t'2uropean Journal of Pharmacology - Molecular Phannacology Section, 227 (1992) 123--129 ~. 1992 Elsevier Science Pablishers B.V. All rights reserved 0922-4106/92/$05J10
123
EJPMOL 90347
Polyphosphoinositide hydrolysis and ,","-":';,, kinase C activation in guinea pig tracheal smooth muscle cells in culture by' leukotriene D 4 involve a pertussis toxin sensitive G-protein l t J 1 ! . ~ li,%,J 1 1 1
S a n d r a H o w a r d , M o i r a C h a n - Y e u n g , L i a n a M a r t i n , S y l v a n e P h a n e u f a n d H a s s a n Salari Department of Medicine, Unit'ersiO"of British CohmTbia, Vancoucer, BC. Canada
Received 27 January 1992, revised MS received 27 April 1992, accepted 26 May 1992
Leukotrienc D 4 (LTD a) at concentrations > 1 nM induced phosphatidylinositol bisphosphate (PiP 2) hydrolysis and protein kinase C (PKC) activation in primary culturc of airway smooth muscle cells. Within seconds of activation, an increase in inositol 1.4,5-trisphosphate (IP3) was obscrved reaching a maximum at 5 rain. Thc level of IP3 decreased after 5 rain and was followed by an incrcase in inositol 1,4-bisphosphate (IP z) and inositol l-monophosphate (IP~). LTDa-induccd PIP~ hydrolysis was inhibited by 1 h pretreatment of cells with 10/xg/ml of pertussis toxin (PTX). LTD.~ activated both solublc and particulate forms of PKC by 2-3-fold, The LTD4-induccd PKC activation was blocked by treatment of cells with PTX, suggesting the involvement of a PTX-sensitive G-protein. To assess the involvemcnt of G i in smooth muscle ccll reccptor activation, tile modulation ol adenylyl cyclasc activity was investigated. LTD 4 did not stimulate cAMP forlnation in smooth muscle cells, and did not inhibit for,d¢oiin-induccd cAMP formation. These data suggest that the LTD.~ rcceptor in airway smooth muscle cells is coupled to a PTX-scnsitive G-protein, possibly G,,. Leukotrienes; Smoolh muscles; Puhnonary system; lnosiml phosphates; Protein kinase C: G-Protein
1. Introduction Leukotrienc D~ ( L T D 4) is one of the major components of the "slow reacting substance of anaphylaxis' (Murphy et al., 1979; Lewis et al., 1980). LTD~ produces prolonged contraction of smooth muscle cells from airways (Dahlfin et al., 1980; Krell et al., 1983) and vascular tissues (Feigen, 1983). In addition, L T D 4 causes mucus secretion in the airways (Marom et al., 1981), and thus has been considered to be an important mediator of bronchial asthma. Early observations demonstrated that the LTD 4 binding to m e m b r a n e recepu~rs i:, inhibited by N a ' and guanine nucleotides ( M o n g c t al., 1984). Anderson et al. (1982) reported that L T C 4 decreases c A M P level in guinea pig trachea, and suggested the involvement of an inhibitory G-protein (Gi). These investigators, however, did not investigate the L T D 4 receptor interaction in their study. G i h;ls traditionally been linked to the inhibition of adcnylyl cyclase (Casey and Gilman, 1988). Torphy ct al. (1986) reported that tile L T D 4
Correslmndcnce to: II. Satari. Dcparlmei~| of Medicine, 2660 Oak Slrccl, Vancou,,'er, BC, V611 3Z6, (hnada. l'el. (604) 875-486B; Fax (604) 875-4497.
receptor activation in opossum trachea did not cause an increase or decrease in c A M P level, suggesting the involvement of another class of G-proteins than G r In all cases, the nature of the G-protein that interacts with the L I ' D 4 receptor appears wtriable and tissue d e p e n d e n t (Mong ct al., 1986; Boucherouche et al., 1990). The aim of this study was to obtain information as to the nature of the G-protein with which this lipid mediator interacts in airway smooth muscles from guinea pigs. Using phosphatidylinositol bisphosphate (PIP,) hydrolysis, protein kinase C (PKC) activation and c A M P assays, we demonstrate that the L T D ~ receptor in airway smooth muscles from guinea pigs is coupled to phospimlipase C via a pertussis toxin (PTX)-sensilive G-protein, but does not involve adenylyl cyciase.
