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Membrane-buffer partition coefficients of a local anesthetic tetracaine monitored by an anesthetic sensor; effects of temperature and pH
Toxicology Letters 100 – 101 (1998) 441 – 445
Membrane-buffer partition coefficients of a local anesthetic tetracaine monitored by an anesthetic sensor; effects of temperature and pH Hiromu Satake a, Takuo Kageyama b, Hitoshi Matsuki b, Shoji Kaneshina b,* b
a Center for Cooperati6e Research, The Uni6ersity of Tokushima, Minamijosanjima, Tokushima 770, Japan Department of Biological Science and Technology, Faculty of Engineering, The Uni6ersity of Tokushima, Minamijosanjima, Tokushima 770, Japan
Accepted 7 May 1998
Abstract Binding of a local anesthetic tetracaine (TC) to dimyristoylphosphatidylcholine (DMPC) bilayer membrane was studied by the potentiomerty with an ion-selective electrode sensitive to TC cation. DMPC membrane-buffer partition coefficient (Kapp) was determined in mole fraction unit as a function of pH for the lamellar gel (at 12°C), ripple gel (at 20°C), and liquid crystal (at 30°C) phases. The partition coefficients of charged (K + ) and uncharged TC (K0) into the DMPC membranes were estimated from the pH-dependence of Kapp. The three states of DMPC membranes were more receptive to the uncharged TC than the charged species. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Anesthetic sensor; Bilayer membrane; Local anesthetic; Partition coefficient; Tetracaine
1. Introduction Anesthetic partitioning into lipid bilayer membranes has been studied by the methods of ultraviolet spectroscopy (Boulanger et al., 1980; Seelig et al., 1988), microelectrophoresis (Ohki, 1984), 2 H-NMR (Kelusky and Smith, 1983, 1984), dualradiolabel centrifuge (Janes et al., 1992), equilibrium dialysis (Fukushima et al., 1993, 1994), * Corresponding author. fax + 81 886 553162.
and ion-selective electrodes (Kitagawa et al., 1995; Satake et al., 1995). However, the contribution of charged- and uncharged-anesthetics to the membrane-buffer partition coefficients has not been elucidated at all. We have developed a small, thin coated-wire electrode sensitive to local anesthetic cations (Satake et al., 1991; Yokono et al., 1992). In this study, partition of local anesthetic tetracaine into a dimyristoylphosphatidylcholine (DMPC) bilayer membrane system is analyzed by using the coated-wire electrode selectively sensi-
0378-4274/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00218-5
442
H. Satake et al. / Toxicology Letters 100–101 (1998) 441–445
tive to tetracaine cation. It is well known that DMPC multilamellar vesicle undergoes the thermotropic pretransition at 14°C from the lamellar gel (Lb% ) phase to the ripple gel (Pb% ) phase, as well as the main-transition at 24°C from the Pb% phase to the liquid crystal (La ) phase. Three states of bilayer membranes are known to have a different receptivity to anesthetic partitioning (Simon et al., 1979; Janes et al., 1992). The present study describes the partition coefficient of tetracaine as a function of pH for the Lb% , Pb% and La% phases of DMPC bilayer membranes. Moreover, we focus our attention on pH-dependence of partition coefficients in order to elucidate the contribution of charged and uncharged-tetracaine to the membrane-buffer partition coefficients for the three states of membranes.
was measured relative to an Ag–AgCl reference electrode in the following cell system: TC electrode/test solution/isotonic NaCl solution (ISCS) agar-bridge/ISCS/Ag –AgCl electrode. The EMF was measured with a digital multi-ion monitor (Yamashita Giken, Tokushima, Japan) at various temperatures. A calibration curve of the EMF against logarithm of TC concentration at 25°C and pH 5.5 showed a linear response with a Nernstian slope over the concentration range of 10 − 6 –10 − 3 mol dm − 3. The pH of solution was measured simultaneously by a pH meter (Denki Kagaku Keiki, Model HPL-40, Tokyo, Japan). All of the solutions were prepared in the ISCS.
