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1992 [Vol. 131

and polarity. Identifying differences between large-vessel and microvessel endothelial cells that could explain their differences in function is likely to prove one of the most challenging tasks. Acknowledgements We are grateful to Dr D. C. Ogden for comments on the manuscript.

References

1 Adams, D. J., Lategan, T. W., Lodge,

N. J. and Van Breeman, C. (1987) J. Physiol. (Land.) 394,45P 2 Colden-Stanfield, M. et al. (1987) Circ, Res. 61,632-640 3 Colden-Stanfield, M. B., SchiUing, W. P., Possani, L. D. and Kunze, D. L. (1990) J. Membr. Biol. 116,227X238 4 Johns, A. et al. (1987) Tissue Cell 19, 733-745 5 Takeda, K., Schini, V. and Stoekel, H. (1987) Pf7iiiers Arch. 410,385-393 6 Lodge, N. J., Adams, D. J., Johns, A., Ryan, U. S. and Van Breeman, C. (1988) in Resistance Arteries (Hatpem, W. et nl., eds), pp. 152-161, Perinatology Press 7 Olesen, S-P., Davies, P. F. and Clapham, D. E. (1988) Circ. Res. 62, 105%1064 8 Ryan, U. 8, Grigorian, G. Y., Avdonin. P. V. and Bregestovski, P. (1988) New Trends Lipid Mediat. Res. 1, 144-158 9 Cannell, M. B. and Sage, S. 0. (1989) 1. Physiol. (Land.) 419, 555-568 10 Silver, M. R. and DeCoursev, T. E.

(1990) J. Gen. Physiol. 96,109& 11 Hoyer, J., Popp, R., Meyer, J., GaBa, H-J. and Gogelein, H. (1991) J. Membr. Biol.

123,55-62 12 Lansman. J. B., Hallam, T. J. and Rink,

T. J. (1987) Nature 325,811-813 13 Vigne, P. et al. (1989) J. Biol. Chem. 264, 7663-7668 14 Fichtner, H., Frobe, U., Busse, R. and Kohlhardt. M. (1987) 1. Membr. Biol. 98. 125-133 .. 15 Hansen, A. 1. and Olesen, S-P. (1990) J. Physiel. (L&d.) 423, 1lOF . 16 Olesen, S-P. and Bundgaard, M. (1992) Acta Physiol. Stand. 144,191-198 17 Bossu, J-L., Feltz, A., Rodeau, J-L. and Tanzi, F. (1989) FEBS Left. 255,377-380 18 Bossu, J-L. et al. (1992) Pflfigers Arch.

420,200-207 19 Busse. R.. Fichtner,H..

Luckhoff, A. and Kohlhardt, M. (1988) Am. J. Phy&ol. 255, H965-H969 20 Rusko, J., Tanzi, F., Van Breeman, C. and Adams, D. J. (1991) Biophys. J. 59, Bla 21 Sauve, R., Chahine, M., Tremblay, J_ and Hamet, P. (1990) J. Hypertens. 8 (Suppl. 7). S193-S201 P., Bakhramov. A., 22 Bregestovski, Danilov, S., Moldobaeva, A. and Takeda, K. (1988) Br. 1. Pharmacol. 95, 42%436 23 Nilius, B. (1990) Pflsigers Arch. 416, 609-611 24 Sauve, R., Parent, L., Simoneau, C. and Roy, G. (1988) Pfliigers Arch. 412, 469-481 25 Adams, D. J., Barakeh, J., Laskey, R. and Van Breeman, C. (1989) FASEB J. 3. 2389-2400 . 26 Freay,A., Johns, A., Adams, D. J., Ryan,

407 U. S. and Van Breeman, C. (1989) Pfliigers Arch. 414,377-384

27 Jacob, R. (1990) J. Physiol. (Land.) 421,

28 29 30 31 32

55-77 Luckhoff, A. and Clapham, D. E. (1992) Nflture 355, 356-358 Jacob, R. (1990) Biochim. Biophys. Actu 1052,427-438 Beny, J-L. and Gribi, F. (1989) Tissue Cell 21,797-802 Beny, J-L. (1990) Am. 1. Physiol. 258, H836-H841 Luckhoff, A. and Busse, R. (1990)

Pffiigers Arch. 4X,305-311 33 Sage, S. O., Adams, D. J. and Van Breeman, C. (1989) J. Biol. Chem. 264,

6-9 34 Tomlinson, A., Van Vliimen, H., Loesch, A. and Bumstock, G: (199i) Cei Tissue Res. 263, 173-181 35 Morel, N. et al. (1989) J. Cell. Physiol.

141,653-659

36

He. P., Pagakis,S. N. and Curry, F. E.

