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Leukotriene receptors in the skin of rats differ from those of mouse skin or rat stomach strip
1f 1992) X33-342 A 1992 Elsevier Science Publishers B.V. All rights reserved oOi4-2~9/92/$0~,()~
E~~r[~p~~r~Joi~r?lt~tof Ftiuri~l~~~~~fc~~,72
333
E3P 52662
Leukotriene receptors in the skin of rats differ from those of mouse skin or rat stomach strip Tom Miura, Naoki Inagaki, Shoichi Goto, Kenji Yoshida, Hiroichi Nagai and Akihide Koda Dcpartrrwt
t of Ptwrtnuccdogy~
Gift
Phannacchd
Ut~iwrsity. S-6- I Mimttorat~i~ast~i, Gifr 502. Japatr
Rt,eived 5 December 1991. revised MS received P,7 June 1992. accepted 30 June f9Y2
To compare the reccpfors for cystcinyl-leuk~~triene (cys-LT) in rat skin with those in other tissues. we investigated the effects and L-648051) on cys-f T-induced cutaneous reactions of specific cys-LT receptor antagonists (FPL 55712. LY 171883, MCI-826 in rats and mice, and on cys-LT-induced contractile responses in rat stomach smooth muscle. WC .~lso studied the effects of thcsc drugs irii h~molago~s pass& cutaneous anaphylaxis. The four cys-LT receptor antagonists dose dependently inhibited cys-LT-induced cutaneous reactions in mouse car, but failed to inhibit passive cutaneous anaphylaxis and the histamine-induced cutaneous reaction. In rats, only MCI-826 inhibited cys-LT-induced ~ut:~neous reactions although the other three drugs failed to inhibit these reactions. In contrast, the ~~-LT-induced contractile n_sjjcnses of rat stomach smooth muscle were inhibited by alf thcsc drugs in a concentration-dependent manner. Thcsc results suggest that cys-LT rcccptors in rat skin have an affinity diffcrcnt from that of rcccptors in mouse skin and rat stomach. They also suggest that cys-LTs arc not involved in passive cutaneous anaphylaxis in mice and rats. Cystcinyl-leukotricnc;
Cystcinyl-lcukotrienc
rcccptor antagonists; Cutaneous anaphylaxis (passive!; (Rat); tMousef
1. Introduction Cysteinyl-leukotrienes including (cys-LTs), leukotriene (LT) C,, LTD, and LTE, are chemical mediators that are metabolitcs of the 5-lipo~genase pathway of arachidonic acid and cause potent and long-lasting contraction of smooth muscles (Samuelsson, 1983). Cys-LTs are also known to cause an increase in vascular permeability (Ueno et al., 1981; Welton et al., 1981) and changes in blood pressure (SmedegHrd et al., 1982). Recently, it has been suggested that cys-LT might play very important roles in several kinds of diseases, especially allergy (Schwartz and Austen, 1982; Kaliner, 1985). Studies of specific antagonists against cys-LTs have therefore been actively undertaken. Sodium 7-[3-14-acetyl-3-hydroxy-2propylphenoxy~-2-hydroxypropyi]4-oxo-8-propyl-4H-lbenzopyran-2-carboxylacetate (FPL 55712) (Augsten et al., 1973) is one of the earliest discovered cys-LT receptor antagonists and has very often been used as a standard cbmpound in the development of anti-cys-LT drugs. it was reported that FPL 55712 inhibited LTC,-
Cifu Pharmaceutical University. S-6-1 Mitehor;~tlig;lshi,Cil’u 502, Japan. Tel. Xl.54X37-3931,fax 8 I.5X2-37-5979. Correspondence to: T. Miura, Department of Plwmacoltrgy,
and LTD.+-induced contractions of human airway (Buckner et al., 1986) and LTD,-induced contraction of guinea pig trachea (Snyder and Krcll. 1984). However, it was also reported that the agent inhibited neither LTC,-induced contraction of guinea pig trachea (Snyder and Krell, 1984) nor LTD~-induced contraction of guinea pig heart and cat gall bladder (Feniuk et al., 1984). This evidence suggests the cxistcnce of subtypes of receptors for cys-LTs in several mammalian tissues. Recently, we found that FPL 55712 did not inhibit the LTC~-induced cutaneous reaction in rats (Koda et al., 1990). In this study, we compared the response of rat skin against cys-LTs with responses of the mouse skin and rat stomach smooth muscle, and investigated the effects of the specific cys-LT antagonists, FPL 55712, 1-~2-hydroxy-3-propyl-4-~4-( I H-tctrazol-~-yl)butoxyl phenyl] ethanone (LYl7~~~3) (Flcisch et al., 19%). (E)-2,2-diethyl-3’-[2-[2-(4-isopropyl)thiazolyl]ethcnyl~ succinanilic acid (MCI-8261 (Takahashi et al., 1987) and sodium 4-[3-(4-acetyl-3-hydroxy-Zpropylphenoxy)propylsulfonyl-~-ox~-benzeneb~tanoaie IL-648051 1 (Jones ct al.. 19BG. on cys-LT-induced cutaneo~ls reactions in rats and mice and contractile responses in rat stomach smooth muscle. In addition, the effects of these drL!gd on homologous passive cutaneous anaphylaxis were iwxstigatcd. ‘i hc role of cys-LTs is also discussed.
