Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C

Biochirnicaet BiophysicaActa, 1095(1991)255-260 1991EIs~ier Sciene~publishers B.V All righl~re~e~ed 0167-4889/91/$0350

255

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Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca z÷ and protein kinase C C h r i s t i a n C. l a n s s o n , Jyrki K u k k o n e n a n d K a r l E . O . ,g,k e r m a n Depanraent of Bit2chemZ~tryand Pharmacy, ~bo AkademL Abe (FinlandJ (U¢~ived27 December 1990) (Revised manuscript received 2~July 1991)

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SYSY human aeeroblastoma cells has been investigated. The dose-response relations of carbachul-induced cAMP synthesis and carbachol-lnduced rise in intracellular free Ca 2+ were similar. Th~ stimulated cAMP synthesis was inhibited by about $0% when cells were entrapped with the Ca z+ chelator BAPTA or in the prescence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca 2. innophore, ionemycin and by TPA, an activator of PKC. When added tngethec TPA and ionemycin had n synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE I or PGE I + pertussis toxin cathachul stimulated cAMP production to the same extent as in control cells. Ca ~'+ and protein Idnase C thus seem to be the mediators of muscarinic.receptor linked e.AblP ~ntbesis by a direct action on adenylate cydase.

The museaxinic acetylchulin¢ receptor in the central nervous system mediating both inhibition and excitation of neurons. Muscariulc receptors are also folllld at the somatic neuromuscular junctions where they are involved in the secretory activity of exocrine glands [1,2]. Activation of muscarinic receptors elicits a wide diversity of physiolog;.eally important biochemical responses. These are categorized in two main groups; those coupled to inhibition of adenylate cyclase [3] and those linked to activation of phosphoinositide hydrolysis [4]. The G.proteins Gi and Gs are thought to mediate inhibition and stimulation of adeuylate cyclase, respectively [5]. The increases in phosphoinositide turnover again results in CaZ+-mobilization and activation of protein kinase C [6,7]. In most tissues different subtypes of muscarinic receptors mediate either inhibition of adouylate cyclase or activation of Abbreviations: mMX. 3-isobuthyl-l-raethyl-xanthine;BAPTA/AM, 1,2-bis(o-aminophenoxy)-eth,ane N,N.N'.N'.tctraacetic acetoxymelhylester; fura-2, fura-2-a~toxym©tl~.j!ester,TPA, t2-o-tctradecanoyl-phorbob13-a~tat¢. Corre,z~edence: C.C. Jansson, Deparlment ot nioch0raistl~ at,,1 Ph~acy, ~bo Akaderoi, Porthanssatan 3, SF-20.,¢00~,bo, Finland.

pbosphoinositide hydrolysis. Recently it has been reported that cloned muscap:nic receptors that are coupled only to phosphoinositide hydrolysis, are linked to a stimulation of adcnylate ¢yclase [8]. In the SH-SYSY neuroblastoma cell line stimulation of musearinic receptors also leads to an increase in the cAMP level [9]. The aim of this investigation was tD find out the mechanisms for the stimulator'/effect on cAMP synthesis through muscarinic receptors. Since one main response to muscarinic stimulation is a mobilization of Ca 2+ and activation of protein kinase C the role of this pathway in the activation of cAMP synthesis in SHSYSY neurohlastoma cells is of interest.

Materials Carbaehol, TPA and cholera toxin were purchased [rein Sigma (St. Louis, Me. U.S.A.). Fura-2/AM and B A P T A / A M were obtained from Molecular Probes (Junction City, U.S.A.), ionomycin from Calbiochem (Behring diagnostics, La Jolla, CA, U.S.A.) and staurosporin from Boehringer-Mannbeim, Germany. Experimental media Na÷-hased medium: 137 mM NaCI, 5 mM KCI, 1 mM CaCl 2, l0 mM glucose, 1.2 m.M MgC12, 0.44 mM KHzPO4, 4.2 rnM NaHCO~, 20 mM 2-[(3/hydroxyl-

