Permeability increase in black lipid membrane induced by compound 4880

Biochimica et Biophysica Acta, 805 (1984) 127-130

127

Elsevier

BBA Report BBA 10023 P E R M E A B I L I T Y I N C R E A S E IN BLACK L I P I D M E M B R A N E I N D U C E D BY C O M P O U N D

48/8o HISASHI SHIBATA,MITSUNOBU MIO and KENJI TASAKA * Department of Pharmacology, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700 (Japan)

(Received February 16th, 1984) (Revised manuscript received June 15th, 1984)

Key words: Black lipid membrane; Conductivity," Compound 48/80; Histamine release," (Mast cell)

When compound 4 8 / 8 0 , a potent histamine liberator, was added in the aqueous phase facing the black lipid membrane, the conductivity of the membrane was remarkably increased. Although valinomycin displayed a distinct selectivity for K + movement, such selection for ionic permeability was not observed in the case of compound 48 / 80.

Since histamine release induced by compound 4 8 / 8 0 (48/80) is similar in som6 way to that observed in allergic reaction, this compound has been extbnsively used to study histamine release from mast cells [1,2]. Hino and co-workers [3] suggested the presence of the receptor for this amine on the surface of mast cells; however, the site of action of 4 8 / 8 0 still remained obscure. Since the uptake and content of Na + and C a 2+ are augmented in histamine release [4-6], the permeability of the mast cell membrane might be increased. The onset of histamine release from mast cells induced by 4 8 / 8 0 rapidly reaches a plateau within 30-60 s at 2 2 ° C [1], so that it can be assumed that biophysical rather than biochemical events may be more important in the process of histamine release. Furthermore, there is some evidence proving that the physical properties of the lipid bilayer in the plasma membrane alter markedly in histamine release [7,8]. The present study was carried out to investigate the interaction between the lipid bilayer and 48/80, using the permeability change of the lipid bilayer as reference.

The electric conductivity of the planar lipid bilayer was measured by the apparatus shown in Fig, 1. A small window was punched in the Teflon wall separating two aqueous phases. Both compartments were filled with salt solution consisting of 154 mM NaC1, 2.7 mM KC1 and 0.9 mM CaC12 and 5 mM Hepes buffer (pH 7.4), and were maintained at 22 o C. Planar lipid bilayer was made by a modification of the method of Hanai et al. [9]. Chromatographically, pure egg yolk phosphatidylcholine (10 mg) and the same weight of cholesterol were dissolved in 0,5 ml of n-decane. This lipid solution was spread over the hole in the Teflon wall through a fine polyethylene tube connected to a microsyringe. The electrical resistance of the black lipid membrane (BLM) was measured by applying 20 mV (d.c.) between two Ag/AgC1 electrodes, one placed in each compartment. As the switch closed, voltage drop through a reference resistor ( R ref ) was recorded as V0. When the switch was opened, V0 decreased exponentially and reached steady-state voltage (Va). The electrical conductance (G) of black lipid membrane was calculated by the following equation:

* To whom correspondenceshould be addressed.

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Groups of five male Wistar rats were used in all experiments. Peritoneal mast cells were collected by the method of Akagi et al. [8]. 10 ml of buffered physiological salt solution were injected into the abdominal cavity and the peritoneal fluid was collected. The cell suspension was preincubated at 2 2 ° C for 5 min to equilibrate and compound 4 8 / 8 0 was added. Histamine release was allowed to proceed for 12 min before the reaction was stopped by placing the test tube in ice-cold water. After centrifugation, histamine in the supernatants was measured by the fluorometric assay of Shore [10] and residual histamine in the cell pellet was determined similarly. The amount of histamine in the supernatant was expressed as a percentage of the total found. When rat mast cells were exposed to 4 8 / 8 0 at concentrations ranging from 0.2 to 2.0 # g / m l , histamine release took place in a dose-dependent fashion, as shown in Fig. 2A. An abrupt increase in histamine release was noticed at low concentrations. The electrical resistance of the black lipid membrane was within the range 2 • 107-7 • 107 ~2. cm 2 and a mean value of 4 - 1 0 7 ~ " cm 2 was determined. The mean electrical capacitance of black lipid membrane was 0.4 # F / c m 2. Fig. 2B shows

Fig. 1. (A) Schematic diagram of apparatus used to measure the membrane conductance of lipid bilayer. The chamber is separated by a Teflon wall which has a 1,5 m m diameter hole. (B) Equivalent circuit of bilayer membrane. (C) Schematic presentation of voltage change.

