D-myo-Inositol Derivatives Alter Liposomal Membrane Fluidity

Transcription

1 Vol. 44, No. 1, January 1998 Pages D-myo-Inositol Derivatives Alter Liposomal Membrane Fluidity Eugen Brailoiu ~, Anca Margineanu ~, Catalin P. Toma I, Catalin M. Filipeanu 2, Valeriu Rusu ~, Dimitrie D. Branisteanu a 1. Department of Physiology andbiophysics, University of Medicine and Pharmacy 'Gr. T. Popa' Iasi, RO-6600, Romania 2. Department of Clinical Pharmacology, Groningen Institute for Drug Studies, Faculty of Medicine, University of Groningen, 9713 AV Groningen, The Netherlands Received June 24, 1997 Received after revision October 31, 1997 Summary We investigated the effect on membrane fluidity induced by D-myo-inositol derivatives (IP3, IP4, IP5, IPr). Fluidity was determined as the anisotropy of fluorescence polarisation from liposome model membranes labelled with DPH (1,6-diphenyl-l,3,5 hexatriene). IP3 (10 q~ to 10-5 M) increased the membrane fluidity with a maximum effect at 10-5 M. For IP4, IP5 and IPr, at concentrations less than 10-6 M these derivatives increased the membrane viscosity (i.e. reduced fluidity). This effect was enhanced when the derivatives were incorporated in the vesicles, rather than added to the vesicle suspension. In this case IP5 and IP6 increased viscosity over the reference values. We conclude that inositol derivatives directly modified membrane fluidity which could play a role in their effects in biological systems, beside the one mediated by binding to specific receptors. Key Words: membrane fluidity; D-myo-inositol derivatives; liposomes Introduction In a large variety of cells, the hydrolysis of phosphatidylinositol 4,5-bisphosphate to 1,4,5-trisphosphate (IP3) and diacylglycerol is a key step for the initiation of the specific response to agonist-receptor coupling [1]. Subsequent metabolic pathways include the generation of D-myo-inositol 1,3,4,5 tetrakisphosphate (IP4) from D-myo-inositol 1,4,5 trisphosphate, which can subsequently be converted to D-myo-inositol 1,3,4,5,6 pentakisphosphate (IPs) and finally D-myo-inositol 1,2,3,4,5,6 hexakisphosphate (IP6). It is well known that IP3 induces Ca 2+ release from the sarcoplasmic reticulum [1]. IP4 could act like IP3 for Ca 2+ release [2]. IP5 and IP6 binding sites were also found at the microsomal level [3]. Author for correspondence: Eugen Brailoiu, Department of Physiology and Biophysics, University of Medicine and Pharmacy,16 Universitatii Street, Iasi, RO-6600, Romania, Fax: , brailoiu@umfiasi.ro /98/010195~) /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved

2 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAl There are many examples in which inositol derivatives regulate plasmalemmal functions. IP3 mediates adrenaline activation of K conductance in mouse macrophages [4], activates plasma membrane calcium current in human T cells [5], regulates the voltage-dependent calcium channels in cerebellar granule neurons [6] as well as the calcium influx in Xenopus oocytes [7]. IP3 stimulates the plasmalemmal Ca2+-ATPase pump and inhibits the Na/Ca exchanger in rat brain [8]. IP4 stimulates the influx of Ca 2+ in Xenopus oocytes [7], modulates the Ca2+-insensitive K + conductance in HL-60 cells [9], activates potassium channels [10] and the non-specific cation conductance in Aplysia neurons [11] and also inhibits the plasmalernmal Ca2+-ATPase in rat brain [8]. Membrane fluidity can be changed experimentally [12] and there is evidence that changes in membrane fluidity affect some receptor-mediated cellular responses. Beta-adrenergic drugs affect [13] the VIP receptor/effector system [14] and nicotinic acetylcholine receptor function [ 15] were affected by changes in membrane fluidity. Variations of membrane fluidity also alter the intermediary steps in agonist inducedresponses such as the activation ofphospholipase A2 [16] through active or passive ion movement. The Na+/K+-ATPase [17] and the sarcoplasmic reticulum Ca 2+ pump [18] are modulated directly by the overall rotational motion ot~this ATPase [19]. Increase in fluidity enhances the activity of Na+-IT exchange [20] and at the vascular smooth muscle level alter the Ca2 K channel kinetics [21]. Given that changes in membrane fluidity can have many effects on plasmalemmal functions, we considered the possibility that IP3 and its derivatives exert some of their biological effects by changing membrane fluidity. To test this hypothesis, we investigated the effect of IP3, IP4, IP5, and IP6 on membrane fluidity, using phosphatidylcholine liposomes as a model membrane. Methods Anisotropy measurements were done with DPH (1,6-diphenyl-l,3,5- hexatriene)-labelled phosphatidylcholine model membrane. The effect of inositol derivatives (IP3, IP4, IPs, or IP6) on anisotropy was determined either by entrapping the derivative in the liposome, or by additing it to the vesicle suspension. Liposome preparation The liposomes used in these studies were prepared from egg phosphatidylcholine (Sigma, 30 mg lipid per ml aqueous phase represented by tripledistilled water) according to the method described by Bangham et al. [22]. The same solution was used to prepare IP3 ( l~ M), IP4 ( "1~ M), IP5 ( ~ M) and IP6 ( "1~ M)-loaded liposomes (all from Sigma) (30 mg/ml aqueous phase). In order to remove the non-incorporated solutes, llposome batches were 196

