Distribution and anchoring of molecular forms of acetylcholinesterase

TiPS - August 1989 [Vol. 101 16 Parker, I. P., Ito, Y., Kuriyama, H. and Miledi, R. (1987) Proc. R. SW. London Ser. B 230,2L7-214 17 Abe, A. and Karaki, H. (1988) Jpn. /. PharmacoL 46,293-301 18 Morgan, J. P. and Morgan, K. G. (1984) J. Physiol. (London) 357,539-551 19 Fukumitw, I’., Hayashi, H., Tokuno, H. and Tomita, T. (1988) Jpn. J. Smooth Muscle Res. 24,475-476 (in Japanese) 20 Karaki, H., Sato, K., Ozaki, H. and Murakami, K. (1988) Eur. 1. Pharmacol. 156, 259-266 21 Morgan, J. P. and Morgan, K. G. (1984) J. Physiol. (London) 351,155-167 22 Rembold, C. M. and Murphy, R. A. (1988) Circ. Res. 63, 593-603 23 Lincoln, T. M., Wear, L. 8.. Cornwell,

24

25 26 27

28 29 30

T. L. and Taylor, A. (1989) FASEB 1. 3, A881 Ambler, 5. K., Poenie, M., Tsien, R. Y. and Taylor, P. (1988) J. Biol. Chem. 263, 1952-1959 Biilbring, E. and Tomita, T. (1987) Pharmacol. Rev. 39,49-96 Kamm, K. E. and Stull, J. T. (1985) Anmr. Rev. Pharmacol. Toxicol. 25, 593-620 Himpens, B., Matthijs, G., Somlyo, A. V., Butler, T. M. and Somlyo, A. P. (1988) J. Gen. Physiol. 92, 713-729 Karaki, H., Sate, K. and Ozaki, H. (1988) Eur. 1. Phnrmacol. 151,325-328 Bradley, A. 8. and Morgan, K. G. (1987) J. Physiol. (London) 385,437-B Karaki, H., Ozaki, H., Ohyama, T., Sato, K. and Kato, N. (1989) Jpn. J. Pharmacol.

Distribution and anchoring_ of molecular forms of acetylcholinesterase Nibaldo C. lnestrosa and Alejandra Perelman Molecular forms of acetylcholinesterase exhibit tissue-specific distribution, and each form is anchored to the cell surface via a particular post-translational modification of the catalytic subunit. Nibaldo Inestrosa and Alejandra Perelman review evidence that heparan sulphate proteoglycans are the extracellular matrix receptors for the collagen-tailed enzyme, and that a glycolipid which contains phosphatidylinositol and a 20 kDa hydrophobic peptide participate in the anchoring of the hydrophobic globular forms of acetylcholinesterase to the cell surface. The physiological role of acetylcholinesterase at nicotinic cholinergic synapses is believed to be the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Since the whole process of synaptic transmission takes place within - 1 ms, it demands a very precise temporo-spatial integration of the structural and functional components involved’. Several molecular forms of acetylcholinesterase exist in synaptic junctional and extrajunctional areas which are distinguishable by their solubility characteristics and hydrodynamic properties. One class of acetylcholinesterase is an asymmetric species formed by three tetramers of up to twelve catalytic subunits linked to a collagen-like tail through disulN.C. Inestrosa is Heud and A. Perehnan is n Research Associate at the Moleculnr Neurobiology Unit, Department of Cell and Melecular Biology, Faculty of Biological Sciences, Catholic Unioersity of Chile, PO Box 114-D, Sanh’ago, Chile.

phide bridges. Asymmetric acetylcholinesterase is concentrated in neuromuscular junctions of mammalian skeletal muscle and in the electric organs of rays and eels. It appears upon synaptogenesis and is regulated by the motor nerve’,‘. A second class of molecular form, commonly called the globular species, exists as monomeric (G1), dimeric {Gz) and tetrameric (G4) assemblies of catalytic subunits (Fig. la). The globular species differ in their degree of hydraphobicity and can exist as both soluble and membrane-associated form&2. Asymmetric acetylcholinesterase and the extracellular matrix Taylor and co-workers3 in 1976 postulated that the collagen-tail subunits were responsible for the association of acetylcholinesterase with the extracellular matrix. Although there was no direct evidence for the localization of the asymmetric forms at the cell surface in viva, evidence had been accumu-

