J. Biochem. 118, 693-699 (1995)

The Significance of Membrane in Exocytosis: Control of Liposome-Evoked Amylase Release from Secretory Granules Isolated from the Rat Parotid Gland1

Masako Mizuno-Kamiya,2 Hiroshi Inokuchi, Yasunaga Kameyama, Koji Yashiro, Sun-Ok Shin, and Atsushi Fujita Department of Oral Biochemistry,Asahi University School of Dentistry, 1851 Hozumi, Motosu, Gifu 501-02

Received for publication, February 14, 1995

We investigated the significance of the plasma membrane composition in exocytosis in an in vitro interaction system using an intact secretory granular fraction (SG) isolated from the rat parotid gland. When various liposomes (as a model of plasma membranes) were added to this assay system, rapid and transient amylase release from the SG was evoked and increased by Ca2+ in a concentration-dependent manner. The extent depended upon not only the amount of liposomes but also their lipid composition. The addition of 1,2-diacylglycerol and phosphatidic acid to egg yolk -liposomes remarkably facilitated the release. On the other hand, that of various free fatty acids had different effects depending upon their molecular species. Furthermore, a fluorescence de-quenching study demonstrated that membrane fusion actually occurred in this interac tion system, and appeared to correlate with the amylase release. These results suggest that the transient alteration of the membrane lipid composition upon cell activation is a modulator of the exocytotic membrane interaction.

Key words: amylase release, fluorescence de-quenching study, lipid composition, lipo some, secretory granule.

Exocytosis is the final stage of the stimulus-secretion artificial systems, membrane fusion can be chemically coupling phenomenon (1, 2). At this stage, intracellular induced by many reagents including various lipids, and its signals, which were chemically transmitted, are linked to a molecular mechanism has been investigated (11-13). physical event, membrane fusion between secretory gran Furthermore, membrane fusibility can be controlled by ules and plasma membranes (1-3). It has been proposed modifying the membrane lipid composition (14-17). that some cellular components such as a particular protein However, it is unclear whether or not these data are with high affinity to secretory granules (2, 4-6), Ca2+ (7 applicable to in vivo exocytosis, since the composition of - 9), and GTP (10), are modulators of exocytosis. We also native membranes is complex. The in vivo investigation of suggested that Mg-ATP is involved at this stage (9), from the significance of the membrane lipid profile in exocytosis the results of studies using a reconstitution system with has progressed little, since it is difficult to alter only the isolated plasma membranes and secretory granules. The membrane lipid composition without affecting other cellu secretory granular and plasma membrane lipids them lar metabolic processes. selves are also thought to be important and more direct We have reported a cell-free interaction system consist effectors of exocytosis, since membrane fusion requires a ing of isolated secretory granules and plasma membranes drastic structural reorganization of membranes (11). In from the rat parotid gland (9). In this system, plasma membranes affected the permeability of granular mem 1 This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of branes, resulting in the evocation of a rapid and transient Japan, and by a grant from Miyata Research Foundation of Asahi amylase release in a time-, temperature-, and Ca2+-de University. pendent manner. Furthermore, phosphatidylcholine-lipo 2 To whom correspondence should be addressed. somes evoked a similar release of amylase. Several reports Abbreviations: 1-acyl-GPC, 1-acyl-sn-glycero-3-phosphocholine; 20: have addressed the correlation between the change of 4, ; DAG, diacylglycerol; di20:4-GPC, 1,2-diarachi membrane permeability and fusion (17-19). Therefore, donoyl-sn-glycero-3-phosphocholine; dil8:2-GPC, 1,2-dilinoleoyI sn-glycero-3-phosphocholine; dil4:0-GPC, 1,2-dimyristoyl-sn-gly our system seems to be a functional model of exocytotic cero-3-phosphocholine; di18:1-GPC, 1,2-dioleoyl-sn-glycero-3-phos interaction and, compared with other artificial systems,

phocholine; egg PC, egg yolk phosphatidylcholine; FFA, free fatty conditions resemble the in vivo conditions. acid; 18:2, ; 18:3, ƒ¿-linolenic acid; lysoPC, lysophospha In this study, we investigated the significance of the tidylcholine; 17:0, margaric acid; Mops-NaOH, sodium 4-morpho membrane-lipid profile during exocytosis, using this cell linepropanesulfonate buffer; R18, octadecylrhodamine B chloride; free reconstitution system. Various liposomes, instead of 18:1, ; 16:0, ; PA, phosphatidic acid; PC, plasma membranes, interacted with isolated secretory phosphatidylcholine; PM, plasma membrane-rich fraction; PAF, platelet activating factor; SG, secretory granular fraction; 18:0, granules and the effect of membrane lipid composition on , the exocytotic interaction was compared by measuring the

