Biochemical characterization of serotonin stimulated phosphoinositide turnover

Pergamon P r e s s

Life Sciences, Vol. 38, pp. 663-669 Printed in the U.S.A.

BIOCHEMICAL CHARACTERIZATION OF SEROTONIN STIMULATED PHOSPHOINOSITIDE TURNOVER P

Jeffrey

Conn and E l a i n e S a n d e r s - B u s h

Tennessee Neuropsychlatric Institute and Department of Pharmacology Vanderbilt Unlversity School of Medlcine Nashville. Tennessee 37232 (Received

in final form December 2, 1985) Summary

Serotonin (5HT) stlmulates phospholnositide turnover in a number of tissues, but it is not known whether this effect is due to activation of a 5HT receptor which is coupled to phosphoinositide hydrolysls or if the effect Is secondary to 5HT stimulated arachidonate metabollsm or to the release of another neurotransmitter In the present study we show that neither indomethacin nor BW 755C inhibits 5HT stimulated phospholnositide hydrolysls in rat cerebral cortex, suggesting that neither cyclooxygenase nor llpoxygenase activity is required for the response to 5HT Protelnase inhibitors do not potentiate the response to 5HT, suggesting that 5HT's effect is not due to stimulation of release of a peptlde neurotransmitter Tetrodotoxin does not inhibit the effect of 5HT and 5HT's effect is additive ~ith that of KCI and veratrine These data suggest that 5HT stlmulated phosphoinositide hydrolysis is not dependent upon release of another neurotransmitter

Serotonln (5HT) interacts with a number of putatlve receptor sites in m~mmalian tlssues The most common classification scheme for 5HT receptors is based upoh data from radioligand bindin~ studles, in which three major 5HT binding sites have been identified (Sla0 S|b, and $2) Whlle a biochemical effector system has not been definltively sho~n to be linked to any of these binding sites, we have shown that 5HT stimulated phosphoinositlde hydrolysis in rat cerebral corticaI slicer is mediated by a receptor which resembles the 82 binding site. and suggested that the second messenger system linked to the S2 binding site involves increases in phosphoinositide hydrolysis (I) Since that time, these findings have been confirmed and extended in brain (2.3) and other tissues (4-6) Agonlst induced hydrolysis of membrane phosphoinositldes is thought to serve as a multifunctional transmembrane transduclng mechanism which results in the liberation of two major second messengers, diacylglycero] (DAG) and inositol triphosphate (IP3) DAG activates proteln kinase C, ~hile IP3 llberates calcium from non-mitochondrlal internal stores (7) While it is tempting to assume otherwise, the f~ndlng that activation of a given receptor stlmulaLes phosphoinosltide hydrolysis does not necessarily mean that that receptor is directly linked to phosphoinositide hydrolysis In platelets, the phosphoinositide response to ADP. epinephrine (Sweatt and Limbird, unpublished findings), the ionophore A23187 (8) and to a lesser extent collagen (9) im blocked by inhibitlon of cyclooxygenase These agents apparently cause

0024-3205/86 S3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.

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phosphollpase A2 mediated arachldonlc acld release resultlng In productlon of arachidonate metabolltes which stimulate phosphoinositide turnover 5HT stimulates phospholipase A2 activity in synaptasomes prepared from guinea plg cerebral cortex (I0) and increases the synthesis of prostacyclin in aortic smooth muscle (II,12) It }s possible, therefore, that 5HT's effect upon phosphoinosltlde turnover Is dependent upon release of arachldonate and its subsequent metabolism to cycloxygenase or lipoxygenase products. Another possible indirect mechanism which may be involved is 5HT stimulated neurotransmitter release 5HT has an excitatory effect ;hen iontophoretlcally applied to some cells in rat CNS (13,14) and ]s known to stimulate release of a number of other neurotransmitters (15.16) Furthermore. the removal of extracellular calcium from cerebral cortical sllces inhlbits 5HT hut not earbachol stimulated phosphoinositlde turnover (17) It has been suggested that an agonlst which requires extracellular calclum for StlmUlatlon of phosphoinositide turnover exerts its effects by releasing another neurotransm~tt~r, ~hich then stimulates phosphoinosltlde hyrolysis (18) The followlng studles were almed at further testlng the hypothesis that phosphoinositlde hydrolysls serves as the transducing system linked co a 5HT receptor in rat cerebral cortex by addressing the possibility that 5HT stimulated phosphoinosiLide hydrolysis is dependent upon arachldonlc acld metabolism by cyclooxygenase or lipoxygenase, or upon 5HT stimulated ~ncreases in neuronal cell flring wlth subsequent neurotransmitter release