passive cutaneous
anaphylaxis,
30-fold
diluted
mouse
antiserum was injected in a volume of 10 ~1 into both ears under cthcr anesthesia. After 48 h, the mice were challenged Female BALB/c mice weighing ahrut 2tt g and female Wistar rats weighing about 150 g were used for preparing antisera. In other experiments, male ddY mice weighing about 30 g and male Wistar rats weighing about 250 g were used throughout. All animals were purchased Japan 1.
from Japan
SLC.
Inc. (Hamamatsu.
with an intravenous
injection
of 0.25 ml of
0.5% Evans blue dye saline solution containing
0.25 mg
of dinitrophcnylatcd bovine serum albumin. In mediator-induced cutaneous reactions, 10 r.tl of mediator solution
(2 x lo-
LTC, or 5 x IO-” cars under ether
g/ml
histamine,
g/ml LTD,) anesthesia.
5 x lo-”
g/ml
was injected into both Immediately after the
injection, mice received an intravenous injection of 0.25 ml of 0.5% Evans blue dye saline solution.
mediator
Thirty min after the injection of dye solution, the mice Dinitrophenylated
Ascoris SIII~II~ extract and dinitro-
phenylated bovine serum albumin were used as antigens. Asruris SIII~III estract prepared according to the method of Strejan and Campbell (1968) and bovine serum albumin were dinitrophenylatcd according to the method of Eisen et al. (1953). Dinitrophenylated .-tst~ris SIIII~I extract was used for preparing antisera, and dinitrophcnylatcd bovine serum albumin served as the antigen for *a _liciting passive cutaneous anaphylaxis.
were killed by cervical dislocation removed. The pair of ears obtained
and their ears were
from
dissolved in 0.7 ml of I N KOH
each mouse was
solution at 37 “C
overnight and 9.3 ml of extraction solvent, a mixture of 0.6 N H3P0, After
solution and acetone (5: 13), was added.
vigorous shaking, the precipitates
off and the amount tally at 620 nm.
of dye was measured
were filtered colorimetri-
2.6. Cutarwous reactiorts it1 rats Asrtrris swat extract Antisera to dinitrophenylated in mouse and rat were prepared according to the methods of Levine and Vaz (1970) and of Tada and Okumura (1971). respectively. The titers of the both preparations measured by 48-h homologous passive cutaneous anaphylaxis were I : 5 12.
Six treatment points were marked on the backs of rats shaved I day before. In concentration-response studies of mediators, 0.1 ml of physiological saline was injected intradermally into one point as a control, and mediator solutions were injected in the order diluted into the other five points. Immediately after the mediator injections, I ml of 0.5% Evans blue dye saline solution was injected intravenously.
FPL 55712 (MW: 56s)). LY 1718X3 (MW: 318). MCIS26 (MW: 122.5) and L-64X051 (MW: 49X) were donated by Fisons Pharmaceutical Co. Ltd. (Leicestershire. U.K.). Shionogi Pharmaceutical Co. Ltd. iOsaka; Japan). Mitsubishi-kasei Co, Ltd. (Tokyo. Japan) and Merck Frosst Canada, Inc. (Quebec. Canada), respcctively. LY 171X83 was dissolved in saline containing 0.05 M NaHCO, in saline.
and the other compounds
were dissolved
into one of the six points as a control, and the same volume of chemical mediator solution (lo-’ g/ml histamine 3 x IO -” g/ml LTC, or 3 x IO-" g/ml LTD,) was injected intradermalty into the second point. The mediator solution containing an antagonist was injected into the other four points. Immediately after this 1 ml of 0.5% Evans blue saline solution was injected intravenously.
