prostaglandin H2 receptors and histamine H1 receptors in cultured Guinea-pig tracheal smooth-muscle cells

Biochimica el Biophysica Aela, 1137(1992) 107-115

107

© 1992Elsevier Science Publishers B.V. A!I rights reserved 0167-4889/92/$05.00

BBAMCR 13232

Characterization of thromboxane A2/prostaglandin H 2 receptors and histamine H mreceptors in cultured Guinea-pig tracheal smooth-muscle cells Ichiro Miki, Hiromi Nonaka

and Akio Ishii

Pltarmaceulical Research Laboratories, Kyowa Hakko Kogyo Co,, Ltd., Nagaizumi. Sunto, Shizuoka (JalTatD

(Received 13 May 1992)

Key words: Thromboxane A 2; Proslaglandin H2; Histamine H t; Receptor; Smooth-musclecell; Trachea; (Guinea pig) We characterized thromboxane A?~/prostaglandin H 2 (TXA2/PGH 2) receptors and histamine Hj receptors in Guinea-pig cultured tracheal smooth-mltscle cells (TSMC). [3H]SO 29548 (a TXA 2 antagonist)-binding sites were saturable and a high affinity ¢vith a dissociation constant of 6.2 ± 0.60 nM (mean ± S.E.) and a receptor density of 46 ± 4.6 fmol/10* cells. [3H]SO 29548 binding was completely inhibited by TXA 2 mimetics or antagonists, intracellular calcium concentration ([Ca2+]i) in TSMC was increased with U46619 stimulation and the increase was attenuated by TXA 2 antagonists, the potencies of which correlated with those inhibiting the activities of the [3H]SO 29 548 binding. [3H]Mepyramine (a H I antagonist)-binding sites were also present in TSMC. [3H]Mel~ramine had a single class of low-affinity-bindingsites with a dissociation constant of 2.6 ± 0.~1 /tM and a receptor density of 10.6 ± 0.1 ! nmol/mg protein. [3H]Mepyramin¢ binding in TSMC membrane was inhibited by H I antagonists, but not by H 2 antagonists. The inhibition constants of mepyramioe in TSMC were 910-times lower than those in tracheal membranes. In contrast, the histamine-induced increase in [Ca2+]i in TSMC was inhibited in the presence of low concentrations of H t antagonists. All these observations provide evidence that TXA2/PGH 2 receptors, mepyramine-binding sites a n d / o r H I receptors are expressed in cultured TSMC.

lnlroductinn T X A 2 and histamine are potent contracting substances of human, Guinea-pig and dog airway smoothmuscle [1-4]. T X A 2 stimulates platelet activation and smooth-muscle contraction through a T X A 2 / P G H 2receptor-mediated process [5-7]. T X A 2, mainly produced by platelets [6,8], is also synthesized by the Guinea-pig lung with the stitnulation of plateletactivating factor [9]. T X A 2 causes bronchoconstriction and plays an important role in bronchial hyperresponsiveness in patients with asthma [10,11]. Histamine induces tracheal smooth-muscle contraction by stimu-

Correspondence to: I. Miki, Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co_ lad., Nagaizumi. Sunto. Shizuoka 411, Japan. Abbreviations: TX, thmmboxane; PTAz. pinane t h ~ n e A2; Ci'A2, carbocyche thromboxane A2; PG. prostaglandin; HBSS. Hanks" baL~cd salt solution: DMEM/FI2, Dulbecco's modified Eagle's medium and Ham's FI2 nutrient mixture; PBS(-), Dulbecco's phosphate-buffered saline without calcium and magnesium; TSMC, trac4aealsmooth-mtts¢l¢cells.

lating histamine H I receptors [12]. The newer antihistamines, teffenadine [13] or azelastine [14], cause b r o w chodilation in asthmatic patients. Activation of both T X A 2 / P G H z receptors and H I receptors elevates intracellular Ca 2+ concentrations ([CaZ+]i) in vascular and tracheal smooth-muscle cells, followed by contraction of these cells [15-17]. The calcium-sensitive dye fura-2 [5,18,19], the calcium-sensitive photoprotein aequorin [16] or voltage clamp metbod [20] were used to measure the transient increase in [Ca'+]i in tracheal smooth-muscle, in cultured vascular smooth-muscle cells, the phenotypic modulation can include changes in specific contractile proteins [21]. Tracheal smoothmuscle cells (TSMC) grown in culture provide the experimental systems for studies on cellular processes [18,19]. There is evidence that Guinea-pig tracheal cells possess contractile activities for 20 or more passages [22]. A search of the literature revealed no report dealing with T X A 2 / P G H z and H~ receptors in Guinea-pig TSMC. W e have now obtained evidence for the presence of functional T X A z / P G H 2 and H, receptors in TSMC in receptor-binding assays and in agonist-induced calcium mobilization.

108 Materials and Methods

Materials Commercial sources of materials and reagents were as follows. [3H]SQ 29548 (i110 GBq/mmol) and [3H]mepyramine (884.3 GBq/mmol) were obtained from Du Pont/New England Nuclear (Boston, MA); 1346619. U44069, pinane thromboxane A 2 (PTA_,), carbocyclic thromDoxane A_, (CTA,), TXB 2, PGD2, PGE, and PGF,~ from Funakoshi (Tokyo): fura-2 AM, histamine HCI and Scintisol EX-H from Wako Pure Chemical (Osaka); fetal bovine serum (FBS) and ant~ioties from Gibeo; collagenase type 1, elastase type IV, Hanks' balanced salt solmion (HBSSL Dalbecco's modified Eagle's medium an~ Ham's FI2 nutrient mixture without phenol red ( D M E M / F I 2 ) from Sigma (St. Louis). SO 29548 was a kind gift from Dr. M. Oglctree at Squibb Institute for Medical Research. KW-3635 (sodium ( E L I l-[2-(5,6-dimethyl-l-benzimidazolyl) ethylideae]-6,11 -dihydru-dibenz[ b,e ]oxepin-2-carboxylate monohydrate) [23-25], BM13177 [26], BM13505 [27] and AA-2414 [28] were synthesized ia our laboratories.

