238 Cell Biology International Reports, Vol. 14, Abstracts Supplement 1990

P637 APEX TO BASE MEMBRANE TRANS- P638 ATI’EMPT OF HETERO=US SECRETION OF LOCATION IN EPITHELIAL CELLS. CHROM&FFfNGRANULBSlNPARAIUECIUM

Kajsa Holmgren, Karl-Eric Magnusson, Dept. of Medical Microbiology, University of Linkiiping, S58185 Linkiiping, Sweden.

To characterize the restrictions of apex to base membrane translocation in epithelial cells, cells were grown on glass or filter supports and labellecl with different fluorescent probes, Confocal Laser Scanning Microscopy and image processing were used for analysis of the distribution of the probes. As epithelial cell lines were mainly used the Madin Darby canine kidney- derived clone MDCK-I, and partly the differentiated human colon carcinoma HT29 cells. Labelling was performed with Wheat Germ Agglutinin (WGA), different phospholipids, or lipid analogs. We found that the restrictions displayed by MDCK-I cells were most apparent in cellular domes, and less rigorous in adjacent, confluent areas of the cell layer. WGA-labelled glycoconjugates and the phospholipids (NBD-PC, NBD- PE) were more restricted to the apical portion of the membrane than the lipid analogs (DiI-Cl6, AFC-12, AFC- 16). However, even to the phospholipids the barrier at the tight-junctional level was not absolute, indicating that the restrictive element(s) may form a defective protein fence. In I-IT29 cells only WGA was restricted. We also observed that the restriction in MDCK-I cells was determinable to a certain depth from the site of contact between adjacent cells. Thii indicates that formation of the junctional complexes in the cells is contact- dependent, and occurs whenever physical contact has been established with surrounding cells.

Ren6 Glas -Albrecht, Birgit Kaesberg, Viola Scblosser, Regina Pape, Karl AUmann, Helmut Plattner, Faculty of Biology, University Konstanz, POB 5560, D -7750 Konstanz., Fed. Rep. Gemmy

Almost all of the -1000 secretory organelles (trichocysts) of a Paramedum cell are normaUy firmly attached at the cell membrane. We found a method for their detachment, followed by synchronous (20-40 trichocysts/ min) reattachment. During the detachment phase, we made the followiag experiments: (1) A method was designed to isolate tricbocysts and then their membranes (for Western blots); (2) chromaffin granules, isolated from bovine adrenal medullae were microinjected into Paramecium cells, so they could compete with trichocysts during the reattachment phase.

On SDS -PAGE the protein pattern is quite different with both types of secretory organelle membranes. On Western blots there are no antigens (synaptophysin, synaptobrevin) recognizd which would be in common to both secretory organelle membranes. A serum against a docking protein (51 kDa) described to occur only at the cell membrane (but not organeUe membranes) of chromaffin c&is (Schweizer et al., 1989, Nature , 339, 709 -712) yielded a positive reaction witb both membrane types, trichocyst membranes and even purified chromafFm granule membranes (showing slightly different molecular weights).

Microinjected cbromaffin granules (identified by x-ray microanalysis after a chromate reaction), however, are not transported to or docked at the cell membrane of Paramecium =lh while this occun with their tricbocysts during the synchronous re- attachment phase (after previous experimental detachment). Conclusion: Despite many features in common to a variety of secretory systems, their components cannot deliberately be exchanged for competitive transport. Heterologous secretion, therefore, is not possible on the evolutionary levels analyzed. - Supported by SFB 156.

P639 CALCIUM DEPENDENT SECRETION IN RBL

CELLS IS ABOLISHED BY DOWN REGULATION OF

PKC AND IS RESTORED BY GTPyS.

P640 A G PROTEIN DIRECTLY CONTROLS EXOCYTOSIS IN CHROMAFFIN CELLS

Maria Antonietta De Matteis, Giuseppe Di Tullio, Albert0 Luini, lstituto di Ricerche Farmacologiche “Mario Negri”, Consorzio Mario Negri Sud, 66030 S. M. lmbaro (Chieti), Italy

We have used SLO permeabilized Rat Basophilic Leukemia (RBL) cells as a model to investigate the role of PKC in the control of exocytotic release of serotonin. The minimal requirements for secretion were: ATP (EG& 3mM) and calcium (EC&: ~FM). A pH between 7 and 7.6 was necessary for optimal responses. The non hydrolyzable analogue of GTP, GTP$ potentiated secretion by affecting the maximal response rather than the sensitivity to calcium. The content of PKC in RBL cells could be reduced to undetectable levels by a pretreatment with PMA (1 OO- 1000 nM PMA for 1-6 hours), as judged by the 3H- phorbol- 12,13-dibutyrate binding technique. Under these conditions the calcium induced secretion was virtually abolished whereas the exocitotic response to combined GTPyS and calcium was maintained. We conclude that activity of PKC is necessary as a preparatory event for calcium induced secretion and that the stimulation of a G protein can rescue the secretory process from inactivation. *This study was supported by FIDIA SpA and CasMez

(PS.35.93/IND).

Marie-France Ba&r, Jean-Marie Sontag, *Bruno Rouot & Dominique Aunis, INSERM U-44, STRASBOURG, FRANCE & *C.C.I.P.E., MONTPELLIER. FRANCE.

The role of GTP-bin&g proteins in the s&etory process in clmmafKu cells was investiaated by studvina the effects of pertussis toxin (IAP) on Eatech&mi& &case and generation of various second messengers. IAP was found to stimulate the eatech~lamine seaetioxt indud by nicotine, 59 mM K+ and veratridine, and also potentiated the calcium- evoked catccholamine release from permeabilizcd cells, suggesting that IAP substrate(s) regulate the exocytotic machinery at a step distal to tht rise in intracellular calcium. We have investigated the Possible intracellular pathways involved in the IAP stimulation of secretion. It seems unlikely that the effect of IAP on secretion is mediated by activation of protein kinasc C (PKC) because IAP did not modify the translocation of PKC to membmncs in intact and permeabilizcd cells; in addition, neither inhibitors nor activators of PKC had any effect on the enhanced catecho- release induced by IAP. The effect of IAP is also CAMP-independent as an increase in secretion was observtd in the v of forskolin and IAP did not change cvtoolasmic CAMP levels. The relation between IAP t&&nt and arachidonic acid release was also examined. IAP did not sipsificantl~ modify the release of arachiinic acid measdin rest& and s&m&ted chromaffin cells, suggesting that the stimulatory effect of IAP on secntion is not mediated by an activation of phospholipase AzTaken together thcscIe-sultss

T tthatanIAI-sensitiveGProtein

maymcdulatethcintrace ularmachineryatastepdistalto the generation of second messcn@crs. A model coupling an inhibitory G protein directly with the exocytotic site in admnalmedullarychmmafBn eells is &?Ioped.