prostaglandin H2 receptor

EurotTean Journal of Pharrnaco!o~" - Molecular PharmagoIoK~: Settlon, 206 ( ] 991 ) ~5 - 21 L 199I E]se~5erScience Publishers B.V. (Biomedical Division) 0922~U06/91/$03 50 ADONIS

~5

r922a10691U'0"9528

EJPMOL 901,27

Interactions of dihydropyridine Ca 2+ channel agonists with the human ptatelet thromboxane a z / prostaglandin H z receptor Philip R. M a y e u x , D a l e E. M a i s a n d P e r U V. H M u s h k a Departments of Pharmacoto~, and Exper~memat Therapeuucs and Medwine. Medical L;n¢fers~ of South Carohn~ Char elzr~ SC 2~425. U S 4

Received 7 At~gust ] 9~. accepted 1] SeWea~b~ i~9<)

The specific interactions at the human platelet thromboxane Ajprostaglandin H a (TXA2/PGH2t receptor b 3 four I-4 dihydropyridine (DHP) agonists were studied. Using competition equilibrium binding assays with the T X A z / P G H : receptor agonJst [I~IIBOP and the antagonist {~-'Sl]PTA-OH, the affinities of racemSc BAY K 8644 tBAYL raven-dr CGP 28392 (CGP) and ( + ) and ( - ) SDZ 202-791 (SDZ} for the T X A , / P G H 2 receptor ,~ere determined. The rank order potencie~ far competition were BAY > ( - ) S D Z > CGP/> ( + )SDZ. BAY. CGP and SDZ (stereoseiectively} inhibited specific ir~eorp,~ration of the TXA j P G H z receptor phomaffinity probe [lZsl]PTA Azido into three proteins associated with the T X ' A , / P G H : receptor with M~ of 43. 39 and 27 kDa as assessed by sodium dodeeyl sulfate-po!yacrylarnide gel electropboresis amoradiograph 5. Using the fluore.cem Ca:* probe Fura-2, it was obsereed that I-BOP failc..d to stimulate ctasaical divatem cation channel,. However. S D Z siereo~eleco~el? inhibited the rise in [Ca~+], induced E,y l-BOP while not affecting that induced by Ihrombin Although DHPs specifically and stereoeelectively interact with the ' I X A 2 / P G H , receptor on human platelets, the T X A z / P G H : recepto~-mediat~'.d rise in {Ca z *1~ is not :hrough stimulation of a c[assieal divalent cation channel. T X A z / P O H 2 receptors: I-PTA-OH: I-BOP: Dihldropy~dines: P~atelets ~human)

1. Introdu~cllon Several classes of agonists and antagonists of vottage dependent C a z+ channels on cardiac and smooth mttscte (Nayler, 1988; R o g g et al.. 1985: Bechem e t a L . 1988) have been described. A m o n g the various classes are the 1-4 d i h y d r o p y r i d i n e s ( D H P ) which are g a i n i n g widespread use in the treatment of coronary arteQ' disease and hypertension (Nayler. 1988). D H P , both agonists and antagonists, have been shown to inl,5,bil platelet function (Murphy c~ el., 1983; Mehta. 1985: Johnson et a l . 1986; Sunkel etal.. 1988) in spite of the fact that vollage-dependent C a ~'+ channels ha~e no:. yet been identified in the platelet. The mechanism by which this class of compounds exerts its anti-plate~et acrid'try is not well understood but. such acti~4ty m a y add to the therapeutic benefit of these drugs in the treatment of certain cardiovascu|ar diseases. T h r o m b o x a n e A : ( T X A z ) and ~ts immediate precursor p r o s t a ~ a n d i n H z ( P G H 2 ) are synthesized and

Corresponde.nce to: Perry. V. H'Aushka. Ph.D.. M [), DepL of Pharmacology and Experimental "I'herapeudes. Medical Un:versily of S-at~h CaroLina, 171 Ashley Avenue. Chart~ton. SC 29z~25, U.S.A.