Histamine tdihydrochloride: Nacalai Tesque. Inc., Kyoto. Japan), scrotonin (creatininc sulfate: Merck, NJ. USA). LTC,. LTD,. LTE, IWako Pure Chemical Industries. Ltd.. Osaka, Japan) and acetylcholine tchloride: Nacalai Tesque. Inc.) were used as mediators for eliciting cutaneous reaction and smooth muscle contraction. All mediators were prepared in saline. 2.X Cutaticou~ rcactiorls iti momc
To examine the effects of locally administered antagonists, 0.1 ml of vehicle was injected intradermally
ear
When passive cutaneous anaphylaxis and cutaneous reactions by mediators were induced simultaneously in the same rats (Koda et al., 1990), 0.1 ml of saline was injected intradermally into one point as a control for passive cutaneous anaphylaxis, and 40-fold diluted rat antiserum was injected into the second point. After 48 h, 0.1 ml each of saline as a control for cutaneous reactions, IO-’ g/ml histamine, 3 x IO-” g/ml LTC, and 3 x IO-” g/ml dermally
Passive cutaneous anaphylaxis and mediator-induced cutaneous reactions were elicited in ddY mice as reported hy lnagaki et al. (1986). Briefiy. in the case of
LTDj
solutions were injected intra-
into the 3rd, 4th, 5th and 6th points, respec-
tively., Then, 0.5 ml of I% Evans blue dye solution containing 1 mg of dinitrophenylated bovine serum albumin was injected intravenously.
In all experiments, the rats were killed by bleeding 30 min after the reactions were induced, and the skin containing each reaction spot was excised. The amount of dye leaked within the reaction. sites was measured according to the same method as used in mice except that the volumes of IN KOH and extraction solvent were 1 ml and 8.3 ml, respectively. Each group consisted of six rats, and injection orders were rotated in consideration of biases in reactivity among the six spots.
dye
Amount of
(~g;
20 -
10 -
o-
2.7. Contractile response of smooth muscle strips of rat stomach Rat stomach smooth muscle strips were prepared according to the method of Offermeier and Ariens (1966). After killing the rat by bleeding, the stomach fundus was removed and carefully cleaned with Tyrode solution at 31°C. The fundus was halved by careful cutting in the direction of the longitudinal muscle fibres. Alongside this incision, two strips about 3 mm wide were taken, one from each half cf the fundus. Each strip was placed in a 5-ml Magnus bath and suspended in Tyrode solution. After a l-h equi!ibration period, responses were recorded isotonically with an initial load of 0.3 g. Cumulative concentration-response curves for each mediator were made for the tissue by successive increases in the bath concentration of the reagent. For testing the effects of antagonists on LTC,and LTD,-induced contractile responses, one of the two strips was used for LTC,, and the other for LTD,. The curve for IO- "'-lo-'g/ml LTC, or LTD, was generated twice, and the antagonist (or saline as a control) was added into the organ bath 5 min before the second curve was made. There was no tachyphylaxis observed against LTC, or LTD, in the second response. The data were expressed as a percentage of the maximum contraction obtained at the first response. pAz values were calculated according to the method of Arunlakshana and Schild (1959).
20 r
10 -
OI
I
9
Concentration
Fig. I. lncreasz LTD.,
in vascular
and LTE,
,
7
8
6
of mediator
permeabihy
in mice (upper)
tions were injected
5
4
(-log.
inducc~
3
LIJ
ti$ histamine.
and rats (lower).
Mediator
!.TC;.
solu-
into the mouse ear and the rat dorsal skin just
before injecting Evans blue dye solution. Each point and vertical bar represents
the
mean
and S.E.M.
Histamine.
�
LTC,.
of S-10 A LTD,.
mice �
and
12 rats.
o
LTE,.
After confirmation of the significance of differences (P < 0.05) by Kruskal-Wallis’ analysis, the differences between individual pairs of means were analyzed using Williams-Wilcoxon’s analysis.
3. Results 2.8. Statistical arlalysis 3.1. Increase k lqascular permeability induced by cys-LTs The data are reported as means and S E.M. Results for cutaneous reactions were calculated after subtraction of the corresponding control value from the individual value. Multiple comparisons were made for statistical analysis. When uniformity of variances in the data was identified by Bartrett’s analysis (P < 0.051, I-way analysis of variance was used to test for statistical differences. Significant differences (P < 0.05) were identified, after which the data were further analyzed for significant dtfferences between individual pairs of means using Duncan’s multiple range test. If uniformity was not demonstrated in the variances of the data, multiple comparisons were made non-parametrically.
