erythrocytes were preloaded with 200 mM glucose by four washings in isotonic NaCl solution containing 200 mM glucose. One hundred and fifty microliters of these preloaded cells were incubated a t 20°C in 10 ml medium consisting of isotonic phosphate buffer pH 7.5 with different concentrations of C14-glucoseand local anesthetics as indicated in the various experiments. After five seconds the incubation was stopped by pouring the suspension into 80 ml ice cold stopping solution (2 mM HgCl,, 310 mM NaC1, 1.25 mM KJ). After centrifugation and washing the inside of the tube with the stopping solution at O"C, the erythrocytes were hemolysed in aqua bidest and the proteins precipitated according to Somogyi ('45). The radioactivity of the C14-glucosewas determined by liquid scintillation counting. For details of the experimental procedure see Lacko et al. ("72). Commercial preparations of the following local anesthetics were used in the experiments: procaine HC1 (Merck, Darmstadt), brufacaine HC1 (Doctor Christian Brunnengraber, Liibeck), dibucaine HC1 syn. cinchocaine (Ciba-Geigy, Wehr/Baden), mepivacaine HCl (ICN Pharmaceuticals GmbH and Co., Eschwege), butacaine-hemisulfate (Sigma, Miinchen), oxybuprocaine HCl (Wander GmbH Frankfurt/Main), baycaine HCl syn. tolycaine (Bayer, Leverkusen) , lidocaine MATERIAL AND METHODS HC1 (Welding and Co., Hamburg) and proHCl, tetracaine HC1, hostaHuman blood of healthy donors was col- cainamide lected in ACD solution (11 gm sodium citrate, caine-phosphate syn. butacetoluid, ultracaine Received Nov. 1, '76. Accepted Jan. 11. '77. 35 gm glucose, 4 gm citric acid with aqua
Local anesthetics are chemical compounds which produce a wide variety of effects on biological processes. First of all, the anesthetic action on excitable membranes by blocking the sodium current in nerve (Ritchie and Greengard, '66) should be mentioned. Additional effects include the induction of membrane expansion (Seeman, '70), the modification of osmotic fragility of erythrocytes (Roth and Seeman, '71), the inhibition of firefly luminiscence (Ueda et al., '76), the effect on phospholipases (Kunze et al., '76) etc. (Browning and Nelson, '76; Poste et al., '75). Some of these effects are tightly linked to the function of cell membranes. Although much attention has been focused on the mode of action of local anesthetics (Papahadjopoulos et al., '75; Mercki et al., '75), this problem has not yet been solved. The assumption can be made, however, that the lipids of the cell membranes play a major role in these processes. According to our findings, membrane lipids are also important for the inhibition of glucose transport by alcohols (Lacko et al., '74) and steroids (Lacko et al., '75). So, one can suppose t h a t local anesthetics will also influence the glucose uptake in erythrocytes. Results on this topic would be of interest not only for developing the concept of the glucose transport in erythrocytes but also for the interaction of local anesthetics with cell membranes (Koblin et al., '75).
.
-
.
.
.
J. CELL. PHYSIOL.,92: 257-264.
.
.
-
.
257
258
L. LACKO, B. WI'ITKE AND I. LACKO
HC1 (Farbwerke Hoechst AG, Frankfurt/ Main). The local anesthetics were dissovled in 0.9% NaCl solution in the described concentrations and added to the incubation medium. To determine the Rf-values thin layer chromatography was carried out on glass plates (20 x 20 ern) coated with a 0.25-mm thick layer of silica gel 60 FZs4(Merck, Darmstadt). The 12 compounds dissolved in water were layered onto the plate (100 pg/spot) and developed for 6.5 hours in a solvent consisting of the upper saturated phase of a mixture of nbutanollacetic-acid/water (4:1:5). a
RESULTS
The effect of the different locl anesthetics on the exchange transport of glucose The investigated local anesthetics contain a lipophilic radical, a n intermediate chain and a hydrophilic secondary or tertiary amino group. Their chemical formulas are shown in table 1. The pK value of the amino group on the benzene ring (if present) is about 2.2 (Killian, '73) which implies that this group is practically not protonized in our experimental conditions. The amino group a t the other end of the molecule has pK values in the range of 8.0 to 9.3 (Buchi and Perlia, '71) indicating that the proportions of the protonized to the non protonized group depend on the pH of the medium. of the The relative inhibition (1 - 3) VO
glucose exchange transport has been studied a t different pH values of the medium. With the glucose concentration at 0.033 mM, the local anesthetic concentrations were adjusted to such values to get reasonable relative inhibitions for the studied pH range, that means the stronger inhibitors were used in lower concentrations and vice versa. It can be seen from table 1that the relative inhibitions of all local anesthetics were smaller a t pH 4 than a t higher pH values. This points t o smaller inhibition effects of the protonized form than of the non-protonized one.