Tracheal cell ctdture Primary cultured TSMC were prepared as described elsewhere [22]. Briefly, tracheae from adult male Hartley Guinea-pig (250 g) were placed in HBSS and cut iongitodinally through the rings opposite the strip of smooth muscle. The tracheae were then incubated with gentle agitation in 2 ml of HBSS containing collagenase (0.2%), elastase IV (0.05%), penicillin (100 U / m l ) and streptomycin (100/~g/ml) at 37°C for 30 rain. The enzyme solution was removed and smooth-muscle cells were collected from the undigested tracheal tissue by suspending it twice in 4 ml of HBSS. The cell suspension was centrifuged and the pellet was resuspended in 10 ml of D M E M / F I 2 with 10% FBS and the antibiotics, then incubated in two 25-cm 2 culture flasks at 3"PC under 5% CO x : 95% air. TSMC were subcultured aRer 7 to 10 days incubation. Cells at confluency were washed sequentially with a large volume of Dulbecco's phosphate-buffered saline without calcium and magnesium (PBS(-)) for 1 rain, and with ! ml of 0.02% EDTA in P B S ( - ) for 1 rain. TSMC were harvested by trypsinization (0.25% trypsin, 1 rain at 37°C) and subcultured at 1 x 10~ cells in an 80 cm 2 flask.

Cryopreservation o f TSMC Trypsin-harvested cells (3rd to 5th passage) were washed with D M E M / F I 2 containing 10% FBS and centrifuged at 200 x g for 6 rain. The cells were resuspended in ice-cold medium containing 10% dimethylsuifoxide, frozen using a programmable freezer Model 700 (Cryo-Med, Mt. Clemens) and stored in liquid nitrogen.

inummoJluorescence staining o f actin TSMC were cultured on Lab-Tek tissue culture chamber/slides (Nunc, Naperviile, IL) for 2 days. TSMC were washed with PBS(- ), immediately fixed with 3% formaldehyde solution for 5 rain at 37°C, then washed 3 times with PBS( - ). The cells were permeabilized for 2 rain in cold acetone (-30°C), washed 3 times with P B S ( - ) and incubated with anti-muscle actin-lgG (against chicken gizzard actin, BT-574, Biomedical Technologies, Stoughton)diluted i :5 in PBS(- ) for I h at 25°C. The ceils were then washed 3 times with PBS{-), and incubated with FITC-cunjugated goat anti-rabbit-lgG (H + L) (Seikagaku Kogyo, Tokyo) diluted ! : 16 in PBS( - ) for I h at 2.5°C. The glass slide was washed 3 times with PBS(- ) and mounted onto a coverslip using 25 /tl of glycerol/ P B S ( - ) ( 9 / i (v/v)). The cells were examined on a OPTIPHOT microscope (Nikon, Tokyo) using epifluorescence optics, and then photographed.

Binding assay o f TXA 2/ PGH 2 receptors in TSMC The TSMC were washed with PBS{ - ) and exposed to PBS( - )containing 0. ! 25% trypsin and 0.02% EDTA to detach the cells, The ceils were then centrifuged at 200 x g for 5 rain at room temperature, washed with HBSS containing 10 /tM of indomethacin (HBSS/INDO) and re.suspended in HBSS/INDO (10 ~' cells/160/zl). Binding studies were performed by incubating 1 × 10~ cells with 5 nM of [3H]SO 29548 in a total volume of 200/zl at 25°(: for 90 rain. BM13505 (100 p.M) was added to determine the nonspecific binding. Four ml of ice-cold 50 mM Tris-HCi buffer (pH 7A) containing 100 mM NaCI was added to terminate the reaction, and the content was passed through a Whatman G F / C glass filter using a MT-24 cell ha~ester (Brandel, Maryland). The filter was washed three times with 4 ml of ice--cold buffer, dried, and immersed in 8 ml of Scintisoi EX-H. The radioactivity on the filter was counted in a liquid scintillation counter. Computer analysis (EBDA, LIGAND and KINETICS) [29] was used to evaluate the dissociation constant (K o value), receptor density ( B ~ value), Hill coefficient (nil), association rate constant (t: I) and dissociation rate constant (k _ t)-

Binding assay o f TXA 2/PGlt2 receptors in platelets Male Hartley Guinea-pig blood was anticoagulated with 1/10 (v/v) of 77 mM EDTA (pH 7.0) containing 100 /.tM indomethacin and centrifuged at 120 × g at room temperature for 12 min. The platelet-rich plasma was collected and centrifuged at 900 × g for 10 rain. Then the platelets were washed in 25 mM Tris-HCI buffer (pH 75)/138 mM N a c l / 5 mM MgCi2/I mM EGTA/10 p.M indomethacin and resuspended in the same buffer. Binding study was performed by incubating 1 x l0 s platelets with 5 nM of [3H]SQ 29548 in a

109 total volume of 200 #1 at 25°C for 60 min. Nonspecific binding was meas~nred in the presence of 100 /zM BM13505. Ice-cold 50 mM Tris-HCI buffer (pH 7.4) containing 100 mM NaCi was added to the tube and the preparation was passed through a Whatman G F / C glass filter. The filter was washed three times with 3 mi of ice-cold buffer and the radioactivity on the filter was counted as stated above.

conditions of stirring at 37°C. The increase in [Ca2+]i was determined using a CAF-100 Ca 2~ analyzer (Japan Spectroscopic, Tokyo). The excitation wavelengths were 340 nm and 380 nm and the emission wavelength was 500 nm. The fluorescence ratio (fluorescence at 340 n m / t h a t at 380 nm) was used to calculate the [Ca-'+]~ concentration [30].