released bF platelet~ .~Hamberg et aL !975). Both stimulate p~ate!et aggrega'don apparemi5 through a c o m m o n T X A z / P G H : receptor (Ma?eux et eL, 1988). Stable structural analogues
2. MateSa3s and methods

2.3. [n.~ra{ellular .free Ca 2 " mea.,;ltre¢?~etll5

2.1. Materia!s

Plate]or-inch plasn:a was incubated in the dark with the fluoreseeat Ca: - probe, Fura-2 A M E ( G ~ n k i e w i c z et al.. t985} (4 a M ) for 30 rain a~ 2 5 ° C . Platelets were isolated ~y centrifugation at 1000 × g for 20 min and the platelet pellet ~as gently resuspended in a Ca2+-free HEPES buffer (mMJ: tlEPES ~0: NaCt 145: KCI 5; glucose 5.5: pH 7.4. at a c,mcentration of 3 × l0 s ptate~ets/ml. Changes in [Ca z~ i, were monitored using an Aminco-Bowman spectrophotofluorometer (Silver Spring. M Dt. Platelet suspensions (1 rot) were placed in a 1 cm z quartz Cnvette (Fisher Scientific) and maintained at 3 7 ° C with constont stirring. The instrument was calibrated before each experiment by addition of 1 m M Ca z" plus 100 # M dtgitonia to a platelet sample to determine maximum fluorescence followed by 20 m M Tris base and 10 mM E G T A to determine m i n i m u m fluorescence as previously described (Morinelli el al., 1989). Test agents were introduced into platelet samples in a volume of 10 p.h The addition of E G T A in some experiments was to remove any contribution to the fluorescence signal caused by leakage of Ca z* out of the platelet and residual extracellular Fura-2. The ptatelets were exposed to an excitatory wavelength of 340 nm and emission was recorded at 500 nm.

The T X A z / P G H : receptor agonist {1S-(tc,.2,8{5Z~, 3a(1E. 3S* ),4a)]-7-[3-(3-hydrox)-4-(4'-~:5 t-phenox3 i 1-butenyt)-7-oxabicycio-[2.2. i J-heptan-2-51 l-5-heptenoic acid ([~:51]~OP~. the T X A j P G H a receptor a r , " g onist 9,1t, dimetbyt-methano-ll.12-mothano-!6-~3 [ L~51|~4-h3~droxyphenyt~-I 3.14-dih)dro- 13-aza- i 5 a B-~tetranort~romboxane A , ([~2:'I]-P]A-OHL and the T X A 2 / P G H : receptor photoaffinity probe 9.1|-dimethylmetha~ao- i !.12-methano- t 6-( 3-[ ~z5I]-4-azidophenyl)- 13.14-dihydro-I 3-aza- l 5a~-w-tetranort hromboxane A 2 ([~'Sf]PTA-Azido, were synthesized ms described previously (Mo6nel~i et al.. i989: Mais et aL. ~984: Mais and Halushka. 1988~. The following compounds were obtained as gifts from their respective companies: ( + ) B A Y K 8644 (Miles Laboratofe~, New Haven. CT); ( ± ) C G P 28392 (Ciba-Gei~" Corp., Summit. NJ): { + ) and ( - ) SDZ 202-79i (Sandoz Ltd.. Basel. Switzerland) and L657925 (Merck Frosst Canada Inc., Point ClaireDorvak QuebecL Fgra-2 pentacetoxymethyl ester and Ionomycin were pt~rchased from Behring Diagnostics (LaJol~a. CA). a-Thrombin "aas purchased from Sigma Chemical Co. fSt. Louis. MO).