3 subjected to dialysis (Sigma dialysis sacks) in triple-distilled water (150 min, 1:600, V/V, the dialysing medium being exchanged every 30 min). Experimental protocol Fluorescence anisotropy was measured for all batches of fiposomes containing inositol derivatives, and for control liposome suspensions to which inositol derivative solutions [P3, IP4, IP5 or IP6 were added (99:1, V/V) in order to obtain the same extraliposomal concentrations corresponding to the ones of entrapped drugs. Fluorescence polarisation studies 20 ~tl of all liposome batches (including control liposomes) were diluted into a volume of 3 ml of phosphate buffer saline (PBS) at ph 7.4 and labelled with 1,6- diphenyl-l,3,5-hexatriene (DPH; Sigma). The stock solution we used was 2raM DPH in tetrahydrofuran for UV-spectroscopy (Fluka) and the final concentration of DPH for fluorescence labelling of liposomes was 10.6 M. The anisotropy was estimated at 22 ~ using a PTI (Photon Technology International, France) spectrofluorimeter adapted for fluorescence polarisation measurements. The excitation wavelength was set at 350 nm and the emission wavelength at 430 nm, with a 5-nm slit for both excitation and emission. The measurements were carried out in steady-state conditions. Fluorescence was sampled for 30 s at 5 Hz. The anisotropy was calculated from the equation: Ivv - GIvn t-- I w + 2GIvH were Ivv and Ivn are the fluorescence intensities recorded with polarization analyzer oriented, respectively, parallel to and perpendicular to the direction of the polarised excitation beam and G was calculated as IHV/IHu. The anisotropy of each batch was calculated as the mean of 150 measurements. Statistics Student's t-test for group values was used to evaluate the significance of differences (P < 0.01 were considered as significant). All values were expressed as mean + SEM, six experiments for each series of batches were done. The concentration dependence of inositol derivative effects on membrane fluidity was characterised by second degree polynomial regression. Results Irrespective of the protocol used, e.g. entrapped or not in liposomes, IP3 (10 ~~ -10 ~ M), increased the membrane fluidity up to a maximum at 10.5 M (Fig. 1 and Fig. 2). For IP4, IPs, and IP6, at concentrations lower than 10 "6 M an increase in fluidity was observed. At doses higher than 10-6 M (except with IP4) these derivatives increased the membrane viscosity (Fig. 1 and Fig. 2). This effect was more evident with their intraliposomal administration. In this case IP5 and IP6 caused an increase in rigidity above control reference values. Discussion Fluidity of the phospholipid bilayer, has major effects upon the lateral mobility of enclosed/attached proteins and therefore interferes with plasmalemmal fimctions 197

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL O.0684 I o.o4,.b.,,.~,, ~ -~ ~ ~ -3 -~ Concentration (log M) Fig. 1. Fluorescence anisotropy modification by addition of inositol derivatives to a liposome suspension. The meaning of symbol is shown in the panel. Controls represent empty liposomes. Each point represents the mean + SEM. Polynomial regression analysis of the curves showed P < [19]. We used fluorescence anisotropy to characterise membrane fluidity; it has been previously shown that anisotropy is closely correlated to the fluidity of phosphatidylcholine vesicles [23]. In our case, anisotropy characterises the degree of organisation of membrane lipid molecules in a fluid medium. This is directly proportional to the rigidity of the phospholipid bilayer. In other words, the rigidity is associated with increased anisotropy, while decreased anisotropy represents an increase in fluidity. As a model for phospholipid bilayers we used multilumellar liposomes (passive trapping capture), in order to avoid expunging the drug content (inositol derivatives) during intermediary steps (such as chemical or ultrasonication treatments). The anisotropy measured for control liposomes, filled with triple-distiued water, was used as reference value. 498