2. Materials and methods 2.1. Materials
Fetal bovine serum (FBS), tissue culture medium, and antibiotics were purchased from Gibco Laboratories (Grand Island, NY, USA). LTD 4, histone H1 (type III-S), p r o t a m i n e chloride, 4 - m o r p h o l i n c p r o p a n e -
124 sulfonic acid (MOPs), cAMP-dependent protein kinase inhibitor peptide (PKiP), 3,isobutyl-l-methylxanthine (IBMX), pertussis toxin (PTX), Nonidet P-40 (NP4~), sodium pyruvate, taurine, creatine, trypsin, carbachol, collagenase, elastase (type iV), anu c ~AM~rJ , wcrc pHr_ , . chased from Sigma Chemical Co. (St. Louis, MO, USA). Smooth muscle-specific monoclonal antibodies to desmin and a-actin were purchased from BioGcnex Laboratories (San Ramon, CA, USA). [y-~'-'P]ATP (3000 Ci/mmol), myo-[2-3H]inositol (10-20 Ci/mmol), and cAMP assay kits were purchased from Amersham Radiochemicals (Arlington Heights, IL, USA). Dowcx-1 resin was purchased from Bio-Rad Laboratories (Richmond, CA, USA). Anion exchange Mono Q column was purchased from Pharmacia Biotechnology (Montreal, Canada). 2.2. Tracheal smooth muscle cell culture Airway smooth muscle cells from guinea pig trachea were isolated as reported (Devore-Carter et al., t988). Guinea pigs (150-300 g) were killed by a saturating dose of pentobarbital sodium (5{)0 mg/kg). Tracheae from five animals were excised aseptically, combined and rinsed twice in ice-cold Hanks' balanced salt solution (HBSS), supplemented with pcnicillin (100 units/ml), streptomycin (100 units/ml) and nystatin (10 units/ml). The connective tissues were carefully stripped off by blunt dissection with frequent rinsing of the tissue in HBSS to prevent desiccation. The tracheae were cut longitudinally on both sides of the strips (through the rings opposite the strip of muscle). The tracheae were incubated with an enzyme solution containing 0.2% collagenase and (1.05% elastase at 3TC for 30 rain. The dissociated cells that were predominantly epithelial cells and other loosely attachcd non-smooth muscle cells were discarded. The tissue fragments were washed in HBSS once to rcmove all the dissociated non-smooth muscle cells. The rcmaining tissues were put in a solution of 0.2% collagenase and 0.05% elastase, and digested for 1-1.5 h in an atmosphere of 5% CO 2 at 37°C. The enzymatically digested tracheae were then passed through a nylon mesh to remove the large debris and fragments. The cells were pelleted by centrifugation at 260 × g for 5 rain. The supernatant was removed by aspiration and the cells were resuspended in 1-5 ml of modified Dulbecco's modified Eagle's m e d i u m / H a m ' s FI2 ( D M E M / F 1 2 ) (1:1, v / v ) with I% FBS with(~ut growth factors for 24 h to eliminate the non-smooth muscle cells, since epithelial cells and fibroblasts' growth are highly dependent on the growth factors present in the serum. After 24 h the cells were washed with culture medium and fresh meditnn containing 10% FBS was added, When cells were seeded at 1 × 104 cells/ml, they became confluent, usually after 10-14 days. Using
immunocytochemical techniques with monoclonal antibodies to smooth muscle desmin and a-actin, the cultured ceils were characterized as 95% smooth muscles. For polypho~phoinositide hydrolysis experiments, the cells were cultured in 60 mm Petri dishes and directly challenged, while for PKC experiments, the cells were cultured in 75 cm flasks and wher~ the;y reached conflue~cy were detached from the plastic surface by the use of non-enzymatic cell dissociating buffer (Sigma Chemical Co.). The dissociated cells were then challenged with the agonists. 