MeLhods Measurement of phospholnositlde turnover The procedure used for measurement of agonlst induced Increases in phospholnositide hydrolysis in cerebral cortex was a modification of the method of Berridge et al (19) as described by Conn and Sanders-Bush (2) B r i e f l y . cross chopped c e r e b r a l cortical slices (350 um x 350 um) were Incubated in Kreb's bicarbonate buffer containing I0 mM glucose (KRBG) and [3-H]myo-inosltol (5uCi/ml) for 90 minutes Prelabelled sllces .vere washed thoroughly with ~arm KRBG containlng 5 mM myo-inositol and aliquots containing about I mg proteln .':ere added to tubes containing 10 mM LiCI. I0 uM pargyline, and other drugs xhere appropriate (le proteinase inhibltors, tetrodotoxln, indomethacln or BW 755C) Tubes sere gassed, capped and incubated in thls medlum for 15 minutes prior co addition of agonlsts (final volume : 300 ul) Slices "~:ere incubated for 45 minutes In the presence of agonlsts In experiments in ~hich the effect of KCI Aas tested. prelabelled slices '~ere added to tubes containing KRBG in which 15 mM Nat1 v,as replaced ~iLh 15 mM KCI (flnal concentration : 20 mM) Also present .vas 10 mM LiCI, I0 uM pargyline, and :.:here approprlate 100 uM 5HT or other agonists (final volume = 300 ul) The procedure used for measurement of phosphoinosltide hydrolysls in aorta ~las identlcal except that 3 mm aortic rings were prelabelled (5 rings per ml incubation medium) arld 1 prelabel]ed aortic ring was added to each tube contalnlng appropriate drugs All Incubatlons were performed under an atmosphere of 02/C02 (95 5) at 37 C Water soluble inositol phosphates were extracted a[,d radloactlvlty present in [3-H]inositol phosphate (IP) determined as descrlbed previously (2) Drugs TetrodoLoxln, veratrlne HCI, and 5HT creatlnine sulfate were from Sigma Chemlcal Co (St Louis. MO) and were dissolved in KRBG BW 755C was a gift from Walter Hubbard (NIH) and was dlssolved in K R B G All experiments wlth BW 755C were performed in conditlons of low lighting Indomethacln (Sigma Chemical Co ) was dlssolved in I N potassium blcarbonate, diluted l to SO in water and then dlluted I to 12 in the flnal Incubatlon medium A cocktall of protelnase inhlbitors was used whlch consisted of phenylmethylsulfonyl fluoride (PMSF), leupeptln, and pepstatin dlssolved in ethanol (all from Sigma Chemlca] Co ) This solutlon ~as dlluted 1 %o I000 Into the flna[ incubatlon medium (final concentratlons PMSF. 0 2 mM~ leupeptln, l ug/ml, pepsLatrn. I uM)

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5HT S t i m u l a t e d

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Results Effect of indomethacin nor phospholnositide possible effect neither compound

inhibltion of srachldonzc acid metabolism. Neither I0 uM I00 uM BW 755C had a significant effect upon 5HT stimulated turnover (fiGure I ) Both compounds were tested for a upon phosphoinositide turnover in the absence of 5HT and had am effect by itself

E~(ect of t~tr9do~o$1q, Droteinase inhlbltors, and addztlvitv with deDolarlzlDg a~ents Tetrodotoxln (luM) did not block the phospholnositlde response to I00 uM 5HT (figure 2), nor did tetrodotoxin have an effect upon phospholnositide turnover by itself The concentration of tetrodotoxin used is capable of completely inhibiting the phospholnosltlde response to a maximally stlmulablng concentration of veratrlne (data not shown) The addition of a cocktail of proteinase inhibitors was also without significant effect both in the presence and absence of 5HT (figure 2). 2000

I

1800 1600 1400

T

T

T

1200 =E a.