Vol. 118, No. 4, 1995 693 694 M . Mizuno-Kamiya et al.

amount of evoked amylase released. Furthermore, the was 2.0•~10-1M (9)], then incubated with various lipo correlation between liposome-evoked amylase release and somes (final lipid concentration was 500ƒÊM) or an equal membrane fusion in our cell-free reconstitution system was volume of buffer C as a control. The final volume of the confirmed by a fluorescence de-quenching study. assay mixture was 0.1ml. After incubation for 15s, 9 volumes of ice-cold buffer C was added to stop the interac

EXPERIMENTAL PROCEDURES tion, then intact granules were removed by centrifugation at 10,000•~g for 3 min. The release of granular contents Chemicals-Egg yolk phosphatidylcholine (egg PC), resulting from the interaction between SG and liposomes synthetic with various kinds of acyl was determined by measuring amylase activity in the chains, platelet activating factor (PAF), and phosphatidyl supernatant. Liposome-evoked amylase release was calcu serine were obtained from Sigma Chemical (St. Louis, lated as the percentage of amylase activity present in the MO). 1,2-Diacylglycerol (1,2-DAG) derived from egg PC intact granules, using the following formula: and phosphatidylinositol (from pig liver) were obtained Liposome-evoked amylase release (%) from Serdary Research Laboratories (London, Canada). =100•~(A1-Ac)/(At-Ac) Palmitic acid (16:0), margaric acid (17:0), oleic acid (18:1), and arachidonic acid (20:4) were also purchased from At represents the total amylase activity, and A, and A, are Serdary Research Laboratories. Linoleic acid (18:2) and the released amylase activities in the presence and absence ƒ¿-linolenic acid (18:3) were obtained from Nacalai Tesque of liposomes, respectively (9). The value of Ac is usually (Kyoto) and stearic acid (18:0) was from Wako Pure less than 30% of the total amylase activity, which may Chemical Industries (Osaka). The lipids described above result from granular lysis during their preparation and/or were of the highest purity available. 1,3-Diacylglycerol the removal of intact granules after incubation. Net release (1,3-DAG) was prepared by the isomerization of 1,2-DAG during the incubation was negligible when liposomes were derived from egg PC and purified by thin-layer chromatog absent (9). raphy (TLC) (20). Phosphatidic acid (PA) and 1-acyl-sn Fluorescence De-quenching Assay for Membrane Fu