(1990) Am. J. Physiol. 258, H1366-Hl374 37 He, P. and Curry, F. E. (1991) Am, J. Physiol. 261, Hl246-H1254 38 Olesen, S-P., Clapham, D. E. and Davies, P. F. (1988) Nature 331, X8-170 39 popp. R.. Hoyer, J.. Meyer,J., GaBa, H-J.

and Giigelein, H. in Quinckc Armizer_

suy Symposium (Felg&rauer, F, ed.), Springer Verlag (ii press) 40 Shen, J., Luscinskas, F. W.. Connolly, A., Dewey, C. F.. Jr and Gimbrone, M. A., Jr- (1992) Am. J. PhyhL 262, c384-C39tI

41 Yoshima, M. et al. (1989) Biochem. Biophys. Res. Cmnmun. 161. -Z&8& BayK8644r methyl-l&dihydro-2,6dimethyl-3-n&o-4-(2-triBuoromethyIphenyl)-pyridine-Sxarboxylate

The fall and rise of neuronal wbungarotoxin binding proteins Paul B. S. Clarke Although neuronal [‘2511-at-bungarotoxiinbinding proteins are similar in many respects to muscle nicotinic acetylcholine receptors, their functional significance has eluded researchers for the past fifteen years. Over this period, their status became increasingly doubtful, as almost all attempts failed to demonstrate that ar-bungarotoxin could block neuronal nicotinic responses. Recently, these enigmatic proteins have been cloned and expressed in oocytes, and have been examined afresh in their native state. As Paul Clarke explains, it is time to recognize neuronal a-bungarotoxin binding proteins as distinct members of the nicotinic acetylcholine receptor gene family, even if perhaps they do not function quite like other members. ar-Bungarotoxin (arBTX) blocks neurotransmission at the neuromuscular junction by binding to nicotinic acetylcholine receptors in a remarkably potent, specific and persistent manner. Although binding sites for arBTX have also been found in autonomic ganglia, adrenal gland, spinal cord and brain, their physiological significance has been widely questioned. Perhaps for this reason, molecular, genetic and functional rucotinic characterization of acetylcholine receptors ha!: concentrated mainly on receptors that do not bind (wBTX (Ref. 1). The story of neuronal [‘251]-arBTX binding sites is itself serpentine, P. B. S. Clarke is Assisfant Professor in the Department of Pharmacology and Therupeutics, McGill University, 3655 Drummond St, Room 1325, Montreal, Canada H3G lY6.

but thanks to recent advances, it seems at last to be uncoiling. The 1970s - a golden age Nicotine exerts diverse pharmacological actions in the CNS: it can release, transmitter enhance depolarize neurons and increase glucose utilization. It can also reinforce the voluntary inhalation of carcinogens. These actions all reflect an activation of CNS nicreceptors. acetylcholine OtilliC However, before 1975 the only well-characterized example of a CNS nicotinic acetylcholine receptor was in the spinal cord’. Subsequently, reports of high-affinity [i~&arBTX binding sites in brain began to appear. The biophysical, biochemical and pharmacological characteristics of the binding sites were strongly reminiscent of acetylcholine muscle nicotinic Q1992, Elsevier SciencePublishersLtd (UK)

TiPS - November

40s receptors (for reviews see Refs 3,4 and 5). The late 1970s and 198Os- the

dark ages Differences between brain and muscle aBTX binding proteins also emerged, particularly with respect to binding kinetics and antigeni&y+s. These differences could have been dismissed as reflections of receptor heterogeneity, but other evidence suggested that [=I]-oBTX binding might, in certain tissues, have nothing to do with nicotinic acetylcholine receptors: &bough sympathetic ganglion neurons were kn0Wn t0 express iizsl]-arBTX sites that had a nicotiuic binding profile, several investigators in the late 1970s found that arBTX would not attenuate nicotinic transmission in these preparationP. Analogous results were reported in rat PC12 cells’, where it was further shown that antibodies raised against nicotinic acetylcholine receptors purified from electric eel greatly reduced nicotinic agonist-induced Na+ flux but failed to precipitate the [1251]aBTX binding component. This indicated that the [1251]-oBTX binding component, and the functional nicotinic acetylcholine receptors detected in the Na+ flux assay, were completely different proteins. These results tended, as the authors commented, ‘to limit the use of arBTX in exploring CNS nicotinic acetylcholine receptors’. The same conclusion was drawn from electrophysiological experiments in the CNS. The synaptic link between motoneuron collaterals and Renshaw cells represents the best-established example of nicotinic acetylcholine neuroixansmission in the CNS. However, Duggan and colleagues’ reported that synaptically-evoked nicotinic acetylcholine responses were refractory to local application of arBTX. However, studies of the suprachiasmatic nucleus of the hypothalamus gave the story a new twist- This nucleus contains a major circadian pacemaker that, in rats, controls a number of diurnal rhythms including that of pineal 5-HT-N-acetyltransferase. At night, the acetyltransferase activity rises dramatically, and this rise can be rapidly reversed by brief pulses of light. The acute effect of light was

