Angiotensin II and Related Peptides Alter Liposomal Membrane Fluidity

Transcription

1 Vol. 44, No. 1, January 1998 Pages Angiotensin II and Related Peptides Alter Liposomal Membrane Fluidity Eugen Brailoiu 1'2, Anca Margineanu 2 and Michael D. Miyamoto 1 1. Department of Pharmacology, East Tennessee State University, James H. Quillen College of Medicine, Johnson City, Tennessee, USA 2. Department of Physiology and Biophysics, University of Medicine and Pharmacy "Gr. T. Papa", Iasi, Romania Received October t6, 1997 Received after revision October 31, 1997 Summary We investigated the effects of angiotensinogen (Ang), angiotensin I (Ang I), and angiotensin II (Ang I1) on the fluidity of phosphatidylcholine vesicles. Changes in fluidity were assessed by changes in anisotopy values calculated from fluorescence polarization measurements. All three compounds produced an increase in membrane fluidity when localized inside the phosphatidylcholine vesicles. When placed outside the vesicles, Ang II increased bilayer rigidity (decreased fluidity), whereas Ang and Ang I produced no effect. These results suggest the possibility that these peptides may alter the fluidity of cell membranes by a direct action on the phospholipid bilayer, which may in turn interfere with receptor-mediated effects. Key Words: membrane fluidity; angiotensin derivatives; liposomes Introduction The lipid fluid environment is an important determinant in the functioning of membrane proteins due to its effect on the lateral mobility of these proteins within the membrane [1]. Alterations in phospholipid membrane fluidity (e.g., fluidity of plasmalemma and intracellular membranes) may be produced by many endogenous substances, including compounds with established roles in intercellular signalling (hormones, neurotransmitters). For example, well known chemical mediators, such as noradrenaline and Ang II, have been shown to increase the lipid fluidity of isolated rat liver plasma rnembranes [2]. Effects on plasmalemmal fluidity have also been observed with thyroid hormones on skeletal muscle sarcolemma [3], ACTH on Author for correspondence: Michael D. Miyamoto, Department of Pharmacology, Box East Tennessee State University, James H. Quillen College of Medicine, Johnson City, Tennessee , USA, Fax: , miyamoto@access.etsu-tn.edu /98/ /0 Copyright by Academic" Press Australia. All rights of reproduction in any form reserved.

2 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL synaptic plasma membranes [4], prostaglandin I2 on liver membranes [5], and prostaglandins E2 and F2~ on myometrial sarcolemma [6]. The importance of Ang II in the regulation of cardiovascular function is widely accepted. The customary explanation is that these effects are mediated by interaction with specific plasmalemmal receptors [7] and activation of the phosphoinositide system [8]. On the other hand, it has also been shown that Ang II may promote calcium influx [9] through plasmalemmal calcium channels [10], modulate Ca 2+activated K + channels [11] and resting K + conductance [12], and stimulate Na-K-CI cotransport [ 13] and Na+/K+-ATPase [ 14]. Furthermore, there is evidence that Ang I and Ang II may affect cellular function by interacting with intracellular receptor sites, e.g., (a) intracellular dialysis with Ang II produces a decrease in junctional conductance in myocardial syncytium [15], (b) intracellular administration of Ang, Ang I and Ang II via liposomes results in contraction of rat aorta [16], and (c) intracellular injection of Ang II into vascular smooth muscle cells induces an increase in cytoplasmic and nuclear Ca 2+ due to Ca 2+ influx from the extracellular space [ 17]. The above findings indicate that Ang II and its congeners have actions beyond their traditionally held roles. The aim of the present study was to examine whether Ang, Ang I and Ang II could produce changes in fluidity by a direct action on the membrane. We used phosphatidylcholine liposomes as the membrane model and fluorescence polarization (anisotropy) as the indicator of changes in membrane fluidity. Materials and Methods Liposome preparation. Anisotropy measurements were made on 1,6-diphenyl- 1,3,5-hexatriene (DPH)-labeled phosphatidylcholine model membranes. The effects of Ang, Ang I, and Ang II on anisotropy were determined under conditions in which the peptides were either entrapped in liposomes or added to the vesicle suspension. Accordingly, two types of liposomes were used in this study: (a) multilamellar vesicles (MLV), to test the effect of encapsulated peptides on membrane fluidity and (b) small unilamellar vesicles (SUV), to test the effect of non-encapsulated peptides on the same parameter. Liposomes for testing the intravesicular effects of angiotensin derivatives were prepared from egg phosphatidylcholine (Sigma; 30 mg lipid per ml aqueous phase represented by triple-distilled water), according to the method described by Bangham 204