2.2. Binding of [Je-~I]PTA-OH and [~::I[BOP to waahed human platetets tncubatmns were performed in dapheate in a volume of 200 tLi containing 5 × l0 ~ washed haman ptatele~s for the ! t b l t P r A - O H assays (Mais et aL. 1985) or t "z t07 platelets for the [~-'SlJBOP assays (Moricelti et aL. 1989) in sitanized (12 × 75 mmJ glass tubes at 3 7 ° C for 30 nan. The incubation media consisted of Tris (50 m M ) / N a C I (100 raM) buffer and - 0.1-0.2 nM ( - 0 5 to 1 × 105 cpm) [:2~IIPTA-OH or ~ 0J)5-0.1 nM ( 0.25-0.5 x 10 s cpm) [~2st]BOP per tube and various concentrations of BAY K8644, ( + ) o~ ( - ) SDZ 202-791 or C G P 28392. Each D H P was dissok'ed in DMSO and serial diZutions were made in T r i s / N a C t buffer. Stock solutions were stored in the dark at - 4 ° C and not used after three days of storage. The reaction was terminated by the addition of 4 ml of ice-cold T r i s / N a C l buffer at p H 7.4. followed by rapid filtration through W h a t m a n G F / C glass fiber filters (Whatman, Inc.. Clifton, NJ). The filt~s v,'ere washed three more ~imes with 4 ml o~ ice-cold buffer, The filtration procedure was complete within 10 s, Nonspe~cific bimling was defined as ihat amount of radioactivity bound in the presence of 10 t~.M L657925, a T X A z / P G H z receptor antagonisl (Mais et al., 1989). Specific binding was - 65% for [~z-~I]PTA-OH and - 90% for [t~I]~BOP.

Z 4. Photoaffou 0 labeling of the T J ( A 2 / P G H 2 receptor Photoaffinity experiments were conducted as previously described (Mais et al., 19891 by incubating 0.5 ml of washed platelets (5 × 10s/roll with approximately 2-4 `aCi of [125I]PTA-azido both in the absence and presence of various DHP. L657925 (250 nM) was used to define specific labelling of T X A a / P G H z receptor associated proteins. Incubations were for 30 rain at 3 7 ° C and were followed by a 2 rain phGtolysis with a hand held ultra-violet lamp (Ultra-Violet Products, Inc., San G~briel, CA) at a distance of 5 cm. The samples were diluted to 4 ml and centrifuged at 2000 × g to obtain the ptatelet pellet. The pellets were dissolved in sodium dode~'l sulfate (SDS) buffer for use in polyac~4arnide gel electrophoresis (PAGE). Electrophoresis under reducing conditions ',,,'as conducted according to the method of Laemmli (1970) using a 4,% stacking gel and 12% running gel. After application of approximately 100 ~g t~f protein in a 50 ld volume, and low ~aoiecular ~eight standards (BioRad. Richmond, CA) electrophoresis was carried out at 4 " for 5 h at a constant current of 45-50 mA. The gel was stained for protein with Coomassie blue, destained and dried onto fi~ter paper. Autoradiography was performed at - 70 QC using Kodak X-Omat film and DuPont Cronex cassettes with intensifying screens for 2-5 days.

2.5. Data analysis IC5o v a l u e s for t h e v a r i o u s D H P s were d e t e r m i n e d f r o m a log-toBit t r a n s f o r m a t i o n of t h e c o m p e t i t i o n b i n d i n g assays. T h e IC5o is d e f i n e d as the c o n c e n t r a t i o n of D H P p r o d u c i n g a 50% i n h i b i t i o n of specific l i g a n d b i n d i n g . T ~ e d i s s o c i a t i o n c o n s t a n t ( K e ) was d e t e r m i n e d f r o m the ICgo v a l u e by the m e l h o d o f C h e n g a n d P r u s o f f (1973).

3.

