Acta Physiol Scand 1990, 140, 393400

Calcium and calmodulin stimulate phospholipase A, and fusion of H,K-ATPase-containing membrane vesicles isolated from pig gastric mucosa H. O L A I S S O N , S. M A R D H and G. A R V I D S O N Department of Medical and Physiological Chemistry, University of Uppsala, Sweden

OLAISSON, H., MKRDH,S. & ARVIDSON, G. 1990. Calcium and calmodulin stimulate phospholipase A, and fusion of H,K-ATPase-containing membrane vesicles isolated from pig gastric mucosa. Acta Physiol Scand 140, 393400. Received 26 March 1990, accepted 11 June 1990. ISSN 0001-6772. Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.

Fusion of pig gastric H,K-ATPase- and phospholipase A,-containing vesicles in z'itro was studied by electron microscopy and by monitoring the change in fluorescence of octadecyl rhodamine B-labelled vesicles. Ca2+stimulated fusion of the vesicles, and the fusion rate showed a positive correlation with the activity of the phospholipase A,. Both the Ca2+-stimulatedfusion rate and the Ca2+-dependent phospholipase A, activity were further enhanced by the presence of calmodulin. The present results supported our previous findings (Olaisson et al. 1990) and further indicate that the phospholipase A, associated with the H,K-ATPase-containing membranes might play a central role in membrane fusion processes in the stimulated parietal cell. Key words : calcium, calmodulin, fusion, H,K-ATPase membrane, parietal cell, phospholipase A,

T h e H,K-ATPase-containing vesicular membranes are derived from the secretory membrane system of the HC1-producing parietal cells of the gastric mucosa (Ganser & Forte 1973a, b, Lee et al. 1974, Chang et al. 1977). A resting parietal cell contains numerous tubulovesicles. Stimulation leads to a morphological transformation which is characterized by a large increase in the apical membrane area and the formation of a large number of microvilli in the secretory canaliculi at the expense of the tubulovesicular surface area. T h e morphological change has been interpreted as being due to fusion of the H,K-ATPase-containing membrane vesicles (Forte et al. 1977, Gibert & Hersey 1982, Jiron et al. 1984, Vial et al. 1985), although some investigators have considered the expansion of a Correspondence : Sven Mirdh, Department of Medical and Physiological Chemistry, Biomedical Centre, University of Uppsala, Box 575, S-751, 23 Uppsala, Sweden.

collapsed tubular system into canaliculi as more probable (Berglindh et al. 1980). Ca2+ has been shown to play a pivotal role in the induction of membrane fusion (De Lisle & Williams 1986). In most secretory cells a rapid elevation of the cytosolic [Ca"] occurs during stimulation by secretory agents (Rubin 1982). This also applies to the parietal cell where either histaminergic, cholinergic or gastrinergic stimulation increases the intracellular [Ca"] (Muallem & Sachs 1984, Chew & Brown 1986, M i r d h et al. 1987). T h e Ca2+-binding protein calmodulin has been proposed as having a regulatory function in membrane fusion (DeLorenzo 1980, Trifaro et al. 1989). Calmodulin increases the activity of a number of Ca2+-dependent enzymes by increasing their sensitivity to Ca2+(Cheung 1980). Recently we found that an H,K-ATPase-containing membrane fraction prepared from pig gastric mucosa exhibits a Ca"-dependent phospholipase A, activity which was increased in the presence of calmodulin (Olaisson et al. 1990).

