Ahnert-Hilger

Bader

Baker

Bhakdi

Bittner

Brooks

Dunn

Gilman

Grant

Greene

Howell

Knight

Lind

McEwan

Neher

Wilson

English

Ahnert-Hilger, G.

Gratzl, Manfred

Open Access LMU ( Ludwig-Maximilians-Univ. München)

TlPS - June 1988 {Vol. 91 How environment affects conditioned drug tolerance Repeated drug administration in a given stimulus context may induce the development of a con-ditioned response, and this response may occur in anticipa-tion of I, and interact with 2, the regular 'unconditioned' effect of the drug. Very often, the con-ditioned response is a 'compensa-tory' response; Le. one that con-sists of an effect opposite to the one that would be expected from the drug (e.g. an increase instea<! of a decrease of locomotor activ-ity). Siegel and his co-workers have expanded these early observations into a conditioned-reflex model of drug tolerance, and have indeed demonstrated a role of condition-ing in the development of toler-ance for the analgesie effect of morphine, and for the hypother-mie effects of ethanol, among other drugs3• The problem is of practical as weil as theoretical importance. A lot of the drug-seeking behavior of drug abusers is said to be conditioned, partly to the environment and/or the sodal context; and it is common for drug abusers to switch from a given drug or drug combination to another (usually cheaper or more socially acceptable) in order to procure the same 'high'. Many die in this quest, either because they succeed or because they don't. Two papers bearing on this point have recently appeared. Pal-letta and Wagner4 reported on the development of context-spedfie to]erance to both the analgesie and the activity-depressant effects of morphine in rats: conditioned 'compensatory' responses occur-red only in the activity measure. Krank and Bennetts reported on the development of context-specific conditioning to the locomotor-stimulant effect of (+)-amphetamine, also in rats: pre-and post-drug activity levels increased over a number of daily sessions but only in the box where :: mJijijffftßiip~iJJIIllI I. [LtlfiJ_ Controlled manipulation of the cell interior by pore-forming proteins Experimental analysis of intracel-lular processes is hampered by the limiting plasma membrane. Thus data from intact cells often pro-vide only indirect evidenee on the nature and regulation of intracel-lular events. Isolated enzymes and purified subcellular organelles can be eharacterized biochemically but this gives no information about their cellular function. In contrast, permeabilization of the plasma membrane by appro-priate techniques allows access tO, and contro} over, the cytosol while leaving the intracellular organiza-tion intact. Such permeabilized cell preparations permit study of the regulation of exoeytosis in secretory cells, a process which i5 eontrolled by intracellular events and involves the fusion of secre-tory vesicles with the plasma membrane. The chain of events leading to exocytosis and its mod-ulation are still not completely understood. Techniques for membrane permeabilization Exoeytosis can be studied in permeabilized secretory cells, pro-vided that neither the vesicle membrane nor the plasma mem-brane has been disturbed by the permeabilization procedure. Per-meabilization of secretory cens has hitherto been performed by physieal or ehemieal techniques. High voltage discharge, first used with adrenal medullary ehro-maffin eells1 allows dialysis of small molecules from the ceU interior, whereas soluble cytoplas-mie proteins remain within the ce1l2• Electrical permeabilization is especially suitable for cells in sus-pension. The whole eell patch elamp technique, which records the 195 the rats reeeived the erug. More importantly, Krank and Bennett showed that this response is transferred to morphine, even when morphine is given at a dose that normally should induee a deerease, rather than an increase, in activityS. The implications of these find-ings for human addietive behavior are obvious, particularly in relation to environment or contextual effects and drug switches. IV AN IZQUIERDO Centro de MemDria, Departamento de Bio-quimica, Insti/uto de Biociencias, UFRGS, 90049 Porto Alegre, RS, Brazil. References 1 PavJov, I. P. (1927) Conditioned Reflexes, Oxford University Press 2 Bykov, K. M. (1959) The Cerebral Cortex and the Internal Organs, USSR Foreign Languages Publishing House 3 Siegel, S. and MacRae, J. (1984) Trends Neurosei. 