Phosphatidic acid as a calcium ionophore in large unilamellar vesicle systems

Biochimica et Biophysica Acta, 777 (1984) 343-346 Elsevier

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BBA Report BBA 70186

P H O S P H A T I D I C ACID AS A CALCIUM I O N O P H O R E IN LARGE UNILAMELLAR VESICLE S Y S T E M S RAJIV NAYAR, LAWRENCE D. MAYER, MICHAEL J. HOPE and PIETER R. CULLIS

Department of Biochemistry, University of British Columbia, Vancouver, B.C. V6T 114/5 (Canada) (Received April 20th, 1984)

Key words: Dioleoylphosphatidic acid," Ca: ÷ ionophore; Phospholipid vesicle

The ionophoretic capabilities of dioleoylphosphatidic acid (DOPA) for transporting calcium across phospholipid bilayers have been investigated. Calcium uptake by large unilamellar vesicles is shown to depend on the presence of DOPA. This uptake is sensitive to the nature and concentration of calcium chelators in the vesicle interior, indicating that accumulation results from DOPA-mediated translocation of calcium across the membrane. Further, it is shown that characteristics of DOPA-mediated Ca 2+ uptake are similar to those observed for the fungal calcium ionophore, A23187.

Considerable controversy surrounds the possibility [1] that phosphatidic acid can act as a Ca 2+ ionophore in transmembrane signalling events. Initially, it was demonstrated that phosphatidic acid can facilitate Ca 2+ transport between two aqueous compartments separated by organic solvent [2] and across the bilayers of multilamellar liposomal systems [3]. In biological membrane systems it has been shown that an increase in phosphatidic acid content due either to addition of exogenous phosphatidic acid [4-6] or phospholipase D treatment [7] results in physiological effects associated with an influx of Ca 2÷. However, a recent report [8] indicates that phosphatidic acid (derived from egg PC) is unable to facilitate Ca 2+ transport across bilayer phosphatidylcholine (PC) systems. In this work we characterize the ability of dioleoylphosphatidic acid (DOPA) to act as a Ca 2+ ionophore in large unilamellar vesicle systems. We show that the presence of DOPA results in Ca 2+ uptake which is sensitive to the nature and concentrations of Ca 2+ chelators trapped inside the large unilamellar vesicle systems. 1,2-Dioleoyl-sn-glycero-3-phosphorylcholine (DOPC) was synthesized employing oleic acid as 0005-2736/84/$03.00 © 1984 Elsevier Science Publishers B.V.

described elsewhere [9]. Dioleoylphosphatidic acid (DOPA) was prepared from DOPC employing standard procedures utilizing phospholipase D [10] and was purified by carboxymethyl cellulose chromatography [11]. No lysophosphatidic acid or other contaminants were detectable by two-dimensional thin-layer chromatography (2D-TLC) and the ionophoretic properties observed were insensitive to extended storage in CHC13 under N 2 (4 weeks). Very similar results were observed for four different preparations of DOPA. Phosphatidylserine (PS) was prepared from egg phosphatidylcholine by similar methods. The negatively charged phospholipids were converted to their sodium salt form. All phospholipids were greater than 99% pure as indicated by 2D-TLC and were stored under N 2. Large unilamellar vesicles were prepared by repetitive extrusion of multilamellar vesicles through a polycarbonate (Nucleopore) filter (0.1 ~m pore size) as described elsewhere [12]. These vesicles exhibited a size distribution of 700 + 100 ~, as detected by freeze-fracture procedures [12]. Experiments employing entrapped 22Na indicated trapped volumes of 1.0+0.05 /~l//~mol phospholipid. The untrapped buffer was replaced with