2.3. cAMP measurement Smooth muscle cells werc incubated in 24-well plates with LTD4 (1 /.tM) or forskolin (50 # M ) for 5 min. Unless otherwise stated, samples contained 0.5 mM 1BMX to inhibit phosphodiesterase activity. The reactions were stoppcd by addition of 25 p.l concentrated trichloroacetic acid and the supernatant was collected. The concentration of cAMP in the samples was assessed by a cAMP-[3H] radioassay kit as directed by Amersham Radiochemical Co. 2.4. Polyphosphohlositide hydrolysis Polyphosphoinositide breakdown products were measured according to the method of Watson ct al. (1984). To thc culturcd Pctri dishes 2.5 ~ C i / m l of [3H]inositol was added and the incubation was continued for 48 h at 37°C with 5% CO 2 in an inositol-frce mcdium and 1% FBS. After 48 h, tlae cells were washcd 3-5 times with HBSS to remove the free inositol. 10 ml PBS containing 10 mM LiCI was added to the flasks prior to the addition of LTD 4. For positive control, AIE~ (10 /xM) and G T P y S (20(] ~I~~) were used (Sasagari ct al., 1985). Cells were permeabilized with 18 ~ g / m l of saponin tor 2 rain prior to the ~ddition of AIF4 and GTPyS. The incubation was tcrminated by the addition of 24 ml of denaturing buffer ( m c t h a n o l / H C I ; 200:2, v / v ) followed by addition of 12 ml chloroform. Inositol phosphates wcre is(ffated as reported earlier (Salari ct ai., 1990). 2.5. Ptz,paratiolz o f extracts .fbr protein khtase C assay Smooth muscle cells in culture flasks were dissociated by treatment with dissociation buffer. The cells were washed in Hanks' balanced salt solution (HBSS) and challenged with LTD 4 (1 ~ M ) for 5 min. Cells were centrifuged at !5,000 × g for 15 s. The cytosolic and microsomal fractions were separated and assayed for PKC activity as rcported earlicr (Pelech et al., 1990).
125 3. Results
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3.1. Formation of inositol phosphates Initial experiments examined the maximal release of [3HllP in p,i,,,a,y""" .... I~ . . . . of 'a;. . . . . . . . m n v b l h musolo cells. Myo-[2-3H]inositol-labelled cells stimulated with various concentrations of LTD 4 for 1 rain were shown to generate [3H]IP~ approximately 3-fold greater than the controls. Analysis of the concentration-response characteristics for the effects of LTD a on iP3 formation indicated that LTD 4 was effective at concentrations > 10 nM. The maximal release of 1P3 was observed with 1 #M LTD 4 (data not shown). Using maximal (I tzM) concentration of LTD 4, the time course for generation of various inositol phosphates was investigated. Fig. 1 A - C demonstrates the kinetics of [3H]IPt,
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Fig. 2. Effects of pertussis Ioxin (PTX) treatment of smoofl~ muscle cells on LTD4-induced PIP e hydrolysis. Various inositol phosphate mctabolilcs of untreated cells (control), ~timulated with 1 # M LTD~ for 5 rain, or incubated with 10 # g / m l of PTX for I h, prior to the addition of 1 p.M I.'I'D.~ (LTD 4 + PTX) arc shown, Resulls arc lhe mean of tllree experiments in triplicate, ± S,D,
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Fig, I. Effect!; of I.TD 4 on polyphosphoinositide hydr~lysis in t,acheal smooth muscle cells in culture. Cells were labelled with myo-[23ll]inosilol and then incubated for various lengths of time with 1 ,aM LTD 4 ( I ) , or 10 p.M A I E I + 2 0 0 /xM G T I ' y S ( ~ ) , unstimulalcd level ( [ ] ) . The iimsitoi plmsphates were exlrach:d and resolved im a Dowex-I c o l u n m chromatography. The r M oae ivitv incorporated into the wlrious "polyphosphoinositktc metabolites was counlcd in a B-liquid scintillation counter. Results arc lhe nlcan of five experiments in triplicate, ± S.D.