I000

-I600 600

T _2:

-!-

4O0 ~'00

%o.%% -o. FIG

%

1

Effect of I00 uM BW 755C (BW) and I0 uM indomethacin (Indo) upon lO0 uM 5HT stimulated phosphoinositide turnover in cerebra] cortex Data are means of 2 separate experiments each done in triplicate (N = 6) aIRd are expressed as cpm in [3-H]IP in drug treated and no drug (ND) s~.ples. Vertlcal b a r s represent S.E M

The depolarizing agents KCI and veratrlne were both capable phosphoinositlde turnover (figure 3). presumably by stimulating endogenoum neurotransmitters Maximum responses to veratrine oht&iDed with 15 ug/ml Bad 20 mM respectively KCI did phosphoinosi~ide hydrolysis in rat aorta (Table i) In

of stimulating the release of and KCI were not sblmulate brain slices,

666

5HT Stimulated

Phospholnosltide

Turnover

Vol.

38,

No.

1500 1400

,

i

I

1300 1200 I100 I000 900 ~E 8 0 0 n (D 700

T T 600

T

500 400 300 200 I00

FIG

2

Effect. of p~ o L e l n a s e '.nhibl tors (Pr lnh ) and 1 uM tetrodotoxln ( T e t ) u p o n 100 uM 5HT s t i m u l a t e d phospholnosit, lde turnover in cerebral cortex The cock L~] I of prote] llase Inhlblt.ors cofl.qlsted of PMSF (0 2 mM). leupeptln (| ug/ml), aIld p e p s t a t l n ( l uM) Dabs.are expressed as cpm in [3-H]IP The V@''t.] C~ ] h ~ r ~ ~ p r o , ~ o n t . .q V. M T h e ~ ' ds.t.a r e p r e s e n t , mP~i,~ or" g separate e × p e r l m e n b ~ ; do,le l,~ q u a d r u p l t c a t e (N = 8)

7, 1986

Vol.

38, No. 7, 1986

5HT Stimulated Phosphoinosltide

Turnover

667

T

3500

3000

ZSO0

I ZOO0 Q.

t

1500

I000

I

*°%%%\

•o %+o,

FIG

%

%%

3

E f f e c t of I00 uM 5HT upon phosphoinositide turnover in the presence of 20 mM KCI and 15 ug/ml v e r a t r l n e (vera) The data are means of 2 (vera) or 4 (KCl) s e p a r a t e experiments each done ~n t r i p l i c a t e (N = 6 and 12 r e s p e c t i v e l y ) and are expressed as cpm In [3-H]IP The v e r t i c a l bars r e p r e s e n t S E M The shaded bars r e p r e s e n t the predicted additlve e f f e c t of 5HT plus KCI or veratrine for

comparison

TABLE [ Effect of 5HT and KCI In Aorta Basal

106

, p
5H'F

~ 27

429

•

KCI

61,

138

* 80

sallne

The e f f e c t of 5HI (I00 uM) and KCI upon [3-H]IP r e l e a s e ~as measured In 3 mm r i n g s of r a t a o r t a In tubes in ~hlch KCI ~as added. 25 mM NaCl has replaced zlLll 2b m~ KCI ( t o t a l KC1 c o n c e n t r a t l o n = 30 mM)

Data

are

rep~e~ehsative

t r l p l l c a L e (N - 6).