R

~

.0t

.1

1

10

1o0

3.1. Radioligand binding studies ~oo

Competition for binding by the DHPs with [~25I]PTA-OH, a T X A , J P G H 2 r e c e p t o r a n t a g o n i s t a n d [~z~I]BOP, a T X A z / P G H ~ r e c e p t o r a g o n i s t in w a s h e d human platelets was performed. BAY, (-)SDZ, ( + ) S D Z , a n d C G P w e r e e v a l u a t e d for t h e i r a b i l i t y to c o m p e t e w i t h [~:511PTA-OH a n d [~-~5I]BOP for b i n d i n g to w a s h e d h u m a n p l a t e l e t s (fig. 1). B A Y was the m o s t potent DHP in c o m p e t i t i o n b i n d i n g assays w i t h [t~SI]BOP a n d [~2SI]PTA-OH, c o m p a r e d to C G P a n d S D Z . C o m p e t i t i o n for T X A 2 / P G H ; receptor binding by S D Z d e m o n s t r a t e d stereoselectivity. T h e K d v a l u e s for ( - ) S D Z a g a i n s t [~511BOP a n d [ ~ z ~ t ] P T A - O H are 2 . 3 _ 0.3 a M a n d 0 . 9 4 + 0 . 2 2 ~ M respectively w h i l e ( + ) S D Z at c o n c e n t r a t i o n s u p to 5 0 ~ M f a i l e d to c o m p e t e for m o r e t h a n 50,% o f the b i n d i n g s~tes for e i t h e r l i g a n d ( t a b l e 1). T h e p s e u d o - H i l l c o e f f i c i e n t s for the compounds were not significantly different from -1 i n d i c a t i n g a n a p p a r e n t i n t e r a c t i o n w i t h o n e class o f T X A 2 / P G H _, receptors.

°

0 -1

........

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DHP t = M ]

Fig. I. CornF~ti~ion for specific binding b; BAY. ~- !SDZ. CGP and (*)SDZ ~Upper panet~ Representative (of four9 c~mpe~.iuon curves for B.~kY~ I . 9 - 9SDZ ¢=9.CGP (1119and t + ~SDZ 9-3( to compete for specific [~[]BOP binding Io "~a~hed human pla~ele~.s. Binding a.ssay~ ~ere performed in duplicate in a ~9~mc of 2CKifal containing 1 × !0 ~ platelets al 37°C fcr 30 mm in the presence of g,05 to t #M [I:gIIBOP and incre.asmg conceatratioas of competing agenL Noa~pecific binding ~as defined as ~.heamount bound in the presence of I0 gM L657e'25. fLo',~er paneb Repre>enlafive for h}ur) compefilion cur~,e~ for BAY 011,), ~-~SDZ ~c~L CGP 11119and 9*gSDZ 1o9 lo compete fer sp~ific ~:'I]PFA OH binding to washed heinar~ pgatetetg, Biadmg a,~a3s ~ere performed iu d~plica~e in a volume of 209 ~t comaimng 5 x ~0v pizle]¢l~ at 37 ~ C for 30 rain in ~he presence of 0.1 1o 0.2 nM [L'gI~PTA-OH and increasing concentratmus of competing agem. Nonspecific binding ~xas defined ~ the amount bour~d in the pre~nce of 10 gM L657925.

3.2. Photoaffinity labeling of the TXA: / PGH. receptor i a a d d i t i o n to the b i n d i n g studies, the specific i n t e r a c t i o n o f the D H P s ~5th the T X A ~ / P G H ~ r e c e p t o r was d e m o n s t r a t e d by t h e i r a b i l i t y to i n h i b i t i n c o r p o r a t i o n o f the p h o t o a f f i n i t y a n t a g o n i s t [ ~ S l ] P T A - A z i d o (fig. 2). B A Y (10 # M ) , ( - ) S D Z (10 /aM) a n d C G P ( ! 0 0 #.M)

TABLE

i

t

Fhe K a values and p~'cudo-Hitl coefficients (nil) for BAY. CGP and SDZ for tb ~ glaleget TXA:/PC-H 2 receptor. Data are expressed as meant ± S E..M. N = 4 for each compound. l'ne K~, value5 were calcuiated from the 9C5~values determined from a 9eg-9o~t transformation of the compellllon binding data. All pseudo-Hill coefficlents were no~ signihcam]y d.qferem from - t A p~eudo-Hd9 c~eff~clen~ could r~oI be calcugated tot ( ~ )SDZ. Ligand 1~"~51]BOP llz~I]PTA-OH

BAY

CGP

I - ~SDZ

(+)SDZ

K~ (/~M)

nH

K d l/aM}

nH

Ka {#M)

v.H

K a (;aM)

&Tgg ±0.019 0.542_+0.025

- 0.85 ± 0,0~ -0.89_+0.06

79+_22 56± 15

-0.96±0.02 - t.l ±0~9

2.3 +0.3 094±0.22

-0.89.-,__0.07 - 1.2 =0.t'~

>59 > 50

nH

ro

97.4

--

66-

42.7

-.