49 (Suppl.), 96P 31 Sato, K., Ozaki. H. and Karaki, H. (1989) Jpn. J. Pharmacol. 49 (SuppI.), 97P 32 Endoh, M. and Blinks, J. R. (1988) Circ. Res. 62,247-265 33 Jiang, M. 1. and Morgan, K. G. (1987) Am I. PhysioL 253, H1365-H1371 34 Hwang, K. and Van Breemen, C. (1987) Eur. j. Phormacol. 134, 155-162 35 Francis, S. H., Noblett, B. D., Todd, B. W., Wel1s.J. N. and Corbin, J. D. (1988) Mol. Pharmncol. 34,506-517 36 Pfitzer, G., Merkel, L., Riiegg, J. C. and Hofmann, F. (1986) Pfriigers Arch. 407, 87-91 37 Hori, M., Shimizu, K., Nakajo, S. and Urakawa, N. (1989) Jpn. /. Plrarmacol. 49, 540-543

lating to suggest that this might be the case4z5. This was confirmed in 1982 using the surface of myotubes derived from a mouse muscle cell line termed C2, from which asymmetric acetylcholinesterase patches, embedded in a rich extracellular matrix (Fig. lb), were easily removed by collagenase“. In 1978 Massoulie and coworkers7 suggested that polyanions such as chondroitin sulphate were involved in the association of acetylcholinesterase to innervated membranes of the electric organ of Electrophorus. In 1983 it was shown that low concentrations of heparin were able to solubilize acetylcholinesterase activity from the endplate regions of rat diaphragm’. Other glycosaminoglycans such as chondroitin sulphate or hyaluronic acid were not able to release acetylcholinesterase from skeletal muscles. Velocity sedimentation analysis of the acetylcholinesterase molecular forms solubilized by heparin confirmed that this glycosaminoglycan selectively released asymmetric forms of the enzyme. These observations indicated that acetylcholinesterase has a ‘heparinbinding domain’ in its structure, presumably in its collagen-like tail. This hypothesis was confirmed by affinity chromatography on columns or heparin-agarose heparan sulphateproteo$ycansq*‘O. Indeed, an intact collagenous tail appears to be required for ‘the interaction between acetyicholinesterase and heparin since binding to the columns was abolished by pretreatment of the enzyme with collagenase’. These reSults support the idea that heparin binds to a specific domain in the collagenlike tail of the asymmetric form of acetylcholinesterase.

0 1989, Elsevierkience

Publishers

Ltd. (UK)

Olffi- 6147/791503.50

‘m’s - ~ugusf

326

I. Molecular Fig. forms of acetytcholineslerase and cellular localization of asymmetric form. a: Sediunction anatysis of a muscle cell extract in sucrose gradient distinct separates forms of acetykholinesterase acwrding to their size and shape. These are sfabfemotecutar form& possess equivatent catalytic activities and can be divided into globular forms G,. G2 and G,, and asymmetric (A) forms. Arrows indf cate positions of marker enzymes in the gradient! &ptactosidase 06.1 S. teftl and alk&ne pho& phatase (6.7 S. right). b: C, mouse myoFractionnumber lubes express the asymmetrk form of acetyl&oiinesterase on their surface, where it is assoctated with the extraceffutar matrix. The aspmet& (A) form was extmtectwith hepadn and run on a sucrose gmdient in high salt conditions. ff~~~rni~t staining of &, myotubes shows focal patches of acetykholinesterase activity on the ceti surface (inset). These patches consist of asymmettic acetykholinesterase because wttagenase treatment, which removes only the asymmetric Fan. abolishes the staining pattern.

fn-uiao interactions between asymmetric acetylcholinesterase and heparin/heparan sulphate moieties have been studied by binding the enzyme to intac’ ,rd!s rich in cell-surface heparan sulphate proteoglycans. Asymmetric a~e~lcholines~rase binds in a specific manner to either endothelial or skeletal muscle cells, showing time de endence and saturation kineticsg. Pretreatment of the asymmetric acetylcholinesterase with colIagenase to remove the collagenous tail abolished the interaction. Scatchard analysis showed a single class of binding sites with an apparent dissociation constant of 0.65 x lob7 M. and the binding was reversed by heparing. Direct evidence for an interaction between the asymmetric forms of acetylcholinesterase and the heparan sulphate proteoglycans at the nehromuscular junction came from experiments using specific ~ycosaminogly~ hydrolases*‘. Purified eiectric organ extraceIIuIarmatrix fractions were incubated with heparitinase, an enzyme that degrades heparan sub phate, and with chondroitinase