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Fig. 1. Self-quenching of C,,RhB fluorescence in H,K-ATPase-containing membrane vesicles. The fluorescence was determined before and after addition of Triton X-100 ( 1 O b , v/\-). From the ratio the percentage of fluorescence self-quenching was calculated and plotted against the concentration of C,,RhB in the membranes (molO, of the membrane lipids). (A) Emission spectra (excitation wavelength = 522 nm) of C,,RhB incorporated in the vesicle membranes was recorded before (solid line) and after (dotted line) addition of Triton X-100 (B). Llannheim (Mannheim, FRG), octadecyl rhodamine B chloride was from Molecular Probes (OR, USA), pbromophenacyl bromide from Sigma (St Louis, MO, US.-\), and silica gel H was from Merck (Darmstadt, FRG). Other chemicals were of analytical grade and commercially available. Deionized, doubly quartzdistilled water was used throughout the experiments. Preparation of H,K-ATPase-containing membrane rrsislrr and mashing procedure. Microsomal membranes containing the H,K-ATPase as the dominating protein (about 70°0 of the total protein content) were prepared from homogenates of pig gastric mucosa essentially as previously described (Ljungstrom et at. 1984). One millimolar EGTA was included in all solutions used in the preparation procedure. The purified H,K-ATPase-containing vesicle membranes were then washed according to previously established procedures (Olaisson et ul. 1990). The membrane vesicles were stored at -70 "C in 0.25 M sucrose containing 1 mM EGTA. In a few experiments as indicated, either unwashed vesicle membranes were used, or membranes washed once in 0.25 M sucrose containing 50 mM Tris-HCI, pII 7.5, and 1 mM EGTA. Phospholipase - 4 , assu,y. In some experiments M A T E R I A L S AND M E T H O D S phospholipase t\. activity and fusion rate were meas+fateriais l-Palrnit0~l-2-[1-'~CCJarachidono~l-sn- ured simultaneously in the same sample. The assay of gJj cero-3-phosphocholine was from Veu England phospholipase A, was carried out in accordance with Yuclear Chemicals (Dreieich, FRG) Calmodulin and previous results showing that the phospholipase A, Triton Y-100 uere purchased from Boehringer actkit!- may be measured by adding a trace amount of

Electron microscopy ma>-be used for studying membrane fusion, but severs1 other methods, employing various fluorescent probes, are also available today for such investigations. T h e rate of fusion can be determined both by measuring the rate of membrane lipid mixing (probe mixing or probe dilution) and bl- measuring the rate of intermixing of the interior aqueous contents. Duzgunes et al. (1987) obtained different rates of liposome fusion depending on the method used (membrane probe mixing > membrane probe dilution > contents mixing). T h e membrane probe-mixing technique was sensitive to aggregation, in contrast to the probe dilution technique. I n the present paper, electron microscopy, as well as a membrane lipid-mixing (probe dilution) assay using the probe octadecgl rhodamine B, was employed in order to investigate the fusion of isolated membrane vesicles which contain both H,K-ATPase and phospholipase A,.

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Fig. 3. Morphometric analysis of vesicle size. Morphometric analysis, as described in Materials and Methods, was performed on electron micrographs of the same vesicle preparations as shown in Fig. 2. Vesicles were incubated in the presence of 1 mM EGTA (e),or in the presence of 1 mM CaC1, plus 2.0~14calmodulin (0). Symbols represent mean fSE, where n is 300 vesicles at 0 and 60 min incubations and 50 vesicles at 30 min. Experimental data were analysed for statistical significance by ANOVA using the Statview 512f program. Asterisks (") indicate data significantly different from controls at 0 min (P < 0.01).

F+,,. 2. Electron micrographs of H,K-ATPase-containing membrane vesicles. Vesicles were incubated in the presence of 1 mM EGTA (A) or 1 mM CaCI, plus 2.0 p M calmodulin (B) at 37 "C for 60 min in 50 mM Tris buffer, pH 7.5, containing 0.25 M sucrose. After incubation, samples were prepared for electron miscroscopy as described in Materials and Methods. The bars represent 0.5 pm.

radioactive phosphatidylcholine to the vesicles (Olaisson et al. 1990). 1-Palmitoyl-2-[ l-'4C]arachidonoylsn-glycero-3-phosphocholine (1.2 x 10" d.p.m. mmol-l) was dispersed by sonication in 50 mM Tris-HC1 buffer, pH 7.5, in a sonication bath for 30 min at room temperature. Ten microlitres of this dispersion containing 0.45 nmol ['4C]phosphatidylcholine was added to the vesicles in the cuvette at the start of the fusion assay. After 20 min a sample of the incubation mixture was removed for lipid extraction and determination of phosphatidylcholine hydrolysis by thin-layer chromatography as described (Olaisson et al. 1990).