the changes in electrical conductance of black lipid membrane by the addition of 4 8 / 8 0 into one side of the aqueous phases. Final concentrations of 4 8 / 8 0 were adjusted to those employed in histamine release. Membrane conductance increased along with an increment of the concentration of 4 8 / 8 0 in the medium as seen in histamine release. The dose-response curves in these two experiments A

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Fig. 2. (A) Histamine release induced by compound 4 8 / 8 0 from isolated rat peritoneal mast cells. Means + S.E. from five experiments. (B) Concentration-response relationships between m e m b r a n e conductance and the concentrations of compound 48/80. Mean 4- S.E. from twelve experiments.

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were quite similar. It should be noted that an increase in membrane conductance takes place even at the low concentration of 0.2 # g / m l . When the conductance change due to 4 8 / 8 0 was studied, it was revealed that the conductance increase occurred very rapidly in much the same way as seen in histamine release (data not shown). These facts suggest that 4 8 / 8 0 is able to interact with the lipid bilayer of the mast cell membrane so as to increase ionic permeability. To investigate whether or not the ion selectivity participates in the permeability increase elicited by 48/80, the membrane conductance of black lipid membrane bathed in a variety of single electrolyte solutions was determined before and after addition of 48/80. When valinomycin was used as a reference compound, it evoked the concentrationdependent increase of membrne conductance only in the KC1 solution, as shown in Fig. 3A. Although the concentration of KC1 was altered from 70 m M to 150 mM, the effect of valinomycin appeared consistently. However, such a change was not induced in either NaC1 (70 m M to 150 mM) or CaC12 solution (10 m M to 100 mM). On the other hand, the addition of 4 8 / 8 0 into any of the test solutions augmented the conductance dose-dependently (Fig. 3B). The concentration of NaC1 and that of KC1 was fixed to 100 mM. In the case of the CaCl 2 solution, the effect of 4 8 / 8 0 was significantly suppressed at concentrations higher A

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Fig. 3. Changes in black lipid m e m b r a n e conductance brought about by 48/80. (A) Changes in m e m b r a n e conductance produced by valinomycin: determined in 100 m M KCI (e); determined in 100 m M NaC1 (A); determined in 100 m M CaCl 2 (I). (B) Changes in m e m b r a n e conductance evoked by 48/80. 100 m M KCl (e); 100 m M NaCI (A); 10 m M CaC12 (I).

than 20 raM, so that the determination was carried out in 10 m M CaCl 2 solution. At the Ca 2+ concentration of 50 mM, changes in membrane conductance associated with NaCl and KC1 due to 4 8 / 8 0 were abolished completely. Compound 4 8 / 8 0 is easily soluble in water but it also provides some hydrophobicity. The partition coefficient of 4 8 / 8 0 in octanol/water phases was - 0 . 7 5 , so that about 15% of the 4 8 / 8 0 dissolved in water can penetrate into the lipid bilayer membrane. When phosphatidic acid (up to 10% of phosphatidylcholine) was added in n-decane in addition to the regular components and the black lipid membrane was prepared in aqueous phase containing 0.1 m M E D T A instead of calcium, the increase in membrane conductance was induced by 4 8 / 8 0 dose-dependently as seen before. This indicates that the interaction between 4 8 / 8 0 and the negatively charged head of lipids is not necessary to augment the membrane permeability. Instead, the penetration of 4 8 / 8 0 into the lipid bilayer may take plac,a and this seems to be important in affecting the membrane permeability. Ortner et al. [11,12] reported that the action of 4 8 / 8 0 does not elicit a marked change in the fluidity of the lipid membrane, and it may bind to the membrane proteins of mast cells. However, it became evident that 4 8 / 8 0 definitely interacts with the membrane lipids in the present study. Also, we reported previously that 4 8 / 8 0 decreases the order parameter of liposomes [7], indicating that penetration of 4 8 / 8 0 into the lipid bilayer takes place to produce perturbation of the bilayer membrane. Since the movement of ions across the membrane is equivalent to the movement of an electrical charge, ionic permeability can be measured by determining membrane conductance [13]. Fewtrell et al. [14] mentioned that substance P, one of the polycationic histamine releasers in which 4 8 / 8 0 was included, releases Ca 2+ from intracellular stores and makes the cell membrane permeable to C a 2+, and, in the following step, C a 2 ÷ released intracellularly or entering from outside induces histamine release. In relation to this, it has been reported that sodium ions which have entered into the secretory cell promote Ca 2+ release from intracellular stores [15]. It can be assumed that the perturbation of lipid bilayer due to 4 8 / 8 0 not only increases Ca 2÷ permeability but also it elicits the