5 VOI. 44, NO. 1, , o ~ / ~ I 9 9 P4 fib 9 control 0.o4.,. _~.,,,,.h.h -11 -;o -b -8 ~ -s -4 Concentration (log M) Fig. 2. Fluorescence anisotropy modification by entrapped inositol derivatives into phosphatidylcholine liposomes. Controls represent the empty liposomes. Each point represents the mean + SEM. Polynomial regression analysis of the curves showed P< In many types of cells the hydrolysis of phosphatidylinositol 4,5-bisphosphate to IP3 and diacylglycerol is a key step for the initiation of the specific response to the agonist-receptor coupling. Diaeylglycerol activates protein kinase C [24] but does not change the anlsotropy value in phosphatidylcholine vesicles [25]. IP3 is the best described messenger of this series; in physiological concentrations [1], it decrease the anisotropy in both cases. When incorporated into liposomes at 10-5 M, the concentration which activates the IP3-gated Ca 2+ channel reconstituted into planar lipid bilayer membrane [26] it had the strongest effect on fluidity increase. Low doses (<10-6 M) of all inositol derivatives tested, either incorporated in liposomes or present in the external medium only, reduced membrane anisotropy, corresponding to the increased fluidity of the lipid bilayer. For IP3 and IP4, the increase in fluidity was significantly higher when the drugs were trapped into 199

6 Vol. 44, No. I, 1998 BIOCHEMISTRYclnd MOLECULAR BIOLOGY INTERNATIONAL fiposomes than when present in the external medium. For IP5 and IP6, there were no significant differences between the two situations. This increase in membrane fluidity (at concentrations between 10 -~~ and 10-6 M) diminished at higher phosphoinositol concentrations. This effect was most obvious for intraliposomal IP5 and IP6, which increased rigidity over control reference values at high concentrations. Except IP3, a well described second messenger, our data indicate that at physiological doses [1] these polar compounds decrease membrane fluidity, directly related to the number of negative charges they carry. Thus, highly charged inositol molecules (IP5 and IP6) at high concentrations increased membrane rigidity significantly. This is in agreement with the data ofrendu et al. [27] who observed that platelet plasma membrane stimulation with thrombin induced a concomitant phosphatidylinositol breakdown and an increase in membrane fluidity. In conclusion our data indicate that inositol derivatives have a direct effect on membrane fluidity, which could play a role in their cellular effects, in addition to the actions mediated by their coupling to specific receptors. Acknowledgement We would like to thank Dr. Christopher Kushmerick, Department of Physiology and Biophysics and of Pharmacological Sciences, Health Sciences, SUNY, Stony Brook, USA for helpful discussions about the manuscript. This work was supported by the Romanian Ministry of Science and Education (CNCSU grant) through the Romanian Ministry of Research and Technology (C-D grant). References 1. Berridge, M.J. (1986) Biol. Chem. Hoppe. Seyler 367, Wilcox, R.A., Whitham, E.M., Liu, C., Potter, B.V., Nahorski, S.R. (1993) FEBS Lett. 336, Cullen, P.J., Irvine, R.F. (1992) Biochem. J. 288, Hara, N., Ichinose, M., Sawada, M., Maeno, T. (1993) Pflugers Arch. 423, McDonald, T.V., Premack, B.A., Gardner, P. (1993) J. Biol. Chem. 268, De Waard, M., Seagar, M., Feltz, A., Couraud, F. (1992) Neuron 9, DeLisle, S., Pittet, D., Potter, B.V., Lew, P.D., Welsh, M.J. (1992) Am. J. Physiol. 262, Fraser, C.L., Sarnacki, P. (1992) Am. J. Physiol. 262, : 9. Wu, LT., Gong, Q.H., Chou, R.H., Wieland, S.L (1991) J. Biol. Chem. 266, Molleman, A., Hoiting, B., van der Akker, J., Nelernans, A., Den Hertog, A. (1991) J. Biol. Chem. 266,

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