the non-inhibited reaction v, was estimated a t various [SI concentrations, too. The kinetic data were plotted in three ways: as a Lineweaver-Burk plot 1or -.?.- respectively "I
versus -,1 as a Dixon plot IS1
is1
1 Vo
-
versus [I], and
VI
as - versus [I], as proposed by CornishVI
Bowden (Cornish-Bowden, '74). By this procedure the types of inhibition and the dissociation constants of all investigated local anesthetics (except tetracaine and oxybuprocaine) could be determined unambiguously. The kinetics and K, values of the latter two were clarified by further kinetic analysis. As an illustration of the three types of plots, the inhibitions by procaine, lidocaine and tetracaine are shown in figures 1-3.I t can be seen that procaine produces a competitive inhibition; lidocaine, a non-competitive inhibition of the glucose transport. Tetracaine (and oxybuprocaine-not shown in the figure) is a competitive inhibitor according to the Lineweaver-Burk plot. The other two plots did not give straight lines but curves bending towards the ordinate a s observed with other inhibitors, too (Lacko e t al., '74, '75). The result of the graphical analysis for all local anesthetics investigated are summarized in table 2. The lipid solubility corresponds neither with the apparent affinity nor with the inhibition kinetics of the local anesthetics t o the transport system (table 2), as judged from the Rf-values of the thin layer chromatography (Buchi e t al., '75).
Kinetics of the inhibition by tetracaine and oxy buprocaine We wanted to test whether the bending of the curves (fig. 3) is in agreement with the assumption that two molecules of the inhibitor [I] are bound to one transport site [Cl. In this case, for competitive inhibition the following is valid: IS1 "I = Vm,, iil [I]' ' (1) ISI+& (1 + -+ -1
Kinetics of the inhibition ofglucose exchange K, (2) To characterize the inhibition by local anes- where Ki = 1c1 . [I1 qi = lCIl . I11 ICIl ' ICIIl ' thetics, the relation between the uptake velocity vi and glucose concentration [Sl a t Transforming this relationship a t two concenconstant local anesthetic concentration [I1 trations [I,] and [I,] of a (given) inhibitor and between v, and [I1 a t constant [SI was (similarly as was done for non-competitive studied; the velocity of the glucose uptake of inhibition, Lacko et al., '75) and plotting ,
~
~
259
INHIBITION OF GLUCOSE TRANSPORT BY LOCAL ANESTHETICS TABLE 1
Inhibition ofglucose exchange transport by diferent local anesthetics S t r u c t u r a l .oea1.nesthe t ic s
Aromatic residue
I
oncenration
F o r m u l a s
Intermediate chain
1
Amino group
(mM) . . -
45.0
36.0
'rocainarnide
Relative Inhibition In 9
40
PH
PH 75'
PH 9.0
55
59
2s
45
54
1
46
52
7
43
az
11
15.0
lepivacaine
12.0
'rocaine
.idocaine
8.0
9
40
64
Hostacaine
6.0
1
48
80
Jltracaine
4.5
0
42
60
Iletracaine
1.5
0
41
81
3.0
3
43
65
1.2
4
40
78
1.6
5
42
80
35
44
,CH,-
Baycaine
CH,
kybuprocaine
\
c y- cH,
PIltacaine
Xbucaine
C 0 - N H - C ;H CHa-
t, calculated from Ki, except tetracaine and oxybuprocaine.
CH;
CH,
0.35
5
260
L. LACKO, B. WITTKE AND I. LACKO
A
-viS
-1
1
V
6
Vi
5-
-v1
c
5-
A
-1
B
vi
5-
100
-S
C
vi
-
5-
:k -
rl "i
3
2
1
w
@
&-tMGLucose)-4
12
2P
I (mM Tetracaine)
w
1,2 2,0 I ( m M Tetracaine)
Figs. 1-2-3 Kinetics of procaine, lidocaine and tetracaine-inhibition of the glucose transport. Erythrocytes were preloaded with 200 mM glucose. The incubation was carried out for five seconds a t 2 0 T in phosphate buffer (pH 7.5) a t various concentrations of glucose and inhibitors, as described in A,B,C. A Double reciprocal plot of glucose uptake. The incubation medium contained five different C "-glucose concentrations between 0.5 and 2.5 mM with ( 0 )and without (0) the inhibitor. The inhibitor concentrations were: figure 1: 12 mM procaine; figure 2: 8 mM lidocaine and figure 3: 1.2 mM tetracaine. The reciprocal value of t h e uptake velocity, sec. mM I , of the glucose is plotted against the reciprocal value of the glucose concentration in the incubation medium. B The dependence of glucose exchange flux on the inhibitor concentrations [I].The incubation medium contained the following glucose and inhibitor concentrations: in figure 1: W 1 mM glucose, A 3 mM glucose and t h e procaine concentrations between 5 and 20 mM; in figure 2: 0.033 mM glucose, A 0.066 mM glucose and lidocaine concentrations between 3 and 15 mM; in figure 3: A 0.5 mM glucose, 1.0 mM glucose, 0 2.0 mM glucose and the tetracaine concentrations between 0.4 and 2.0 mM. The reciprocal value of the uptake velocity, sec. mM-', of glucose is plotted against the inhibitor concentration 111. C The reciprocal value of the relative flux (@-I against the inhibitor concentration [I]. Values and symVi
bols for figures 1 and 3 as in B and for figure 2: A 0.066mM,
+ 1.0 mM,
3 mM glucose.