glycero-3-phosphocholine (1-acyl-GPC) were prepared by sion-Membrane fusion was monitored in terms of the means of the cabbage phospholipase D (21) and snake relief of self quenching of the fluorescent probe R18, venom phospholipase A2 (22) hydrolysis of egg PC, respec essentially by the method of Maclean and Edwardson (27). tively. The lipid-soluble fluorescent probe, octadecylrho The probe (1ƒÊ1 of the 20mM solution in ethanol) was damine B (R18) was purchased from Molecular Probes added to 300ƒÊl of SG suspension (2 mg protein/ml). SG (Oregon, OR). Other reagents were of the highest purity was incubated with the probe in 20mM Mops-NaOH, pH available from commercial sources. 7.0, containing 0.3M sucrose, 1mM MgC12, 1mM ATP, 5 Preparation of Liposomes-Lipids were dried under a mM EGTA, and 4.92mM CaCl2 at 30•Ž for 10 min, then nitrogen stream for about 15 min, then in a vacuum collected by centrifugation at 1,900•~g for 10 min. Labeled desiccator for about 45 min. They were hydrated with an SG was resuspended in 10 times the original volume of appropriate volume of buffer C [20mM sodium 4-morpho buffer C. In the assay, 25ƒÊ1 of SG was added to 2ml of the linepropanesulfonate buffer (Mops-NaOH), pH 7.0, con incubation buffer (20mM Mops-NaOH, pH 7.0, containing taining 0.3M sucrose] to give a 5mM lipid concentration 0.3M sucrose, 1mM MgCl2, 1mM ATP, 5mM EGTA, and and vigorously mixed with glass beads to obtain a complete 4.92mM CaC12) at 30•Ž. Fluorescence was measured ly dispersed suspension (23). All procedures were per continuously at 30•Ž in a spectrofluorophotometer (Model formed at room temperature. RF-540; Shimadzu, Kyoto) at an excitation wavelength of Preparation of Other Samples-A purified secretory 560nm and an emission wavelength of 590nm. After a 1 granular fraction (SG) and a plasma membrane-rich frac min preincubation, liposomes were added. The fluorescence tion (PM) were separated from male Wistar rat (9-13 was monitored for 2-6 min. The light scattering by lipo weeks old) parotid glands by differential and Percoll somes was also monitored under the same conditions except gradient centrifugation as described previously (9). Judg for using unlabeled SG. Then, its intensity was subtracted ing from the distribution of various marker enzymes, the to give the net increase of de-quenching. De-quenching was maximal extents of contamination with mitochondria, expressed as a percentage of that occurring after solubiliza lysosomes, endoplasmic reticulum, and plasma membranes tion of the labeled SG in 0.2% Triton X-100. in SG were less than 0.9, 3.5, 0.9, and 0.05%, respectively Biochemical Analysis-Amylase activity was assayed (24). with a commercial assay kit using a blue starch polymer Total polar lipids derived from PM (L-PM) were separat (Pharmacia Diagnostics AB, Sweden) essentially as de ed by TLC as described previously (25) from the total scribed by Ceska et al. (28). One unit (U) of amylase was lipids extracted from PM by the method of Bligh and Dyer defined as the amount of enzyme that catalyzed the forma (26). tion of reducing sugar equivalent to 1ƒÊmol of glucose per Liposome-Evoked Amylase Release from Secretory minute at 37•Ž. The protein concentration was determined Granules-The interaction between SG and liposomes was by the method of Lowry et al. (29) using bovine serum usually estimated by measuring amylase release evoked by albumin as the standard. The composition of the Ca2+ liposomes. The standard procedure was essentially as EGTA buffer for the free Ca2+ concentration was calculated described in our previous report (9) with a slight modifica as described by Harrison and Bers (30) with slight modi tion as follows. SG (2.5 U of amylase/ml) was preincubated fications of the incubation temperature (30•Ž) and ionic for 1 min at 30•Ž in 20mM Mops-NaOH, pH 7.0, contain strength (about 35mM) (9). Phospholipid phosphorus was ing 0.3M sucrose, 1mM MgCl2, 1mM ATP, 5mM EGTA, determined as described by Bartlett (31) and modified by and 4.92mM CaCl2 [the calculated free Ca2+ concentration Marinetti (32). and diacylglycerol concentra

J. Biochem. Liposome-Evoked Amylase Release from Secretory Granules 695 tions were measured by gas-liquid chromatography (25) PC and increased depending on the amount of liposomes. using margaric acid methyl as the internal standard. The release was enhanced with increasing concentration of Ca2+ (Fig. 2), like the release evoked by PM (9). These RESULTS results suggest that secretory granules could interact with the membranes without any plasma membrane protein in To investigate the significance of the membrane lipid this system. properties in the exocytotic interaction, liposomes were Often, especially in vitro, a change in membrane perme prepared based upon phosphatidylcholine (PC) as a model ability accompanies membrane fusion (17, 18). We con of plasma membranes. This is because PC is predominant firmed the relationship between the liposome-evoked amyl in the L-PM (about 50%) (33). When SG was incubated ase release and the fusion event itself (Fig. 3). When R18 with liposomes prepared from egg PC, a rapid and transient -labeled SG was incubated with egg PC-liposomes under the release of amylase was evoked (Fig. 1). This was roughly same conditions as those of the assay for amylase release similar to that using PM, although it was greater than that evoked by PM (9). Amylase was released at over 10 nmol

Fig. 3. The relationship between de-quenching and the lipo somal concentration. SG (5ƒÊg of protein, i. e., 0.045 U of amylase) labeled with R18 was incubated with various amounts of egg yolk Fig. 1. Amylase release from secretory granules evoked by phosphatidylcholine-liposomes under the conditions described in "EXPERIMENTAL PROCEDURES liposomes. SG (0.25 U of amylase) was incubated with various ." The fluorescence intensity was amounts of egg yolk phosphatidylcholine-liposomes under the condi traced. The arrow represents the time at which liposomes were added. tions described in "EXPERIMENTAL PROCEDURES." The amounts The basal fluorescence at the time of liposome addition is indicated on of liposomes added to the individual incubations were 10 (•£), 25 (•œ), each trace by a bar. The final concentrations of liposomes added in and 50 nmol of egg PC (•¡). Data are means•}SD of five determina individual incubations were 0 (a), 10 (b), 50 (c), 100 (d), and 150 tions. nmol of egg PC (e).