2. Experimental
3.1. Membrane-buffer partition coefficient
Tetracaine hydrochloride (TC HCl), 2(dimethylamino)ethyl-4-(butylamino) benzoate hydrochloride, was obtained from Sigma (St. Louis, MO) in the crystalline form and was used without further purification. Synthetic DMPC, 1,2ditetradecanoyl-sn-glycero-3-phosphocholine, was purchased from Sigma. Water was purified by triple distillation, once from alkaline potassium permanganate solution. All reagents used in the preparation of the electrode and the solutions were of the analytical grade. The DMPC multilamellar vesicle was prepared by suspending DMPC in buffer solution using a Branson model 185 sonifier and cup horn. The DMPC concentration was 5.0 mmol dm − 3. The coated-wire electrode selectively sensitive to tetracaine (TC) cation was prepared by the same method described previously (Satake et al., 1991). A copper wire (0.55 mm diameter) was dipped in the coating mixture and coated with sensor membrane of 0.4 mm in thickness. The solution of the coating mixture contained poly(vinylchloride) membrane matrix (110 mg), o-nitrophenyloctyl ether plasticizer (100 mg), TCdodecatungstophosphate ion-pair (2 mg) and tetrahydrofuran solvent (1.5 ml). The electromotive force (EMF) of the TC cation-selective electrode
Typical EMF curves as a function of pH at 30°C are shown in Fig. 1. In the absence of DMPC vesicle (Curves 1 and 2), the EMF in tetracaine solutions at a constant concentration is almost constant at below pH 7.0, and then goes down as the pH increases. Since the TC-electrode is sensitive to TC cation and not sensitive to uncharged TC, the depression of EMF at above pH 7.0 is attributable to the decrease of TCcation by the dissociation equilibrium. In the presence of DMPC vesicle (Curve 3 in Fig. 1), the EMF at the same concentration of TC goes down forward the lower concentration of TC, which means the decrease of the concentration of free TC-cation due to the binding of DMPC bilayer membranes. By using a pair of EMF versus pH curve (Curves 1 and 3 in Fig. 1) and calibration curves of the electrode response at respective values of pH, we can evaluate the concentration of free TC cation, Cf (mol dm − 3), in binding equilibrium with DMPC vesicles at the total concentration, Ct (mol dm − 3). Thus, the amounts of tetracaine bound to DMPC vesicle can be determined at various values of pH and temperatures. The mole fraction of TC in the DMPC bilayer W membrane, X m A , and in the buffer solution, X A , are given by
3. Results and discussion
H. Satake et al. / Toxicology Letters 100–101 (1998) 441–445
Xm A=
Ct −Cf (Ct − Cf)+Cm
(1)
Cf 55.5+Cs +Cf
(2)
XW A =
443
where Cm and Cs are the concentrations of DMPC and buffer electrolytes in mol · dm − 3. Here, we can define the apparent partition coefficient of TC between DMPC bilayer membrane and buffer solution as Kapp =
Xm A XW A
(3)
The apparent partition coefficients of tetracaine between DMPC bilayer membrane and buffer solution are shown in Fig. 2 as a function of pH at 12, 20 and 30°C. It is well known that the DMPC bilayer membrane undergoes the thermotropic main transition and pretransition at 24 and 14°C,
Fig. 2. Apparent partition coefficients (Kapp) of tetracaine into DMPC bilayer membranes in the states of lamellar gel (at 12°C), ripple gel (at 20°C) and liquid crystal (at 30°C). Kapp is shown as a function of pH.