of

2

separate

experiments

each

done

and arc expressed as cpm in [3-H]IP . SEM

In

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5HT Stimulated Phospholnosltlde Turnover

Vol. 38, No. 7, 1986

simultaneous addition of maxlmal concentrations of veratrine or Eel and lO0 uM 5HT resulted in a phosphoinositide response ~hlch ~as approximately additive (figure 3) DIscusslon It is ~ell establlshed that 5HT stlmulates phospholnositlde hydrolysis ill a number of tissues (I-6,19,20) and the original flndlng that the pharmacology of this response is slmllar %o that of the $2 binding site (1) has been confirmed In rat cerebral cortex (2,3). as nell as rat aorta (5,6) and human blood platelets (4). The results of the present study support the hypothesis that phospholnosltide hydrolysls is the biochemical effector system linked to this receptor in rat cerebral cortex, and rule out the possiblliLy that 5HT's effects are medlated by 5HT stimulated arachldonate metabollsm by cyclooxygenase or llpoxygenase or by neurotransmltter release Indomethacin and BW 755C do not modify 5HT stlmulated phosphoinosltlde hydrolysis in concentrations which completely Inhibit cyclooxygenase (22) and llpoxygenase (23) respectively These results suggest that 5HT stimulated phosphoinositide hydrolysis is not dependent upon arachidonate metabollsm by these enzymes. If 5HT stimulated phosphoinositlde hydrolysis Tere dependent upon 5HT induced increases in cell flring and subsequent neurotransmitter release, it would be expected that the sodium channel blocker tetrodotoxin would reduce thls response However, tetrodotoxin has no effect on 5HI stlmulated phosphoinosltlde hydrolysis at a concentration v;hich completely abolishes the phosphoinosltlde response to veratrlne These results suggest that inhibition of neuronal cell flring does not reduce the response to 5 H T However, it is posslble that SHY could stlmulate release of another neurotransmitter in a tetrodotoxin insensltive manner Selective antagonists of HI histaminergic. alpha adrenergic, or m u s c a r i n i c receptors do not block the response to 5HT (2,3) suggestlng that 5HT's action is not dependent upon activatlon of any of these receptors Furthermore. the present data shot that addition of a cocktail of protelnase Inhlbltors does not potentiate the response to 5HT. suggesting that 5HT's effect is not medlated by stlmulatlon of release of a peptlde neurotransmitter To Lest further the possibility %hal 5HT stimulated phosphoinoslblde turnover is medlated by 5HI ~nduced neurotransmibter release. we examined the effect of 5HT in the preseuce of the depolarizing agents, KCI and veratrine If SHY stimulated phosphoinositlde turnover ~ere dependent upon neurotransmltter release, it should be abollshed in the presence of a supramaxlmum concentration of veratrine or KCI Since the effect of 5HT plus KCI or veratrlne ~as addltlve, 5HT stimulated release of another neurotransmltter seems unlikely Finally, 5HI stlmulates phospholnosltide turnover in rat aorta with a pharmacology that resembles that found in rat cerebral cortex (5,8) However. in rat aorta. KC] does not increase phospholnosltide turnover This lack of effect of KCI suggests that endogenous neurotransmlLters are not stored in this relatlvely pure preparatlon, which further argues agalnst a role of neurotransmltter release in the action of 5HT In conclusion, the present, data rule out the p o s s i b i l i t y that 5HT's effects upon phosphoinositide hydrolysis in cerebral cortex are medlated by 5HT stimulated neurotransmitter release or 5HT stlmulated arachldonate m e t a b o l i s m Published data suggest that there are differences between the pharmacological profiles of the phosphoinositlde response and the S2 blndlng site (2,3) However, when antagonlst potencies are determined by Schlld regression analyses, these discrepancies are no longer evident (Corm and Sanders-Bush, unpublished flndlngs) Therefore, we conclude that phospholnositide hydrolysls is the transducing mechanism of the $2 serotonerglc r e c e p t o r