The activities of three kinds of cys-LTs to increase vascular permeability in mice and rats were examined and compared with that of histamine (fig. 1). In mice, the cutaneous reactions induced by histamine, LTC, and LTD, increased concentration dependently and their intensity was in the order: LTC, > LTD, > histamine, but LTE, failed to increase vascular pcrmeability even at a concentration of 2.28 X lo-’ M. In rats, LTC, and LTD, also caused a concentration-dependent increase in vascular permeability and were stronger than histamine. The cutaneous reaction to LTE., was observed only at a concentration of 2.28 X
33fi TABLE I Effects of FPL 55712. LYI71!!!!3. MCI-!!2fi and L-M8051 on cutaneous reactions induced by antigen. histamine. LTC 4 and LTD4 in mice. Drug"
Control FPL 55712
Dose mg/kg
1 2 5
Control LYI7IS!!3 3
10 Control MCI-S2fi
IWI 0.1 I
Control L-MS051
Amount n of dye J.Lg (% inhibition) Passive cutaneous anaphylaxis
Ilistamine
LTC 4
2!!.M±2.99 29.29±3.47 (-2.3) 24.1l5 ± 2.5fi (13.2) 21.fiH 1.!!7 (24.3)
12'<)( ±0.43
\3.59± 1.7!! S.O!!± 1.39 c 7.33±0.77 c 4.fi3±0.51 d
(40.5) (4fi.1) (65.9)
14.40± 1.84 13.38± 1.30 (7.0 1O.72±O.70 (25.6) 7.94 ± 0.95 d (44.9)
22.40± 2.44 23.9fi ± :1.3S ( -7.0) (S.fi) 20.4!! ± 2.21 (1.5) 22.0fi±4.94
7.5S± 1.44
17.22±2.lfi 15.M± 1.3fi \3.43±2.45 fi.32±0.fiO
(9.2) (22.0) (63.3)
13.50±0.40 13.IH 1.42 (2.4) 11.37 ± 0.88 (I5.8) 8.31 ±0.71 d (38.4)
4.2HO.70 5.21 ± I.fi9 ( -22.0) (15.0) 3.fi3±0.84 1.5!!±0.2!! ,. (63.0)
6.16±0.96 4.1 t± 0.52 (33.3) 5.3HO.79 (12.8) 2.3HO.71 d (fiI.5)
10
ROfi ± 1.23 ( - 6.3)
2!!.M±2.99 (9.0) 2fi.07 ± 3.3fi 34.fiS ± 3.57 ( - 21.1) 23.3S±4.23 (1S.4) 2S.M±2.99 24.21 ± 1.74 24.13 ± 2.01 23.3fi±3.23
3
10.74 ± 1.09 ( 10.6)
!!.06± 1.47
7.90±0.98
(2.3)
12.01 ±0.43 (15.5) (t5.7) (l8.4)
14.06± 1.07 14.06± 1.33 13.91 ± 1.19 9.64± 1.26
1O.2HO.77 (14.5)
Drugs were administered intravenously just before the reactions were elicited. mice. ,. P < 0.05. d P < lUll. II
10- 5 M. the intensity was thus weaker than that of the other mediators.
3.2. Effects of cys-L T antagonists on cutaneous reactions in mice The effects of FPL 55712, LY171883, MCI-826 and L-648051 on passive cutaneous anaphylaxis and cuta neous reactions induced by histamine, LTC 4 and LTD4
I>
LTD4
d
(0)
(1.1) c
(31.4)
12.16±0.S5 11.21 ± 1.60 1O.27± 1.60 6.32 ± 1.06
d
(7.S) (15.5) (4!!.0)
Each value represents the mean and S.E.M. for seven to nine
in mouse ear were investigated. Table 1 shows the results of these drugs administered intravenously im mediately before eliciting the reaction. All drugs signif icantly inhibited LTC 4- and LTD4-induced cutaneous reactions in a dose-dependent manner. MCI-826 proved to be the most potent of the four drugs. However, these drugs had no effect on passive cutaneous anaphy laxis and histamine-induced cutaneous reaction. The effects of local administration of these drugs on
TABLE 2 Effects (If FPL 55712. LYI7IS!!3. MCI-!!2fi and L-MS051 on cutaneous reactions induced by histamine. LTC 4 and LTD4 in mice. Drug
II
Control FPL 55712
Control LYI71!!!!3
Cone. -Ing. glml
Amount
I>
of dye J.Lg (% inhihition)
Ilistamine
LTC 4
13.59±2.M fi 5 4
IS.96±2.73 (-39.5) 13.59± 2.64
fi
5 4 Control MCI-!!2fi
Control L-M!!051
15.49 ± 2.97 ( - 14.0) 13.59±2.M
fi 5 4
11.54 ± 2.48
(l5.])