7, 140-143 4 Palletta, M. S. and Wagner, A. R. (1986) Behav. Neurosci. 100, 611-623 5 Krank, M. D. and Bennett, D. (1987) Behav. Neural Biol. 48, 422-433 increase in capacitance of the plasma membrane during exocy-tosis, is a method for permeabili-zation of single cells. It allows the introduction of not only small molecules but also high molecular weight pro teins into the cello Furthermore it is possible to analyse the fusion of single vesi-eies with the plasma membrane3• Detergents can be used to per-meabilize cultured cells in sus-pension or attached to eulture plates. In the past, saponin and digitonin have been used for the permeabilization oE adrenal medullary ehromaffin and other secretory cells2,4-6. However, detergents may destroy intracelJu-lar membranes2,7. To overcome these and other difficulties inherent in the pre-vious teehniques we have employed baeterial toxins for the stable, selective and prolonged permeabilization of the plasma membrane of seeretory cells7- 10• Two pore-forming proteins, CY-toxin from Staphylococcus aureus and streptolysin 0 from ß-hemolytic streptococci, permit the permeabilizath:m of secretory cells ~ \988. 1l1 •• ~leI' I'lIhll.,UO"l. C.ntbrldse 0165 - 6147/88/$02,00 196 either for smaller molecules, i.e. up to 1000 Da, or for large proteins such as lactate dehydrotenase cr immunoglobulins, respectively7-12. Since the pores formed in the membrane by the hexamerization of the IX-toxin molecules are too small to allow the monomer (34 kDa) to pass13, its attack is dearly restricted to the plasma membrane. Streptolysin 0 pores are large enough for the free pas-sage of the monomer (64 kDa). However, the experimental pro-tocol (which involves binding to the extracellular side of the plasma membrane in the cold followed by warming to 30"C to trigger pore formation) makes an attack of intracellular membranes rather unükely13. In contrast, membrane permeabilization by digitonin is insensitive to the incubation tem-perature and therefore difficult to control (G. Ahnert-Hilger, M-F. Bader, S. Bhakdi and M. Gratzl, unpublished). The main features of permeabilization obtained by IX-toxin, streptc.jysin 0 and digi-tonin are compared in Fig. 1. Exocylosis by chromaffin cells For the study of the final events during secretion by exocytosis we used the rat pheochromocytoma celliine PC 12 and adrenal medul-lary chromaffin cells in primary culture. Both take up catechol-amines and release them on stimulation9.14.15. Since primary cultures of adrenal medullary chromaffin cells also contain other cell type:> (cortical and endothelial ceIls, as weIl as fibroblasts), the donal ceIlline PC 12 is more suit-able for the study of secretion since it consists of a homogeneous rell population14.15. IX-Toxin-per-meabilized cells release catechol-amines by exocytosis more than an hour after toxin treatmentll, and cells permeabilized by strep-tolysin 0 do so for at least 30 min12. The basic properties of catechol-amine release by both permeabil-ized chromaffin types are summarized as folIows: • The release occurs by exocyto-sis. This has been proven for bovine adrenal medullary cells by the parallel release of noradrena-line and the vesicular protein ch.romogranin A, while cytoplas-mlC lactate dehyrogenase is retained under these conditions9. TIPS - June 1988 [Vol. 91 ALPHA - TOXIN STREPTOLVSIN 0 DIGITONIN /---... /--.. ( \ . (~O ) 0, ) , • e: \ /.... \'~ J \ I " . '---",:,,/,& ~;. '--_ .. :./'&:'~~ ' ..... _"':"/~ .. \• 0 ~ '.. \'~' ~ •• ' • \ .. ' ~ e . " . . ,. . " . '. '.': , ' . . ' \1lI •... ,;.. .' ,,_ ,:...