Protein assay. The protein concentration was measured according to Lowry et al. (1951). Electron microscopy. Vesicle membranes were fixed for electron microscopy by the addition of 20 volumes of 2.5% dutaraldehyde in 0.125 M cacodylate buffer, pH 7.2. The suspensions were centrifuged at 72000 g for 30 min at 4 " c . The membranes were then washed twice with the glutaraldehyde-containing buffer and left overnight at 4 "C. The samples were treated with 1% OsO,, dehydrated with ethanol and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate (Nilsson et al. 1981). A continuous series of ultrathin sections was made, and electron micrographs were taken every fifth section in a JEOL-100 B. The thickness of the sections was 5&70 nm. Vesicle membranes were randomly selected on the micrographs and the cross-sectional area was analysed with a graphic pen-data tablet device for planimetry (MOP, Kontron). Fusion assay using octadecyl rhodamine B-labelled vesicles. Fluorescence was monitored and recorded in a Hitachi F-4000 fluorescence spectrophotometer. The method of Hoekstra et al. (1984) was used to label vesicles with octadecyl rhodamine B (C,,RhB), which was dissolved in ethanol. The final concentration of ethanol in the incubation mixture was 1 yo (v/v). The fluorophore was mixed with membrane vesicles

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Fusion betn-een labelled and unlabelled vesicles resulted in an increased fluorescence as a result of the dilution of the fluorophore and subsequent relief of fluorescence self-quenching (Hoekstra e t al. 1984). Maximal fluorescence, F,,,, was recorded at the end o f each incubation after the addition of Triton X-100 (1 O0, v/v), which dissolved the membranes and thus diluted the probe enough to abolish self-quenching. The rate at which the fluorescence increased at the beginning of the incubation was used as a measure of the initial rate of vesicle fusion. T h e initial slope was expressed as AFoh min-', where AF% is the increase in fluorescence in per cent of F,,,,. Each fusion experiment was started by adding non-labelled vesicles to C,,RhB-labelled vesicles (ratio of 16: 1 and 300 pg total protein). T h e vesicles were suspended in a 50 mxr Tris-HCI buffer, pH 7.5, containing 0.25 M sucrose and additions as indicated. T h e final incubation volume was 2.0 ml.

Fig. 4. Fusion of H,K-.1TPase-containing membrane \esicles in the presence of Ca'- and calmodulin. (;,,RhB-labelled and non-labelled vesicles (ratio of 1 : 16) were mixed in 50 ms3 Tris-HCI buffer, pII 7.5, containing 0.2.5 XI sucrose, and incubated at 37 "C in the presence o f either 1 mxi CaCI, plus 2.011~1 RESULTS calmodulin (solid line), or 1 mXj EGT.1 (dotted line). Fusion is indicated by an increase in fluorescence Vesicles were incubated at 37 "C, either in the which was continuously monitored. presence of 1 mM CaCI, plus 2.0 ,UM calmodulin, comprising 0.8 mg protein and 1.6 pmol lipid (phospholipids + cholesterol glycosphingolipids) in 50 mXi Tris-IICI buffer, pH 7.5, containing 0.2.; XI sucrose and 1 mxi EGT.4. The final incubation volume \+as O.4ml. After 60 min in the dark at room temperature all non-incorporated fluorophore was removed by Sephadex G-75 chromatograph!-, The percentage uptake of various amounts of C,,RhB into the vesicle membranes was determined after extraction of the incorporated C,,RhB with chloroform/ methanol containing IlCl (Hoekstra et ul. 1984). At the fluorophore concentrations tested ( W 0 / i l l ) , there was an incorporation of 75-85 O O . Fluorescence quenching increased as the concentration of the fluorophore in the membranes increased (Fig. 1 .\). Quenching increased linearly up to about 0.4 mot", C,,RhB. hlaximal fluorescence was determined in the presence of lo, (v/v) Triton X-100. Doubling the Triton X-100 concentration did not increase the fluorescence further. Nor did the detergent alter the emission wavelength yielding the highest value for the C,,RhB incorporated in the vesicle membranes (Fig. 1 B). The excitation spectrum obtained from labelled tesicles exhibited two maxima, one at 5 5 5 nm and a smaller one at 522 nm. Similar quenching curyes were obtained at the two different excitation maxima for membrane-incorporated C,,RhB. An excitation a-avelength of 522 nm was selected in this study, since at this wavelength the light recorded with unlabelled vesicle membranes in the cuvette was negligible at the maximal emission wavelength of 577 nm.