mimicked by infusion of the agonist receptor acetylcholine carbachol adjacent to the supraand was chiasmatic nucleus, blocked by close infusion of arBTX (Ref. 9). The use of a physiological stimulus (light) suggested that crBTX might actually be blocking an effect of endogenous acetylcholine. At the time, the results seemed clear-cut. [1251]-(uBTXand CNS nicotinic binding sites In the early 198Os, the first convincing demonstrations of saturable [3H]nicotine and [3H]acetylcholine binding to rodent brain homogenates were reportedrW1’. Extensive comparisons have since indicated that 13H]nicotine and [3H]acetylcholine bind to the same population of nicotinic sites. Like the radiolabelled [l*s~]-orBTX agonists bound with high affinity (nanomolar Kd) and the binding sites showed a selective affinity for nicotinic agents. But there were differences. Most strikingly, aBTX failed to inhibit [3H]-agonist binding, and whereas nicotine inhibited t3H]-agonist binding with a nanomolar Ki, inhibition of [“51]-atBTX binding required micromolar concentrations. These findings might have been attributed to the existence of distinct toxinand agonistbinding sites on the same nicotinic acetylcholine receptor macromolecule, but for a key observation: the relative densities of [3H]-agonist and [1251]-arBTX binding sites varied widely across different microdissected brain regions10,12. Later, autoradiographic comparison of adjacent brain sections13 and affinity chromatography14 confirmed that high-affinity sites for [3H]agonists and [i251]-aBTX binding had to be on completely different proteins. More recent evidence from molecular genetic and other approaches places [3H]-agonist and [1251]-~BTX binding proteins in different branches of the nicotinic acetylcholine receptor subunit gene family’. (uBTX blocking actions in CNS Toxin-sensitive nicotinic acetylcholine receptor-mediated responses in rat brain remained elusive. The realization that different commercial lots of aBTX varied considerably in their

1992 [Vol. 131

ability to block nicotinic transmission in an autonomic ganglion ied to the isolation of a contamivariously polypeptide nant termed bungarotoxin 3.1, toxin F, K-bungarotoxin and, by current neuronal bungaroconsensus, toxin6*15*16. The suspicion naturally arose that some previous reports of oBTX blockade could have been due to contamination by neuronal bungarotoxin. Subsequently, the reported blocking action of CVBTXin the suprachiasmatic nucleus’ was called into question by a new study in which purified arBTX was used”. to block Several attempts effects on nicotine-induced transmitter release and neuronal firing with (uBTX were also unsuccessful; somehow, the fact that most researchers were looking in brain areas with sparse [1251]rwBTX binding was lost in the argument. But worse was to come: in vivo administration of nicotine produced a pattern of cerebral activation in rat brain (shown by 2deoxyglucose uptake) that looked uncannily like the distribution of t3H]nicotine binding and quite unlike that of [1251]-arBTX binding”. The solitary finding that [125~]-arBTX binding proteins could be regulated by chronic in vivo nicotine administration” provided cold comfort to those still persisting with the notion that [‘251]-arBTX might label a of brain nicotinic population acetylcholine receptors. Until 1987, the central actions of nicotine, with rare and less than convincing exceptions, were reported to be blocked by classical nicotinic receptor antagonists of the ganglionic type (e.g. mecamylamine) and not by 0rBTX. Subsequently, cerebellar interneurons were found to respond to nicotine in an uBTX-sensitive manner; neither mecamylamine nor neuronal bungarotoxin were effective antagonists*‘**‘. These findings remain puzzling, for two reasons: first, virtually all interneurons demonstrated these properties, whereas [1251]-oBTX binding sites are sparse and form widely dispersed clusters in cerebellum’3~22, and secondly, the basal firing rate of inter-neurons was reduced by aBTX and recovered upon its removal, whereas toxin-induced blockade of nicotine excitation showed little sign of recovery.