13.59±2.M fi 5 4
12.9fi±2.69
(4.6)
II Drugs were mixed with mediator and injected intradcrmally. " P < (1.01.
I>
LTD4
5.06± 1.50 1.74± 0.22 3.32± 1.26 2.37±0.79
(65.6) (34.4) (53.2)
10.11 ±2.34 8.06± 1.55 fi.32±2.02 3.32± 1.01
(20.3) 07.5) (67.2)
11.21 ± 1.53 9.4!!± 1.39 6.95 ± 1.12 3.95 ± 1.14
(15.4) (3S.O) (64.S)
16.59±2.54 15.1fi±2.21 12.95 ± 1.52 9.4!!±0.68
(S.6) (21.9) (42.9)
6.16±0.77 8.8Si 1.04 ( - 43.7) 5.06± 1.56 (17.9) 1.2H 0.95 d (79.4)
S.37±O.96 9.00 ± 1.98 6.47± 1.39 2.84 ± 0.93
( -7.5) (22.7) (66.1)
5.06± 1.50 3.32±0.71 3.16± I.M l.74±0.70
C
d
(34.4) 07.5) (65.6)
10.11 ±2.34 9.64 ±2.53 6.oo± 1.26 4.42 ± 1.56
C
(4.6) (40.7) (56.3)
Each value represents the mean and S.E.M. for six or seven mice.
C
P < 0.05.
337
TABLE 3 Effects of FPL 55712, LYi7iX83, MCI-826 and L+64iWl on cutaneous reactions induced by antigen. histamine. LTC, and LTD, in rats. Drug a
Control FPL 55712
Dose me/k
Arn~~unt* of dye pg <% inhibition)
0.3
1 3
Chmrl LY1718X3
Control MCI-826
I 3 10 O.lil 0.1
1 Control L-h4HOSI
1 3 10
Passive cutaneous ana~hyiaxis
histamine
LTC,
LTD,
26.30 & 6.82 28.44_t3.36 (-8.1) 25.93 & I .94 (1.4) 12.5Xf 4.40 (52.23
iS.25t I.54 15.93Lt:il.92 (-45) i3.WXfti.91 (9.0) il.81 f 1.62 (22.6)
13.9s rf:I .xs 14.X7kO.69 (-6.6) 15.59~0.98(-11.X) 15.~~~0.~3 (-7.71
itl.hc)* I.23 13.142tO.67 (- 22.9) 11.03~0.83 i-3.2) 12.04&0.73 I- 12.61
29.92 -r_4.67 31.19*s.37 (-4.2) 31.32&2.?6 (-4.7) 25.11 k4.49 (ih.i)
19.962 1.70 20.74 f 1.hS t - 3.Y) 25.00 *I .OS( - 25.3) 22.14k3.07 i - 10.9)
l&69* 1.47 17.2xlt 1.39 f-3.5) 17.82-f 1*OS t - 6.8) ih.22I 1.73 (2.X)
II.271 I.22 1X62& 1.30 i - 12.0) i3.34 &fl.X(i f - 1X.4) 13.181 I.55 i - 16.9)
19.09-r_3.30 17.45 * 3.37 W) 23.13 e ?.tlOf - 21.2) 17.04 i 4.29 f lfl.7i
24.70+ I.12 22.81 * 1.68 (7.71 23.57 f 2.70 (4&f 24.02 & 3.73 i -0.9)
l&54* 1.3s 13.63rt i.tui (17.6) 13.67a 1.77 (17.4) 7.91+ 11.76c 62.2)
13.54f 0.96 lZ.lhlt: I.16 ( 10.2I ff.llh* 1.15 fiX.3l 7.91 *ti.fiJ E (41.61
26.ti8 $z3.21 23.87 jl4.07 I x.79 & 3.37
28.94 & I A:! 34.45 f 2.52 ( - 19.0) 30.13k2.79 (-4.1) 28.73 f I .73 in.71
20.41+ 2.22 22.36 & 1.25 ( - 9.6) 2l.hOk 1.22 (-S.8) i9.ssz5:2.17 (4.2)
18.14*0.94 10.09~ I.44 ( - i0.h) IX.25& 1.29 ( -0.h) 17.17* I.61 (5.3)
It%3 ,+ 3.15
’ Drugs were administered c P < 0.01.