1,1-bis (hydroxymethyl)ethyl]amino)ethane sulphonate (TES) adjusted to pH Z4. Cell cultures SH-SY5Y neuroblastoma cells [10] were obtained through Dr. S. P~ihlman (Department of Pathology, University of Uppsala, Sweden). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibeo, U.IC) supplemented with 10% foetal calf serum (Gibco, U.K.), 50 ~ g / m l streptomycin and 100 I U / m l penicillin (Nordvaee media) at 3 7 ° C in 5% CO z in an air ventilated humified incubator. The cells were passaged when confluent by using E D T A (0.02% in phosphate-buffered saline), and the medium was changed twice a week. Cells for experiment were washed once with phosphate buffered saline and then harvested with EDTA. Measurement o f intracellular Ca z + The intracellular free Ca 2+ in cells was determined using fura-2 [ l i ] as described previously [12]. Briefly harvested ceils were collected by centrifugation (800 × g, 5 rain), washed once in the Na÷-based medium and resuspemled in the same medium supplemented with 10 mM glucose. After 5 rain at 3 7 ° C 1 m M CaCI 2 and 2 t~M fura-2/AM were added. The cells wore loaded with fura-2 with constant agitation for 20 rain at 37 ° C. Thereafter they were separated by centrifugation and washed once in Na+-based medium containing 10 mM glucose and 1 mM CaCI 2. The cells were subsequently suspended in the same medium in the abscence of Ca 2+ and were pelleted in different tubes by centrifngatinn at 1 0 0 0 0 × g for 30 s. The medium were carefully sucked off and the ceils were subsequently stored on ice. No differences in ceil response could be detected on ceils stored on ice for 4 h. Fluorescence measurements were initiated by resuspendlng one pellet into the experimental medium containing 1 m M CaCI 2 and 10 m M glucose at 3 7 ° C . The fluorescence was recorded at 340 nm (ex] and 505 nm (era) in a Hitaehi F-2000 fluorescence speetrofotometer with constant stirring. The dye responses were calibrated by sequential addition of 0.02% digltonin and 100 ~ M M n S O 4 at the end of the experiment to obtain maximal and minimal fluorescence values, respectively [13]. Entrapment of the Ca 2+ chelator BAPTA into the cells was performed by incubation of ceils for 20 rain in the Na+-based medium containing 10 m M glucose, 1 m M CaCI 2 and 10 p M B A P T A / A M at 3 7 ° C . Thereafter the cells were washed once in the same medium, pelleted and stored on ice as above. Measurement o f intmcellular cAMP Detached ceils were washed once in Na+.based medium. Thereafter they were rcsuspended in Na +based medium containing 10 m M glucose and incu-

bated for 5 min at 3 7 °C. 1 mM CaCI 2 was added and after a 20 rain incubation at 3 7 ° C the cells were pelleted in different tubes by centrifugatinn at 10{100 × g for 30 s and stored on ice. The cells were stored on ice as short a time as possible, not more than 2 h. The reaction was started by resuspending one pellet into the experimental medium containing 10 mM glucose, 1 mM CaCI2,1 mM 3-isobuthyl-l-metbyl~:anthine (IBMX) and various effeetors used. After 10 min the reaction was terminated by centrifngation of the cells for 1 rain at 10000 × g and application of ice-cold 0.1 M HCL The samples were freeze-dried and the content of cyclic A M P was determined using a RIA-ldt (Amersham, U.K). In experiments using TPA (100 nM) and staurosporlne (100 nM) the ceils were preineubated with these agents for 2 h before the experiments. When using the Ca 2+ chelator BAPTA the cells wore incubated in the same way as in the CaZ+-mobilization experiments, in experiments using cholera toxin the toxin was activated by treatment with 20 mM dithiotreitol for 30 rain at 370C. The ceils were treated with 1 p~g/ml cholera toxin for 3 h before the experiment. Cells treated with pertussis toxin were incubated with 100 n g / m l for 3 h before the experiment. Protein was determined according to Lowsy ot al. using bovine serum albumin as standard [!4]. Results The dose-response relation for the carbaehol-induced increase in cAMP and intracellular Ca 2+ as measured with fnsa-2 is shown in Fig. 1. The EDso for the increase in cAMP synthesis was 17 :t: 2 v,M (n = 3) while that of the rise in eytosolic Ca 2+ was 12 + 1 I~M

0

s

2~

22

1.0e [CARBACHOLI(M) Fi~. t, Dos=-response relationshipof ¢arbachol-induc~d increase in t2ytosolicCaz* and in eytosolic cAMP. "rile responses, from 134 nM to 315 nM Ca2+ or from 4.86 pmol cAMP/ms protein to 7.25 pmol cAMP/ms protein, was taken as 100%. Carbachol.ioduced increase in cytosolic Ca2+ (O) was determined with fura-2 and ealbachol-induced increase in cytnsoliecAMP (o) ~¢asdetermined with a RIA kit. See Materials and Methods for eXlxximental de~ails, Each e~pe.rimental point represents an average from four indeponrtent experiments performed in quadriplicate.

257

O.

b.