[31-I]IP=, and [3H]IP3 release in smooth muscle cells stimulated with 1 p.M LTD 4 in the presence of 10 mM LiCI. When the cells were treated with 1 /.,M LTD 4, a rapid formation of 1P~ was observed (fig. IA). Formation of IP 3 was delectable as early as 30 s after addition of LTD 4. The fomwtion of IP3 was transient, reached a maximum at about 5 rain, and then declined. As positive control, the permeabilized cells were stimulated with AtF4 and G T P y S . Formation of IP3 in response to stimulation with AIF4 and G T P y S was rapid and transient, reaching maximum at about 5 min. A l i a / G T P y S released approximately 2-fold more IP 3 dmn LTD~ did (fig. IA). The decrease in tP~ was accompanied with an increase in IP,. The formation of IlL increased steadily with both LTD 4 and G T P y S , approaching a plateau at about 5 min. The A I F a / G T P y S , and lSrD4-stimu!ated accumulation of [3HIlPI is shown in fig. tC. The LTD4-induced IPj accunmlatkm was 5(lC4 less than the amount lk~rmcd by ,,;fimulation with A I t : ~ / G T P y S (fig. IC).
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Fit;. 3. Mtmo O column chrornatoglaphy of protein kinase C from guinea pig tracheal snloolh muscle cells, control (A and B). Ireated with i # M I.TD 4 (C and D), treated with 10 /zg/ml of PI'X for 1 h. prior t~ LTD,a (1 pM) for 5 rain (F and F). Mono O ehruma|ography of 0.5 mg NPa.-solubilizcd particulate extracts (A, C and E). Mono Q chromalogrums of 0,5 mg c?~h~mlic proteins (B. I) and }:). The hismne 111 phosphop.,!aling activity in the absence of Ca z ' and lipids (H), histone I{I phosphurylating aclivity in Ihe presence of lipids and ('a ~ ' ( I ) , and pmtamine plmsphoD'lating aclivily in the absence of lipids and ('a 2~ (~). Chrt~matogranls are laken I'mm a representative of at Icasl five experlnlenls.
3.2. Pertussis toxin effi'cl T h e possible i n v o l v c m e n t o f a P T X - s c n s i t i v e G - p r o tein in m e d i a t i n g L T D , t - i n d u c e d p h o s p h o l i p a s e C activation was i n v e s t i g a t e d . S m o o t h m u s c l e cells w e r e t r e a t e d with 1{} / , g / m l o f a c t i v a t e d P T X for 1 h at 30°C p r i o r to t h e a d d i t i o n o f L T D 4. Fig. 2 s h o w s t h a t cells t r e a t e d with P T X did not r e s p o n d to LTD~ by g e n e r a t i n g inositol p h o s p h a t e s . T h e 1.5-fold i n c r e a s e in IP 1 was i n h i b i t c d by P T X . Similarly t h e f o r m a t i o n o f IP 2 a n d IP 3 was i n h i b i t e d by t l ' e a l m c n l o f cells with P T X , s u g g e s t i n g that the L T D , r e c e p t o r in g u i n e a pig t r a c h e a l s m o o t h m u s c l e cells is c o u p l c d to a P T X - s e n silive G - p r o t e i n .