Fig. 2. Concentration-response curve demonstrating the Fig. 4. Concentration-response curve demonstrating the effects of the free Ca2+ on the liposome-evoked amylase release. effects of the free Cal2+ on the fluorescence de-quenching. SG (5ƒÊg SG (0.25U of amylase) was incubated in the presence of various of protein, i. e,, 0.045 U of amylase) labeled by R18 was incubated concentrations of free Ca2+ under the conditions described in "EX with egg yolk phosphatidylcholine-liposomes (150nmol) in the PERIMENTAL PROCEDURES." Data are means•}SD of 4-6 deter presence of various concentrations of free Cal2+ for 2 min. Data were minations. obtained from a typical experiment.

Vol. 118, No. 4, 1995 696 M. Mizuno-Kamiya et al.

except for the concentrations of SG and liposomes, de -3-phosphocholine (dil4:0-GPC) and 1,2-dilinoleoyl-sn quenching clearly occurred and was enhanced with increas glycero-3-phosphoccholine (dil8:2-GPC) had slightly lower ing amount of liposomes. In addition, as well as amylase (about 80% of egg PC) or similar ability (about 90%), release, de-quenching was stimulated by Ca2+ in a concen respectively. tration-dependent manner (Fig. 4). This stimulation of Since SG has phospholipase A2 activity (24), it is unclear de-quenching by Ca2+ is in good agreement with that of whether the results in Table I are due to the acyl moiety or amylase release. Ca2+ at 10-3M stimulated either by about to the free fatty acid (FFA) produced enzymatically during three times, compared with that at 0M. Thus, although the incubation with SG. Therefore, the effects of various free percentage of de-quenching was less than that of amylase fatty acids in PC-liposomes upon amylase release were release evoked, both were affected by the same factors studied (Table II). In this assay system, added FFA (15 (amounts of liposomes and Ca2+) and showed similar mol%) is much larger than endogenous FFA since the responses (9, Figs. 1-4). These results indicated that granular phospholipase A2 activity is low (24). Therefore, membrane fusion and change of membrane permeability the effect of FFA produced enzymatically may be negligible were correlated to some degree in our cell-free reconstitu in this experiment. The addition of saturated FFA such as tion system. Therefore, the extent of amylase release palmitic acid (16:0), margaric acid (17:0), and stearic acid evoked by various liposomes was compared as a parameter of the exocytotic interaction between SG and liposomes.

In Table I, the effects of the acyl chain composition on the TABLE III. The effect of phosphatidylcholine metabolites on interaction between SG and liposomes are shown. Although evoked amylase release. SG (0.25 U of amylase) interacted with all PC-liposomes studied evoked amylase release from SG, egg PC-liposomes containing 15 mol% 1,2-diacylglycerol, 15 mol% they did so at different levels depending upon their fatty phosphatidic acid, 17 mol% 1-acyl-sn-glycero-3-phosphocholine, or acyl molecular species. Compared with egg PC, 1,2-dioleo 10 mol% 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) under the conditions described in "EXPERIMEN yl-sn-glycero-3-phosphocholine (di18:1-GPC) had higher TAL PROCEDURES." Data are means•}SD from the number of ability (about 160%), whereas 1,2-dimyristoyl-sn-glycero determinations shown in parentheses.

TABLE I. The effect of the acyl chain composition of phospha tidylcholine-liposomes on evoked amylase release. SG (0.25 U of amylase) was interacted with liposomes prepared from various kinds of phosphatidylcholine (50 nmol) under the conditions de scribed in "EXPERIMENTAL PROCEDURES." Data are means•}SD from the number of determinations shown in parentheses.