I'Ccl2+] i (nM) 300

[C°2+] i ,300 _

12Or-

120 CARB

(nM)

CARB

Fig. 2. Effect of BAPTA entrapment on carbachol-induced rise in cytosolic Caz *. Add±don of 100 !tM carbachot (CARB) was made to control cells (a) and to ceils treated with 10 #M BAPTA/AM for 20 rain 1b1. Loading of cells with fur~-2 and calibration of the response were ca~ied out as described in Materials and MerTtods.

( n ffi 3). T h i s gives a n i n d i c a t i o n a b o u t a fairly direct c o u p l i n g t h r o u g h t h e s a m e e f f e c t o r system. I n o r d e r to investig ate t h e role o f C a 2. in carbac h o l - i n d u c e d c A M P s y n t h e s i s t h e cells w e r e e n t r a p p e d with t h e C a 2+ c h e l a t o r B A P T A w h i c h is e x p e c t e d t o b u f f e r cytosolic C a ~+ a n d a t t e n u a t e C a 2+ t r a n s i e n t s . I n

Fig. 2a 100 / *M c a r b a c h o i c a u s e d a rise in t h e i nt ra cellular C a -'+ c o n c e n t r a t i o n f r o m 1 3 4 + 1 2 . 2 n M t o 315 + ~3.9 a M . Fig 2b shows t h a t B A P T A e n t r a p m e n t totally i n h i b i t e d t h e rise in cytosofic C a : ~ i n d u c e d by c a r b a c h o L T a b l e I d e m o n s t r a t e s t h a t B A P T A e nt ¢a pm e n t r e d u c e d c a r b a c h o l - i n d u c e d c A M P -ojm.tllesis by

TABLE I Effecls o1"diJferent agents on the intraeellular let'et o f c A M P in presence and a b ~ c e o,¢ I ~ ~ M cadmehol

The cells were incubated with l0 /xM nAPTA/AM for 20 rain. with 100 nM staumsporine for 2 h and with ICO nh,I TPA for 2 h before the etcetiment. The ionomyein concentration was 0.8/zM. For other details see Malerials and Melhods section. The values are the means -+S.E. of four independent experiments performed in quadrplieate. The values in brackets in Ihe leR column indicate percent stimulation by TPA only and the values in the right column indicate percent stimulation bY carbaehol where the stimulation in control ceils is laken as 100%. Treatment

pmol cAMP/rag protein control

Control + Ionomycin + BAPTA + Stauros!~orlne + BAPTA and stauroslmrine +TPA +TPA and BAPTA +TPA and staurosporlne ÷ TPA and ionomycin

4.86±0.85 15.10+ 1.40 3.495:0.27 3.46±0.43 2.34_+0.22 8.87±0.48 t (82) 8.54±0.63 h (76) 3.93-+0.27 i (14) 28.67 5:2.84

a p < 0.02 compared to control. P < 0+01 compared to cDr.trol. ¢ P < 0.02 compared to coatrol. d p < 0.05 compared to control ¢ e > 0.2 compared 1o control; not significant. f P < 0.001 compared to control control. B p < 0.82 compared to control. h p > 0.2 compared to TPA control; not significant. i p < 0.2 compared to staumsperine control; not s~gnificant. J P<0.05 compared to ~ntroL

carbachol

% S:Jumlation by carbachol

7.255:0.8O ~ 22.60± 2 2g b 4.39 ± 0.34 ~ 4.37±0.44 a 2.57 _+0.24 ~ 10.91 -+0,83 ~

180 10C 52 53 20 47

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Fi~. 3. Intracetlular cAMP as a function of intraeellular free Ca z÷. (h) lntracellular free Ca ~* was determined with fura-2 in the presence of differenl conc~ntratlolls (0.l-I ~M) of ionomycin. The amount of intracellular cAMP wa~ measured in parallell in cells treated with the same concentrations of [onomycin as aboee. (O), ~ntml ceils; to), TPA treated cells. Each point represents an average from tour independent experiments perfo~cd in quadriplicate. (n) The synergistic effect between TPA and innomyelu. The sum of the effecl of TPA alone and ionomycin alone are reduced from the effect of both compounds together.