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Subunit composition of P%]CVBTXbinding sites Putative nicotinic acetylcholine receptors that bind arBTX have been isolated by affinity chromatography and characterized by SDS-PAGE. N-terminal amino acid sequencing of tuBTX binding protein from chick optic lobe provided early evidence for sequence homology between brain [‘251]-cuBTx sites and muscle nicotinic acetylcholine receptors=. Depending on the study, anywhere from one to four major polypeptide components were detected in both chick and rodent brain. Estimated molecular masses also varied between studies, but tended to fall between 44 and 65kDa (rat) or 48 and 72 kDa (chick), reminiscent of the muscle nicotinic acetylcholine receptoPzs. These findings, though divergent, provide evidence that putative nicotinic acetylcholine receptors that bind aBTX are probably composed of several non-identical subunits. This conclusion has recently been supported by reconstitution Affinity-purified experiments26. (uBTX binding protein from neonatal chick brain bound [iZI]arBTX with a pharmacological profile resembling that of PC12 cells and brain; three components (52,57 and 67 kDa) were identified on SDS-PAGE. Insertion of the macromolecule into lipid bilayers yielded carbachol-gated cation channel openings which were blocked by the classical nicotinic receptor antagonist (+)-tubocurarine. This study, albeit somewhat preliminary in its electrophysiological aspects, showed for the first time that proteins that bind [1251f-~BTX in brain can act as nicotinic acetylcholine receptors.

Molecular cloning of CvBTX binding proteins A bre~th~ugh occurred with the molecular cloning of brain oBTX binding proteins in chick. In the first such report, Schoepfer et aLz7 screened a chick brain cDNA library with synthetic derived from oligonucleotides previously determined terminal peptide sequences of an arBTX binding protein subunit, and initially isolated a cDNA clone (&BTX binding protein ~111).Lowstringency screening then revealed a second cDNA clone en-

coding a second subunit (aBTX binding protein (u2) with somewhat similar features. Soon after, Couturier et aL2* also identified a clone from a chick cDNA library, identical to that encoding binding protein oil, which they termed ru7. common agreementtg, BY (wBTXbinding proteins arl and o2 are now termed rw7and o8 proteins, respectively. These proteins share moderate sequence homology with other cloned nicotinic acetylcholine receptor a! subunits that do not bind cuBTX. More particularly, they share several important features, including two adjacent cysteines located close to positions 192 and 193 - a halhnark of agonist-binding nicotinic acetylcholine receptor subunits so far encountered. The main difference between alI at subunits so far cloned is in the third putative cytoplasmic loop, and indeed the or7 and o8 clones both possessed unique sequences here. The two proteins are much less homologous to other identified members of the ligand-gated ion channel superfamily, but share certain general features, notably four hy~ophobic domains (MlM4), with a long loop between the third and fourth, and certain conserved residues. The M2 hydrophobic domain is thought to line the ion channel as it spans the membrane and is highly conserved among members of the nicotinic receptor family. It differs between nicotinic acetylcholine receptors (which form cation channels} and glycine and GABA receptors (which form anion channels). The lw7 and ar8 clones possess M2 domain sequences almost identical to those of other nicotinic ace~lcholine receptors, but differ in one amino acid, prompting Schoepfer et a1.27 to suggest - perhaps presciently that cation selectivity or conductance might also differ {see below). Given the uncertainties surrounding attempts to characterize the subunit composition of brain [*251]-arBTX binding proteins {see above) and the possibilities of to related cross-hybridization clones, it was important to test whether the ot7 and rut3proteins bound [1251]-~BTX. A bacterial fusion protein encompassing the predicted agonistand toxinbinding sites of 017 was found to

bind I’251]-arBTX {Ref. 28). However, expression of the o8 subunit in Xenopus oocytes did not yield[‘2511-~BTX binding siteP, even though an approach using shorterlength synthetic peptides predicted that the a8 protein might also bind [*g-oBTX (Ref. 3’1). Hence, at present it is undear whether native ar8 binds rn?J(UBTX. There are also data to suggest that the previousiyidentified o5 subunit mav also bind [u51]-arBm j:Z&. 311, but the pattern of 05 mRNA exI?re&;;n in brain bears little relation to &at of [=I]-aBTX binding, suggesting that most [‘25J1-&I’X binding gins do not contain this sub- 2 Schbepfer et al.” have taken the biochemical characterization of ar7 further. Two monoclonaI antibodies, raised against recombinant peptide fragments from nonconserved regions of cu7 and &, respectiveIy, were tested against ~~~-pu~~ed arBTX binding proteins on western blots. The monoclonals identified subunits of approximately 57 and &&Da, respectively, within the range encountered in previous protein purification studies. Several lines of evidence indicate that most, and perhaps all, (UBTX binding proteins in brain contain ar7 subunits. Firstly, antiar7 monoclonal antibodies recognize at least 98% of chick brain arBTX binding proteins in depletion and immunoprecipitation assays27, Secondly, in situ hybridization maps of ar7 mRNA closely resemble the pattern of fU51](UBTX binding in rat brainzrJ3. Thirdly, Britto et ~2.~~ have described in detail the disiribution of a17- and &-Eke immunocytochemical staining in postnatal chick brain. With the exception of one or two areas, the of ar7-like immuno~~~~ resembled that of [“I]arBTX binding. o&Isubunits seem to occur less commonly, and when they do occur, they tend to be associated with the more abundant o7 subunits27. Thus, anti-&l antibodies smaller much recognized a fraction (15%) of [‘251]-arBTX binding macromolecules, and this fraction was also recognized by an&o7 antibodies. This was the first firm indication that a: subunits of more than one type may