-•• _ J:. ~ • \.". '0, '. ; •••• '. ~. ' •• ' .. '.. .. -, \'.', ~ .• - '.1"~': '.... ,~'; .:. .' e... :. . 8 .... '. ' ••. '. 0.· .• · ••.. . .... ":~ .~ ·· .. r .•• · -, •..•.•• ~ • . .~. ,. ,0, y' ... . . ". , .. , ., .. :. . .....•.•.. '. ':'1.' .. •• ....• . • • • .' \;..,. e. . . ~. ' ................ :.~ ... ' .•. J'~'" .... ··t.·:· .. ····:· .. ·· ". .. ', .:.:.: .. ·.!Tt{;:·:\:?J~:: .. ·.·: .. ~~~::·:r.;rt:r::·.·;:.(~~:·: ~~~~.~·~·;:· .. tt~~~:;:.~~;·\ A. Small molecules.-.;;·Y' A ~:"::: + (e.g. ions, nucleo- B. Large moleeules :.!~ . A + B + tides up to 1000 dalton I (e.g. enzymes, immunoglobulins) C. Cell organelles .: (e.g. sec re tory vesicles, mitochondrial Fig. 1. Properties of cells permeabilized by the pore-forming toxins, a-toxln (ReIs 7, 10, 11,13 and 23) and streptolysln 0 (ReIs 7, 12, 13,22 and 23), and by dlgltonln (ReIs 7 and 10). Also, the lack of catecholamine metabolites (which would be formed if the transmitter had access to the cytoplasm of permea-bilized PC 12 cells) stron:nly indi-cates an exocytotic event . • In permeabilized PC 12 cells exocytosis is exdusively triggered by Ca2+ (Refs 7, 8, 11 and 16). In contrast, cateeholamine release from adrenal medullary chromaf-fin cells re~uires both Ca2+ and Mg2+/ATp1 .. 4-6.9.12. It is probable that the random distribution of the vesides in adrenal medullary chromaffin cells necessitates ATP-driven organelle transport during exocytosis which precedes the Ca2+ -induced membrane fusion15. Such a transport may be not required in PC 12 cells where the vesides are preferentially located near the plasma membrane17. Modulation of exocylosis by permeabilized PC 12 cells Since exocytosis in permeabil-ized PC 12 cells ean be triggered by micromolar concentrations of Ca2+ alone, provided that pH and osmolarity are maintained within a physiological range, studies eon-ceming the regulation and mod-ulation of exocytosis are feasible. Thus the roles of Mg2+, protein kinase C and GTP-binding pro-teins during the final steps of exocytosis have been investigated using IX-toxin-permeabilized PC 12 cells. In permeabilized PC 12 eells, free eoncentrations of Mg2+ of apgroximately 1 mM increase Ca + -induced exocytosis1, where-'as, in adrenal medullary chromaf-fin ceIls, Mg2+ alone has no effect but must be com;lexed to ATP to elicit exocytosis1 • Further investi-gations are required to aseertain the precise role of Mg2+ during exocytosis. Activation of protein kinase C by the diacylglycerol analogue OAG (1-0Ieyl-2-acetylglycerol) or the phorbol ester TPA (1-0-tetra-decanoylphorbol-13-acetate) ameli-orates the Ca2+ -induced exocy-tosis by both permeabilized PC 12 cells16 and adrenal medullary chromaffin cells (G. Ahnert-Hil-ger, M-F. Bader, S. Bhakdi and M. Gratzl, unpublished) due to an increase in the Ca2+ sensitivity of the release process. In PC 12 cells 16 this effect is absolutely deeendent on the presence of Mg"'+/ATP, while in adrenal medullary chromaffin cells the ATP depen-dence of protein kinase C activa-tion cannot be clearly separated from the ATP-dependent process of exocytosis. GTP-binding proteins (G pro-teins) are specialized membrane-associated proteins involved in the transduction of diverse sig-TIPS - June 1988 EVol. 91 nals19• Activation of G proteins by the GTP analogue GTPyS partially inhibits the Ca2+ -induced exo~­tosis by PC 12 cent> in a Mg2+-dependent manner. Pretreatment of the cells with pertussis toxin has no direct effect on Ca2+ -in-duced exocytosis but overcomes the observed inhibition by GTPyS. This probably indicates the regulatory control of exocyto-sis by PC 12 cells through a per-tussis toxin-sensitive G protein16• Similar results have been ob-tained with freshly isolated bo-vine adrenal medulIary chromaf-fin cens permeabilized by high voltage discharge20• In contrast, a stimulatory effed of GTPyS on exocytosis has been reported for chicken adrenal medullary chrom-affin cells20, bovine adrenal medullary ceDs in primary cul-turel1, mast ceIls3•22 and neutro-phils22• These contradictory re-sults