344 the desired external buffer b y passage of the vesicle p r e p a r a t i o n over a 1.5 × 15 cm S e p h a d e x G-50 c o l u m n equilibrated with the a p p r o p r i a t e buffer. In all cases, internal and external m e d i a of equal o s m o l a r i t y were used. N o d e t e c t a b l e leakage of e n t r a p p e d 22Na o c c u r r e d u n d e r all c o n d i t i o n s used over the e x p e r i m e n t a l time-course (4 h). C a l c i u m u p t a k e was m o n i t o r e d b y i n c u b a t i n g the vesicles ( 3 - 5 ~tmol p h o s p h o l i p i d / m l ) in a buffer c o n t a i n i n g 2 m M CaC12 a n d 45Ca (1 ~tCi//~mol Ca2+). A l i q u o t s (100/~1) were r e m o v e d over a 4-h time course a n d the external 45Ca 2+ was removed b y passage over a 1 ml S e p h a d e x G - 5 0 column. T h e vesicles were subsequently analyzed for 45Ca on a Philips PW-4700 liquid scintillation c o u n t e r and for lipid p h o s p h o r u s [13]. In some cases d u a l - l a b e l e x p e r i m e n t s were used to m o n i t o r 45Ca and p h o s p h o l i p i d s i m u l t a n e o u s l y by employing trace amounts of [3H]dipalmitoylphosphorylcholine (NEN, Canada) and a 3H/45 Ca d u a l label channel. Initial e x p e r i m e n t s were designed to d e t e r m i n e whether D O P A - c o n t a i n i n g vesicles could accum u l a t e exogenous calcium. P o t a s s i u m p h o s p h a t e (20 m M ) was included in the vesicle interior to sequester C a 2 ÷ in the event of a m e m b r a n e transp o r t process. As shown in Fig. 1, the presence of D O P A does result in increased levels of vesicle associated Ca 2÷ which is d e p e n d e n t on the a m o u n t of D O P A present. In particular, increasing the D O P A c o n t e n t from 5 to 20 percent increases the a m o u n t of vesicle associated Ca 2÷ from 1.8 to 10.0 n m o l C a 2 + / / ~ m o l p h o s p h o l i p i d . The presence of 20 mol percent egg PS d i d not result in d e t e c t a b l e C a 2+ u p t a k e (results not shown). In order to d e t e r m i n e whether this calcium uptake was due to b i n d i n g to p h o s p h a t i d i c acid on the vesicle exterior, or reflected actual translocation of C a 2 + across the m e m b r a n e , various c o n c e n t r a t i o n s a n d types of calcium chelators or ' s i n k s ' were e n t r a p p e d in the vesicle interior. A s shown in Fig. 2, calcium u p t a k e increases as the a m o u n t of p h o s p h a t e in the vesicle interior is increased. In addition, different t r a p p i n g efficiencies of the calcium sinks e m p l o y e d are observed, with E G T A being m o r e effective than oxalate or p h o s p h a t e . T h e a m o u n t of Ca 2+ u p t a k e o b s e r v e d e m p l o y i n g E G T A indicates that > 90% of the e n t r a p p e d E G T A is c o m p l e x e d to Ca 2+ at 4 h.

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Time(hr) Fig. 1. Uptake of Ca2+ into large unilamellar vesicle systems containing: (O) 5 mol% DOPA, 15 mol% egg PS and 80 mol% DOPC; (11) 15 mol% DOPA, 5 mol% egg PS and 80 mol% DOPC; (A) 20 mol% DOPA, 80 mol% DOPC. The large unilamellar vesicle systems were prepared by an extrusion technique (see text) in the presence of a buffer containing 125 mM KCI, 20 mM KH2PO4 (pH = 7.4) and the untrapped buffer was exchanged for a buffer containing 125 mM KCI, 20 mM Hepes (pH = 7.4) by gel filtration employing Sephadex G-50. These vesicles were incubated (20 o C) in the presence of 2 mM CaCI/(1 /~Ci//~mol 45Ca), aliquots removed at various times and exterior (non-sequestered) Ca 2÷ removed by gel filtration. The eluate was then assayed for 45Ca2+ and lipid phosphorus.