( 10.5) (29.h) (37.7)
int~ven{3usiy just before the reactions were elicited. ’ Each value represents the mean and S.E.M. fitr six rats.
mediator-induced cutaneous reactions are shown in table 2. The drugs were mixed with mediator solutions and injected ~ntradermaliy. All drugs ~~h~bjted LTC4and LT~~-induced cutaneous reactions in a ~oncentration-dependent manner. The inhibition was significant at concentrations of lo-’ and lo-’ g/mi in LY1718X3
on LTC,-induced cutaneous reaction and at a concentration of NJ-” g/ml in ~Cl-82~ on both reactions. The percent i~b~b~tion~of IO-’ g/ml FI?L 55712 and L-6~051 on LTC, anti LT~~-induced reactions were greater than 50% although the inhibition was not statistically significant. However, these four drugs did not
TABLE 4 Effects of WC 55712. LYl71tW. MCi-~2~ and t_-~4~ilSl on cutane~~us re~tcti~~nsinduced by his~mine, LTCJ and LTI), in r&s. Drug ”
Control FPL 55712
Cont. -log. s/ml
Amn:mt ” of dye pg (‘;j. inhibition) -_ LTC, Histamine
7 6 s 4
is.23 f I .Y2 17.hOt 1.80 t - iS.hf IS.98 * ff*?h f - 4.9) 14.21,* ii.96 fh.lf i3.2Xk I.70 (12.X)
LTD,
is.ss* 1x.47* I.61 (-25.7) lfi.S2& i.S2(- iZ.Sf
i2.42+ I.11 12.1nte.4s 12.74&0.71) i2.xs *fl.Xtl ii.77i 1.03
(2.h) t - 2.6) t ,:;: . ._
19.76_+157 ih.OYf I .3J 16.42 + 2. i I 17.39+0.X7 211.2t(1* Il.67
(it&% i ih.Yf f i3.Of ( - 2.2)
14.6Yk I.37 ih.lf*
I.44 f- 10.3) I.13 f -5%
Control LY 171883
24.52 f I .49 23.44 f I .43 23.54+ Lib 22.36 f I .4Y 28.62 & I .s7 f -
(4.4) (4.0) tS.81 16.7)
2l.hO~2.91 (2.5) 2 1.Oh* 1hh 21.0,,~2.‘33 1- i.S) 23.54 f 2.hl ( - 9.0) X&14+2.21 (-2l.fii
Control MCI-WY
19.12+ I.11 i7.60+ 2.08 18.14&“O.h9 19.01_t(1,9s 1x.79* 1.36
(7.9) (5.1) (O.hf (1.7)
is.23 + 0.9S Ui.7) 15.12*0.x4 (2.X) 14J!o*ti.91 ls.hh+o.3X f -2.81 3.78* f).SXrt (75.2)
Wh _t0.39 Y.hl+l.?X (--KS) 9.50& 1.33 (- 7.2) IX.hl X.iOji*li 2.4x + (3.48(’ (72.0)
13.39*:0.93 iX.i4f2.37~-35.5f 18.58+.2.47 ( - 3X.X) 20.20 rt i.76 ( - 50‘9)
12.85t WI4 13.2X~rO.69 (-3.3) i3.39* 1.no (-4.2) i4,yo* 1.62 t - lh.0)
17.42&il.82 12.53f 1.07 I-0.9) ( - 0.9) 12.53_t0.9I lS.SSf. I.21 c t-25.2)
Control L-h4WS I
h 5 4
” Drugs were mixed with medi~tl)r and injroted intmd~r~l~y.
” E;&
v&g
r~~~sents the mean and S.E.M. for six rats,
’ P < tt.OS,’ P < tl.tii.
IOO-
80 -
AS shown in table 3, MCI-826 significantly inhibited LTC,- and LTD,-induced cutaneous reactions at a dose of I mg/kg when administered intravenously immed~tely before eliciting of the reactions, but did not affect passive cutaneous anaphylaxic and histamine-induced cutaneous reaction elicited simultaneously in the ume rats. The other three drugs did not yield any significant inhibition in any of the cutaneous reactions at doses sufficient to inhibit cys-LT-induced cutaneous reactions in mouse ear. Table 4 shows the effects of loeat administration of these drugs on histamine-. LTC,- and LTD,-induced cutaneous reactions in rats. FPL 55712 and LYl71883 did not inhibit any of these cutaneous reactions. MCI-826 significantly inhibited LTC,- and LTD,-induced cutaneous reactions at a ~n~entration of IO-’ g/ml. but did not affect histamine-induced cutaneous reaction. L-648U51 dose dependently enhanced all reactions tested.