31-

21-

14-

Fig. 2. Pbotoaffinitylabellingof washed human platetet~. Pre~ented is a representative (of threet autoradingram of an SDS-PAGE ge~ of ~.,asbed human platelels photoaffinity labelled ~,~th [t'~llFYA-AzJdo in the presence and abset~ce of competing heralds. Lanes: I. control: 2, L657925(250 riM); 3. ~AY 110 t~Mt: 4. ( - )SDZ It0 tLM~;5. ( + } SDZ (tO ~tM); 6. CGP (I00 #Mr. L657925. BAY. I - ~SDZand CGP inhibited incorporation into tt,me protein bands (indicated by the arrows) with molecular weights of 43. 39 and 27 kDa. t + )SDZ was unable to inhibit incorporation indicatingstereosetective competition by ( - ~and ( + }SDZ for [i2~I|PTA-AzIdoincorporation_

fluorescence signal by Mn z* when it binds to the indicator (Ffesketh et aI.. 1983) provides a means of monitoring the m.3vemcnt of Mn ~-+ through divalent cation channels. The: present studies w~th Fura-2 employed this property of .tin 2- to quench the Fura-2-Ca 2 fluorescence, lonomycin (125 nM). a cationophore, elicited a maximum rise in [Ca~'*], that ,.,;as not inhibited by 100 nM iloprost (Skuballa and Vorburggen. 1983). a PGI 2 analogue which stimttlates c A M P production and can block platelet activation (fig. 31- The addition of 1 mM Mn 2" produced an initial fall in baseline fluorescence, The Fura-2-Mn 2* complex produces less fluorescence than the Fura-2-Ca z* complex or Fura-2 alone, thereft;re, the immediate fall in fluorescence upon addition of Mn -~* is due to residual extracelhilar Fura-2. The constant steady decrease in signal after addition of Mn z" is likely due to passive movement of Mn 2+ into the platetet and quenching of the intraplatelet Fura-2C a ' * signal or quenching of the extracellular Fura-2Ca-" * signal resulting from leakage of Fura-2 out of the platelet. Stimulation by ionomycin (125 nM) elicited a transient rise in [Ca-'+], followed by a fall in fluorescence to below the extrapolated baseline suggesting that ionomycin stimulated Mn 2÷ entry. Pretreatment with iloprost (100 nM) did not alter the entry of Mn 2+ after ionomycin. Thrombin (0.05 U / m l ) elicited a rise in [Ca2*], and also enhanced the entry of Mn 2. as evidenced by the fall in signal to below the extrapolated baseline produced by the slow passive influx of Mn 2÷ (fig. 4). Unlike the cationophore ionomycin, thrombin failed to elicit a rise in [Ca~'+]~ or Mn 2+ entry after pretreatment with iloprost. The T X A 2 / P G H 2 receptor agomst I-BOP (2.5 nM) elicited a rise in [Ca2+], but failed to promote Mn ~'* entry (fig. 5, upper panel) as A

=. inhibited incorporation into three proteins with molecular weights of 43 kDa, 39 kDa and 27 kDa. Since L657925 (250 nM) also inhibi:,ed incorporation of [JzSIJPTA-Azido into these proteins, they are believed to be proteins associated with the T X A 2 / P ( 3 H z receptor (Mais et at., 1989). Stereoseleetivity was demonstrated by the inability of ( + ) S D Z (t0 p,M) to inhibit incorporation.