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AcknowledKements T h i s work was s u p p o r t e d by N a t i o n a l I n s t l t u t e s o f H e a l t h T r a x n i n g G r a n t GM 07628 from National Institute o f G e n e r a l M e d i c a l S c i e n c e s ; A l c o h o l , Drug A b u s e and M e n t a l H e a l t h A d m i n i s t r a t i o n R e s e a r c h G r a n t MH 34007 f r o m t h e N a t i o n a l Institute o f M e n t a l H e a l t h ; a g r a m t from L i l l y R e s e a r c h L a b o r a t o r i e s : and t h e Tennessee Department of Mental Health and Mental Retardation References I. P.J. CONN and E. SANDERS-BUSH, Neuropharmacology 23 993-996 (1984) 2. P.J CONN and E SANDERS-BUSII, J Pharmacol. Exp Ther 254 1 9 5 - 2 0 3 (1985) 3. D A KENDALL and S . R NAHORSKI, J Pharmacol Exp Ther 233 4 7 3 - 4 7 9 (1985). 4. D. DE CHAFFOY DE COURCELLES, J E. LEYSEN. F DE CLERCK, H. VAN BELLE and P A . J . JANSSEN, J Biol Chem 260 7 6 0 3 - 7 6 0 8 (1985) 5. B.L. ROTH, T NAKAKI. D -M CHUANG and E. COSTA, N e u r o p h a r m a c o l o g y 2 3 1223-1225 (1984) 6 B L ROTH, T NAKAKI, D -M CHUANG, B CHERNOW and E. COSTA, Fed. Proc 44 1244 (1985) 7. M J. BERRIDGE, Biochem J 220 345-360 (1984) 8 S E RITTENHOUSE, Biochem J 222 103-110 (1984) g S.P. WATSON, B REEP, R T MCCONNELL and E G LAPETINA. B i o c h e m J. 226 8 3 1 - 8 3 7 ( 1 9 8 5 ) . lO. R.J. GULLIS and C E. ROWE, Biochem J 148 1 9 7 - 2 0 8 , ( 1 9 7 5 ) . ii S.R. COUGHLIN, M.A MOSKOWITZ, H.N. ANTONIADES a n d L LEVINE, P r o c Natl Acad Sci. U S A. 7 8 7 1 3 4 - 7 1 3 8 (1981). 12 S R. COUGHLIN, M A MOSKOWITZ and L LEVINE, Biochem Pharmacol 33 692-695 (1984) 13 R.B. MCCALL and G.K. AGHAJANIAN, B r a i n R e s lfi9 11-27 ( 1 9 7 9 ) . 14 C DE MONTIGNY, P BLEIR and Y CHAPUT, N e u r o p h a r m a c o l o g y 2 3 1 5 1 1 - 1 5 2 0

(1984). 15 K -H BUCHHEIT, E GUNTER, E MUTSCHLER AND B RICHARDSON, Naunyn-Schmiedeberg's Arch. Pharmacol 329 36-41 (1985) 16. S. GLUSMAN and E.A KRAVITZ, J Physiol 325 223-241 (1982) 17 E. BROWN, D.A. KENDALL and S R NAHORSKI, J Neurochem 42 1379-1387 (1984) 18. P P. GODFREY, S . J MCCLUE, M C.W MINCHIN and M YOUNG, Br J Pharmacol. 84 II2P (1985). 19. M J BERRIDGE, P C DOWNES and M R HANLEY, Biochem J 206 587-596 (1982). 20. M.J. BERRIDGE and J . P HESLOP, Hr. J. Pharmacol 73 729-738 (1981). 21. A JANOWSKI, R LABARCA and S M PAUL, L i f e S c i 3_~5 1953-1961 ( 1 9 8 4 ) 22. T M CONNOLLY and L E LIMBIRD, P r o c . Nat Acad Scl U S A 80 5320-5324 (1983) 23 D J. HAWKINS, A.H. ROSS. R. fiERZER and J.G HARDNAN. Fed. Proc 44 1817 ( 1 9 8 5 )