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410

conw

a

100 pi acetylcholine -I7

P

!ji 2C

100 nruu 67X, 30 min 100 pi acetylcholine

a-bungarotawinsensib responses demonstrahxi in m in occytesproduced fmnsiien whole-cell n+prmse to acelylchdie that were blocked by 30 min pre-incubation with nanomoktr concenbations of (uBTx were suhici..nt to PkmgerolaanWWb: to 600~ acetylcholine. See Ref. 28 for further details. P@=bkdtadeof=PJ== heprcduced wfth pemiksion. from Neuron 5,647-656. Fig. 7. Fig. 1. a: Rapkliy dese&Mg

=;7

coexist in the same neuronal nicotinic acetylcholine receptor.

Recentelectrophysiological shdies

The chick a7 subunit clone has been expressed in Xenopus oocytes and studied under wholecell voltage clampB3. Whereas previously cloned nicotinic acetylcholine receptor (Y subunits require coexpression of p subunits, oocyte expression of homooligomexs composed of a7 subunits xesulted ir. clear inward cumznt responses to acetylcholine and nicotine. These responses were blocked by low nanomolar concentrations of CUBTX, were voltage-sensitive, and highly decayed rapidly in the continuing presence of agonist (see Fig. 1). Fast desensitization, should it prove to be a common characteristic of aBTX binding proteins in the CNS, could help to explain why toxin-sensitive nicotinic receptor responses have rarely been found. Two groups have recently reported nicotinic responses in brain tissue that are strikingly reminiscent of the oocyte data-’ Significantly, both groups if investigators employed a method providing rapid drug delivery and examined cultured fetal or postnatal cells from hippocampus, a brain region with one

of the highest densities of [1251]aBTX binding. Administration of acetylcholine and other nicotinic receptor agonists induced wholecell inward currents that possessed several characteristics in common with oocytes expressing a7 subunits: low sensitivity to acetylcholine (EC, > ~OO~M), strong inward rectification, blockade by arBTX and extremely rapid and profound desensitization (see Fig. 2). Interestingly, arBTX-sensitive nicotinic responses displayed by oocytes and cultured hippocampal neurons differ from those observed in vivo in the cerebellum of adult rats, where nicotineinduced excitations did not show fast desensitization20. This dif-

ference may be due to additional subunits whose presence in adult nicotinic acetylcholine native significantly could receptor modify pharmacology and channel properties’. It is also possible that the cerebellar nicotinic acetylcholine receptors studied to date do not possess a7 subunits. It is also conceivable that oocytes differ in post-translation processing or in short-term regulatory processes such as phosphorylation, or lack key allosteric modulators. Finally, the ionic species mediating these clrBTX-sensitive nicotinic receptor responses have yet to be identified in neurons; this problem is not helped by the occurrence of rectification, which has prevented determination of reversal potentials28*36. Subunit deletion experiments Although IX: subunits can funstion as homooligomers in oocytes, they probably do not do so in the brain. The protein purification data tend to rule this out, and Schoepfer’s evidence suggests that some a7 subunits assemble with a8 subunits (see above). What other subunits might coassemble? Coinjection of mRNAs encoding previously cloned nonagonist-binding subunits from neuronal and muscle nicotinic acetylcholine receptors do not alter the properties of the a7 response28. Perhaps this means that the oocyte is simply not equipped to assemble the a7 subunit efficiently with the appropriate subunits, or that more than two types of subunit are required. It is also possible that the appropriate nicotinic acetylcholine receptor subunits remain to be discovered.

a

1 mM dimethylphenyl-

50 ms

100 ms

Fig. 2. Rat hippocampal neurons in vitro showad two types of response to nicotinic receptor agonists: a. a rapidly desensitizing response that wasblocked by &KY, and b, a slower response that was not &TX-sensiiive. See Ref. 36 for further details. Reproduced, with permission, from MO/.Pharmacol 41, 931-936, Fig. 6.