3 Vol. 44, No. I, 1998 et al. [18]. The same solution was used to prepare Ang (10-4 to M), Ang I (10-4 to M) and Ang II (10-4 to 10 "12 M)-loaded liposomes (all from Serva; 30 mg/ml aqueous phase). To remove non-incorporated solute, liposome batches were dialyzed (Sigma dialysis sacks) in triple-distilled water (150 rain, 1:600 V:V, the dialyzing medium being exchanged every 30 rain). Liposomes for testing the extravesicular effects of angiotensin derivatives were prepared from control multilamellar liposomes (obtained under the same conditions as described above). This suspension was sonicated tbr 30 min and subsequently passed through 0.45 ~tm Millipore filters. The small unilamellar vesicles obtained were labeled for 1 h (described below under "Fluorescence polarization studies"). Solutions containing the different (10-12 to 10-6 M) extraliposomal concentrations of Ang, Ang I and Ang II (all from Serva) (99/1 V:V) were prepared 5 min before each measurement. Fluorescence polarization studies. Twenty gl of all liposome batches (including MLV and SUV control liposomes) were diluted in a volttme of 3 ml of phosphate buffer saline, ph 7.4, and labeled with DPH (Sigma). The stock solution contained 2 mm DPH in tetrahydrofuran for UV - spectroscopy (Fluka), and the final concentration of DPH for fluorescence labeling of liposomes was 10-6 M. Anisotropy was estimated at 22 ~ using a PT1 (Photon Technology International, France) spectrofluorimeter adapted for fluorescence polarization 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. Measurements were carried out under steady-state conditions, and fluorescence sampled for 30 s at 5 Hz. Anisotropy was calculated from: lvv - GIv~ F-- Ivv + 2GIv, where r is anisotropy, and Ivv and IvH are the respective fluorescence intensities recorded with polarization analyzer oriented parallel to, and perpendicular to, the direction of the polarized excitation beam. G was calculated as 1HV/IHH. The anisotropy of each batch was determined as the mean of 150 measurements. Statistics. Student's t-test was used to test for significant differences between groups of values (P < 0.01 indicating a significant difference). All values were expressed as means + SEM, with six experiments for each series of batches. Results We observed a significant reduction in fluorescence anisotropy (P < 0.001) for all three peptides incorporated into MLVs. Ang I produced a moderate reduction in anisotropy that was independent of the Ang I concentration (Fig. 1). Ang and Ang II, on the other hand, produced decreases in anisotropy that were inversely related to peptide concentration. In the case of SUVs, where the peptides were present only in the extravesicular compartment, Ang and Ang I produced modest reductions in anisotropy for peptide 205

4 >, O~ i Angl 9 Ang II 9 control i I I ' I I Concentration (log M) Fig. 1. Modification of fluorescence anisotropy by angiotensin derivatives entrapped in phosphatidylcholine liposomes. The code for each symbol is shown in the panel. Control points represent measurements obtained with empty liposomes. Anisotropy values below control points indicate decreases in membrane rigidity, while values above control reflect increases in rigidity. Each point represents the mean + SEM. concentrations between to 10-9 M (P < 0.01) but had no significant effect (P > 0.05) for concentrations >10.9 M (Fig. 2). Ang II, on the other hand, produced a biphasic effect: there was a small reduction in fluorescence anisotropy for peptide concentrations between 10 "12 and 10 ll M and a large, significam increase for concentrations > 10 "9 M. Discussion We used fluorescence anisotropy to monitor membrane fluidity since anisotropy is closely correlated with the fluidity of phosphatidylcholine vesicles [19]. In our case, anisotropy reflects the degree of organisation of membrane lipid molecules in a fluid medium which is directly proportional to the rigidity of the,-206

5 a~ o.3o ' s 0.24 O om ~ Ang Ang I Ang II control [ [ I I I I I Concentration (log M) Fig. 2. Alterations in fluorescence anisotropy produced by addition of angiotensin derivatives to liposome suspensions. Controls represent values obtained for empty liposomes. Each point represents the mean + SEM. phospholipid bilayer. In other words, an increase in anisotropy is associated with an increase in rigidity, while a decrease in anisotropy reflects an increase in fluidity. To simulate the intra- and extracellular effects of the peptides, we used multilamellar liposomes (passive trapping capture) and small unilamellar vesicles as phospholipid bilayer models, respectively. The anisotropy measured for control liposomes filled with triple-distilled water was used as reference. It is generally accepted that Ang and Ang [ do not have direct effects on plasmalemmal receptors and, in physiological concentrations, do not alter bilayer fluidity. When incorporated into liposomes, however, Ang, Ang I and Ang II all produced increases in membrane fluidity (Fig. 1). As mentioned, it has already been shown that Ang II can act at intracellular sites to modulate tight junctions of myocardial syncytium [15] or induce Ca 2+ release from internal stores in vascular 207