may be due to the different ceU preparations used and/or re-fleet the regulation of exocytosis by different G proteins. PC 12 cells permeabilized for either small or large molecules o <======== o ,,'11-===== "'-G <~=== may be an ideal tool to salve some of these discrepancies and to gain more insight into the molecular events that occur during exocyto-sis. They are easl to handle and require only Ca2 for exocytosis. Figure 2 summarizes current data conceming the regulation and modulation of exocytosis by PC 12 cells. Further applications of permeabilized cells The unique role of ea2+ as the promoter of exocytosis is weIl established, but little is known of the exact mechanisms involved. In chromaffin ce11s there is evidence that exocytosis is not a calmodulin-dependent process since antagonists and antibodies against calmodulin are without effect on either streptolysin 0 - (G. Ahnert-Hilger, M-F. Bader, G. Bhakdi and M. Gratzl, unpub-lished) or ~-toxin-permeabilizedll cells. Application of antibodies against intracellular proteins through the weil defined strep-tolysin 0 pores can help in the search for key proteins involved modulated by protein kinase C - diacylglycerol (phorbot ester) GTP-binding protein -- GTPj'S (pertussis toxin sensitive) Fig. 2. ModulatiOn 0' Cs2+.jnduced exocytosis in «-toxin permesbillzed pheochromocyloma cells (PO 12). (From Aefs 7, 11 and 16.) 197 iu exocytotic membrane fusion. However, the use of cell per-meabilization is not restricted to the study of exocytosis. IntracelIu-lar Ca2+ movements can be stud-ied giving a more precise picture of Ca2+ regulation during exocyto-sis and Ca2+ sequestration within secretory cells. Questions conceming compart-mentalization, biosynthesis and metabolism of catecholamines can also be addressed using per-meabilized cells16• Furthermore, the intracellular handling of fuels like glucose23 or the action of drugs and their metabolism may easily !:>e studied with these cells. G. AHNERT-HILGER AND M. GRATZL Abteilung Anatornie und Zellbiologie, Univer-sität Wrn, Oberer Esselsberg, Postfach 4066, 0-7900 Wrn. FRG. References 1 Baker, P. and Knight, D. (1978) Nature 276, 62D-622 2 I<night, D. and Sautton, M. (1986) Blochern. ,. 234,497-506 3 Neher, E, (1988) I. Physiol. 395, 193-215 4 Brooks, J. and Treml, S. (1983) ,. Neurochern. 40,468-474 5 Dunn, L. and Holz, R. (1983) J. Biol. (,h11m. 258, 4983-4993 6 Wilson~S. - and Kirshner, N. (1983) 1. Biol. ehern. 258, 4994-5000 7 Ahnert-Hilger, G., Bhakdi, S. and Gratzl, M. (1985) I. Blol. ehern. 260, 12730-12734 8 Ahnert-Hilger, G., Bhakdi, S. and Gratzl, M. (1985) Neurosci. Lett. 58. 107-110 9 Bader, M-F., Thierse, D., Aunis, D., Ahnert-Hilger, G. and Gratzl, M. (1986) ,. Biol. Chern. 261, 5771-5783 10 Lind, I., Ahnert-Hilger, G" Fuchs. G. and Gratzl, M. (1987) Anal. Biochern. 164, 84-89 11 Ahnert-Hilger, G. and Gratzl, M. (1987) 1. Neuroehern. 49, 764-770 12 Sontag, J-M •• Aunis, D. and Bader, M·F. Eur. 1. Ce" Biol. (in press) 13 Bhakdi. S. and Tranum-]ensen, J. (1987) Rev. Physiol. Blochern. Pharmacol. 107, 147-223 14 Greene, L. and Tischler, A. (1982) Mv. Cell. Neuroblol. 3, 373-407 15 Grant, N., Aunis, D. and Bader, M·F. (1987) Neuroscience 23, 1143-1155 16 Ahnert-Hilger, G., Bräutigam, M, and Gratzl, M. (1987) Blocllemislry 26. 7842-7848 17 Brocks, J. and Cannichael, S. (1983) Mikrosk"/Jie 4Ö, 34'1-356 18 Knight. D. and Baker. P. (198;1) I. Membr. Biol. 68. 107-140 19 GiJman, A. (1987) Annu. Rev. Biochem. 56,615-649 20 Knight, D. and Baker, P. (1985) FE85 Lelt. 189, 345-349 21 Bittner, M., Holz, R. and Neubig, R. (1986) 1. Biol. ehern. 261, 10182-10188 22 Howell, T. W., Cockroft, S. and Gom-perts, B. D. (1987) 1. Cell Biol. 105, 191-197 23 McEwan, B. F. and Arion, W. J. (1985) ,. Cell Blol. 100, 1922-1923 

Controlled manipulation of the cell interior by pore-forming proteins

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Controlled manipulation of the cell interior by pore-forming proteins

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