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x

3,3. Studies with Fura-2 Extracellular Mn ~-* has been used to assess the activation of divalent cation channels in cells loaded with the fluorescent calcium probe Quin-2 (Hesketh et al., 1983: Hallam and Rink, 1985: Avdonin et al.. 1987). The higher affinity of Mn z÷ than Ca 2+ for tt~e calcium fluorescent indicators, coupled with a quenching of the

Fig. 3. Eftcc~ of M n ~ " on ~onomycin-mduced rl~e in [Ca ~* ~,. Iloprost (rio. tOO n M ) fad~:d to inhibit tbe rise m [Ca ~ " J, induced by innomy-

cin (lono. 125 nM). I mM Mn-'* produced a constant rate of fall in the basal Fura-2 signal. In the pre~erme of Mn2~. [ono produced a transient rise in [Ca"* ], that fell lo a level below the extrapolated baseline represented by the dashed line indicating ionomycin stimulated ent~ of Mn2.. The tracings are representative of two separate e~periments.

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=-2

x

g xx

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g=-2

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=

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Fig. 4. Effect of Mn2" on thrombindnduced rise in [Ca-" ],. Thrombin {Thr. 0.05 U/ml) produced a transient rise in [Ca 2~ l, a, indicated by the increase in fura-2 fluorescence. 1 mM Mn:" produced a constant rate of fall in basal fluorescence. In the presence of Mn 2 ~. Thr pr~xtuced a transient rise in [Ca:" 1~which fell to a level belo~ the extrapolated Mn 2+ baseline Irepresented by the dashed hnej indicating thrombin stimulated entry- of Mn2 +. lloprost tllo. 100 riM} inhibited both the thrombin induced rise in [Ca'* ] and ~hc slimtalaled entD' of Mn 2 +. Tracings are represenlative of three separate experi ments.

t,_ a

a~

m .a,

o=

Fig. 6..q~ereoselettAe inhih~l~m of i-BOP÷reduced iura-2 P,.uorc~eencc b F SDZ. in the presence r~f 1~) mM EGTA. t~BOP I2.5 nM) induced a ribe m Fur,~-2 fluorescence thal zas stere~-~ei~cti~el2, inhibited by { - i S D Z I2 nMI but no~ b, t * }SDZ 12 #MI. Vehicle (VEH) contained 0.02~ D M S O Tra,'mg~ are represen~auve of ~wo experiment-

e v i d e n c e d by the r e t u r n of the signaI ~:o the e x t r a p o l a t e d b a s e l i n e p r o d u c e d by the slow p ~ s i v e i n f l a x o f M n 2-. Both i l o p r o s t ( I 0 0 n M ) a n d -:he T 2 C 4 . : / P G H : r e c e p t o r a n t a g o n i s t L 6 5 7 9 2 5 ( 1 0 0 n M ) i n h i b i t e d the t - B O P s t i m u l a t e d rise in [ C a : - L (hg. 5, l o ~ e r p a n e b . S D Z demonstrated s t e r e o s e l e c t i v e i n h i b i t i o n o f the l - B O P d n d u c e d rise in [ C a 2 - 1 , (fig. 6). ( - ~SDZ (2 ~ M ) i n h i b i t e d the l - B O P r e s p o n s e whi~e { -~ ) S D Z at ~he s a m e c o n c e n t r a t i o n (2 p.M) d i d not. T h e i n h i b i t o w a c t i v i t y of I-)SDZ was specific for ~he T X A : / P G H : receptor since { - i S D Z at a c o n c e n t r a t i o n o f 2 ,uM failed to inhibit the rise in [ C a : - ], in r e s p o n s e to t h r o m b i n (fig. 7).