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The fact that single neurons can express several different types of nicotinic acetylcholine receptor subunit greatly complicates the study of native nicotinic acetylcholine receptors. Sympathetic ganglion neurons from chick embryo might seem particularly difficult to analyse, since they express a daunting number of nicotinic acetylcholine receptor subunit genes (most strongly cf3, cu7 and 84) and four classes of nicotinic acetylcholine receptor channels=. Using a novel approach, Listerud et ~1.~ investigated alterations in the electrophysiological responses of newly formed nicotinic acetylcholine receptors in cells that had been treated with antisense oligonucleotide probes designed to block the expression of specific subunits. Functional deletion of ar3 subunits profoundly reduced responses to acetylcholine, revealing novel classes of nicotinic acetylcholine receptor whose responses were greatly reduced by oligonucleotide-mediated deletion of (~7, and by arBTX.These channels did not occur in control cells that were permitted to express o3 subunits, suggesting that some nicotinic acetylcholine receptors contain both ar3 and ar7 subunits. Certain caveats should be borne in mind. Firstly, the antisense approach is novel, and its limitations are not yet fully understood. For example, deletion of (~3 subunits may have altered the expression of the remaining subunits. Secondly, administration of ar7antisense oligonucleotides into otherwise normal cells reduced macroscopic acetylcholine-gated currents, whereas (wBTXdid not. This result is puzzling, but could be explained if a7-containing nicotinic acetylcholine receptors are rendered insensitive to &TX when certain other subunits are presenP. This suggestion has yet to be tested experimentally, but a precedent does exist: the ability of neuronal bungarotoxin to block nicotinic acetylcholine receptor function depends on the identity of subunits that do not bind agonist as well as on those that do’. Such an interpretation, if correct, may also account for an earlier and potentially troubling observation, namely that antisera raised against a nicotinic acetyl-

20 -

Conditions Fig. 3. In chickciliaryganglionneumnsin vitro,nicotine(0. l-l.Oph#; E&i) and 50~ K+ (n) increasedintracellularfree Ca”, shownby qua&attve fluorescence? mirroscopy. Responses to nicotine were dependent on extracellularCa2+ and Mocked by the nicotinicreceptorantagonists(+)-tubocumdhe,cu-bungamtoxin (a87JfI, ekm&aconitine and neumnal EiTx; responses were unafkcted by the muscadllicmceptor antagonist &opine or by tetmdotoxin f7TX). See Ref. 42 for further d&k Reproduced,with permission,from Neumn 8,353-W?, Fig. 2.

choline receptor (lr3subunit fusion protein did not recognize oBTX binding proteins in chick ciliary ganglia39. SignaIling pathways Muscle and neuronal nicotinic acetylcholine receptors induce membrane depolarization mainly by conducting Na+ ions. In addition, oocytes expressing (~7 homooligomers and hippocampal neurons manifest appreciable inward currents in response to nicotinic receptor agonists, although the permeant species have yet to be identified2*,3E37. Ganglion cells possess an abundance of cell-membrane [‘“I]aiBTX binding proteins, and it is therefore curious that in these neurons, arBTX does not consistently attenuate agonist-induced membrane depolarization6. Two recent reports suggest that certain neuronal nicotinic acetylcholine receptors conduct considerable amounts of Ca2+ (Refs 40, 41). These studies did not examine ar7containing nicotinic acetylcholine receptors, but the implication was

clear: perhaps toxin-sensitive responses in brain have been hard to demonstrate because the wrong responses have been measured! These suspicions are strengthened by a report that chick ciliary ganglion cells express toxinsensitive nicotinic acetylchohne receptc-1; hat raise intracellular free Ca” (Ref. 42) (see Fig. 3). These neurons possess two classes of putative nicotinic acetylcholine receptor, of which the more abundant binds [=?j-arBTX. Although arBTXdid not significantly reduce inward currents evoked by nicotine, the toxin greatly attenuated increases in nicotine-evoked intracellular free Ca2+. Whatever the mechanism, it would seem that CWBTX-sensitive nicotinic acetylcholine receptors perturb Ca2+ levels much more than they perturb membrane potential. This conclusion, if substantiated, holds major implications for the function of these proteins-. The nicotine-induced increases in Ca2+ fluorescence were greatly attenuated by several agents that block voltage-dependent Ca2+