ABC, which digests both chondroitin sulphate and dermatan sulphate. Only the asymmetric forms of acetylcholinesterase were released to the assay medium, and this orrurred following exposure to hep-titinase but not to chondroitinase ABC (Ref. 11). Moreover, a stable variant of the rat pheochromocytoma PC12 cell line, which lacks a heparan sulphate proteoglycan on the cell surface, exhibits an atypical distribution of asymmetric ace~l~o~n~~ra~: in normal PC12 cells. virtually all the asymmetric acetylcholinesterase is localized externally in the cell surface, whereas in the mutant cells the enzyme is found in an intracellular compartment’2. This suggests that a cell-surface hepanm sulphate proteoglycan is necessary to externalize the asymmetric forms of acetylcholinesterase at the neuronal cell surface (Fig. 2a). Recent studies have also shown that the glycosaminoglycan dermatan sulphate can solubilize the asymmetric ace~I~~~linesterase from muscle extracellular matrixr3. Since a dermatan sulphate proteoglycan is solubilized from the rat neuromuscular junction when

1989 [Vol. l@J

asymmetric acctylcholinesterase is released by heparin14, it is possible that dermatan sulphate proteoglycans also mediate the anchorage of asymmetric acetylcholinesterase in the synaptic extracellular matrix. Thus, although the exact nature of neuromuscular junction proteoglycan-acetylcholinesterase interactions is not totally understood, a new avenue of research has been opened with the use of the anticoagulant molecule heparin. Gagiobular acetylrholinesterase and the plasma membrane Dimeric G2 acetylcholinesterase is a membrane-bound enzyme form which behaves as an integral membrane protein and is solubilized by detergent. It is anchored to the plasma membrane via a covalent linkage to a glycophospholipid containing phosphatidylinositol’*‘5 (Fig. 2b). The evidence that phosphatidylinositol acts as an ace~lcholinesterase hyd~phobic anchor comes from two experimental designs: (1) release of acetylcholinesterase by phosphatidylinositol-specific phospholipase C; and (2) chemical analysis of the membrane-anchoring domainr5. Dimeric acetylcholinesterase has been solubilized from various sources, including bovine erythrocytesr5, Torpedo electric organ 15, Xenoplrsmuscle16 and rat liver”. The hydrophobic domain of the Ga acetylcholinesterase can be specifically labelled with the photoactivatable hydrophobic reagent ‘251-labelled trifluorophenyldiazirine (1251-TID), an affinity reagent that selectively labels the membrane hydrophobic domains of proteins. This reagent partitions into the hydrophobic phase of membranes or into detergent micelles, and following photoactivation it covalently reacts with lipid or protein sequences in the hydrophobic phase. The labelled fragment has been identified after proteolysis of T~~ed~18 ace~lcholinesterase. Recent studies on acetylcholinesterase forms in rat hepatocytes demonstrated that most of the G2 acetylcholinesterase activity was localized at the celI surface facing the extracellular medium and that the majority of this ‘aftivity was released after treatment with phosphatidylinositol-specificphospholipase C (Ref. 17). Tissue fractionation studies aimed at

TiPS -August

1989 [Voi. 201

,127

The assembly and cell su ce d@~~sitionQf 8s ace~lc~~~ines~@~ase Two of the most sfxikkg features to emerge from recent studies of the neuromuscular junction are its rich biochemical complexity and specialiiation. Fresynaptically, the nerve terminal contains synaptic vesicles and specialized sites of transmitter release. Postsynaptically, the most conspicuous feature is the elaborate folds of the postsynapticmembrane which contain the acetykholine receptors, packed in quasi-crystalline array, on their CEEtS. Extending between pre- and postsynaptic membranes is the extracefhrlarmatrix or basal lamina, part of the continuous sleeve that ensheathes the muscle fibre. At the endplate, this matrix also has distinctive features: the asymmetric form of acetylcholinesterase (which has a collagen-like tail}, a heparan sulphate proteoglycan, and several components that have been identified by irnrn~o~oche~~ criteria’ are concentrated there. AR three specialized structures are in exact register, with the active zones positioned in direct apposition to the extensions of the acetylcholinesterase-enriched extracelhdar matrix into the postsynaptic membrane folds. How are the assembly and maintenance of such a compIex structure achieved? In neuronat and muscle celfs in cuhure, the asymmetric ace~~o~nestemse is mainly located at the extraceUukr matrix or ceil surface; it was therefore of interest to determine whether this taiied form is assembled intra- or extraceWarly. An internal pool of asymmetric acetylchohnesterase has been identified by the use of acetylcholinesterase inhibitors of differing membrane permeability. After irreversible inhibition of all celh&r e&erase by ~-i~~py~uo~pho~honate (DFP). the newly synthesized asymmetric acetylcbahnesterase appears in an intracelhdar compartment within about 90 min, but does not appear on the cell surface until after 2.5