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or in the presence of 1 mM EGTA, and then prepared for electron microscopy. T h e vesicles appeared to be larger after incubation in the presence of Ca2+ plus calmodulin than after incubation with E G T A (Fig. 2 ) . This impression was verified by morphometric analysis of randomly selected vesicle membranes. T h e crosssectional area of the vesicles remained unchanged during incubation with E G T A for 60 min, but in the presence of Ca2+ plus calmodulin a significant increase was observed after incubation periods of 30 and 60 min (P < 0.01; Fig. 1). These data demonstrate that Ca2+plus calmodulin induces fusion of the H,K-ATPasecontaining membrane vesicles. I n the fluorescence assay of vesicle fusion the fluorescence was stable in the presence of E G T A for more than 60 min but increased in the presence of Ca2+ plus calmodulin (Fig. 4). Vesicles incubated in the presence of 1 mM Ca2+ were found to fuse at varying rates. This variation could be correlated to variations of the endogenous phospholipase A, activity of the to 10-' M Ca2' the membranes (Fig. 5 ) . At rate of fusion was about twice as fast as that at M Ca" both in unwashed membranes (phospholipase A, activity not determined) and in washed membranes with a phospholipase A, activity around 10 nmol mg-' h-' (Fig. 6). Fusion did not occur in the presence of pronase (Fig. 6).

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Fig. 5. Correlation between fusion rate and endogenous phospholipase A, activity. Results from various vesicle preparations, in which both phospholipase A, activity and fusion rate had been determined in the presence of 1 mM CaCI, but in the absence of calmodulin, were compiled and plotted as shown. The curve fitted to the data is an exponential regression curve 0,= 0.036455 x 10 0.0874742; R2 = 0.834 using the Statview 512+ program).

The phospholipase A, activity of the H,KATPase-containing membranes is stimulated by calmodulin and inhibited by treatment of the membranes with p-bromophenacyl bromide (Olaisson et al. 1990). As shown in Fig. 7(A), both fusion rate and phospholipase A, activity increased in the presence of calmodulin, but the stimulatory effect on the fusion rate appeared to be more pronounced than the stimulation of phospholipase A, activity. Treatment of the vesicles with p-bromophenacyl bromide inhibited both fusion and phospholipase A, activity in parallel (Fig. 7B). DISCUSSION Two models have been proposed for the hydrochloric acid secretory cycle. One includes fusion, retrieval and recycling of membrane vesicles (Forte et al. 1977, Gibert & Hersey 1982, Jiron et al. 1984, Vial et al. 1985). The other postulates the osmotic swelling of a membrane system which is collapsed in the resting state of the parietal cell (Berglindh et al. 1980). The present investigation demonstrates that isolated H,K-ATPase-containing membrane vesicles may undergo fusion in nitro, thus supporting a fusion model for the hydrochloric 16

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Fig. 6. Rate of fusion at different concentrations of Ca2+. Fusion rates were determined in different preparations in the presence of various concentrations of CaCI, but in the absence of calmodulin. The figures within parenthesis are the phospholipase A, activities of these preparations. Open symbols represent fusion rates in washed membrane vesicles and filled symbols represent fusion rates of a preparation of unwashed vesicles which were frozen after C,,RhB labelling and then stored for 1 ( 0 ) or 3 (m) days before measurement of the fusion rate. The rate of fusion was also assayed at 1 mM Ca2+ for vesicles which were pretreated with pronase (1 mg ml~') at room temperature for 15 min (X). The Ca2+ levels were maintained with CaZ+/EGTA buffers (Bartfai 1979). The Ca2+concentration was determined with quin-2 (Tsien et al. 1982) in three experiments ( 0 , in samples in which the expected concentrations of free Ca2+ were < 1 0 - 6 ~ .