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mobilization of Ca 2+ from the internal stores. In the presence of valinomycin, potassium ions have been shown to pass through the bilayer membrane very selectively, but passage of sodium ions as well as calcium ions is not affected. Similar results have been obtained with monactin in black lipid membrane [16], however, such selectivity of ion passage was not seen in the case of 4 8 / 8 0 . Although phosphatidylcholine and cholesterol, used to prepare black lipid membrane, are both common constituents of the cell lipids in various cells, little action of 4 8 / 8 0 on cells other than mast cells has been shown. However, Lenney et al. [17] reported that 4 8 / 8 0 provides antimicrobial action, and as a conceivable reason for this phenomenon the permeability increase of Escherichia coli provoked by 4 8 / 8 0 has been reported [18]. It has been shown that [Ca]o at higher concentrations exerts an inhibitory effect on histamine release from rat mast cells treated with 4 8 / 8 0 [19] as well as in the anaphylactic reaction [20]. In accordance with this, Nakai et al. [21] reported that histamine release by 4 8 / 8 0 was completely repressed in the medium containing 50 mM [Ca]o. The correlation between histamine release from mast cells and the membrane conductance in black lipid membrane exposed to 4 8 / 8 0 indicates that the black lipid membrane can be used as a model for analysing the mechanism of histamine release. References I Bloom, G.D., Ferdholm, M. and Haegermark, O. (1967) Acta Physiol. Stand. 7 1 , 2 7 0 - 2 8 2 2 Tasaka, K. and Yamasaki, H. (1973) Acta Dermatovener (Stockholm), 73, Suppl. 167-174

3 Hino, R.H., Lau, C.K.H. and Read, G.W. (1977) J. Pharmacol. Exp. Ther. 200, 658-663 4 Foreman, J.C., Hallett, M.B. and Monger, J.L. (1977) J. Physiol. 271, 193-214 5 Garland, L.G. and Payne, A.N. (1979) Br. J. Pharmacol. 65, 609-613 6 U v n ~ , B. (1974) Fed. Proc. 33, 2172-2176 7 Mio, M., Akagi, M., Sakuma, Y. and Tasaka, K. (1982) Advances in Histamine Research ( U v n ~ , B. and Tasaka, K., eds.), pp. 7-23, Pergamon Press, Oxford 8 Akagi, M., Mio, M. and Tasaka, K. (1983) Agents Actions 13, 149-156 9 Hanai, T., Haydon, D.A. and Taylor, J. (1965) J. Theoret. Biol. 9, 433-443 10 Shore, P.A., Burkhaher, A. and Cohen, V.H., Jr. (1959) J. Pharmacol. Exp. Ther. 127, 182-186 11 Ortner, M.J. and Chignell, C.F. (1981) Biochem. Pharma° col. 30, 1587-1594 12 Ortner, M.J., Turek, N. and Chignell, C.F. (1981) Biochem. Pharmacol. 30, 277-282 13 Bangham, A.D. (1975) Cell Membranes: Biochemistry, Cell Biology and Pathology (Weissmann, G. and Claiborne, R., eds.), p. 27, HP Publishing Co., New York 14 Fewtrell, C.M.S., Foreman, J.C., Jordan, C.C., Oehme, P., Renner, H. and Stewart, J.M. (1982) J. Physiol. 330, 393-411 15 Lowe, D.A., Richardson, B.P., Taylor, P. and Donatsch, P. (1976) Nature 260, 337-338 16 Eisenman, G., Ciani, S.M. and Szabo, G. (1968) Fed. Proc. 27, 1289-1304 17 Lenney, J.F., Siddiqui, W.A., Schnell, J.V., Furusawa, E. and Read, G.W. (1977) J. Pharm. Sci. 66, 702-705 18 Katsu, T., Yoshimura, S. and Fujita, Y. (1984) FEBS Lett, 166, 175-177 19 Atkinson, G., Ennis, M. and Pearce, F.L. (1979) Br. J. Pharmacol. 65, 395-402 20 Foreman, J.C. and Mongar, J.L. (1972) J. Physiol. 224, 753-769 21 Nakai, S., Furuta, K., Akagi, M., Uokawa, M. and Tasaka, K. (1979) Jap. J. Pharmacol. 29, 69p