Binding assay of HI receptors in TSMC monbrane Cells detached by trypsinization were washed with 50 mM Tris-HCI buffer (pH 7.5)/120 mM NaCI/5 mM MgCI_, (Tris-buffered saline) and homogenized in a Polytron homogenizer (Brinkman Instruments, Westbury) for 20 s at 4°C in Tris-buffered saline 007 cells/ml). The homogenate was centrifuged at 41000 × g for 10 min at 4°C. The pellet was resuspended in Tris-buffered saline and used as the membrane fraction. For the binding study, we incubated the membrane fraction (0.15 mg protein) prepared from 0.5 x 10~' cells with 2 nM of [3H]mepyramine in a total volume of I ml at ff'C for 60 rain. Mepyramine (1 mM) was added to determine the nonspecific binding. Icecold Tris-buffered saline (6 ml) was added to terminate the reaction and the content was immediately passed through a Whatman G F / C glass filter. The radioactivity on the filter was counted in a liquid scintillation counter, as described above. Binding assay o f H r receptors in Guinea-pig tracheal membrane Guinea-pig tracheal membranes were prepared as described elsewhere [12]. Binding studies were done by incubating tracheal membranes (280/tg protein) with 2.0 nM of [3H]mepyramine in a total volume of 1.1 ml of Tris-buffered saline at 25°C for 30 min. Histamine (10 mM) was added to determine the nonspecific binding. Ice-cold Tris-buffered saline (4 ml) was added to the assay tube, and the reaction mixture was passed through a Whatman G F / C glass filter. The filter was washed three times with 5 ml of ice-cold buffer, and the radioactivity was counted as described above. Measurement o f / C a ~ *L TSMC detached by trypsinization were washed with Eade's BSS and resuspended in Earle's BSS (107 cells/ml). These cells were incubated with 3/zM fura-2 AM (final dimethylsulfoxide concentration was 0.3%) for 30 min at 3"PC. The fura-2-1oaded cells were washed once with Earle's BSS and resuspended in the same buffer at 10~ cells/ml. The cells were preincubated for 5 rain az 37°C. and stirred at 1000 rpm for 2 min at 3TC ia a glass cuvette (Z2 mm in diameter). TSMC were preincubated with the vehicle alone or with various concentrations of drugs for 1 rain, and stimulated with 10 -7 M U46619 or 10 -5 M histamine, under

:. : : ~ . . ~ .

~

-.

_ _

< Fig. I. Morphological characterization of cultured Guinea-pig TSMC. (a) Cells in the third passage were grown for 48 h in DMEM/FI2 and were photographed with a phase-contrast microscope ( x 4 0 magnification). Cells in the sixth passage were cultured for 3 days, fried with acetone and stained with anti-actin-lgG and FITC-labeled

secondantibody.The cellswerephotographedusinga (b) phase-contrast microscopeor (c) fluorescence microscopeix200 magnification).

110

6,000 ,o 5.000 ! i ,~ 4,000 3.000 nn2.000 1.000 0

~

0

5

10

15

20

25

CH]SQ29,S,~(riM) Fig- Z Characterization of saturation-bioding properties of [3H]SQ 29548 in TSMC. Guinea*pig TSMC (! x 10~ ceils) were incubated with [3H]SQ 29548 in a total volume of 200 p.I at 25°C for 90 min. Specific binding (e) was defined as the difference between binding in the presence (El) and absence ( o ) o f 100/.tM BM 13505.(ln;et) Scutchard plot of [3H]SO 29548 binding in TSMC. E~ch datum represents the mean + S.E. of three different e: periments done in duplicate. Results

l c show p h a s e - c o n t r a s t a n d fluorescence microscopies o f the cells, respectively. I m m u n o f l u o r e s c e n c e o f actin filaments w a s o b s e r v e d in all t h e cells. T S M C a t c o n f l u e n c e (3rd passage) w e r e frozen in m e d i u m c o n t a i n i n g 10% dimethylsulfoxide, using a p r o g r a m m a b l e freezer. T h e recovery o f stock cells was a b o u t 9 0 % , as assessed b y the exclusion o f t r y p a n blue. T h e s t o r e d cells h a d t h e s a m e a p p e a r a n c e , a n d a n increase in [CaZ+]i a n d r e c e p t o r - b i n d i n g p r o p e r t i e s w e r e t h e s a m e a s t h o s e in p r i m a r y cultures. T h e T S M C at the 3 r d t o 15th p a s s a g e f r o m p r i m a r y c u l t u r e o r stock cells w e r e u s e d f o r r e c e p t o r - b i n d i n g assays a n d [Ca2+]i m e a s u r e m e n t s .