Such a sensitivity of C a 2 + ' u p t a k e ' to vesicle inner contents is clearly consistent with a D O P A - d e p e n d e n t t r a n s p o r t of calcium to the vesicle interior, r a t h e r than b i n d i n g of Ca 2+ to o u t e r m o n o l a y e r lipids. This conclusion was c o r r o b o r a t e d by passing aliquots of vesicles ( c o n t a i n i n g sequestered C a 2 +) over 1 ml gel filtration c o l u m n s which were p r e - e q u i l i b r a t e d with cold calcium (2 mM). A n y 45Ca b o u n d to the vesicle exterior would be exp e c t e d to exchange with n o n - r a d i o a c t i v e calcium d u r i n g this procedure. This w o u l d result in a decrease of vesicle-associated 45Ca2+ if the u p t a k e process reflects C a 2+ b i n d i n g to the vesicle exterior. In contrast, such a p r o c e d u r e consistently resulted in a vesicle-associated level of 45Ca 2 + that was 20% higher than 45Ca2+ associated with vesicles that were p a s s e d d o w n c o l u m n s that h a d

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Time(hr) Fig. 2. Uptake of Caz÷ into large unilamellar vesicle systems composed of 20 mol% DOPA and 80 mol% DOPC. The interior of the vesicles contained a variety of concentrations and types of Ca2÷ chelators: (O) 125 mM KCI, 20 mM KH2PO4 (pH = 7.4); (ll) 120 mM KCI, 40 mM KH2PO4 (pH = 7.4); (O) 100 mM potassium oxalate, 50 mM KC1, 20 mM Hepes (pH = 7.4); (A) 100 mM EGTA, 50 mM KCI, 20 mM Hepes (pH = 7.4). The vesicle were prepared in the presence of these various buffers and the untrapped buffer exchanged for KC1, 20 mM Hepes buffer (pH = 7.4) of equal osmolarity by gel filtration. For further details, see Fig. 1 legend and text.

not been pre-equilibrated with unlabelled Ca 2÷. This suggests that when Ca 2+ free c o l u m n s are employed, some of the sequestered Ca 2+ is transported out of the vesicles during the gel filtration procedure. The preceding results d e m o n s t r a t e that the presence of 20 mol% D O P A results in uptake of Ca 2÷ into the interior of the vesicle systems employed here. It is of interest to c o m p a r e the rate a n d extent of such uptake with that observed for the fungal Ca 2÷ i o n o p h o r e A23187 in vesicle systems which do n o t c o n t a i n D O P A . As shown in Fig. 3, n o significant Ca 2+ uptake into D O P C vesicle systems is o b t a i n e d in the absence of A23187 or i n t e r n a l oxalate. In the presence of 100 m M o x a l a t e - c o n t a i n i n g vesicles, the rate and a m o u n t of Ca 2÷ uptake is similar to that observed in D O P A - c o n t a i n i n g systems (see Fig. 2). Again, p r e - e q u i l i b r a t i o n of the gel filtration c o l u m n s with

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Ti me (h r/ Fig. 3. Uptake of Ca2÷ into large unilamellar vesicle systems composed of DOPC; (O) DOPC vesicles containing 100 mM potassium oxalate, 50 mM KCI, 20 mM Hepes (pH = 7.4); (A) DOPC vesiclescontaining 175 mM KC1, 20 mM Hepes (pH = 7.4) where the Ca2÷ ionophore A23187 was added (in ethanol) prior to incubation with Ca2÷ to achieve a 1:500 (mol/mol) A23187 to DOPC ratio; (ll) DOPC vesicles containing 100 mM potassium oxalate, 50 mM KCI, 20 mM Hepes (pH = 7.4) where A23187 (1 : 500) was present during the incubation with Ca2+. The vesicleswere prepared in the presence of the various buffers, and untrapped buffer was exchanged for 175 mM KC1, 20 mM Hepes (pH = 7.4) by gel filtration. Uptake of Ca2÷ at subsequent times was determined as indicated in the legend to Fig. 1 and text.