Fig. 1. EMF curves of TC electrode (against Ag–AgCl electrode) as a function of pH at 30°C. The concentration of free anesthetic cation (Cf) in binding equilibrium with vesicle at the total concentration of tetracaine (Ct) can be determined by a calibration curve of electrode response at any pH. EMF curves: (1) at 10 − 4 M TC (in ISCS); (2) at 10 − 5 M TC (in ISCS); and (3) at 10 − 4 M TC in the presence of DMPC vesicle (in ISCS).
respectively (Mabrey and Sturtevant, 1976; Stu¨mple et al., 1981; Lewis et al., 1987). Therefore, DMPC bilayer membrane is in the state of the lamellar gel phase at 12°C, the ripple gel phase at 20°C and the liquid crystalline phase at 30°C. As is seen from Fig. 2, each of the three states of DMPC bilayer membrane exhibits a different receptivity to tetracaine partitioning. The value of Kapp at any pH increased in the order for the three states: Lb% B Pb% B La. Some of the partition coefficients so far studied have been determined indirectly from the depression of the phase transition temperature based on the thermodynamic colligative property (Hill, 1974; Ueda et al., 1977; Lee, 1978; Kamaya et al., 1981; Rowe, 1983; Kaminoh et al., 1988, 1989). Thermodynamic treatment has been sometimes assumed that the anesthetic molecules are completely excluded from the gel phase of lipid membranes. However, non-zero partition of anesthetics into the gel phase of lipid bilayer membranes has been reported by the direct measurement of partition coefficients (Simon
H. Satake et al. / Toxicology Letters 100–101 (1998) 441–445
444
et al., 1979; Janes et al., 1992). Present method using anesthetic sensor is very useful for the direct determination of partition coefficients of anesthetics into various membranes.
3.2. Effect of pH on partition coefficients As is seen from Fig. 2, the apparent partition coefficient increases with an increase in pH at all of the temperatures. The amounts of tetracaine bound to the DMPC bilayer membrane may include both the positively charged and uncharged species. Since the concentration of the uncharged tetracaine increases with an increase of pH in accordance with the acid – base equilibrium of anesthetic tetracaine, the contribution of uncharged tetracaine to the partition coefficient is more significant at higher pH. Now we estimate the partition coefficients of the charged and uncharged anesthetics into the lipid bilayer membrane from the pH-dependence of the anesthetic binding to the lipid bilayer membrane. The partition coefficients of charged anesthetic (K + ) and uncharged anesthetic (K0) are defined as follows: K+ = K0 =
m X+ W X+
Xm 0 XW 0
(4) (5)
where the subscripts + and 0 denote the charged and uncharged species of anesthetic. Therefore, the apparent partition coefficient can be rewritten by the following equation: Kapp =
m X+ +Xm K +K + · b 0 = 0 W X+ + X W 1 +b 0
(6)
where W X+ b= W =10(pKa − pH) X0
(7)
Consequently, the pH-dependence on Kapp can be written by the following equation: Kapp(1 +b)= K0 +K + · b
(8)
which includes two kinds of partition coefficients for the charged and uncharged anesthetics.
Fig. 3. Plots of Kapp (1 +b) against b. The partition coefficients of uncharged anesthetics (K0) and charged anesthetics (K + ) can be obtained from the intercept of the ordinate and the slope for each line, respectively.
In order to calculate the values of b, we employed the values of ionization constant pKa for tetracaine to be 8.63 at 12°C, 8.51 at 20°C and 8.37 at 30°C (Kamaya et al., 1983). The plots of Kapp (1+b) versus b gave a good linear relationship as shown in Fig. 3. A set of partition coefficients, K + and K0, were determined at different temperatures, which are summarized in Table 1. The partition coefficient of uncharged tetracaine into DMPC bilayer membrane is larger than that of charged tetracaine at all of the temperatures. In other words, the DMPC membrane is more receptive to the uncharged anesthetics than the charged anesthetics. The membrane-buffer partition coefficients for both the charged and uncharged tetraTable 1 Partition coefficients of charged and uncharged tetracaine into DMPC bilayer membranes in the states of lamellar gel (at 12°C), ripple gel (at 20°C) and liquid crystal (at 30°C) Temperature/°C
Membrane
K+/103
K0/105
12 20 30
Lamellar gel Ripple gel Liquid crystal
0.19 2.79 3.62
0.34 0.43 1.16
H. Satake et al. / Toxicology Letters 100–101 (1998) 441–445
caine increase stepwise as the bilayer membrane is transformed from the Lb% phase into the Pb% phase, and sequentially into the La phase. Tertiary amine local anesthetics exist as uncharged and charged species in physiologic solution. These forms interact in various modes with phospholipids and proteins in nerve membranes: uncharged anesthetics preferentially partition into hydrophobic membrane interior, whereas cationic anesthetics adsorb at the negatively charged membrane surface, through hydrophobic and electrostatic interactions. As is shown in the present results, the largest partition coefficient of uncharged tetracaine into the liquid crystalline phase of DMPC bilayer membrane is consistent with the idea for the molecular mechanisms of anesthetic action.