Preparation and characterization of TSMC Primary cultured TSMC of Guinea-pig tracheae were p r e p a r e d by enzymic digestion. T h e c u l t u r e d T S M C f o r m e d 'hills a n d valley" a characteristic a p p e a r a n c e o f s m o o t h - m u s c l e cells [22]. A t confluence, the cells w e r e e l o n g a t e d with a c y t o p l a s m a n d longitudinal ridges a n d f o r m e d m u l t i l a y e r e d hillock.~ (Fig. ia). T h e s a m e a p p e a r a n c e w a s p r e s e r v e d at 15th passage. A f t e r the 20th p a s s a g e , the cells b e c a m e slightly e n l a r g e d a n d did not f o r m a multilayer. C u l t u r e d T S M C w e r e s t a i n e d at the 3rd p a s s a g e by a n a n t i b o d y against actin. Figs. l b a n d

loo -~s

~ "6 g

so

25

-

1

10

9

8

DrugIlog(Mll

-4

Fig. 3. Displacement of the specific binding of [3H]SQ 29548 in TSMC. TSMC (I x 106 ceils) were incubated with 5 aM [3HJSQ 295.48 in the presence of various concentrations of SQ 29548 (o), KW-3635(*), BM 13505(~), BM 13177( • ) , U46619 et• ), U44069 (<>),PGD2 (E3), PGF2a ( x ) or PGE 2 ( + ). Nonspecific binding is defined in the presence of 100/.tM BMi3505. Each datum represents the mean + S.E. of thre~ different experiments done in duplicate.

111 A

5

"

___4

% o E c "0

o

E

0

20

40

60

Mepyramine

1

=

e(.m0.10"==.,,)

80

100

(p.M)

Fig. 4. Characterization of the saturation-binding properties of [3H]mepyramine in TSMC. TSMC membranes (0.15 mg of protein, prepared form 0.5 x 106 cells) were incubated with 2 nM [3H]mepyramine and various concentrations of uplabcled mepyramine in a total volume of I ml at 0~C for 60 rain. Non-specifu: binding is defined in the presence of i mM unlabeled mepyramine. (Inset) Scatchard plot of laH]mepyramine binding to TSMC membrane~. Each datum represents the mean +_S.E. of three different experiments done in duplicate.

TXA z / PGHz receptor-binding assay T S M C h a d specific b i n d i n g sites f o r [ 3 H ] S Q 2 9 5 4 8 (a T X A 2 antagonist), a n d the b i n d i n g activity w a s m a i n t a i n e d even a t t h e 15th passage. T h e specific b i n d i n g o f [ 3 H ] S Q 2 9 5 4 8 linearly c o r r e l a t e d with cell n u m b e r b e t w e e n 0.2 a n d 2.0 x 106 cells, a n d r e a c h e d a steady-state p l a t e a u a f t e r 60 rain a t 25°C. T o exclude t h e possibility t h a t the specific b i n d i n g o f [ 3 H ] S Q 2 9 5 4 8 r e p r e s e n t s t h e u p t a k e o f t h e ligand by the cells, dissocia t i o n o f the b o u n d [ 3 H ] S Q 2 9 5 4 8 w a s assessed. T h e specifically b o u n d [ 3 H ] S Q 2 9 5 4 8 w a s immediately dis-

sociated linearly by the addition o f u n l a b e l e d 1 0 0 / z M BM13505, thereby indicating a single class o f binding sites. T h e km a n d k _ j values w e r e 0 . 0 1 5 9 + 0 . 0 0 4 6 n M - t m i n - i ( m e a n + S.E., n = 3) a n d 0.0175 + 0.0029 min - t , respectively. T h e kinetically d e t e r m i n e d K d value was 1.1 nM. A S c a t c h a r d t r a n s f o r m a t i o n o f the s a t u r a t i o n curve is s h o w n in Fig. 2. T h e S c a t c h a r d plot is linear, thereby indicating a single class o f binding sites, with a n a p p a r e n t K d value o f 6.2 + 0.60 nM, Bm~ value o f 46 _+ 4.6 f m o l / 1 0 ~ cells, a n d n H o f 0.93 + 0.023 ( m e a n + S.E.,

100

"o

g

m = >, ~" E o c .= ¢

75

50

25

0 -8

-7

-6

-5

Drug

-4

-3

-2

(log(M))

Fig. 5. Displacement of the specific binding of [3H]mel~,ramine in TSMC membranes. TSMC membranes (0.15 mg of protein) wcre incubated with 2 nM [3H]mepytamine in the 9resence of various oancentrations of astemizole (o), diphenhydramine ( ,a ), ketotifen ( ~ ), mepyramine (@), terfenadine (*) or histamine (o). Specific binding is defined in the presence of i mM mepyramine. Each datum represents the mean+S.E, of three different experiments done in duplicate.

112 TABLE I

H t receptor-binding assay

Intubition o f [3t1]$Q 29o~48bimting to Guim'a-pig TSMC mul piutch,ts

T S M C m e m b r a n e s b a d specific binding sites for [ " H ] m e p ~ a m i n e a n d this specific binding r e a c h e d a steady-state p l a t e a u a f t e r 45 rain at 4°(::. T h e specifically b o u n d [-;H]mepyramin¢ w a s rapidly dissociated by 9 8 % within 30 s by a d d i n g o f 1 m M o f u n l a b e l e d m e p y r a m i n e . T h e dissociation was t o o r a p i d to evalua t e the k , value. S c a t e h a r d analysis s h o w e d a saturable, single class o f low-affinity-binding sites with a K d value o f 2.6 +_ 0.081 ,aM a n d Bm~~ value o f 1 0 . 6 _ 0.1 ! n m o l / m g p r o t e i n (2.9 +_ 0.058 n m o l / 1 0 * cellsL respectively (Fig. 4). T h e binding w a s completely inhibited by high concentratio:~s o f H j a n t a g o n i s t s such as astemizole, d i p h e n b y d r a m i n e , ketotifen, m e p y r a m i n e a n d t e r f e n a d i n e (Fig. 5). T h e binding w a s not displaced by H,_ a n t a g o n i s t s s u c h as cimetidine a n d ranitidine (Table IlL Oui~;dine, quinine a n d imidazolc, c o m p o u n d s which i o b ~ i t the [ 3 H ] m e p y r a m i n e - b i n d i n g in rat liver m e m b r a n e s [32,33], also h a d w e a k activities in TSMC. A single class o f high-affinity [ 3 H ] m e p y r a m i n e - b i n d ing sites w a s o b s e r v e d in G u i n e a - p i g t r a c h e a m e m branes. T h e K j value w a s ! . i +_ 0.061 n M ( m e a n +_ S.E., n = 3) in t r a c h e a l m e m b r a n e s . T h e B,,~, a n d n H values w e r e 79 ± 7.0 f m o l / m g p r o t e i n a n d 0.79 _+ 0.012, respectively. T h e specific binding o f [3H]mel~yramine was potently inhibited by H I a n t a g o n i s t s s u c h as ketotifen a n d m e p y r a m i n e with K~ values o f 1.0 n M a n d 2.3 nM, respectively. T h e inhibitory activities o f ketotifen a n d m e p y r a m i n e in T S M C w e r e a b o u t 1000 times lower t h a n those for t r a c h e a l m e m b r a n e s (Table ll). T h e log