unlabelled Ca 2+ resulted in an increase of vesicleassociated 45Ca 2+ levels similar to that observed for D O P A - c o n t a i n i n g vesicles. I n summary, these studies provide strong evidence that D O P A can act as a Ca 2÷ i o n o p h o r e in large unilamellar vesicle systems. Calcium can be a c c u m u l a t e d when D O P A is present, whereas no such uptake is observed in the absence of D O P A . M a x i m u m calcium uptake levels o b t a i n e d here yielded C a 2 ÷ / p h o s p h a t i d i c acid ratios of approximately 0.5. While this value could be achieved if Ca 2+ uptake were the result of b i n d i n g to exterior phosphatidic acid, experiments performed with unlabelled Ca 2÷ in the gel filtration c o l u m n s indicate that uptake is the result of active Ca 2÷ accum u l a t i o n a n d not phospholipid binding. Furthermore, the a m o u n t of Ca 2 ÷ accumulated is depen-

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dent on the amount and type of the Ca 2+ chelator employed. Clearly, in order for C a 2+ to be sensitive to material trapped inside the vesicle, it must be transported to the vesicle interior. This observation also indicates that sequestered calcium is predominantly associated with the trapped chelator and not with phosphatidic acid in the vesicle inner monolayer. Finally, the characteristics of Ca 2+ uptake observed in DOPA-containing vesicles are similar to those observed for pure D O P C vesicles in the presence of the Ca 2÷ ionophore A23187. The discrepancy between these results and previous observations for egg phosphatidic acid [8] could reflect the more saturated nature of egg phosphatidic acid, the concentrations of phosphatidic acid employed or the presence of 10% diacetylphosphate in the previous study [8]. It should be noted, however, that these studies do not necessarily prove a role for phosphatidic acid as a Ca 2÷ ionophore in vivo. In particular, the levels of D O P A employed here greatly exceed phosphatidic acid levels which may be expected to occur in biological systems. It remains to be determined whether variables such as phosphatidic acid unsaturation or localization can reduce the levels required to biologically relevant concentrations.

This research was funded by the Medical Research Council (MRC) of Canada. L.D.M. is a Fellow of the MRC, whereas P.R.C. is an MRC Scientist. References 1 Allan, D. (1982) Cell Calcium 3, 451 464 2 Tyson, C.H., Zande, H.V. and Green, D.E. (1976) J. Biol. Chem. 251, 1520-1332 3 Serhan, C., Anderson, P., Goodman, E., Dunham, P. and Weissman, G. (1981) J. Biol. Chem. 256, 2736-2741 4 Salmon, D.M. and Honeyman, T.W. (1980) Nature 284, 344-345 5 Putney, J.W., Jr., Weiss, S.J., Van de Walle, C.M. and Haddas, R.A. (1980) Nature 284, 345-347 6 Hanis, R.A., Schmidt, J., Hitzemann, B.A. and Hitzemanm R.J. (1981) Science 212, 1290-1291 7 Philipson, K.D and Nashimoto, A.Y. (1984) J. Biol. Chem. 259, 16-19 8 Holmes, R.P. and Yoss, N.L. (1983) Nature 305, 637-638 9 Farren, S.B., Sommerman, E. and Cullis, P.R. (1984) Chem. Phys. Lipids 34, 279-286 10 Comfurius, P. and Zwaal, R.F.A. (1977) Biochim. Biophys. Acta 488, 36-42 11 Nayer, R., Schmid, S.L., Hope, M.J. and Cullis, P.R. (1982) Biochim. Biophys. Acta 688, 169-176 12 Hope, M.J., Bally, M.B., Webb, G. and Cullis, P.R. (1985) Biochim. Biophys. Acta 812(1), in the press 13 B6ttcher, C.J.F., Van Gent, C.M. and Pries, C. (1961) Anal. Chim. Acta 24, 203-204