A> the first step in evaluating the effects of cys-LT receptor antagonists on .cys-LT-induced contractile responses in rat stomach, WC studied the relationships between the concentration of various mediators and the intenstty of the contractile responses. LTC, and LTD, caused concentration-dependent contractions and their potencies at lower concentrations were greater than those of ?.cetylcholine and serotonin. AIthough LTE, caused contraction in a concentrationdependent manner, the intensity was weaker than those of LTCJ and LTD,, histamine also caused cont~ction at high concentrations (over 9 x lo-’ M), but the contraction induced by 9 x IO-’ M histamine was only about 20% of that produced by acetylcholine (fig. 2). Figure 3 shows the results of FPL 55712, LY171883, MCI-826 and L-6480,51 on LTC,-induced contractile response in rat stomach. All drugs inhibited LTC,-induced contraction in a concentration-dependent manner. The intensities were in the following order: MCI826 (PA, 7.81) > FPL 55712f6.86) > L-648051 (6.38) > LY I71883 (6.28). The slopes calculated by Schild analysis for LY 171883 and L-64805 1 were close to 1 (LY 17 18% 0.96 and L-648OSl: 0.84) and showed competitive antagonism against LTCJ. FPL 55712 and MCI-826 however, antagonized the contraction non-competitive!y as the slopes were not close to 1 (FPL 55712: 0.59 and MCI-826 0.65). All these drugs also inhibited LTI),-induced contraction in a concentration-depen-
(10 -
40 -
20 -
oIO
a
7
Gmccntr8tion
of
9
6
mediator
5
t-log.
3
4
I41
Fig. 1. Dose-response curves of contractile responses induced by acetylcholine, LTC,, LTD4. LTE,. histamine and serotonin in isolated stomach smooth muscle of rats. Each point represents a percentage of the maximum contraction obtained with acetylchoiine. 0 Acrtylcholine. 0 LTC,. A LTD.,. A LTE,, 0 histamine. � serotonin.
dent manner (fig. 4). The intensities wcrc in the following order: MCI-826 (pA2: 7.61) > LY 171883 (6.36) > L-648051 (6.20) > FPL 55712t6.14). The slopes for all drugs were close to 1 (FPL 55712: 1.06, LY171883: 0.94, MCI-826: 1.03 and L-648051: 1.09) and contractions of rat stomach induced by LTDj were inhibited competitively, None of the drugs had any effects on contractile response induced by lo-’ g/ml acetylcholine at the highest concentrations studied here (MCI-826: IO-’ g/ml and FPL 55712, LYl71883 and L-648051: IO-” g/ml).
4. Discussion In the present study we first compared the sensitivity of mouse ear and rat dosal skin against cys-LTs. LTC, and LTD, caused dose-dependent increases in vascular permeability to almost the same extent. These cys-LTs were more potent than histamine in both species. LTE,. however, had little or no effect on vascular permeability in either mice or rats. Although it is generally known that LTE, causes an increase in vascular permeability as well as contraction of smooth muscle (Welton et al., 1981j, the activity in mice and rats might be less than that of LTC, or LTD,. In this
339
study, therefore, the effects of four anti-cys-LT drugs on cutaneous reactions in mice and rats induced by cys-LTs except LTE, were investigated. All anti-cys-LT drugs dose dep~~dentiy inhibited LTC,- and LTR~-induced cutaneous reactions in mouse ear when administered intravenously immediately before the reaction. In rat dorsal skin, although MCI-826 inhibited LTC,- and LTD,-induced cutaneous reactions, the other three drugs had no effect on these reactions at doses sufficient for inhibiting them in mice. Therefore, we investigated the effects of these drugs injected intradermally with mediator since it was possible that sufficient drugs may not have reached the reaction sites in rats. However, FPL 55712, LY171883 and L-648051 showed no effect on cys-LT-induced reactions in rat dorsal skin when administered locally as well as systemically. These three drugs did not affect LTC,-induced cutaneous reaction in rat ear at a con-
FI’L 100
centration of 10-j g/ml (data not shown), suggesting that the low efficacy of these drugs in rats was independent of the site on the skin. In addition, none of the drugs inhibited histamine-induced cutaneous reaction in either mice or rats. These results indicate that the inhibitory action of anti-cys-LT drugs on cys-LT-induced cutaneous reactions in mice and rats is quite different, suggesting that cys-LTs cause a vascular permeability increase in mice and rats through different types of cys-LT receptors. Cys-LT receptors of guinea pig skin may be similar to those of mice and different from those of rats since it is reported that FPL 55712 and LY 171883 inhibit LTD,-induced cutaneous reactions in guinea pigs (Fleisch et al., 1985; Krell et al., 1989). Recently, Gardiner et al. (1991) have proposed that cys-LT receptors can be classified into two large groups, LT, and LT,, depending on the action of some cys-LT antagonists LT, receptors are predominantly
LY171883
55712
Schild
analysis
Schild
analysis
Slope:
0.59
Slope:
0.96
P
SD
c :
0
.-I Y :: :
l-648051
MCI-826
z u 100
Schild
analysis
Srhild
aoalyeis
Slope:
0.65
Slope:
0.84
Pk.2
:
7.81
50
0
Concentration
of LTG
(-log.