Data are expressed as mean_+S.E. for thre~ experiments. Drugs TXA. antagonisLs SO 29548 KW-3635 BMI3505 AA-2414 PTA z BMI3177 CTA., TXA 2 agontsts U44069 U46619 Eicosanokh PGD 2 PGF2~ PGE 2

TXB2

K, (nM) TSMC

1.8_+ 0_~) 6.1)_+ ! 4 12 + 1.5 13 +_ ~ 66 +_ 25 150 ± 5.8 170 _+ 18 3.8+_ 0_~5 15 +_ 5.3 170 +- 511 620 + 15 IEqll +_46[) 601}0 +_4611

platelels 1.8+_ 0.(~ 3.2_+ O-~;q 39 +- I-~ 14 +_ O.Ig't8 340 ± 35 780 +- 30 4911 + 15 4.4+_ 7Z

0.23 11.113

4IX) + 12 3 IIRRI + 29110 +- 52 14{$111 _+l(RH)

n = 3), respectively. T h e specific binding w a s not influe n c e d by 10 m M C a C i , ( 1 0 7 _ + 5 % o f controlL 10 m M MgCi 2 (109 4- 6 % ) o r 1 m M E G T A (89 _+ 2%). T S M C w e r e i n c u b a t e d with [ 3 H ] S Q 2 9 5 4 8 with vario u s c o n c e n t r a t i o n s o f T X A 2 r e c e p t o r antagonists. S O 2 9 5 4 8 , KW-3635, BM13505, AA-2414, P T A 2, B M I 3 1 7 7 a n d C T A 2 , o r agonists, U44069 a n d U46619 (Fig. 3). T h e c o n c e n t r a t i o n o f d r u g r e q u i r e d to r e d u c e specific S Q 2 9 5 4 8 - b i n d i n g by 5 0 % ( I C ~ ) was d e t e r m i n e d from Fig. 3, a n d the IC~, values w e r e used to calculate the i n h ~ i t i o n c o n s t a n t ( K t ) by the C h e n g - P r u s o f f relationship [31] (Table D. T h e r a n k o r d e r o f affinity d e t e r m i n e d f r o m the K i values w a s S Q 2 9 5 4 8 > U44069 > KW-3635 > B M ! 3 5 0 5 > A A - 2 4 1 4 > U46619 > P T A 2 > B M I 3 1 7 7 > C T A , > PGs. G u i n e a - p i g platelets h a d a single class o f specific b i n d i n g sites for [ 3 H ] S Q 2 9 5 4 8 with the Ka value o f 3.0 _+ 0.22 n M ( m e a n _+ S.E., n = 3), Bm~ value o f 228 -+ 7 f m o l / l 0 s platelets a n d n H o f 0.96 _+ 0.012. Association a n d dissociation e x p e r i m e n t s s h o w e d a single class o f b i n d i n g sites with the k I a n d k _ I values o f 0.0103 r i M - ~ r a i n - * ( n = 2) a n d 0.0238 m i n - ' (n = 2), respectively. T h e kinetically d e t e r m i n e d K d value ~ a s 2.3 nM. A s n o t e d in T S M C , the specific b i n d i n g o f [ 3 H ] S Q 2 9 5 4 8 in platelets w a s not affected by the p r e s e n c e o f 10 m M CaCI 2, 10 m M MgCI 2 o r 1 m M E G T A . S Q 2 9 5 4 8 a n d KW-3635 w e r e also p o t e n t inhibitors in platelets (Table I). A linear regression analyses o f the d a t a in T a b l e ! s h o w e d t h a t the log o f the K~ values o b t a i n e d in T S M C highly c o r r e l a t e d with t h a t for platelcts ( r = 0.972).

TABLE II bdu'bilion of/~H[mel~'ramine-binding to Guinea-pig TSMc~ and trachea

[~!llMel~.'ramine concentrations were tLSedat 1.71 to 3.08 nM for TSMC honmgeuates, and ZI2 to "~49nM for tracheal homogen:~tes. respectively. Data are expressed as mean_+S.E, for three exporimenUs.Correlation coefficient ~-as calculated using the K i values of astemizole, diphenhydramine, ketotifen, mepyramine, terfenadine quinidine and histamine. Drugs

Ki ( n M )

AstemizoIe Diphenhydramine Ketotifen Mepytamine Terfenadine Quinidine

1700_+ 88 1900+ 200 2000+_ 410 2100+_ 670 2600+_ 270 4000+58 6400+- 1300 ~'301]0_+ 560 3200000+_220000 > 100000

15 _+ 2.1 31 + 6.2 1.0+_ 0.04 2.3+_ 0.78 140 + 17000 +- 880 > III0000 > 100(gl0 45000 +_3000