References Boulanger, Y., Schreier, S., Leitch, L.C., Smith, I.C.P., 1980. Multiple binding sites for local anesthetics in membranes: characterization of the sites and their equilibria by deuterium NMR of specifically deuterated procaine and tetracaine. Can. J. Biochem. 58, 986–995. Fukushima, K., Someya, M., Shimozawa, R., 1993. Binding of dibucaine to phospholipid mixed vesicles. Bull. Chem. Soc. Jpn. 66, 1613 – 1617. Fukushima, K., Someya, M., Shimozawa, R., 1994. Binding of local anesthetic tetracaine to phospholipid mixed vesicles. Bull. Chem. Soc. Jpn. 67, 2079–2085. Hill, M.W., 1974. The effect of anesthetic-like molecules on the phase transition in smectic mesophases of dipalmitoyllecithin I. The normal alcohol up to C= 9 and three inhalation anesthetics. Biochim. Biophys. Acta 356, 117– 124. Janes, N., Hsu, J.W., Rubin, E., Taraschi, T.F., 1992. Nature of alcohol and anesthetic action on cooperative membrane equilibria. Biochemistry 31, 9467–9472. Kamaya, H., Kaneshina, S., Ueda, I., 1981. Partition equilibrium of inhalation anesthetics and alcohols between water and membranes of phospholipids with varying acyl chainlengths. Biochim. Biophys. Acta 646, 135–142. Kamaya, H., Hayes, J.J. Jr., Ueda, I., 1983. Dissociation constants of local anesthetics and their temperature dependence. Anesth. Analg. 62, 1025–1030. Kaminoh, Y., Tashiro, C., Kamaya, H., Ueda, I., 1988. Depression of phase-transition temperature by anesthetics: nonzero solid membrane binding. Biochim. Biophys. Acta 946, 215 – 220. Kaminoh, Y., Kamaya, H., Ueda, I., 1989. Differential afffinity of charged local anesthetics to solid-gel and liquid-crystalline
445
states of dimyristoylphosphatidic acid vesicle membranes. Biochim. Biophys. Acta 987, 63 – 68. Kelusky, E.C., Smith, I.C.P., 1983. Characterization of the binding of the local anesthetics procaine and tetracaine to model membranes of phosphatidylethanolamine: a deuterium nuclear magnetic resonance study. Biochemistry 22, 6011 – 6017. Kelusky, E.C., Smith, I.C.P., 1984. Anesthetic-membrane interaction: a 2H nuclear magnetic resonance study of the binding of specifically deuterated tetracaine and procaine to phosphatidylcholine. Can. J. Biochem. Cell Biol. 62, 178 – 184. Kitagawa, N., Takasaki, M., Totoki, T., Kamaya, H., Ueda, I., 1995. Binding of charged local anesthetics to charged and uncharged lipids. Prog. Anesth. Mechanism 3 (Special issue), 253 – 256. Lee, A.G., 1978. Effects of charged drugs on the phase transition temperatures of phospholipid bilayers. Biochim. Biophys. Acta 514, 95 – 104. Lewis, R.N.A.H., Mak, N., McElhaney, R.N., 1987. A differential scanning calorimetric study of the thermotropic phase behavior of model membranes composed of phosphatidylcholines containing linear saturated fatty acyl chains. Biochemistry 26, 6118 – 6126. Mabrey, S., Sturtevant, J.M., 1976. Investigation of phase transitions of lipids and lipid mixtures by high sensitivity differential scanning calorimetry. Proc. Natl. Acad. Sci. USA 73, 3862 – 3866. Ohki, S., 1984. Adsorption of local anesthetics on phospholipid membranes. Biochim. Biophys. Acta 777, 56 – 66. Rowe, E.S., 