determining the subcellular local-

ization of acetylcholinesterase showed that the enzyme was local-

ized at the plasma membrane and the Golgi apparatus. The association of the G2 form with organelle membranes was characterized by studying the effect of phosphatidyl-

h (Refs 2 and 31. Rotundo has also anaiysedacetyhhohne~terase assembly after DFP inactivation usjng lecttns that recognize sugars attached in the rough endoplasmic reticulum and Golgi apparatus, and has concluded that asymmetric acetylcholinesterase is assembled in the Golgi apparatus. The iutracelhrlar pathway probably involves the packing of asymmetric acetylcholinesterase into transport vesicles (coated vesicles), and subsequent Eortirtgto the surface of the ceii where a~~Ic~~n~terase would be secreted to the extracelbrlar matrix and anchored to its receptor, the heparan suiphate proteoglycan. Moreover, because the asymmetric acetylcholinesterase and the acetylcholine receptor are transported to the cell surface with similar kinetics, it is possible that they follow a single intracelhrlartransport pathways (see Fig.). The intracellular assembly of the asymmetric acetylcholinesterase would imply some coordination between svnthesis of the collagenous-tail subunits and acetyb cholinesterase catalytic subunits. Recent progress on the cloning of the acetylcholinesterase gene6*’has set the stage for a rapid advance in the understandingof the

biogenesisof this importantcomponentof the synaptic extracelhk matrix. References 1 lnestrosa, N. C. (1988) in Newe-Muscle Cd i’kaphic Communication (Femtidez, H. L., ed.), pp. 147472, CRC Press 2 Inestmsa, N. C., Matthew, W. D., Redness, C. G., Hail.2. W. and Reichardt, L. F. (1985) J. Neurochem. 45,86 3 Inestrosa, N. C. (1984) Biochem. I. 217,377-M 4 Rotunda, R. L. (1964) Proc. Natl Acad. Sci. USA Sl,G’9-483 5 Rotundo, R. L. and Fambrough, D. M. (1980) CM 22,595-W 6 Schumacher, M., Maolet, Y., Camp, S. and Taylor, P- (WE8) J. Biol. Cfzern.263,18979-18987 7 Sikorav, J-L. et nI. (198!3)EMBO i.298?-2993

inositol-specific phospholipase C on acetyicholinesterase activity. most all the G2ace~~cholinesterase activity was removed from Golgi membranes by phospholipase C (A. Perelman ef al., unpublished). This result is consistent with a very rapid transfer of the

glycophospholipid to the nascent catalytic subunit polypeptide during biosynthesis of the dimeric GZ ace~~~o~este~se*‘. Thus, a C-terminal glycolipid on the processed enzyme is responsible for the association of the dimeric G1 form of acetylcholin-

TiPS - August

2989 [Vol.

IO]

20 kDa component of brain enzyme contains a glycolipid similar to that observed at the C-terminus of the Ga form of acetylcholinesterase. Chemical data indicate that there are significant differences in the composition of these domains. In fact, bovine brain acetylcholinesterase contains no inositol, eth~olamine or glucosamine with free amino groupszl. Extended pronase treatment of the Gq form and hydrophobic chromatography on Sephadex LH-60 showed that the 20 kDa fragment contains a pronase-resistam hydrophobic polypeptide of 7 kDa which contains fatty acids and thus probably possesses a non-polypeptide domain”. The hydrophobic G4 form of acetylcholinesterase can be converted to a catalytically active hyd~phi~c enzyme after protease treatmenta3.When ‘22-TID-labelled acetylcholinesterase was treated with proteinase K (Fig. 2b), more than 85% of the enzyme activity was resistant and the G4 acetylcholinesterase would not form aggregates in the absence of detergent. However, almost ail the “‘ITID radioactivi~ associated with the G4 ace~lcholinesterase was released. The label remamed associated with a 13 kDa fragment, indicating that proteinase K released most of the hy~phobic domain from the eruymez4. These observations led to the postulate that at least three different subdomains exist in the hydrophobic anchor of the G4 acetylcholinesterase: firstly a proximal subdomain containing disulphide bridges involved in the binding of the hydrophobic domain to the 68 kDa catalytic subunit; secondly, a proteinase K-sensitive intermediate subdomain; and thirdly a pnmaseresistant 7 kDa subdomain which contains fatty acids and is probably directly involved in Gq acetylcholinesterase binding to the external leaflet of the neuronal membrane bilayer. the

asymmetric acetytcholinesterase

b

dimericacetylcholinesterasa

tetramericacetylcholinesterase

ss 1

s-s

0 c0prot. Q

0

K

‘t?