> 100000

> 100000

Quinine

Imidazole Histamine Cimetidine Rauitidine

> I001~0

113 o f the K i values in T S M C a n d t h a t for the t r a c h e a did not correlate ( r = 0.708),

a

[Ca'- +]i increase in TSMC U46619 (Fig. 6a) a n d histamine (Fig. 6b) induced a d o s e - d e p e n d e n t increase in [Ca2+L. L146619 at 10 7 M o r histamine at 10 - s M i n d u c e d a r a p i d rise in [Ca2÷]~ within 10 s, a n d [Ca-'+]i fell to the p l a t e a u level I min a f t e r stimulation (Fig. 6, inset). T h e m a x i m u m increase in [Ca2+]i c a u s e d b y 10 - ? M U46619 w a s 94 + 14 n M in the p r e s e n c e o f i m M o f extracellular C a 2+ a n d 1.7 _+ 0.57 n M in the p r e s e n c e o f 0.03 m M E G T A , respectively. T h e m a x i m u m increase in [Ca2+]i c a u s e d by 10 - s M histamine waz 56_+7.9 n M a n d 2.7_+0.63 n M in the presence o f extracellular C a -'+ o r E G T A , respectively. W e m e a s u r e d the inhibitory effects o f d r u g s in t h e p r e s e n c e o f I m M C a 2+, since the m a j o r p a r t o f the Ca2+ elevation in T S M C w a s a t t r i b u t e d to a n influx o f extracellular C a : + . T h e U46619-induced [Ca2+]i increase w a s inhibited by various T X A 2 a n t a g onists (Fig. 7a). T h e I C ~ values f o r S O 2 9 5 4 8 , K W 3635, BM13505 a n d B M I 3 1 7 7 w e r e 21, 3 6 1080 a n d 17080 nM, respectively. T h e ICso values for the inhibition o f [ca2+]i increase w e r e o f the s a m e m a g n i t u d e as those for t h e inhibition o f r e c e p t o r b i n d i n g by S O

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Fig. 7. Effect of antagonists on intraccllular Ca-'+ mobilization in U46619- or histamine-stimulated TSMC. (a) Fura-2-1oaded TSMC tl x 16" eells/ml) were incubated for I rain with various concentrations of SO 29548 (o). KW-3635 (eL BMI3~)5 (A) or BMI3177 (IlL then TSMC were stimulated by 10-? M U46619 and the maximum increase in [Ca-'+ li was monitored. (b) Fura-2-loaded TSMC were incubated with various concentrations of mcpyraminc (IL astemizole (ok diphenhydraminc t • ) or imidazole t x ). then TSMC were stimulated by II) -'s M histamine. Each datum represents the mean _+S.E. of four different experiments.

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2 9 5 4 8 a n d KW-3635, but not by B M ! 3 5 0 5 a n d BM13177. T h e histamine-induced [Ca2+]i increase w a s red u c e d by H I antagonists (Fig. 7b). T h e IC.,~ values for m e p y r a m i n e , astemizole a n d d i p h e n b y d r a m i n e were 6.1 nM, 32 a n d 96 nM, respectively. T h e IC5, values for the inhibition o f [Ca2+]i increase w e r e o f the s a m e m a g n i t u d e as the K i values o f [ 3 H ] m e p y r a m i n e binding in tracheal m e m b r a n e s b u t not T S M C (Table I1). Cimetidine a n d ranitidine at 10 -4 M did not inhibit the histamine-induced increase in [Ca2+]~. lmidazole inhibited the [ 3 H ] m e p y r a m i n e - b i n d i n g in r a t liver m e m b r a n e s [33], but did not inhibit the [Ca2+]i increase in T S M C (Fig. 7b).

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Fig. 6. Dose-dependent r ~ ) m t s e o f U 4 f ~ l g - i n d u c e d o r histamine-induced initial lea -*+]i transients increase. Fura-Z-Ioaded TSMC (l x

Discussion

l06 cells/ml) ~cre preincubated for 5 rain at 3"FC and .qirred for 2 min. The TSMC were stimulated by various concentrations of (a) U46619 or (b) histamine. Tim maximum increases in [Ca2+ |i were monitored. Each datum represents the reean_+S.E., of four to five different experiments. (Inset) 'Vane dt.pendency of [Ca2+ ]i increase.

W e o b t a i n e d evidence for the presence o f the highaffinity [ 3 H ] S Q 2 9 5 4 8 - b i n d i n g sites in c u l t u r e d T S M C . T h e K d value o f [ 3 H ] S Q 2 9 5 4 8 in T S M C (6.2 r i M ) w a s c o m p a r a b l e to t h a t in G u i n e a - p i g platelets (3.0 riM)