1983. Lipid chain length and temperature dependence of ethanol-phosphatidylcholine interactions. Biochemistry 22, 3299 – 3305. Satake, H., Miyata, T., Kaneshina, S., 1991. Coated wire electrodes sensitive to local anesthetic cations and their application to potentiometric determination. Bull. Chem. Soc. Jpn. 64, 3029 – 3034. Satake, H., Yamamoto, T., Matsuki, H., Kaneshina, S., 1995. Bilayer membrane-buffer partition coeffficient of a local anesthetic dibucaine by a method of anesthetic sensor. Prog. Anesth. Mechanism 3 (Special issue), 241 – 246. Seelig, A., Allegrini, P.R., Seelig, J., 1988. Partitioning of local anesthetics into membranes: surface charge effects monitored by the phospholipid head-group. Biochim. Biophys. Acta 939, 267 – 276. Simon, S.A., McIntosh, T.J., Bennett, P.B., Shrivastav, B.B., 1979. Interaction of halothane with lipid bilayers. Mol. Pharmacol. 16, 163 – 170. Stu¨mple, J., Nicksch, A., Eibl, H., 1981. Calorimetric studies on saturated mixed-chain lecithin-water systems. Non equivalence of acyl chains in the thermotropic phase transition. Biochemistry 20, 662 – 665. Ueda, I., Tashiro, C., Arakawa, K., 1977. Depression of phase-transition temperature in a model cell membrane by local anesthetics. Anesthesiology 46, 327 – 332. Yokono, A., Satake, H., Kaneshina, S., Yokono, S., Ogli, K., 1992. Local anesthetic-sensitive electrodes: preparation of coated-wire electrodes and their basic properties in vitro. Anesth. Analg. 75, 1063 – 1069.
Membrane-buffer partition coefficients of a local anesthetic tetracaine monitored by an anesthetic sensor; effects of temperature and pH Hiromu Satake a, Takuo Kageyama b, Hitoshi Matsuki b, Shoji Kaneshina b,* b
a Center for Cooperati6e Research, The Uni6ersity of Tokushima, Minamijosanjima, Tokushima 770, Japan Department of Biological Science and Technology, Faculty of Engineering, The Uni6ersity of Tokushima, Minamijosanjima, Tokushima 770, Japan
Accepted 7 May 1998
Abstract Binding of a local anesthetic tetracaine (TC) to dimyristoylphosphatidylcholine (DMPC) bilayer membrane was studied by the potentiomerty with an ion-selective electrode sensitive to TC cation. DMPC membrane-buffer partition coefficient (Kapp) was determined in mole fraction unit as a function of pH for the lamellar gel (at 12°C), ripple gel (at 20°C), and liquid crystal (at 30°C) phases. The partition coefficients of charged (K + ) and uncharged TC (K0) into the DMPC membranes were estimated from the pH-dependence of Kapp. The three states of DMPC membranes were more receptive to the uncharged TC than the charged species. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Anesthetic sensor; Bilayer membrane; Local anesthetic; Partition coefficient; Tetracaine
1. Introduction Anesthetic partitioning into lipid bilayer membranes has been studied by the methods of ultraviolet spectroscopy (Boulanger et al., 1980; Seelig et al., 1988), microelectrophoresis (Ohki, 1984), 2 H-NMR (Kelusky and Smith, 1983, 1984), dualradiolabel centrifuge (Janes et al., 1992), equilibrium dialysis (Fukushima et al., 1993, 1994), * Corresponding author. fax + 81 886 553162.
