Cell Biology International Reports, Vol. 14, Abstracts Supplement 1990 19

s57 INTRACELLULAR SIGNALLING AND CONTROL OF EXOCYTOSIS M SECRETORY CELLS

W.T. Mason, SK. Sikdar. J. Hoyland. S. ALerma& M. Kate, R. Bun- ting and R. Zomc*, Dept. of Neumendocrlnology, AFRC Inst. of Animal Physiology and Genetics Research, Babraham, Cambridge UK, *Inst. of Pathophysiology, University of Ljubljana, W

We have utilised dynamic video hnaghtg (Magical, Joyce Locbl) on Fura2-loaded pnuactin and growth hormone-secreting cells to investi- gate intracelhrlar ionised calchun regulation by peptldes and transmit- ters acting on intracell~messenger pathways. A flwtescent antibody probe has also been employed to reveal recently released hormone. To investigate exocytosis directly, dynamic measurements of membrane capacitance have been made.

These new technologies have demonstmted that prolactin and growth hormone secretion am calcium-dependent, this calcium deriving from both intracellular and extra&Mar sources according to the signal pathway stimulated. CAMP pathways, for instance, appear to regulate intlux across the plasma membrane, whereas IF’3 governs intracellular homeostasis. These signal pathways also interact hi a complementary fashion. Voltage-dependent and independent pathways are important

Direct measurements of membrane surface area have revealed that CAMP and non-hydrolysable GTP analogues also modulate Ca- dependent exocytosis. The site of action is most probably close to the membrane fusion site, and may involve modulation of cytoslceletal integrity as well as membrane fusion. CAMP shifts the ‘equilibrium’ of membrane turnover towards exocytosis, whereas GTP--r-S, for instance, promotes membrane recovery or inhibitionof exocytosis, both acting on short time scales. Imaging of a fluorescent probe for recently released hormone has suggested these cells may possess specialised plasma membrane sites at which hormone secretion can occur. These release sites may also be correlated with local&d rises in calcium within the cell cytosol.

Acknowledgments. We thank AFRC, Joyce Loebl, MRC, the Nuf- field Foundation, the Wellcome Trust and KabiVitrum (Stockhohn) for their generous financial support of our work.

s59 STIMULATION-SECRETION COUPLING IN CHROMAFPIN CELLS.

Dominique Atis, Jean-Marie Sontag, Tristan Sarafiin, Dani& ‘Driers6 & Mar&Prance Bader, INSERM U-44, 67084-Strasbou& Prance.

PemeaWon of chromaffm cells with bacterial toxins has been used tb study the role of cytoskeleton, G-proteins and protein kinase C in oatahlamine secmtion. Expuiments using cyto&eleton-pemtrbing agents in SLG-permeabilized cells have mwaled that aectetion is in Part contmlled by the subplasmalemmal cytoskeleton together with actin binding proteins. The dissolution of F-a&n filaments which is associated with exocytosis requires Ca as the intracellular signal at a concentration which is one order of magnitude higher than that necessary to trigger exocytosis in . . pcmd&zd cells. Howcvor this cytoskoletcm-rcgulatcd step does not seem to be under the control of G-proteins. Stable GTPlldOgOSpO&!.lltiatCdUtki- release from alpha- toxin-pmm&ilixed cells ; however GTP-regulatory protein alone does not trigger exocytosis in these cells since calcium and ATP were both necessary for secretion even in the presence of GTF analogues. TPA was found to mimic the effects of the GTP analogues. while inhibitors of protein kinase C (PKC) completely abolished the stimulatory effects of both TPA and GTP analogues. These findings, together with the obsmvation that GTP analogucs are able to enhance the calcium-induced banslocation of PKC suggest that GTP modulate secretion by activating PKC-liked events in chrom&ftn cells. Treatment of cells with IAP induced a massive potontiation of secretagogue-evoked secretion from intact cells and calcium-, ATP-evoked release from alpha- toxin-tleatcd Ells wggeahg a role of lAP substrates at a step distal to [Cali rise. The effects of IAP did not seem to be mcdiatd by PKC, CAME-~ steps, actin filaments nor ly~Aipase A2.Owr teffa qqd;cl suggests *that m

cells an IAP-scnntwe mhlbrtlng G protem may colltroltherekaseofcatecholamines.