114 and in cultured rat vascular smooth-muscle cells (!.72 riM) [34], Specific binding of [3H]SQ 29548 was completely inhibited by the T X A 2 antagonists KW-3635, BM13505 or SQ 29548, and the agonists U46619 or U44069. In TSMC, PGD 2 also inhibited the [3H]SQ 29548-binding. PGD 2 was found to have an agonistic effect on the TXA2/PGH_, receptors in Guinea-pig trachea [35]. The log of Ki values of compounds in TSMC correlated well with those in Guinea-pig platelets (r = 0.972) (Table 1) and with reported values for rat vascular smooth-muscle cells [34]. T X A 2 is a potent mediator which induces contraction of Guineapig tracheal smooth muscle [2,361, lung strips [37] and human airway smooth muscle [1]. Although we found high-affinity~bind.;ngsites of [3H]SQ 29548 in TSMC, a specific binding of [3H]SQ 29548 was not detected in Guinea-pig and porcine tracheal smooth-muscle homogenates (Data not shown). It seems that even a small amount of the T X A 2 / P G H z receptor is sufficient to induce airway contraction. Thus, cultured TSMC can be. validly used to examine T X A 2 / P G H 2 receptors in the trachea. Divalent cation such as Ca 2+ and Mg 2+ were found to enhance the specific binding of [3H]S-145, a T X A z / P G H 2 receptor antagonist, to human platelets [38] and only Mg 2+ enhanced the binding of [3HIU466i9 in rat plate!et membranes [39]. In Guineapig "PSMC, the specific binding of [3H]SQ 29548 did not increase in the presence of Ca 2+, Mg 2+ or EDTA. We also observed that the specific binding of [3H]SQ 29548 in Guinea-pig platelets was not affected by these cations. These results indicate that there may be species differences in ion requirements. The notion of species difference was also suggested by Northern blot analysis using human placenta eDNA of T X A 2 / P G H 2 receptors [40], We found that TSMC and Guinea-pig tracheae pussessed [3H]mepyramine-binding sites. The K d values of [3H]mepyramine in TSMC were much lower than those for the Guinea-pig trachea. The K i values of compounds in TSMC and that in the trachea had poor correlation. Other investigators noted only high-affinity ~3H]mepyramine-binding sites in Guinea pig [41] and porcine [12] tracheal tissue. Low-affinity [3H]mepyramine-binding sites were reported in the cultured aTmoth-mascle cell line DTI'=MF 2 with a K d value of 7A9 nM [42] and BC3HI with a K j value of 276 nM 42,43]. The K o value of [3H]mel~ramine-binding in ISMC was of the same magnitude as seen in DTT=MF 2 md BC3Hi cells. In Guinea pig TSMC, the binding ;ires were not reduced by homogenization, but the ~inding sites for DTTIMF 2 disappeared with homogerization [43]. A n o t h e r n o n - H t r e c e p t o r o f ~H]mepyramine-binding sites (K d value of 7.7 nM) vas noted in rat liver plasma membranes and that i: tad a high affinity for imidazole and imidazole-4-acetic

acid, both histamine metabolites [331. The '~i values of mepyramine, imidazole and histamine were 0.0049,13.3 and 120 ptM in rat liver plasma membranes, respectively. The K d values of mepyramine and the potencies of the histamine metabolites in inh~iting the mepyramine-binding in TSMC were dissimilar to those in rat liver. These findings indicate that the lower binding sites in Guinea-pig TSMC are close to those for DTTIMF 2 and different from those in liver plasma membranes. U46619 and histamine induced an influx of extracellular Ca 2+ in Guinea-pig TSMC. Histamine-induced increases in [CaZ+li in canine cultured airway smoothmuscle cells were not dependent on extracellular Ca 2+ [18], while U46619- and histamine-induced increases in [Ca2+]i in TSMC was dependent on the extracellular Ca 2+. KW-3635, SO 29548 ir.hibited the [3H]SQ 29548 binding to TSMC. KW-3635 and SO 29548 were also potent inhibitors of U46619-induced increase in [Ca" +]t- In contrast, the inhibitory activities of BM 13505 and BM13177 on the U46619-induced [Ca2+]i increase were weak in comparison with the K i values of other drugs for [3H]SQ 29548 binding. The maximum increase in [Ca2+]i with histamine was about 10-fold lower than that with U46619. The concentration of histamine which induced an increase in [Ca2+] i in canine TSMC [18], was similar to findings in Guinea-pig TSMC. The [Ca2+]i increase was induced by lower concentrations of histamine than that which inhibited the [3Hlmepyramine-binding in TSMC. The [CaZ+]i increase by histamine was potently inhibited by the H i antagonists. IC5o values of mepyramine, astemizole and diphenbydramine for the inhibition nf histamine-induced increase in [Ca 2+ ]i were comparable to K i values of these drugs for [3H]mepyramine-binding in tracheal membrane, but not to those in TSMC. This may indicate that only a small number of H t receptors with a high affinity for mepyramine were present in the TSMC but were not detected in the receptor-binding assay due to over-expressed low-affinity binding sites in TSMC. Another possibility is that the low-affinity mepyramine-binding sites could stimulate an increase in [Ca2+]i. Occupancy of a small fraction of the cell-surface-binding sites may be sufficient to trigger a maximal functional response, as was described in the case of the H= receptor in mouse brain slices [44]. The precise mechanisms involved in the increase in [ca2+]i by histamine in TSMC is under investigation. in conclusion, high aWmity of T X A z / P G H 2 receptors and low*affinity [3Hlnlepyramine-binding sites were detected in TSMC. Both T X A 2 agonist and histamine triggered the ligand-induced [Ca2+]i increase. These cells provide a useful system to investigate spasmogenic and hyperreactivity reactions caused by T X A 2 and histamine.