8 Ratio of the amylase release evoked by various liposomes to that evoked by egg PC-liposomes. *p<0.05: Significance of difference between the amylase release evoked by various liposomes and that by egg PC-liposomes as judged by Student's t test.

a Ratio of the amylase release evoked by various liposomes to that by egg PC-liposomes. **p<0.01: Significance of difference between the amylase release evoked by various liposomes and that by egg PC-liposomes as judged by Student's t test.

TABLE II. The effect of the free fatty acid component in lipo somes on evoked amylase release. SG (0.25 U of amylase) inter acted with egg PC-liposomes containing 15 mol% free fatty acid under the conditions described in "EXPERIMENTAL PROCEDURES." Data are means•}SD of five determinations except for egg PC-lipo somes (10 determinations).

Fig. 5. The effects of diacylglycerol isomers and phosphatidic acid in egg yolk phosphatidylcholine-liposomes on amylase release from secretory granules. SG (0.25 U of amylase) was incubated with egg PC-liposomes containing 1,2-diacylglycerol (•œ), 1,3-diacylglycerol (•Z), or phosphatidic acid (•£) under the conditions described in "EXPERIMENTAL PROCEDURES." Data are express ed as a ratio of the amylase release evoked by various liposomes to a Ratio of the amylase release evoked by various liposomes to that by that by egg PC-liposomes (13.6%). Values are means•}SD of five egg PC-liposomes. b Fatty acids are expressed as carbon chain length: determinations. Values at 10 and 15 mol% 1,2-DAG, and at 15 and 25 degree of unsaturation. **p <0.01: Significance of difference between mol% PA were significantly different from the control (egg PC only) the amylase release evoked by various liposomes and that by egg as judged by Student's t test (10 mol% 1,2-DAG and 25 mol% PA, p< PC-liposomes as judged by Student's t test. 0.01; 15 mol% 1,2-DAG and PA, p

J. Biochem. Liposome-Evoked Amylase Release from Secretory Granules 697

TABLE IV. The effect of acidic phospholipids on evoked amylase release. SG (0.25 U of amylase) interacted with egg PC-liposomes containing 17 mol% various acidic phospholipids for 120s under the conditions described in "EXPERIMENTAL PROCE

DURES." Data are means•}SD of three determinations .

a Ratio of the amylase release evoked by various liposomes to that evoked by egg PC-liposomes. *p<0 .05: Significance of difference between the amylase release evoked by various liposomes and that by egg PC-liposomes as judged by Student's t test.

Fig. 6. The effects of 1,2-diacylglycerol and phosphatidic acid (18:0) to egg PC-liposomes completely suppressed lipo in egg yolk phosphatidylcholine-liposomes on the fluorescence de some-evoked amylase release and stabilized the SG-mem quenching. SG (5ƒÊg of protein, i. e., 0.045 U of amylase) labeled branes. This tendency was similar to that in the case of the with R18 was incubated with egg PC-liposomes containing 1,2-diacyl