S58 CHARACTERIZATION OF A FAMILY 0 F SMALL GTP-BINDING PROTEINS INVOLVED IN TIIE REGULATION OF THE SECRETORY PROCESS

BrumGatd,Unit6deGMtiqueSomatioue,h1stitutFasteur,I’aris, FnitK%.

GTP hydrolysis appears to he used by eulrrvyotic cells to regulate VC?SiCUbWtrafticiUCXUCytosis8lld~Thebaot-COlU~

fromsmdieaonyeestSaccharornyce~cctcvisicuwhichbavereaukediu tbe i&nti6cation of two nnall ras-telmxl GTP-biudlu8 praeias, Se&p andYptlp,whichcontrol~t~ofthe&crstory~~y.In mammalian cells, the observed effects of a non-hydrolysable GTP analog, GTP-yS, on differeat transport events bave suggested the ex&enceofpmtehufunctlonallymlatedtoyeaatSeo4paadYptlp.lhe rabpItX&S~gCllC.ShlWCkenrrc#rtl~ClOllCdd~iUnU and human have in commcn with Sec4p and Yptlp l&hly conmrved domains (including a putative effector bindiug site) and therefore are. god candidates for GTP-binding pro&s involved ia iatrace.llular transpofiinmammpliancelLs(l).Oneofthcmbpotdas(Rblp)isthe mammahaa counteqart of Yptlp. Usin polycloual antibodies raised against therabproleinsexprcssedinE.coli,~havcscaradtolocalize these proteins in various cell types by immunofluorescence, immunoelectron llkXCWp~lUldSUb-CSll~-Rab6pseanS tObe~U~-a;itedOllIhSUlCdiSl~lrmu~Ci- and disibuted over their entire surface (2). Rablp .and rab2p are also found in the Gold region of the cells. We have developed an in virro traqmt assay usiug “9 cells permeebllized w&b m-eptolysin 0 aad we are cmreutly hwaqahq dte effect of the autibodias agaimt rabl, 2 and6ponthe~~pocess.Another~hweareusingto addressthefunctioaortherabproteinsistoseewhetherthelevelof expmssioaoftheaeproteinsva&swheasomecelltypes(suchasBcells stimulatedwithLPS)&velopasecremryappamms.

(1) Zahraoui et al., J. Biol. Chem., 264, 12394-12401, 1989. (2) Goud u al., Nature, 1990 (in press).

S80 Synaptic vesicle-specific -xv- teins: their possible role in the

regulation of ‘tter release. Plavie Valtorta’, Pablo Eenfenati’, Ranossca 7orri Tarelli’, rim-tin

Baur- ard Paul al-eqd-. ‘m ceccarelli center, Dept. of lledid

PhalEcolagy, univ. of nilan, Italy; Ynstitute of wlrvl Physiolagy,

univ. of noam, Italy; -lb Rockefeller univdty, NW narlr, USA.

Second messengers are thought to modulate neuro- transmitter release by activating specific enzymes, the protein kinases, which in turn phosphorylate specific protein substrates. Among the phosphoproteins present in nerve terminals, a key role in the process of neurotransmitter release is probably played by those which are located on the membrane of synaptic vesicles. We have studied the family of synapsins, which comprises four phosphoproteins (synapsin Ia, Ib, IIa and IIb) associated with the cytoplasmic side of synaptic vesicles, and synaptophysin, an integral component of the vesicle membrane. All four synapsins are major substrates for CAMP-dependent protein kinase and Ca/calmodulin- dependent protein kinase I. In addition, synapsins Ia and Ib are also the best substrates known for CaJcalmodulin-dependent protein kinase II. These proteins are able to interact in vitro both with synaptic vesicles and with act-rare thought to play a role in neurotransmitter release by modulating the availability of synaptic vesicles for exocytosis, possibly through the phosphoryla- tion-dependent regulation of the cross-linking of synaptic vesicles to actin. Synaptophysin has been shown to be a substrate for a protein tyrosine kinase (pp60-), which is also present on synaptic vesicles. The significance of synapto- physin phosphorylation in signal transduction remains to be elucidated.