115 Acknowl, e d g m e n t s W e a r e g r a t e f u l t o M . S a n o a n d A . M i h a r a f o r cell c u l t u r e s , to E . O h s h i m a a n d H . O b a s e f o r s y n t h e s e s o f drugs, to T. Hirala and T, Oka for support for experim e n t s , t o M . O n o a n d N . O k u b o f o r t e c h n i c a l assislance and to M. Oharu and Z. Honda for helpful comments. References I Armour, C.L, Johnson. P.R.A.. Alfredson. M.L. and Black, J.L. (19891 Ear. J. PharmacoL 165, 215-222. 2 Svenssono J., Strandberg, K., Tuvemo. T. and Hamberg, M. (19771 Prostaglandins 14, 425--436. 3 Ahmed. F , Foster. ILW, Small, R.C. and Weston. A.H. (19841 Br. J. Pharmacol. 83. 227-233. 4 Drazen, J.M. Fanta, C.H. and Lacouture, P.G. (19831 Br. J. Pharmacol. 78, 687--691. 5 Katsuyama. H , Suzuki. S. and Nishiye. E. (19901 Br. J. Pharmacol. 100. 41~,8. 6 Arila. H., Nakano, T. ~n,'l H,naaaki, K. (19891 Psog. Lipid Res. 28, 273-301. 7 Lefort, J., Rotilio. D. and Vargaflig, B.B. (19841Br. J. Pharmacol. 82. 565-575. 8 Yoshimito. T . Yamamoto, S~ Okuma. M. and Hayaishi, O. (1977) J. Biol. Chem. 252. 5871-5874. 9 Lefer. A.M. and Darius. H. {19871 Fed. Proc. 46. 144-148. 10 Aizawa, H , Chang, ILF, Leikanf, G . D , Ueki. I., Belhel, R.A., O'Byrne, P.M., Hiros¢, T. and Nadel, J.A. {1985} J. Appl. Physiol. 59.1918-1923. I ! Fujimura. M~ Nishioka, S~ Kumabashiri. I.. Matsudao T.. and Mifune, J. 0990) Chest 98. 276-279. 12 Driver, A.G. and Mustafa, SJ. (19871 Eur. J. PharmacoL 139, 287-295. 13 Patel, K.IL (19871J. Allergy Clio. Immunol. 79, 355-358. 14 Gould, C A . L , Oilier, S. Aurich, R. and Davies, R.J. (1988) Br. J. Clio. Pharmaoa4.26, 515-525 15 Fukuo. K., Morimoto. S. Koh. E , Yukawa. S., Tsoohiya. H., !manaka, S. Y~mamoto, H . Onishi, T. and Kumahara, Y. (1986) Biochem. Biophys. Res. Commun. 136, 247-252, 16 Ta~uwa. Y . Takuwa, N. and Rasmus~sen, H. 0987) Am. J. Physiol. 253, C817-C827. 17 Hill, SJ. 0990) PharmacoL Rev. 42, 45-83. 18 Kotlikoff, M.I. Murray. R.K. and Reynolds, E.F.. (1987) Am. J. Physiol. 253, C561-C566. 19 Taylor, D.A. and Stull, $.T. (1988) J. Biol. Chem. 263. 1445614462.

20 Kotlikoff, M.I. (19881 Am. J. Physiol. 254, C'/93-C801. 21 Chamley-Campben. J.H.. Campbell. G.R. and Ross, R. (1979) Physiol. Rev. 59,1-61. 22 Devote-Carter, D., Morway V. P.F. and Weiss. E.B. (1988) Cell Tissue Res. 251. 325-331. 23 Karasawa, A, Taylor !11. P.A. and Lefer, A.M. (1990) Eur. J. Pharmacol. 182, I-8. 24 ishii, A., Miki, !.. Ohsbima. E. and Obase, H. (1990) Eur. J. Pharmacol. 183. 1555. 25 Miki. |. and Ishii. A. (199D Japan. J. Pharmacol. 55. Suppl. L 121. 26 Patscheke, H. and Stegmeier. K. (1984)Thromb. Res. 33, 277-288. 27 Bitterman, H.. Yanagisawa, A. and Lefer, A.M. (1986) Circ. Shook 20. I - I I . 28 Shiraishi, M.. Kato, K., Terao, S., Ashida, Y.. Terashita, 7_.. and Kito, G. (1989) J. Med. Chem. 32. 2 2 1 4 - ~ ! . 29 McPherson, G.A. (1985) J. Pharmacol. Methods 14. 213-228. 30 Grynkiewicz, G., Poenie. M. and Tsien. R.Y. (1985) J Biol. Chem. 260. 3440-3450. 31 Cheng. Y.-C. and Psosoff, W.H. (19731 Biochem. Phz,-macol. 22. 3099-3108. 32 Fukui. H . Mizuguchi. H., Liu, Y.U.. leaps, R., Kangawa, K.. Matsoo, H. and Wada, H.. (19901 Eur. J. Pharmacol. 183, 17271728. 33 Leurs, R., Bast, A. and Timmerman. H. (19901 Agents Actions 30, 157-160. 34 Hanasaki, K., Nakano, ~tL.Kasai. H. and Arita, H. ( 19891 Biochem. Pharmcol. 38, 2967-2976. 35 Featherstone, R.L., Robinson, C., Holgate, S.T. and Church. M.K. (1990) Nannyn-Schmiedeberg's Arch. Pharmacol. 541,439443. 36 Oglctree, M.L., Harris, D.N., Grcenberg, R., Haslangcr, M.F. and Nakane, M. (19851 J. Pharmacol. Exp. Ther. 234, 435-441. 37 Coleman. R.A., Humphrey, P.P.A., Kennedy. I.. Levy, G. P. and Lumley. P. (1981) Br. J. Pharmacol. 73, 773-778. 38 Hanasaki, K.. Nagasal~i. T. and Arita, H. (19891 Bioohem. Pharmacol. 38, 2007-2017. 39 Hanasaki, K. and Arita, H. (19881 Bioohem. Pharmacol. 37, 3923-3929. 40 Hirata, M., Hayashi, Y., Ushikubi, F., Yokota, Y., Kageyama. R~, Nakanishi, S. and Narumiya, S. (1991) Nature 349, 617-620. 41 Carswell, H. and Nahorski, S.R. (19821 Eur. J. Pharmacol. 810 301-307. 42 Mitsuhashi, M. and Payan, D.G. (1988) L Cell. Physiol. 134, 367-375. 43 Mitsohashi, M. and Payan, D.G. (19891 MOt. Phamtacol. 35. 311-318. 44 Quach, T.T., Duchemiu, A.-M., Rose, C. and Schwartz. J.-C. (1980) Mol. Phar,~acoL 17, 301-308.