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acid secretory cycle, or a combination of fusion and osmotic swelling. In the present work a correlation was found between fusion rate and phospholipase A, activity which, however, varied in different membrane preparations (Olaisson et al. 1990). Consistently, deactivation of the phospholipase A, by p-bromophenacyl bromide abolished fusion, while calmodulin seemed to enhance fusion more than the phospholipase A, activity. Phospholipase A, products such as unsaturated fatty acids and lysophosphatidylcholine are fusogenic (Lucy 1978) and might be involved in exocytotic secretion. Thus unesterified cis-unsaturated fatty acids are ascribed an essential role as membrane-perturbing agents in the fusion of chromaffin granules (Creutz 1981, Drust & Creutz 1988), and lysophospholipids are proposed to promote the release of histamine from ACT 140

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Fig. 7. Initial rate of fusion and phospholipase A, activity. Initial rate of fusion (filled symbols) and phospholipase At activity (open symbols) were determined simultaneously. Vesicles from three different preparations were used. Each preparation is identified by its symbol. The concentration of CaCI, \yas 1 msi in all samples. (A) Effects of calmodulin on initial rate of fusion and phospholipase A, activity. Values obtained in the absence of calmodulin but in the presence of 1 msf CaCI, represent 100”,. (B) Effects of pretreatment of the vesicles with p-bromophenacyl bromide; assays of phospholipase A, activity were performed in the presence of 1 mM CaCI,; no calmodulin was added. Both C,,RhB-labelled and non-labelled vesicles were preincubated with different concentrations of p-bromophenacyl bromide in .50 mM Tris buffer, pH 7.5, containing 0.25 M sucrose and 1 mM EGTA. p-Bromophenacyl bromide was dissolved in acetone. The final concentration of acetone in the incubation mixture was 1 O O (v/v). After 30 min at room temperature the incubation mixtures were passed through a Sephadex G-75 column. Initial rate of fusion and phospholipase A, activitj- of the recovered vesicles were expressed as the per cent of the values obtained without pretreatment with p-bromophenacyl bromide.

rat basophilic leukaemia cells (McGivney et al. 1981), insulin release (Metz 1986) and the release of neurotransmitters (Moskou-itz et ul. 1982, Bradford et al. 1983, Nishikawa et a / . 1989). Although calmodulin has been shown to be important in hydrochloric acid secretion in the parietal cells (Im e t a / . 1984, Schepp et al. 1987, 1989), its key function(s) has not yet been defined. T h e extensive morphological transformation of the parietal cell upon stimulation and the subsequent participation of various membranebound and cytoskeletal proteins (Vial & Garrido 1976, Stewart & Kasbekar 1981, Black et a / . 1982, Albinus & Mayer 1985) most likely involve a multitude of interdependent reactions in which La” and calmodulin are important regulators. T h e present and our previous results (Olaisson et a/. 1990) indicate that one of these reactions may be the activation of phospholipase A,. We are grateful to Anna-Carin Ojteg and Jing Yie 41a for skilful technical assistance.

This work was supported by the Swedish Natural Science Research Council, the Swedish Medical Research Council, Project 4X-4965 and Ulf Widengren’s Memorial Foundation.

REFERENCES ALBINUS, M. & MAYER,B. 1985. The effect of antimitotic and microfilament disrupting agents on functions of isolated guinea-pig parietal cells. Agents .4i-tions 16, 199-201. BARTFAI,T. 1979. Preparation of metalkhelate complexes and the design of steady-state kinetic experiments involving metal nucleotide complexes. In: G. Brooker, P. Greengard & G.A. Robison (eds.) Adrances in Cyclic Nucleotide Reseurch, vol. 10, pp. 219-242. Raven Press, New York. BERGLINDH, T., DIBONA, D.R., ITO, S. & SACHS,G. 1980. Probes of parietal cell function. Am 3 Ph,ysiol 238, G16-5-6176. BLACK,J.A., FORTE,T.M. & FORTE,J.G. 1982. The effects of microfilament disrupting agents on HCl secretion and ultrastructure of piglet gastric oxyntic cells. Gustroenterology 83, 595-604.