saturated fatty acyl chains of PC-liposomes (Table I). Oleic glycerol (•œ) or phosphatidic acid (•£) under the conditions described acid (18:1) and linoleic acid (18:2) also had a tendency to in "EXPERIMENTAL PROCEDURES." Data were obtained from a reduce egg PC-liposome-evoked release by 42 and 39%, single typical experiment and are expressed as the ratio of the de respectively, although their esterified forms did not elicit quenching by various liposomes to that by egg PC-liposomes. Values for no addition (i. e. egg PC only) were 0.187 and 0.123% in the assays such effects (Table I). In contrast, polyunsaturated fatty of the effects of 1,2-DAG and PA, respectively. acids such as a-linolenic acid (18:3) and arachidonic acid (20:4) did not cause such suppression. Thus, FFA do not necessarily show a similar effect to the esterifled form. ing as well as they did the evoked amylase release. Table III shows the effects of other PC metabolites on the Therefore, the two lipid classes seemed to enhance the interaction with SG. All lipids except for platelet activating membrane fusion between egg-PC liposomes and SG. In factor (PAF) were enzymatically derived from egg PC. The addition, their concentration-response curves were roughly addition of 1,2-diacylglycerol (1,2-DAG) and phosphatidic similar to those in the case of amylase release. Namely, the acid (PA) to egg PC-liposomes remarkably facilitated maximal fusion occurred at either 10 mol% DAG or 15 amylase release, but that of 1-acyl-sn-glycero-3-phospho mol% PA, at which the maximal amylase release was choline (1-acyl-GPC), which is a detergent-like molecule evoked. A decline of the effect at higher concentrations of (15, 18), exerted no effect. PAF has structural similarity to DAG was also observed. Thus, the fusion seemed to PC and 1-acyl-GPC, and reportedly induces amylase re correlate with the amylase release. lease from the parotid gland (34). However, the addition of PAF into egg PC-liposomes rather reduced the extent of DISCUSSION release in our system. Figure 5 shows the effect of the 1,2-DAG and PA concen We showed that various liposomes (as a model of plasma tration in liposomes upon amylase release. 1,2-DAG and membrane) can interact with SG to evoke amylase release PA showed roughly similar concentration-response curves. in a cell-free system. We also showed that liposomes caused The maximal amylase release was evoked at 10 mol% de-quenching of a fluorescent probe added to SG in this 1,2-DAG or 15 mol% PA and the extent of the maximal system. The percentage of de-quenching was about 10-fold release was roughly similar. However, at a higher concen lower than that of evoked amylase release. However, the tration (15 mol%) of 1,2-DAG, the facilitating effect was de-quenching values were reported to underestimate the rather decreased, whereas the effect of PA hardly declined extent of membrane fusion (27), and can be regarded as up to 25 mol%. The effect of another isomer, 1,3-diacyl essentially arbitrary units. Furthermore, the difference glycerol (1,3-DAG), was also investigated. Although the between the two parameters may also be in part due to the addition of a small amount of 1,3-DAG facilitated the difference in the assay conditions. In the case of fluores amylase release, the extent of the maximal release evoked cence de-quenching, the concentrations of SG and lipo by liposomes containing it was lower than that by liposomes somes were much lower than in the assay for amylase containing 1,2-DAG. In addition, the decline of the effect at release, since the fluorescence intensity of labeled SG higher concentration was more evident. should be adjusted to a suitable range for measuring. Table IV shows the effect of negatively charged phospho Despite these differences in absolute values, the sensitivity lipid on the amylase release evoked by egg PC-liposomes. to Ca2+ and the dependence upon the liposome-concentra Among the three naturally occurring acidic phospholipids tion of de-quenching were similar to those of liposome which are major components of acidic phospholipids of evoked amylase release. Furthermore, DAG enhanced both parotid gland, only PA facilitated the release and the other de-quenching and amylase release at similar concentra two had no effect. tions. Phosphatidic acid also increased both parameters in The effects of 1,2-DAG and PA on the exocytotic interac a similar concentration-dependent manner. These results tion were also investigated by measuring the de-quenching suggest some correlation between fluorescence de-quench of fluorescence caused by liposomes containing them. As ing and liposome-evoked amylase release. Therefore, the shown in Fig. 6, 1,2-DAG and PA facilitated the de-quench liposome-evoked amylase release seems to be one indicator