and ion-selective electrodes (Kitagawa et al., 1995; Satake et al., 1995). However, the contribution of charged- and uncharged-anesthetics to the membrane-buffer partition coefficients has not been elucidated at all. We have developed a small, thin coated-wire electrode sensitive to local anesthetic cations (Satake et al., 1991; Yokono et al., 1992). In this study, partition of local anesthetic tetracaine into a dimyristoylphosphatidylcholine (DMPC) bilayer membrane system is analyzed by using the coated-wire electrode selectively sensi-
0378-4274/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00218-5
442
H. Satake et al. / Toxicology Letters 100–101 (1998) 441–445
tive to tetracaine cation. It is well known that DMPC multilamellar vesicle undergoes the thermotropic pretransition at 14°C from the lamellar gel (Lb% ) phase to the ripple gel (Pb% ) phase, as well as the main-transition at 24°C from the Pb% phase to the liquid crystal (La ) phase. Three states of bilayer membranes are known to have a different receptivity to anesthetic partitioning (Simon et al., 1979; Janes et al., 1992). The present study describes the partition coefficient of tetracaine as a function of pH for the Lb% , Pb% and La% phases of DMPC bilayer membranes. Moreover, we focus our attention on pH-dependence of partition coefficients in order to elucidate the contribution of charged and uncharged-tetracaine to the membrane-buffer partition coefficients for the three states of membranes.
was measured relative to an Ag–AgCl reference electrode in the following cell system: TC electrode/test solution/isotonic NaCl solution (ISCS) agar-bridge/ISCS/Ag –AgCl electrode. The EMF was measured with a digital multi-ion monitor (Yamashita Giken, Tokushima, Japan) at various temperatures. A calibration curve of the EMF against logarithm of TC concentration at 25°C and pH 5.5 showed a linear response with a Nernstian slope over the concentration range of 10 − 6 –10 − 3 mol dm − 3. The pH of solution was measured simultaneously by a pH meter (Denki Kagaku Keiki, Model HPL-40, Tokyo, Japan). All of the solutions were prepared in the ISCS.
2. Experimental
3.1. Membrane-buffer partition coefficient
Tetracaine hydrochloride (TC HCl), 2(dimethylamino)ethyl-4-(butylamino) benzoate hydrochloride, was obtained from Sigma (St. Louis, MO) in the crystalline form and was used without further purification. Synthetic DMPC, 1,2ditetradecanoyl-sn-glycero-3-phosphocholine, was purchased from Sigma. Water was purified by triple distillation, once from alkaline potassium permanganate solution. All reagents used in the preparation of the electrode and the solutions were of the analytical grade. The DMPC multilamellar vesicle was prepared by suspending DMPC in buffer solution using a Branson model 185 sonifier and cup horn. The DMPC concentration was 5.0 mmol dm − 3. The coated-wire electrode selectively sensitive to tetracaine (TC) cation was prepared by the same method described previously (Satake et al., 1991). A copper wire (0.55 mm diameter) was dipped in the coating mixture and coated with sensor membrane of 0.4 mm in thickness. The solution of the coating mixture contained poly(vinylchloride) membrane matrix (110 mg), o-nitrophenyloctyl ether plasticizer (100 mg), TCdodecatungstophosphate ion-pair (2 mg) and tetrahydrofuran solvent (1.5 ml). The electromotive force (EMF) of the TC cation-selective electrode
Typical EMF curves as a function of pH at 30°C are shown in Fig. 1. In the absence of DMPC vesicle (Curves 1 and 2), the EMF in tetracaine solutions at a constant concentration is almost constant at below pH 7.0, and then goes down as the pH increases. Since the TC-electrode is sensitive to TC cation and not sensitive to uncharged TC, the depression of EMF at above pH 7.0 is attributable to the decrease of TCcation by the dissociation equilibrium. In the presence of DMPC vesicle (Curve 3 in Fig. 1), the EMF at the same concentration of TC goes down forward the lower concentration of TC, which means the decrease of the concentration of free TC-cation due to the binding of DMPC bilayer membranes. By using a pair of EMF versus pH curve (Curves 1 and 3 in Fig. 1) and calibration curves of the electrode response at respective values of pH, we can evaluate the concentration of free TC cation, Cf (mol dm − 3), in binding equilibrium with DMPC vesicles at the total concentration, Ct (mol dm − 3). Thus, the amounts of tetracaine bound to DMPC vesicle can be determined at various values of pH and temperatures. The mole fraction of TC in the DMPC bilayer W membrane, X m A , and in the buffer solution, X A , are given by
3. Results and discussion
H. Satake et al. / Toxicology Letters 100–101 (1998) 441–445
Xm A=
Ct −Cf (Ct − Cf)+Cm
(1)
Cf 55.5+Cs +Cf
(2)
XW A =
443
where Cm and Cs are the concentrations of DMPC and buffer electrolytes in mol · dm − 3. Here, we can define the apparent partition coefficient of TC between DMPC bilayer membrane and buffer solution as Kapp =
Xm A XW A
(3)
The apparent partition coefficients of tetracaine between DMPC bilayer membrane and buffer solution are shown in Fig. 2 as a function of pH at 12, 20 and 30°C. It is well known that the DMPC bilayer membrane undergoes the thermotropic main transition and pretransition at 24 and 14°C,
Fig. 2. Apparent partition coefficients (Kapp) of tetracaine into DMPC bilayer membranes in the states of lamellar gel (at 12°C), ripple gel (at 20°C) and liquid crystal (at 30°C). Kapp is shown as a function of pH.