261
INHIBITION OF GLUCOSE TRANSPORT BY LOCAL ANESTHETICS TABLE 2
The mode of inhibition and Ki values ofglucose uptake from different local anesthetics Local anesthetics bound
Local anesthetic
Type of inhibition
Brufacaine Procainamide Mepivacaine Procaine Lidocaine Hostacaine Ultracaine Tetracaine Baycaine Oxybuprocaine Butacaine Dibucaine
Competitive Competitive Competitive Competitive Non-competit ive Competitive Competitive Competitive Non-competitive Competitive Competitive Competitive
against the sum [I,] + [I,] gives a straight line with the intercept (1 on )the ordinate KI
and the intercept
-K,, on the abscissa;
+ Km ). Km From figure 4, one can see that for both tetracaine and oxybuprocaine the inhibition kinetics correspond to the binding of two molecules to one transport site. The values for tetracaine were K, = 5.6 mM, K,, = 0.9 mM and for oxybuprocaine K, = 2.5 mM and K,, = 1.0 mM. Since for both inhibitors K,, < K, it has to be concluded that the binding of the first inhibitor molecule facilitates the binding of the second one (Lacko et al., '74). (f =- IS1
Combined action of a competitive inhibitor and a non-competitive one We wanted to find out whether the competitive inhibitors tetracaine and oxybuprocaine also exhibit a non-competitive component. We estimated the uptake velocity of glucose with the non-competitive inhibitors lidocaine and baycaine (vJ, with the tested competitive inhibitors tetracaine and oxybuprocaine (v,) and under the combined influence of one of the competitive and one of the non-competitive inhibitors (v,,). The non-inhibited glucose uptake (v,) was also determined. The results were inserted in the formerly derived relationship (Lacko et al., '75): -1 + - 1
Ki (mM)
54.0 45.0 18.0 16.0 12.0 6.6 6.3 5.6 4.0 2.5 2.2 0.33
1 1 1 1 1
1 1 2 1
2 1 1
Kii (mM)
Rpvalues
-
0.25 0.22 0.37 0.30 0.36 0.58 0.51 0.38 0.33 0.39 0.54 0.42
-
0.9
1.0
-
the competitive and the non-competitive inhibitors do not influence each others' binding and action: the tested inhibitor shows only one component. If @ < 1and T = 1, the binding of one type of inhibitor is prevented by the binding of the other one. From table 3 it can be seen that oxybuprocaine in contrast to tetracaine shows a non-competitive component, too. This implies that oxybuprocaine also binds to some
/A
-1,5
@
-0,5
I
I
0,s
I
1,5
4,5
Fig. 4 Graphical evaluation to determine the dissociation constants of tetracaine (line 1) and oxybuprocaine (line 2). The glucose concentrations are A 0.5 mM, 1.0 mM and 0 2.0 mM. I + I, is the sum of different concentrations of one inhibitor. f is a constant, calculated from [" Km ; it is for 0.5 mM glucose 1.2, for 1.0 mM glucose +
vj
3,5
1,+ xi (mM I
Km
Vi
23
1.4 and for 2.0 mM glucose 1.8.
262
L. LACKO. B. WITTKE AND I. LACKO TABLE :I
Combined action ofbaycaine or lidocaine respectively with oxybuprocaine or tetracaine respectively in theglucose exchange transport rt,
v 1.05 1.05 1.03 1.05 1.12 1.15 1.10 1.15
Baycaine
+ oxybuprocaine
0.80 0.82
Lidocaine
+ oxybuprocaine
0.82 0.82
Baycaine
+ tetracaine
Lidocaine
+ tetracaine
1.04 0.99 1.00 0.99
The glucose concentration was 0.033 rnM and the inhibitor concentration: for baycaine 3 mM. for lidocaine 8mM, for oxybuprocaine 12 m M and for tetracaine 1.5 mM
extent t o the non-competitive site and prevents the binding of lidocaine or baycaine. DISCUSSION
Most of the investigated local anesthetics exerted (like the steroids, Lacko et al., '75) a competitive inhibition kinetic; only two out of 12 local anesthetics showed non-competitive ones. The non-competitive inhibitors lidocaine and baycaine are characterized by the following features: they have no amino group on the benzene ring, the intermediate chain is - NH - CO - CH2- and the tertiary aminogroup is - N