6 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL smooth muscle [ 17]. It has-also been shown that Ang, Ang I, and Ang II can act at intracellular sites to produce contraction of vascular smooth muscle [16]. Somewhat surprisingly, intracellular administration of Ang I produces a greater contraction (47.8%) than that produced by Ang or Ang II (23.44% and 22.12%, respectively) [16]. This contrasts with results obtained with extracellular administration and may mean that the intra- and extracellular receptor sites for Ang II are different. Another possibility is that the relative effects ofintracellular Ang, Ang I, and Ang II on smooth muscle contraction [16] may be linked to their effects on membrane fluidity (note in Fig. 1 that Ang and Ang II increased liposomal fluidity more than Ang I). In our experiments, physiological concentrations of extraliposomal Ang II produced a decrease in the fluidity of phosphatidylcholine membranes (Fig. 2). Since these membranes are devoid of protein receptor molecules, this suggests a direct effect of Ang II on phospholipids and/or an indirect effect of Ang II due to interaction with saline solutions. In apparent contradiction to our results, Burgess et al. [2] showed that Ang II, by a receptor-mediated effect, increased the fluidity of isolated rat liver plasma membranes. However, it should be noted that she intracellular effects of Ang II are mediated by 1,4,5-inositol trisphosphate (IP3) [20] and that intraliposomal IP3 produces an increase in the fluidity of phosphatidylcholine bilayers [21]. Accordingly, one explanation for this discrepancy may be that Ang II has a dual effect: (a) a direct action to increase membrane rigidity, and (b) a receptor-activated, IP3-mediated, effect to increase membrane fluidity. From a technical standpoint, we can say that fluorescence polarization measurements provide an accurate basis for calculation of anisotropy. Moreover, liposomes appear to provide a suitable model for the study of phospholipid membrane fluidity. We consider this approach to be uniquely suited for discriminating between direct and protein-mediated effects of substances on the plasmalemma and other biomembranes, as well as for understanding the influences exerted by membrane phospholipids on receptor-mediated effects. Acknowledgement This work was supported by the Romanian Ministry of Education (CNCSU A7025/1997 grant and CNCSU C248/1997, grant supervised by World Bank). 208

7 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL References 1. Lenaz, G. (1987). Biosci. Rep., 7, Burgess, G.M., Giraud, F., Poggioli, J., Claret, M. (1983). Biochim. Biophys. Acta., 731, Pilarska, M., Wrzosek, A., Pikula, S., Famulski, K.S. (1991). Biochim. Biophys. Acta., 1068, Hershkowitz, M., Zwiers, H., Gispen, W.H. (1982). Biochim. Biophys. Acta, 692, Dave, J.R. and Knazek, R.A. (1980). Proc. Natl. Acad. Sci. USA, 77, Deliconstantinos, G. and Fotiou, S. (1986). J. Endocrinol., 110, Meyer, P., Papadimitriou, A., Worcel, M. (1970). Br. J. Pharmacoh, 40, 541P. 8. Griendling K.K., Tsuda, T., Berk, B.C., Alexander, R.W. (1989). Am. J. Hypertens., 2, Stauderman, K.A. and Pruss, R.M. (1989). J. Biol. Chem., 264, Ohya, Y. and Speralakis, N. (1991). Circ. Res., 68, Payet, M.D., Bilodeau, L., Drolet, P., Ibarrondo, J., Guillon, G., Gallo-Payet, N. (1995). Mol. Endocrinol., 9, Li, Y.W., Guyenet, P.G. (1996). Circ. Res., 78, Owen, N.E., Ridge, K.M. (1989). Am. J. Physiol., 257, C Gavin, J.L. (1991). J. Am. Soc. Nephrol, 1, De Mello, W.C. (1994). J. Cardiovasc. Pharmacol., 23, Brailoiu, E., Todiras, M., Margineanu, A., Costuleanu, M., Brailoiu, C., Filipeanu, C.M., Costuleanu, A., Rusu, V., Petrescu, G. (1997). Biomed. Cromatogr., 11, Haller, H., Lindschau, C., Erdmann, B., Quass, P., Luft, F.C. (1996). Circ. Res., 79, Bangham, A.D., Standish, M.M., Watkins, J.C. (1965). J. Mol. Biol., 13, Schuler, I., Duportail, G., Glasser, N., Benveniste, P., Hartmann, M.A. (1990) Biochim. Biophys. Acta. 1028, Dasso, L.L., Taylor, C.W. (1994). J. Biol. Chem., 269, Brailoiu, E., Margineanu, A., Toma, C.P., Filipeanu, C.M., Rusu, V., Branisteanu, D.D. (1997). Biochem. Mol. Biol. Int. (In press). 209