was a s s e s s e d by l h e C a - " - , D G - , a n d P S - d e p c n d e n t historic H I p h o s p h o r y l a t i o n a n d by t h e Ca ~''-, D G - , a n d P S - i n d c p c n d e n t p r o t a m i n c p h o s p h o r y l a t i o n (fig. 3). In t h c m i c r o s o m a ! f r a c t i o n s , an a d d i t i o n a l p c a k w a s TABLE 1 Mean values of cytcsI, Sinion, Iq,$8. (i-Prolcin Inullipiicily in cukaryolic trallsdu¢lioll :.,ysicll/S, llio¢Ilc'mislry 27, 4957. Madis;m, J.M. :.!nd / . K !~_rt,,>,~x ]'Jb',~L l)iffi'rettthd h~hlbill,r) cli~:cis of Iklrskolill. Jsoproicrcnol, atid dibutyryl cyclic adenosine mlmophosphalc on phosphoinositldc hydrolysis in canine lrachcal sinoolh muscle. J. Clin. lnvcsl. 82, 14f12.
129 Marom. Z., J.H. Shethamer, M.K. Bach, D.R. Morton and M. Kaliner, 1981, Slow reacting substances, leukolrienes C 4 and D.~, increase the release of mucus from human airways in vitro, Am. Rev. Respir, Dis. 12b, 449. Mong, S.. }I.-L. Wu, G,K. tlogaboom, M.A,, Ciark, J.M, Stadel aml S,T, Crooke. 1984, Regulation of ligand binding to leukotriene D.~ receptors: effects of cations and guanine nuclcotides, Eur. J. Pharmacol. 106. 241. Mong, S., K, ttoffman, It+-L.. Wu and S.T. Crooke+ 1'4~6, Leukotriene-induced hydrolysis of inositol lipids in guinea pig hmg: mechanism of signal transduetion for Icukotriene-D a, IVlol. Pharmacol. 31,351. Moseona-Amir, E., Y.I. Henis and M. Sokol~wsky, 1988, Guanine 5'-triphosphale binding protein (Gi) and two additional pertussis toxin substrates assoeialcd with muscarinie receptors in ra| heart myocyles: characterization and age dependency, BioctlemislEy 27, 4~85. Murphy, R.('., S. tlammcrstrom and B. Samucisson, 1979, Leukotricne C, a slow reacting ~,ubstance (SRS) from mouse mastocymma cells, Proc. Nail, Acad. Sci. USA 79, 4275. Pelech, S.L., D.I . Charest, S,L. Iloward, ll.B. Paddon anti 1t. Salari, 1990, Protein kinase (" activation by platelel activating factor is independent of enzyme lrax:slocation, Biochim. Biophys. Acla 105 I, IO(L
Salari, It., V. Duronio, S. tloward, M. Demos and S,L. Pelech, 1090, Translocation independent activation of prolein kinase C by pialelel activating factor, lhrombin, and prostacycline, 13iochem. Sasagari, T., M. Ilirata and 1t. Kuriyama, 1985, Dependence oI~ ('a:" ol ~he activilics of phosphatidylint~silol 4,5-bisphosphate, phosphodie~,terase and inositol 1,4,5-trisphosphate in ~,mooth muscle of the porcine coronary artery, Bioehem. J. 231,497. Taylor, D.A.B.F, Bowman and J.T. S~ull, 1989. ('ytt~plasmic ('a ~' is ~l primary determinant for nlyo~:;n phosl'~horylati~:l in sm~oth muscle cells, J. Biol. Chem. 2fl4, 6207. Torpiw. T.J., M. Burman, L.W. Schwarts and M,A Wasserman. 1086, Differential effects of melhacholine and leuko~riene D~ on cD, lic micleolidc contenl and isoproterenot-induced relaxation in tile opossum mtehea, J. Pharmacot. Fxp. Ther 237, 332. Waklo, (].L., T, Evans, E,D. Fraser, J.K. Norlhup and M.W. Martin, 1987. hleulifieation and purilication from bovine brah~ ¢fi a guaniue-tmclenlide binding prolein dislinet from Gs, Gi, and Go, Bh~ehcm. J. 246. 431. Watson, S.P.. R.T. McC(mnell and E.G. Lapetina, 1984, The rapid fc,m'~ation of inositol phosphates in human platelels by lhr(m!bin is inhibited by proslacyclin. J. Blot. ('hem. 259. 73191L