about 50%. Since chelation of intraeellular Ca ~+ partially inhibited c A M P production o n e would expect t h a t a rise in intraeellular Ca 2+ would stimulate the production in c A M P . T h e effect of t h e Ca z+ iooophore, ionomycin, was thus tested. lonomycin increased t h e c A M P c o n t e n t of t h e S H SYSY cells. Carbachol was, however, still able to increase c A M P synthesis in the presence of ionomycin. T h e Ca 2÷ d e p e n d e n c y of c A M P accumulation was d e t e r m i n e d t h r o u g h parallel m e a s u r e m e n t s of intracellular free Ca 2+ with ionomycin a n d c A M p accumulation. T h e data s h o w n in Fig. 3a d e m o n s t r a t e that a half-maximal increase in c A M P concentration occurred a r o u n d 0.3-0.4 txM Ca 2+ a n d t h a t the m a x i m a l effect was seen r o u n d 0 . 8 / a M w h e r e a f t e r a fall in the c A M P c o n t e n t was apparent. Since carbachol f u r t h e r stimulated c A M P production in t h e presence of a maximally effective concentration of ionomycin o t h e r m e c h a n i s m s than Ca 2÷ atone seem to play a role in earbaehol-induced c A M P production. As s h o w n in Table I also t h e p h n r b o l ester, T P A , k n o w n to activate protein kinase C, caused a rise in t h e cAIvlP level by about 100%. This rise could be inhibited (to about 85%) by staurosporine, a n inhibitor of protein kinase C. To f u r t h e r test t h e role of protein kinase C in carbachol-lnduced c A M P synthesis t h e effects of staurosporine was tested. T h e earbaehol-induced rise in c A M P synthesis was inhibited by a b o u t 5 0 % in the presence of this alkaloid (Table I). W h e n staurosporine was used in combination with B A P T A e n t r a p m e n t t h e r e was n o significant rise in c A M P in the presence of carbachol. T P A arid ionomycin in combination caused a greater stimulation in c A M P synthesis than the expected additive effect (Fig. 3a a n d b). W h e n ceils were treated with T P A or T P A a n d ionomycin t h e stimulatory effect by carbachol was reduced (Table I). T h e stimulatory effect o n c A M P syn-

thesis caused by T P A was n o t significantly reduced w h e n cells were treated with B A P T A (Table 1). T h e m I muscarinic receptor selective antagonist pirenzepine inhibited carbachol-indueed c A M P production with relatively high affinity (Fig. 4). T h e calculated K i value for p i r e n z e p i n e was 35 + 5.8 n M . This value is very close to the K i value for p i r e n z e p i n e in inhibitirm of carbachol-induced increase in Ca z+, 48 + 9.7 n M (data not shown). P i r c a z e p i n e h a d n o effect o n t h e basal c A M P level (data not shown). C h o l e r a toxin caused a significant stimulation of c A M P synthesiz (Table ll). T h e s t i m u l a t o ~ effect of carbachol was similar in t h e presence of cholera toxin (about 50%), as in control cells, a l t h o u g h the basal c A M P level was 10-fold higher. T h e same stimulator)" effect of carbachol could also b e e n seen w h e n t h e c A M P synthesis already was stimulated by P O E I o r in t h e presence of P G E I a n d pertussis toxin (Table It).

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259 TABLE n Effects o f I00 I~M carbachol on cells treated with cholem tox~n, pemt~r,s toxin a , d PGEp

The ceUs were trealed w0h I /~g/ml cholera lenin and wah 100 ng/ml pertu~sis toxin for 3 h befiare ihe exPeriment. PGE113 t~M) was added at :no beginnin8 of the experiment The cells ~'ere pretreated ~ith la u,~l BAPTA 20 rain or "~ah 100 nM staurosporine 2 h before Ihe application of cholera toxin. The experimenl was performed as described in the methtvJg geclion lhe ~alue~ ire the means~S.E, of four independenl experiments performed in quadr~ptieate. Treatment

pmol cAMP/rag prolein control

Conlrt0 + Cholera t~in +PGE t + Pertussis toxin and PGE t + BAt'TA + St aurosporlne +Cholera toxin and BAPTA +Cholera toxin and staurosporine

5.26::1k55 74 18±5 01 ta.35 ±(L76 23.8O~:3.9-8 3.88 _+O.29 3.79 ± 0.aS 81.23~ ~.94 70.27± 5.61

a " e a

earbachol

% Stimulation by car trachul

T r . 7 ~ . I I ~' 1119.70+ 9±6" 1725_+3.49 ~ 34.84 ± 4.14 J

48.1 47.9 66.7 46.4

P<0.001 compared to conlrol. P<0.01 ~mpared to conlrol. p < 0.02 compared to ~ntrol. p < 0.02 compared to control.

T h e stimulatory effect of cholera toxin was still seen w h e n t h e cells have b e e n p r e t r e a t e d with e i t h e r B A P T A or staurosporine suggesting t h a t these drugs have n o direct effect o n adenylate cyclase (Table Ill.