TiPS - November 1992 Wol. 131

412 throughout the brain4’e4’. The presence of thymopoietin-like immunoreactivity in brain tissue raises the possibility that this peptide may be an endogenous ligand at oBTX-sensitive nicotinic acetylcholine receptors4’. Thymopoietin potently blocks nicotinic receptor-mediated responses in muscle cells, but it is not yet known whether this peptide modulates nicotinic acetylcholine receptor function in brain. Several authors have proposed that [1251]-oBTX binding proteins may play a role in growth and development. In higher vertebrates, most of the evidence is indirect. It is therefore particularly interesting that thymopoietin, which interacts with [‘251]-arBTX binding sites on rat PC12 cells, Acetykholine neurobxnsmission blocks nicotine-induced inhibition Nicotinic acetykholine neuroof neurite outgrowth (M. Quik, transmission is classically viewed pers. commun.). Although the as fast and punctate, serving to trophic mechanism has yet to be transfer frequency-encoded inelucidated, Vijayaraghavan et ~1.~~ formation across synapses. In chick diary ganglion, CXBTX have speculated that in vivo, extrasynaptic arBTX binding proteins binding proteins do not occur serve to monitor extrasynaptic p~~&~~tially at synapsesrs*43. acetylcholine levels and hence However, electron microscopic regulate neurite outgrowth by examination of limited re ‘ons of raising intracellular free Ca2+. rodent brain placed [l2L? I]-arBTX Pauly and colleagues have binding sites primarily at axoestablished an intriguing associsomatic or axodendritic synation between circulating corticoapsesa,45. The transmitter identity steroids, [1251]-~BTX binding proof these synapses was not investiteins and sensitivity to nicotine gated. These studies lacked the resolution to distinguish pre(see Ref. 50 for review). Starting from postsynaptic binding, but from the observation that adrenallesion evidence indicates that ectomy results in corticosteronesome brain [*I]-arBTX binding reversible increases in sensitivity sites to nicotine in a variety of bemay be presynaptie6. Rodent brain [‘25r]-arBTX binding havioural and physiological tests in mice, proteins are regulated in vivo by they showed that chronic nicotinelg, but not, apchronic corticosterone adminisparently, by chronic inhibition of tration selective1 reduced the acetylcholinesterase47. This raises density of brain [’ Y I]-OrBTxbinding. the possibility that a substantial In vitro corticosterone, albeit at high proportion of brain 0rBTX binding concentrations, inhibited [“51]proteins are not, after all, targets aBTX binding to rat brain memfor endogenous acetylcholine. branes and reduced the affinity of nicotine for this binding site, pointOther Iigands ing to a possible allosteric action. What other endogenous ligands These findings suggest that at might interact with [1251]-arBTX least some central actions of nicbinding proteins? Binding sites otine may be mediated via recepfor [1251]-olBTXare regulated by a tors that bind [1251]-crBTX, and number of endogenous submaybe will yield clues to the stances, but only thymopoietin mechanisms underlying the welland corticosterone have been documented interactions between shown to have direct actions. stress and tobacco smoking. Thymopoietin, a thymusderived polypeptide which cl q cl modulates immune function, potently inhibits [1251]-oIjTX Clearly, there is still much to be binding to brain membranes discovered about neuronal aiBTX

channels. Although some sort of cascade effect is thus likely to be involved, it is not yet clear to what extent Caz’ enters the cell, either through toxin-sensitive nicotinic acetylcholine receptors or through voltage-gated channels, and to what extent it is mobilized intracelldarly. If Ca2+ enters in significant amounts, why are inward currents seen in oocytes but not in these ganglion cells? The answer may lie in the existence of Ca’+sensitive Cl- channels; in oocytes, the Cl- reversal potential is around -3OmV, and so activation of these channels normally results in a flow of Cl- ions out of the cell, zt%!ding to the inward

binding proteins. The subunits that coassemble with the (~7 subunit must be identified. The differences electrophysiological between oocyte-expressed homooligomers and native receptors require an explanation. Permeant ion selectivity, and the possibility of receptor heterogeneity, also require examination. It will be important to determine the extent to which arBTX binding proteins in transducing involved are acetylcholine signals, and whether they carry out novel functions. Finally, it is worth bearing in mind that crBTX binding proteins do not occur only on neurons and muscle cells. They also occur on spermatozoa, and as long ago as 1978 (Ref. 51), they were suggested to be nicotinic acetylcholine receptors that serve to translocate Ca”. Plus fu change, plus c’est la me^mechose?

Note added in proof

Since this article went to press, we have learnt of a paper by SCgut?la et al. (1. Neuroscience, in press) reporting that oocyteexpressed rat ar7 homooligomers are selectively permeable to Ca2+, that most of the ACh-gated inward current results from secondary activation of calcium-sensitive chloride channels, and that when Cl- flux is prevented, residual nicdesensitize otinic responses rapidly, suggesting that desensitization is probably an intrinsic property of the receptor.