Fig. 1. EMF curves of TC electrode (against Ag–AgCl electrode) as a function of pH at 30°C. The concentration of free anesthetic cation (Cf) in binding equilibrium with vesicle at the total concentration of tetracaine (Ct) can be determined by a calibration curve of electrode response at any pH. EMF curves: (1) at 10 − 4 M TC (in ISCS); (2) at 10 − 5 M TC (in ISCS); and (3) at 10 − 4 M TC in the presence of DMPC vesicle (in ISCS).
respectively (Mabrey and Sturtevant, 1976; Stu¨mple et al., 1981; Lewis et al., 1987). Therefore, DMPC bilayer membrane is in the state of the lamellar gel phase at 12°C, the ripple gel phase at 20°C and the liquid crystalline phase at 30°C. As is seen from Fig. 2, each of the three states of DMPC bilayer membrane exhibits a different receptivity to tetracaine partitioning. The value of Kapp at any pH increased in the order for the three states: Lb% B Pb% B La. Some of the partition coefficients so far studied have been determined indirectly from the depression of the phase transition temperature based on the thermodynamic colligative property (Hill, 1974; Ueda et al., 1977; Lee, 1978; Kamaya et al., 1981; Rowe, 1983; Kaminoh et al., 1988, 1989). Thermodynamic treatment has been sometimes assumed that the anesthetic molecules are completely excluded from the gel phase of lipid membranes. However, non-zero partition of anesthetics into the gel phase of lipid bilayer membranes has been reported by the direct measurement of partition coefficients (Simon
H. Satake et al. / Toxicology Letters 100–101 (1998) 441–445
444
et al., 1979; Janes et al., 1992). Present method using anesthetic sensor is very useful for the direct determination of partition coefficients of anesthetics into various membranes.
3.2. Effect of pH on partition coefficients As is seen from Fig. 2, the apparent partition coefficient increases with an increase in pH at all of the temperatures. The amounts of tetracaine bound to the DMPC bilayer membrane may include both the positively charged and uncharged species. Since the concentration of the uncharged tetracaine increases with an increase of pH in accordance with the acid – base equilibrium of anesthetic tetracaine, the contribution of uncharged tetracaine to the partition coefficient is more significant at higher pH. Now we estimate the partition coefficients of the charged and uncharged anesthetics into the lipid bilayer membrane from the pH-dependence of the anesthetic binding to the lipid bilayer membrane. The partition coefficients of charged anesthetic (K + ) and uncharged anesthetic (K0) are defined as follows: K+ = K0 =
m X+ W X+
Xm 0 XW 0
(4) (5)
where the subscripts + and 0 denote the charged and uncharged species of anesthetic. Therefore, the apparent partition coefficient can be rewritten by the following equation: Kapp =
m X+ +Xm K +K + · b 0 = 0 W X+ + X W 1 +b 0
(6)
where W X+ b= W =10(pKa − pH) X0
(7)
Consequently, the pH-dependence on Kapp can be written by the following equation: Kapp(1 +b)= K0 +K + · b
(8)
which includes two kinds of partition coefficients for the charged and uncharged anesthetics.