T h e results of this study indicate that b o t h Ca 2÷ a n d p r o t e i n kinase C m e d i a t e t h e muscarininc receptorlinked rise in c A M P in S H - S Y 5 Y n e u r o b l a s t o m a cells. All the e x p e r i m e n t s w e r e carried o u t in the prescenee of I B M X , a n inhibitor o f phosphodiesterase. T h e r e fore, t h e possibility o f a protein kinase C mediated inhibition of this e n z y m e is unlikely [15]. T h e results w i t h cholera toxin a n d P G E I are of interest since carbaehol stimulated cA/rIP production to the same extent w h e n the c A M P production was already stimulated 10-fold. It is t h e r e f o r e obvious t h a t c A M P synthesis coupled to muscarinie receptor-activation occurs at t h e level at the already activated adenylate cyclase. Carbachol will t h e r e f o r e act at a distinct site w h i c h is not linked to t h e G s d e p e n d e n t activation. T h e adenylate eyelase of n e u r o n a l cells is activated by Ca z÷ [I6]. T h e rise in intracellular Ca 2+ therefore seems to be o n e m e c h a n i s m for muscarinic receptorm e d i a t e d c A M P production. Thus the d o s e - r e s p o n s e relation for carbachol-induced Ca2+-mobilization a n d c A M P production was similar a n d t h e Ca 2÷ i o n o p h o r e ionomycin was also able to induce cA/riP synthesis. T h e activation of c A M P production at Ca ~+ concentrations b e t w e e n 0.1 a n d 1.0 t z M is very similar to the Ca 2+ d e p e n d e n c y of a variety o f CaZ+-calmodulin dep e n d e n t enzymes [17]. E n t r a p m e n t of the Ca 2÷ chelat o r B A P T A into t h e cells inhibited carbacbol-induced

c A M P s3'nthe~,is by about 5 0 % a l t h o u g h the carbacholinduced rise in Ca -'+ was totally inhibited. Furtherm o r e , carbachol still stimulated c A M P production in the presence of a maximal effective concentration of ionomycin. The-efore. o t h e r m e c h a n i s m s must be involved as well in carbachol-induced c A M P production. T h e production of c A M P could also be stimulated by the phorbol es!cr, T P A , suggesting that an activation of protein kinase C is involved as well. Activation of c A M P production by phorbol esters has previously been s h o w n in 3'~'3 ceils [18]. Staurcsporiae, a n effe,:tive inhibitor o 8 protein kinase C, inhibited t h e carbachol-induced c A M P synthesis by about 5 0 % giving f u r t h e r evidence for a role of protein kinase C in muscarinic receptor-linked c A M P production. Like in m a n y o t h e r systems protein kinase ,Z a n p e a r s to amplify the effect of C~ :+ [6,71 since w h e n added t o g e t h e r ionomycin a n d T P A had a synergistic effect. T h e effect of carbachol was reduced in t h e combination T P A / ionomycin d e p e n d i n g o n a n almost ma:dmally activated protein kinase C a n d an amplified effect of Ca 2÷. Based o n pharmacological evidence it has b e e n suggested t h a t S H - S Y 5 Y cells mainly express muscarinic receptors of the HM~ (m~) [191 or H M 4 (m 3) [20] s u b , p u s , which subtypes arc linked to inositolphospholipid b r e a k d o w n a n d Ca2÷-mobilization. T h e muscarinic receptor antagonist pirenzepine h a s differe n t affinities for muscarinie receptor subtypes. It is t h o u g h t to have a particular,/ high affinity for H M I receptors (0.03 ,~M) [20]. T h e results with pirertzepine indicate that the stimulatory effect of carbachol should be linked t h r o u g h H M t receptors, since t h e K i for piranzepine in inhibition of calbachol-induced increase in cAIVlP was a r o u n d 0./135 /~M. Production of c A M P

260 seems to be one important consequence of the activation of these ,eceptors since a rise in c A M P is a~so seen when cloned receptor D N A of these subtypes is transfected into target cells [81. In light of the present study it seems that the rise in c A M P seen is a secondary consequence of inositol-lipid breakdown. A rise in c A M P has previously b e e n shown to potentiate electrophysiological effects produced through musearinic receptors in neuroblastoma cells [21] a n d to increase the magnitude and shorten the duration of muscarinic receptor-linked CaZ+-mobilization [1~]. Furthermore, c A M P is required for the function of Ca 2+ channels in neuronal cells [22]. It therefore does not seem unrea.sonable to suggest that c A M P production through mt,scarinie receptor activation is a means for amplifing responses associated with receptor activation. Aclmowledgements This study was aided by grants t'orm the Sigrid Juselius Foundation, T h e A c a d e m y of Finland and T h e Cancer Organisations.

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