AlAAiA;A

id.

rttt’t?t?ttt

AA;AeA;rAAiA

Fii.

2. F&&cu/ar forms of acetytcholiflesferaseand their environment a: The asymmefric hmnis shownhen, interacting through the coflegen-like tail with the g&wsaminoglycan chains of heparan scrphate protecg&cansin the extracellular matrix. IccaW at the C-Mniniis of the catafytfc subunfts (left). Ths tetramerfc G., fom~ fs anchor& by a 20 kl?a @ypeptida to ths plasmamembrane. Plot. K, approximatefccationof proteinase K-sensitivesite.

esterase to the plasma membrane. The identity of the C-terminal peptide of the Torpedo enzyme, after removal of fattv acids has recently been determined by protein sequencing1qto be: Leu-Leu-AsnAla-Thr-Ala-Cys-ethanolamineglycophospholipid. G4glob&r acetylchohesterase and the plasma membrane In mammalian brain nearlv all acetylcholinesterase activity i”sin forms other than the as~met~c species. The bulk of the enzyme is detergent-soluble tetrarneric C4 acetylcholinesteraseZ. However, this form is insensitive to phospha~dy~ositol-spe~~c phospho-

hpase C (Ref. 20}, suggesting that the hydrophobicity does not result from the presence of a glycolipid in the C-terminus of the enzyme. The association of bovine brain acetylcholinesterasewith the neuronal plasma membrane has been studied by labelling the purified G4 forms with the photoa~ni~ reagent =I-TID. The electrophoretic pattern in sodium dodecyl sulphate-pol acrylamide gels shows that 1Y51-TIDlabels a broad 20 kDa bandzXz2which is cleaved from the enzyme after reduction, suggesting that the hydrophobic fragment is bound to the catalytic subunits by disulphide bridges’l. An obvious question is whether

•1

cl

0

Three molecular forms of acetyl~o~~~e contain specific modifications that are used to interact with cell surfaces: (1) three tetramers of catalytic subunits of the asymmetric form attached to a collagen-like tail, which interacts

TiPS - August 1989 [Vol. 201

with heparan and possibly dermatan sulphate proteoglycans in the extracellular matrix; (2) the diacylglycerol moiety of phosphatidylinositol in the C-terminus of each GP acetylcholinesterase polypeptide anchors the enzyme to the outer layer of the plasma membrane; (3) in the brain G4 acetylcholinesterase form there is the covalent attachment of a 20 kDa hydrophobic polypeptide to the plasma membrane. One plausible explanation for the existence of different arrays of anchoring domains in each type of acetylcholinesterase molecular form is probably related to the physiological role of each type of enzyme in a particular type of cell. In fact, it is clear that, for example, the factors required for rapid synaptic transmission at the neuromuscular junction define both an enzyme macromolecular organization and a subcellular location for the asymmetric acetylcholinesterase; in addition, it requires a very specific synaptic heparan sulphate proteoglycan as an extracellular matrix anchor to act as an efficient partner of the acetylcholine receptor at the nicotinic choline@ synapse. Acknowledgements We thank Drs J. Alvarez, M. Bitran, E. Brandan, R. GonzalezPlaza and J. P. Huidobro-Toro for helpful comments on the manuscript. Most of the research carried out in our laborat-y has been supported by DIUC, FONDECYT, Stiftung Volkswagenwerk and Fundacihn Gildemeister. References 1 Taylor,I’., Schumacher, M., bfaulet, Y. 2 3 4 5 6 7 8 9

and Newton, M. (1986) Trends Pharmncol. Sci. 8.321-323 MassouliC. J. and Bon, S. (1982) Annu. Rev. Neurosci. 5, 57-106 Lwebuga-Mukasa, J. S., Lappi,S. and Taylor, P. (1976) Biochemistry 15, 1425-1434 Hall, Z. W. and Kelly, R. (1971) Nature 232, 62-63 McMahan, U. J., Sanes, J. R. and Marshall, R. M. (1978) Nature 271, 172-174 Inestrosa, N. C., SiIberstein, L. and Hail, Z. W. (1982) Cell 29,71-79 Bon, S., Cartaud, J. and MassouIiO, J. (1978) Eur. J. Biochem. 85,1-14 Torres, J, C. and Inestrosa, N. C. (1983) FEES L&t. 154,265-268 Inestmsa, N. C. (1988) in Nerve-Muscle Cell Trophic Communication(Fembdez, H. L., ed.),pp. 147-172,CRCPress