Vol. 118, No. 4, 1995 698 M. Mizuno-Kamiya et al,

of exocytotic interaction (i. e. fusion). acids facilitated membrane fusion. Although these effects In this study, we chose simple egg PC-liposomes as a were not marked in our system, this discrepancy might, in control interaction partner with secretory granules. One part, be accounted for by the membrane conditions. In our reason for this is that PC-liposomes are thought to be system, fatty acids were added in simple egg PC liposomes, physically stable and to have low fusibility, since PC favors whereas in the other systems, they were added to biological a bilayer structure (14, 15, 35). However, simple PC membranes which have a more complex lipid composition - liposomes interacted with SG to evoke amylase release and (14, 45). to relieve self quenching of the fluorescence. This seems to To determine the relevance of our observations to in vivo support our previous suggestion that potential for exocyto saliva secretion, it must be considered whether or not apical tic interaction is inherent in SG (9). Similar results have surface membranes are subject to such lipid conditions been reported by investigators using a cell-free system during in vivo cell activation. We detected both phospho consisting of Golgi membranes (36). In that report, isolated lipase A, and A2 activities in SG (24), and in our prelimi Golgi membranes fused with simple PC-liposomes, and the nary experiment, a similar phospholipase A2 activity was time course and extent of fusion resembled our observa also detected in the apical surface membrane-rich fraction tions in this study. A similar mechanism may operate at from the rat parotid gland (unpublished data). The apical other steps of exocytosis. fraction also had phospholipase D activity (unpublished In our system, the interaction between SG and liposomes data). Therefore, it is possible that these enzymes produce is sensitive to the lipid components of the latter, since the FFA and/or PA at the fusion site. However, we have not extent of interaction varied with the membrane lipid confirmed the response of these enzymes to cell activation. composition. However, the lipid composition can also affect Further investigations will be necessary to provide direct the size and stability of liposomes. Therefore, the individ evidence for the generation of these lipids in activated ual effects of the lipids seen here were compared with those apical surface membranes, to establish the relevance of the using different systems. We found that deleting the base lipids to exocytosis. moiety and polar head group from PC increased its evoking ability, whereas deleting the fatty acyl moiety had no We are very grateful to the late Dr. Yutaka Yokota (former professor effect. Since other acidic phospholipids such as phospha of the Department of Oral Biochemistry, Asahi University School of Dentistry) for his helpful discussions and valuable suggestions during tidylinositol and phosphatidylserine had no effect, the effect this study. of PA is probably not due to its negative charge. Recent studies using artificial membrane systems have also in dicated that the presence of PA and DAG in membranes REFERENCES decreased their lamellar to hexagonal II phase transition temmmerature to induce a non-bilayer structure (17, 37-39) 1. Palade, G. E. (1975) Intracellular aspects of the process of protein secretion. Science 189, 347-358 and facilitated their fusion (17, 40). Our observations 2. De Lisle, R. C. and Williams, J. A. (1986) Regulation of mem support these results. On the other hand, reports concern . brane fusion in secretory exocytosis. Annu. Rev. Physiol. 48, ing the effects of lysophosphatidylcholine (lysoPC) are 225-238 contradictory. Some have indicated that lysoPC is a potent 3. Putney, J. W., Jr. (1986) Identification of cellular activation fusogen (41, 42), whereas others (15, 18) have indicated mechanisms associated with salivary secretion. Annu. Rev. that lysoPC has no fusibility; the latter is consistent with Physiol. 48, 75-88 our observations. This discrepancy may have arisen from 4. Creutz, C.E. and Sterner, D. C. (1983) Calcium dependence of the binding of synexin to isolated chromaffin granules. Biochem. the concentrations of lysoPC used. The former reports used Biophys. Res. Commun. 114, 355-364 about a 1,000-fold higher lysoPC concentration than that 5. Llinas, R., McGuinness, T. L., Leonard, C. S., Sugimori, M., and which was used here and in the latter reports. In our Greengard, P. (1985) Intraterminal injection of synapsin I or system, PAF rather reduced the evoking ability of the egg calcium/calmodulin-dependent protein kinase II alters neuro PC-liposomes, although many investigators have demon transmitter release at the squid giant synapse. Proc. Natl. Acad. strated stimulating effects of PAF and its metabolites on Sci. USA 82, 3035-3039 6. Borregaard, N., Kjeldsen, L., Lollike, K., and Sengelov, H. secretion (34, 43, 44). Since our system did not contain (1992) Ca2+-dependent translocation of cytosolic proteins to plasma membranes with various receptors, our results may isolated granule subpopulations and plasma membrane from represent the direct action of PAF on SG-membranes human neutrophils. FEBS Lett. 304, 195-197 rather than receptor-mediated action. Soling et al. (34) 7. Baker, P. F. and Knight, D. E. (1978) Calcium-dependent exo reported a decline of secretion-stimulating ability at higher cytosis in bovine adrenal medullary cells with leaky plasma (0.1ƒÊM) concentrations. membranes. Nature 276, 620-622 8. Von Grafenstein, H. and Neumann, E. (1981) Interaction of The fatty acyl moiety in membranes is also an important chromaffin granules with plasma membranes mediated by Ca2+ factor. The effect of fatty acyl moiety on the amylase and Mg2+-ATP using self-generating gradients of Percoll. FEBS release did not necessarily correspond with that on the Lett. 123, 238-240 membrane fluidity. The effect of unsaturated species on the 9. Mizuno, M., Kameyama, Y., Yashiro, K., Shin, S. O., and Yokota, interaction between SG and liposomes differed between the Y. (1992) Properties of plasma membrane-induced amylase esterified and free forms. 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Vol. 118, No. 4, 1995