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G. & SCHOLES, P. 1974. An ATPase BRADFORD, P.G., MARINETTI, G.V. & ABOOD,L.G. LEE,J., SIMPSON, from dog gastric mucosa: Changes of outer pH in 1983.Stimulation of phospholipase A, and secretion of catecholamines from brain synaptosomes by suspensions of membrane vesicles accompanying potassium and A23187.3Neurochem 41, 1684-1693. ATP hydrolysis. Biochem Biophys Res Commun 60, H., SACCOMANI, G., RABON, E., SCHACKMANN, 825-832. CHANG, G. 1977. Proton transport by gastric LJUNGSTROM,M., NORBERG, L., OLAISSON, H., WERNR. & SACHS, membrane vesicles. Biochim Biophys Acta 464, STEDT, c . , VEGA, F.V., ARVIDSON, G. & MKRDH,s. 313-327. 1984.Characterization of proton-transporting memCHEUNG, W.Y. 1980. Calmodulin plays a pivotal role branes from resting pig gastric mucosa. Biochim in cellular regulation. Science 207, 19-27. Bioph,ys Acta 769, 209-219. CHEW, C.S. & BROWN,M.R. 1986. Release of LOWRY,O.H., ROSEBROUGH, N.J., FARR, A.L. & intracellular Ca" and elevation of inositol trisphosRANDALL, R.J. 1951. Protein measurement with the phate by secretagogues in parietal and chief cells folin phenol reagent. 3 Biol Chem 193, 265-275. isolated from rabbit gastric mucosa. Biochim Bio- LUCY, J.A. 1978. Mechanisms of chemically induced phys Acta 888, 116-125. cell fusion. In: G. Poste & G. L. Nicolson (eds.) CnEuTz, C.E. 1981. Cis-unsaturated fatty acids induce Membrane Fusion, pp. 267-304. Elsevier/Norththe fusion of chromaffin granules aggregated by Holland Biomedical Press, Amsterdam. synexin. 3 Cell Biol 91, 247-256. MCGIVNEY, A,, MORITA, Y., CREWS, F., HIRATA, F., J.A. 1986. Regulation of DE LISLE,R.C. & WILLIAMS, AXELROD, J. & SIRAGANIAN, R. 1981. Phospholipase membrane fusion in secretory exocytosis. Ann Rev activation in IgE-mediated and Ca2+ ionophore Physiol 48, 225-238. A23 187-induced release of histamine from rat DELORENZO, R.J. 1980. Role of calmodulin in basophilic leukemia cells. Arch Biochem Biophys neurotransmitter release and synaptic function. 212, 572-580. Ann N Y Acad Sci 356, 92-109. METZ, S.A. 1986. Ether-linked lysophospholipids DRUST,D.S. & CREUTZ, C.E. 1988. Aggregation of initiate insulin secretion. Lysophospholipids may chromaffin granules by calpactin at micromolar mediate effects of phospholipase A, activation on levels of calcium. Nature 331, 88-91. hormone release. Diabetes 35, 808-817. DUZGUNES,N., ALLEN,T.M., FEDOR, J. & PAPA- MOSKOWITZ, N., SCHOOK, W. & PUSZKIN, S. 1982. HADJOPOULOS, D. 1987. Lipid mixing during Interaction of brain synaptic vesicles induced by membrane aggregation and fusion: Why fusion endogenous Ca'+-dependent phospholipase A,. assays disagree. Biochemistry 26, 8435-8442. Science 216, 305-307. FORTE, T.M., MACHEN, T.E. & FORTE,J.G. 1977. MUALLEM, S. & SACHS, G. 1984. Changes in cytosolic Ultrastructural changes in oxyntic cells associated free Ca2+in isolated parietal cells: Different effects with secretory function : A membrane-recycling of secretagogues. Biochim Biophys Acta 805, 181hypothesis. Gastroenterology 73, 941-955. 185. GANSER,A.L. & FORTE, J.G. 1973a. K+-stimulated ATPase in purified microsomes of bullfrog oxyntic M k i D H , s.,SONG, Y.-H., CARLSSON, C. & BJORKMAN, T . 1987. Mechanisms of stimulation of acid cells. Biochim Biophys Acta 307, 169-180. production in parietal cells isolated from pig gastric GANSER,A.L. & FORTE, J.G. 1973b. Ionophoretic mucosa. Acta Physiol Scand 131, 589-598. stimulation of K+-ATPase of oxyntic cell microP. & somes. Biochem Binphys Res Commun 54, 69G696. NILSSON,B.O., LARSSON, E., SUNDSTROM, WIDEHN,S. 1981. Electron microscopy of particles GIBERT,A.J. & HERSEY, S.J. 1982. Morphometric similar to type-C virus in human oocytes. Upsala 3 analysis of parietal cell membrane transformations Med Sci 86, 225-232. in isolated gastric glands.3Mernbr Bio167, 113-124. T., TOMORI, Y., YAMASHITA, S. & D., DEBOER,T., KLAPPE,K. & WILSCHUT, NISHIKAWA, HOEKSTRA, SHIMIZU, S.-I. 1989. Inhibition of Na+,K+-ATPase J. 1984. Fluorescence method for measuring the activity by phospholipase A, and several lysophoskinetics of fusion between biological membranes. pholipids: Possible role of phospholipase A, in Biochemistry 23, 5675-5681. noradrenaline release from cerebral cortical synapIM, W.B., BLAKEMAN, D.P., MENDLEIN, J. & SACHS, tosomes. 3 Pharm Pharrnacol41, 45W58. G. 1984. Inhibition of (H++K+)-ATPase and H+ H., ARVIDSON,G., MA,J.-Y. & MRRDH, s. accumulation in hog gastric membranes by tri- OLAISSON, 1990. Occurrence of phospholipase A, and lysofluoperazine, verapamil and 8-(N,N-diethylamino)phospholipase in a gastric H,K-ATPase-containing octyl-3,4,5-trimethoxybenzoate. Biochim Bioph,ys membrane fraction, and the formation of lysoActa 770, 65-72. phosphatidylcholine in stimulated pig parietal cells. JIRON, C., ROMANO, M. & MICHELANGELI, F. 1984. A Acta Ph,ysiol Scand 140, 383-392. study of dynamic membrane phenomena during the gastric secretory cycle : Fusion, retrieval and re- RUBIN,R.P. 1982. In: Calcium and Cellular Secretion. Plenum Press, New York. cycling of membranes. 3 Membr Biol 79, 119-134. 16-2