4. Discussion

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1!

g

1

L.-.A Fig. 5. Effect of Mn2" on I-BOP-induced ns~ m [Ca 2" ], tE:ppcr panel) I-BOP 12.5 nM) produced a tran~aem rise m If'a-" * ], md~c,~ted by an increase in Fura-2 fluorescence. ] trim Mll2" pcodaced a constant rate of fall in basal fluorescence. In the presence of Mn:-. l-BOP produced a transient rise in fluorescence that re~.umed to the extrapolated baseline Irepresented by the dashed line} indicaling that I-BOP did not facilitate the enid of Mn?~. lLov, er paneh In the presence of I mM Mn :~. the rise in fluorescence ~a, mhibiled b 3 bo0l L657925 1925. leo nM} and iloprosl Ilia. leg-,) nM) T~acings are representatave of three separate experlmenl5

T h e pre',ent studies c o n f i r m and e x t e n d the p r e v i o u s o b s e r v a t i o n s ~hat D H P C a : " a g o n i s t s a n d a n t a g o n i s t s may d i r e c t l y i n t e r a c t wi,.h h u m a n p t a t e t e t T X A ~/Pr.3H 2 receptors. In c o m p e t i t i o n b i n d i n g assays p e r f o r m e d at e q u i l i b r i u m . /3A~" a n d C G P competed w i ! b the T X A : / P G t-] _. r e c e p t o r a g o r i st [ ~-~51] B O P a n d a n t a g o n i s t [~'~5t]PTA-OH for ~pecifie b i n d i n g in w a s h e d h u m a n platelets. R a d i o l i g a n d b i n d i n g assays w i t h ( + ) a n d { - ) SDZ demonstrated stereoselective competi,don for specific b i n d i n g for b o t h Iigands w i t h ~ - ) S D Z b e i n g a p p r o x h n a t e t y 50-fo!d m o r e p o t e n t t h a n { + ) S D Z . B A Y d e m o n s t r a t e d a d o s e - d e p e n d e n t i n h i b i t i o n o f the specific i n c o r p o r a t i o n o f ~he T X A : / P G H : receptor photoaffinit> p r o b e { ~ : s l ] P T A - A z i d o i n t o three p r o t e i n b a n d s as.,ociated with the T X A _ , / P G H , r e c e p t o r ( M a i s et al.. t989). B A Y has b e e n r e p o r t e d m i n h i b i t the TXA_- m i m e t i c U 4 6 6 1 9 - i n d u c e d p l a t e l e t secretion, s h a p e change, ag.greg a 6 o n , a n d p h o s p h o r y l a t i o n o f the 4 0 - k D a p r o t e i n s u b s t r a l e for p r o t e i n k i n a s e C as well as c o m p e l e for specific { ~ H ] U 4 6 6 1 9 b i n d i n g ( J o h n s o n et al., t988}.

g e

g

a

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Fig. 7. Effect of t - )SDZ ~n thrombin-induccd Fura-2 ttuoresceacc. In the presence of 10 mM EGTA. thrombin ITHR. nu: U/mlt induced a rise In lhe Fura-2 fluorescence that ~a~ ~lnaff¢cted b~ -JSDZ (2 p-M}. Vehicle IVEH) containeo IL02ci D.MS{~ I~a,-mgs are repre~entati~¢ of ~ao experlment~. Stereoselectivity of the interactions of BAY ','.as also demonstrated since ( + ) B A Y was more potent than ( - ) B A Y or the racemic mixture. In the present studies. the K a values for racemic BAY determined from competition with [~z~llBOP (788 uM) and [~z~I]PTA-OH (542 nM) agree well with the K d t'a]ue of 692 nM determined pharmacologically by Johnson eta'.. (i988). These authors also reported that ( - ) S D Z was approximately one-tenth as potent as ( + ) B A Y . the more potent of the BAY enantiomers. We have found that racemic BAY is approximately twofold more potent than ( - ) S D Z in competition binding assays. The third D H P we have studied. CGP. had one-hundreth the affinity of BAY. T X A e / P G H , receplor agonists and antagonists have been found to inhibit [~:5llPTA-Azido incorporation into three proteins, whh molecular weights of 43. 39 and 27 kDa (Mats et a L 1989). The ability of D H P s to inhibit incorporation of [~2¢l]PTA-azido into the 43-. 39- and 27-kDa proteins is consistent with an interaction with the binding domain of the platelet T X A 2 / P G H z receptor. While ( G P has been reported to produce a slight (78 nM) increase ~,n [CaZ*], in Quin-2 loaded ptatelets which is inhibited by nitrendipine (Erne et ah. 1984) BAY (Johnson et aL. 1988: Doyle and Ruegg. 1985) or S D Z (present study) did not affect resting levels of platelet [Ca 2" I, monitored with Fura-2. The rise in [Ca:*], induced by the T X A 2 / P G H 2 receptor agonist I-BOP is not dependent on extraeellular C a : + since in the presence of 10 m M EGTA. I-BOP still raises [Ca2+], levels to micromolar concentrations with an ECso of 4 nM (Morinelli et al., 1989). Extracellular Mn 2- has been used to demonstrate the opening of divalent cation channels in the platelet (Hallam and Rink, 1985; Avdonin et al., 1987). As Mn 2' moves into the platelet through a divalent cation channel it displaces Ca 2. from Quin-2 or Fura-2 and produces a fall in fluorescence. Rink and Hallam (i985) have employed