Fig. 3. Plots of Kapp (1 +b) against b. The partition coefficients of uncharged anesthetics (K0) and charged anesthetics (K + ) can be obtained from the intercept of the ordinate and the slope for each line, respectively.
In order to calculate the values of b, we employed the values of ionization constant pKa for tetracaine to be 8.63 at 12°C, 8.51 at 20°C and 8.37 at 30°C (Kamaya et al., 1983). The plots of Kapp (1+b) versus b gave a good linear relationship as shown in Fig. 3. A set of partition coefficients, K + and K0, were determined at different temperatures, which are summarized in Table 1. The partition coefficient of uncharged tetracaine into DMPC bilayer membrane is larger than that of charged tetracaine at all of the temperatures. In other words, the DMPC membrane is more receptive to the uncharged anesthetics than the charged anesthetics. The membrane-buffer partition coefficients for both the charged and uncharged tetraTable 1 Partition coefficients of charged and uncharged tetracaine into DMPC bilayer membranes in the states of lamellar gel (at 12°C), ripple gel (at 20°C) and liquid crystal (at 30°C) Temperature/°C
Membrane
K+/103
K0/105
12 20 30
Lamellar gel Ripple gel Liquid crystal
0.19 2.79 3.62
0.34 0.43 1.16
H. Satake et al. / Toxicology Letters 100–101 (1998) 441–445
caine increase stepwise as the bilayer membrane is transformed from the Lb% phase into the Pb% phase, and sequentially into the La phase. Tertiary amine local anesthetics exist as uncharged and charged species in physiologic solution. These forms interact in various modes with phospholipids and proteins in nerve membranes: uncharged anesthetics preferentially partition into hydrophobic membrane interior, whereas cationic anesthetics adsorb at the negatively charged membrane surface, through hydrophobic and electrostatic interactions. As is shown in the present results, the largest partition coefficient of uncharged tetracaine into the liquid crystalline phase of DMPC bilayer membrane is consistent with the idea for the molecular mechanisms of anesthetic action.
References Boulanger, Y., Schreier, S., Leitch, L.C., Smith, I.C.P., 1980. Multiple binding sites for local anesthetics in membranes: characterization of the sites and their equilibria by deuterium NMR of specifically deuterated procaine and tetracaine. Can. J. Biochem. 58, 986–995. Fukushima, K., Someya, M., Shimozawa, R., 1993. Binding of dibucaine to phospholipid mixed vesicles. Bull. Chem. Soc. Jpn. 66, 1613 – 1617. Fukushima, K., Someya, M., Shimozawa, R., 1994. Binding of local anesthetic tetracaine to phospholipid mixed vesicles. Bull. Chem. Soc. Jpn. 67, 2079–2085. Hill, M.W., 1974. The effect of anesthetic-like molecules on the phase transition in smectic mesophases of dipalmitoyllecithin I. The normal alcohol up to C= 9 and three inhalation anesthetics. Biochim. Biophys. Acta 356, 117– 124. Janes, N., Hsu, J.W., Rubin, E., Taraschi, T.F., 1992. Nature of alcohol and anesthetic action on cooperative membrane equilibria. Biochemistry 31, 9467–9472. Kamaya, H., Kaneshina, S., Ueda, I., 1981. Partition equilibrium of inhalation anesthetics and alcohols between water and membranes of phospholipids with varying acyl chainlengths. Biochim. Biophys. Acta 646, 135–142. Kamaya, H., Hayes, J.J. Jr., Ueda, I., 1983. Dissociation constants of local anesthetics and their temperature dependence. Anesth. Analg. 62, 1025–1030. Kaminoh, Y., Tashiro, C., Kamaya, H., Ueda, I., 1988. Depression of phase-transition temperature by anesthetics: nonzero solid membrane binding. Biochim. Biophys. Acta 946, 215 – 220. Kaminoh, Y., Kamaya, H., Ueda, I., 1989. Differential afffinity of charged local anesthetics to solid-gel and liquid-crystalline
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