10 Vigny, M., Martin, G. R. and Gmtendorst, G. R. (1983) 1. Biol. Chem. 258.8794-8798 11 Brandan, E., Maldonado, M., Garrido, J. and Inestrosa, N. C. (1985) J. Cell Biol.

101,98%992

12 Inestrosa, N. C., Matthew, W. D., Reiness, C. G., Hall, Z. W. and Reichardt, L. F. (1985) 1. Neurochem.45,86-94 13 Von Bemarhdi, R. and Inestmsa, N. C. Brain Res. (in press) 14 Brandan, E. and Inestmsa, N. C. (1987) FEBS Lett. 215,15%163 15 Silman, I. and Futerman, A. H. (1987) Eur. 1. Biochem.170,11-22 16 Inestrosa, N. C., Fuentes, M-E., AngIister, L., Futerman, A. H. and SiIman, I. (1988) Neurosci. Lett. 90, 186-190

17 Perelman, A. and Brandan, E. Eur. I, Biockem.(in press) 18 Stieger, S., Bmdbeck, U., Reber, 8. and Brunner, J. (1984) FEBS Lett. 168,231-234 19 Gibney, G. G. et al. (1988) 1. Biol. Ckem. 263,1140-1145 20 Futerman, A. H., Low, M. G., Michaelson, D. M. and Silman, 1. (1985) 1. Neurochem.45,1487-1494 21 Inestrosa, N. C., Roberts, W. L., Marshall, T. and Rosenberry, T. L. (1987) 1. Biol. Ckem. 262, 4441-4444 22 Gennari, K., Brunner, J. and Bmdbeck, IJ. (1987) /. Neurochem. 49,12-18 23 Grassi, J., Vigny, M. and MassouliP, J. (1982) J. Neurockem. 38,457-%9 24 Fuentes, M. E., Rosenberry, T. L. and Inestrosa, N. C. (1988) Biockem.J. 256, 1047-1050

in denervation hemorrhage Motohatsu Fujiwara, Tetsuya Tsukahara and Takashi Taniguchi Vasospasm of cerebral arteries in patients with subarachnoid hemorrhage frequently presents severe clinical problems resulting from cerebral ischemia, but the pathogenesis of vasospasm is still poorly understood. The contractile response of human cerebral arteries to noradrenaline i!: larger than the responses in other species. Neurogenic factors controlling brain circulation may play an important role in pathological conditions such as subarachnoid hemorrhage. Motohatsu Fujiwara and colleagues review species variations of or-adrenocepfors in cerebral arteries and their alterations after surgical sympafhectomy. They compare these changes to those occurring in human cerebral arteries following subarachnoid hemorrhage and discuss their relationship to vasbspasm.-

‘Following subarachnoid hemorrhage caused by cerebral aneurysm, patients may show symptoms of focal cerebral ischemia due to prolonged vasoconstriction of the major cerebral arteries (chronic cerebral vasospasm), even though they seem to be recovering following early operation; indeed the leading cause of death and disM. Fujiwara is Professor in the Departmenf of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, lapan; T. Tsukakara is Senior Researcher in the Cerebrovascular Disease Laboratory, Department of Neurosurgery, National Cardiovascular Center, Osaka 565. ,lapan; and T. Tanigucki is Professor in the Department of Neurobiology, Kyoto Pharmaceutical University. Kyoto 607. lapan.

ability in patients with aneurysmal subarachnoid hemorrhage is cerebral vasospasm’. Intensive clinical and experimental studies have not yet identified the precise pathogenesis of chronic cerebral vasospasmZD, although disturbance of neural control of the cerebral arteries, especially dysfunction of adrenergic innervation, has been considered to be a Histochemical fluorescausee. cence of the adrenernic nerve plexus supplying the- cerebral arteries of humans and animals transiently weakened following hemorrhageG7. subarachnoid Moreover, pial arteries of experimental animals became supersensitive to noradrenaline or 5-m