Quinto demomqrate that ionomycin, thrombin. platelet activating factor and adenosine diphosphate ~4imulate an influx of M n : * suggesting the stimulation of a divalent cation channel. In the present study ionomycin and thrombin both promoted the influx of Mn-'-. However. this activity of thrombin was blocked by iloprost. Thus. it would appear tbat the thrombin receptor mediated rise in [ C a ' ÷ ], is sensitive to the level of intraplatelel cAMP. Zschauer et aL (1988) studied single divalent cation channels from platelct m e m b r a n e vesicles incorporated into a phospholipid bilayer. Vesicles from unstimulated platelets exhibited no detectable divalent cation channels. Vesicles from thrombin-stimulated platelets possessed divalent cation channels which were not present in unstimulated platelets. These channels were not of the classical voltage-dependent type since they were not antagonized by the D H P nisoldipine. In the present study. ( - ) S D Z failed to inhibit the rise in [Ca2+l, produced by thrombin supporting the notion that the rise in [CaZ" 1, elicited by thrombin is not mediated by a DHP-sensitive channel. The requirement of intact platelets for thrombin stimulated expression of these non-DHP-sensitive channels suggested that receptor occupation plus some intermediary steps are required. Indeed. the ability of iloprost to inhibit the rise in [Ca-'*], and the activation of a divalent cation channel suggest that the postreceptor events responsible for the establishment of activated divalent cation channels m a y be under the control of cAMP. The T X A 2 / P G H 2 receptor agonist I-BOP failed to promote the influx of M n 24. This result is compatible with the notion that the T X A 2 / P G H 2 receptor is neither a C a : " channel nor operates one. Clearly, the postreceptor coupling events for thrombin and T X A 2 mimetics are different (Brass et al.. 1987). Both I - B O P and thrombin elicit a rise in [Ca:+]i from internal stores that is inhibitable by cAMP. However, the rise in [Ca 2+ ], elicited by thrombin results in activation of a divalent cation channel while the rise in [Ca~*l, elicited by the T X A e / P G H 2 mimetic I - B O P does not. The nature of the specific interactions of these D H P s with the p[atelet T X A 2 / P G H : receptor remains unknown yet it does not appear to be through a classical receptor-mediated divalent cation channel. Since the platelet and vascular smooth muscle T X A 2 / P G H 2 receptors appear to be different (Mats et a L 1988) it would be of interest to examine the interactions of these D H P s with the T X A 2 / P G H : receptor in other tissues given that the pharmacological activity of BAY, at least in the feline airway, is nut blocked by the T X A : / P G H 2 antagonist SQ29548 (Underwood et aL. 1987). The results of these studies are consistent with the notion that the ptatelet-inhibitory activity of the D H P class of Ca2* agonists and antagonists m a y in part be due to an effect on the T X A 2 / P G H 2 receptor and

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Acknowledgements W e wish to a c k n o M e d g e the secretarial assGtance of Ms. Marie Meadowcroft a n d the assistance of Ms. Janie Nelson in preparing the figures. This ~ r k was supporled by N I H grants H I 36838 a n d HL0726(1 Dr. Halushka is a Burroughs WeBcome Scholar in ('lin,cal Phar-

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