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SCHLPP, W., SCHNEIDER, J., HEM, H.-K., RUOFF, EI.-J., SCEIUSDZIARRA, \-. & c L 4 S S E S , sf. 1987. .4 calmodulin antagonist inhibits histamine-stimulated acid production by isolated rat parietal cells. Regul P e p 17, 209-220. SCHEPP, w., BROSCH,E., TATGE, C., SCHUSDZIARRA, V. & CLASSEN, % 1989. I.Cholinergic stimulation of isolated rat parietal cells: Role of calcium, calmodulin and protein kinase C. Clin Physiol Biochem 7, 137--148. STEWART,E1.E. & K.4SBEKAR, D.K. 1981. Gastric oxyntic cell tubulin : Characterization and possible significance. Am 3 Ph-ysrol 240, G317-G323. TRIFARB, J.-hl., FOURNIER, S. 8. YOVAS, M.L. 1989. The p65 protein is a calmodulin-binding protein

present in several types of secretory vesicles. Neuroscience 29, 1-8. TSIEN, R.Y., POZZAN, T . & RINK,T.J. 1982. Calcium homeostasis in intact lymphocytes : Cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. .7 Cell Bid 94, 325-334. J. 1976. Actin-like filaments VIAL,J.D. & GARRIDO, and membrane rearrangement in oxyntic cells. Proc Natl .4cad Scr U S A 73, 40324036. VIAL, D.V., GARRIDO, J., AND GONZALEZ,A. 1985. T h e early changes of parietal cell structure in the course of secretory activity in the rat. Am 3 Anat 172. 291-306.

Calcium and calmodulin stimulate phospholipase A2 and fusion of H,K-ATPase-containing membrane vesicles isolated from pig gastric mucosa.

Fusion of pig gastric H,K-ATPase- and phospholipase A2-containing vesicles in vitro was studied by electron microscopy and by monitoring the change in...
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