References 1 Deneris, E. 2 3 4

5 6 7 8 9 10

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12 Marks, M. J. and Collins, A. C. (1982) Mol. Pharmacol. 22,554-564 13 Clarke, P. 8. S., Schwartz, R. D., Paul, S. M., Pert, C. B. and Pert, A. (1985) J. Neurosci. 5, 1307-131s 14 Schneider, M., Adee, C., Betz, H. and Schmidt, J. (1985) 1. Biol. Chem. 260, l4505-14512 . 15 Loring, R. H. and Zigmond, R. E. (1988) Trends Neurosci. 11, 73-78 16 Ravdin, P. M. and Berg, D. K. (1979) Proc. Nat1 Acad. Sci. USA 76,2072-2076 17 Miller, M. M. and Billiar, R. B. (1986) J. Pineal Res. 3,159-168 18 London, E. D., Connolly, R. J., Szikszay, M. and Wamsley, J. K. (1985) Eur. 1. Pharmacol. 110,391-392 19 Marks, M. J., Stitzel, J. A. and Collins, A. C. (1985) J. Pharmacol. Exp. Ther. 235, 619-628 20 de la Garza, R., McGuire, T. J., Freedman, R. and Hoffer, B. J. (1987) Neuroscience 23, 887-891 21 de la Garza, R., Freedman, R. and Hoffer, J. (1989) Neuropharmacology 28, 495-501 22 Hunt, S. P. and Schmidt, J. (1978) Brain Res. 157,213-232 23 Conti-Tronconi, B. M. et al. (1985) Proc. Nat1 Acad. Sci. USA 82,5208-5212 24 Kemp, G. E., Bentley, L., McNamee, M. G. and Morley, B. J. (1985) Brain Res. 347,274-283 25 Whiting, P. and Lindstrom, J. (1987) Proc. Nat1 Acad. Sci. USA 84,595-599

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Development of GPllbAlla antagonists as antithrombotic drugs Andrew J. Nichols, Robert R. Ruffolo, Jr, William F. Huffman, George Poste and James Samanen Thrombosis represents a major target for development of drugs to prevent and treat a variety of cardiovascular and cerebrovascular diseases, which are the leading cause of morbidity and mortality in the Western world. This review by Andy Nichols and colleagues focuses on a central process in thrombosis, namely platelet aggregation, and how it can be inhibited by antagonists of the adhesion molecule GPZZblZZZa. Successful and future therapeutic applications of GPZZb/ZZZaantagonists, and their pharmacology, are considered in detail. Thrombosis is the pathological extension of the normal haemostatic process that is required to prevent blood loss following damage to the vascular wall. Red thrombi, which have little or no platelet A. I. Nichols is Associate Fellow in Cardiovascular Pharmacology, R. R. Ruffolo, ]r is Vice President and Director of Pharmacological Sciences, W. F. Huffman is Director of Piptidomimetic Research, -6;. Paste is President and Chairman of Research and Developmenf Technologies, and 1. Samanen is Associate Director in Peutidomimetic Research, SmifhKline Beechan; Pharmaceuficals, 709 Swedeland Road, King of Prussia, PA 19406, USA.

component, are found in areas of low shear stress, such as the deep veins. White and mixed thrombi, which have a significant platelet component, are associated with endothelial damage in regions of high shear stress, such as in the region of a ruptured atherosclerotic plaque in a coronary artery. In such cases of damage, thrombosis is initiated by platelet adhesion to von Willebrand factor, and to collagen and other matrix proteins in the exposed subendothelium. Adhesion of platelets to collagen and

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von Willebrand factor activates the platelets and causes release of second messengers (e.g. thromboxane AZ) and secretion from granules (of, for example, ATP, 5-I-E, platelet-derived growth factor, transforming growth factor and fkhromboglobulin), and catalyses the conversion of prothrombin to thrombin on the platelet surface, alI of which act to produce platelet aggregation in the region of the adherent platelets. Aggregation is associated with further granule release, which amplifies the thrombotic process and may also initiate vascular remodelling in the region of the thrombus. Platelet aggregation is therefore an important target for research into antithrombotic drugs, and one aspect that has recently received much attention is the role of GPIIb/IIla. GPlIb/lIIa is a member of the integrin family of adhesion molecules which are comprised of two glycoproteins, an a-subunit and a g-subunit, joined in a noncovalent complex (Fig. 1). Role of GPIIHIIIa The critical role of Gl?IIb/IIIa in platelet aggregation is exemplified in patients with Glanzmann’s @ 1992, Elaevier

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The fall and rise of neuronal alpha-bungarotoxin binding proteins.

Although neuronal [125I]-alpha-bungarotoxin binding proteins are similar in many respects to muscle nicotinic acetylcholine receptors, their functiona...
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