g/ml1
Fig 7 Effects of FPL 55712, LY171RS3. MCI-826 and L-64X051 on LTC,-induced contructile responsc~ in isnlated stomach smooth muscle of r&.‘i..TC, was added IO the organ bath cumulatively. Drugs were added 5 min hefore the addition of LTCJ. Each point and vertical bar , calculated hy Schild analysis. Drug represents the mczn 2nd S.E.M, of 4-!tt experiments. Each value in the figure represents lhe slope and pA_ IO ’ gfmi. concentration; 0 control, 0 10 -” g/ml, d IO-’ g/ml. A IO- ’ g/ml. � 10 ” g/ml.
both LTD,
and LTE,
on guinea pig ileum
ated
responses
(Garuiner
et al..
1991).
FPL
55712,
al.. IWO) and trachea (Hand et al., 1989) c cys-LTs on guinea pig gall bladder (Fa;Lllexmcand Krell. I991 ) and human bronchial muscle
LY 171883 and L-648051 may belong to the LT, type antagonists because these drugs antagonize LTD.,-, but not LTC,-. induced contraction in guinea pig trachea
(Buckner et al.. IWhI. and are antagonized by most of the existing cys-LT antagonists (e.g. lCll986lS (Snyder
(Hand
et al.. 198> !Krcll et al.. 1987). SK&Flo4!3S3 (Hay et al.. 19S7) and MK.571 (Jones et al., 1989). which are
tion of guinea pig trachea induced by LTDJ (Takahashi et al., 1987) and of human bronchial muscle induced by
novel. wtent and selective cys-LT antagonists). LT, receptors, however. are predominantly activated by
three
LTC,
on guinea pig ileum (Gardincr
ct al.. 1990) and
et al..
1989).
responses through
cys-LTs
LT,
(Habe
MCI-826
also antagonizes
the
receptors, such as the contrac-
et al..
1987).
Furthermore,
we
found that the contraction of guinea pig ileum induced by IO-’ g/ml LTD, was almost completely inhibited by
trachea (Hand et al.. I9S9) and by three cys-LTs on ferret spleen. ferret trachea. sheep ainvay (Cuthbert et
IO-’
g/ml
Iti-’
g/ml LTC,
at.. WI) and human pulmonary vein (Brink and Gardiner. KIWI). but are not antagonized by the above mentioned antagonists. BAYu9773 is the first compound to antagonize both LT, and LT, receptor-medi-
MCI-826 in the presence of L-serine borate (data not shown), suggesting that MCI-826 had no effect on LT,
FPL 55712
LY171883
Schild
analysis
Schild
analysis
Slopr:
I.06
Slope:
0.94
nnalysis
Schild
analysis
Slope:
I.03 -
Slope:
1.09
L
,
9
a
Conccntraiion
ml%. LTDJ
of FPL Wl2.
LYl71XX3.
MCI-X%
by
g/ml
: 7.61
IO
4. Effect*
induced
by even IO-’
L-648051
Schild
PA2
but the contraction
was not inhibited
receptors. These findings lead us to believe that cys-LT receptors in mouse skin (probably guinea pig skin)
MCI -826
Fk
MCI-826
and L-h48tlSi
was added m the organ bath cumulatively.
7
oi
LTD.
on LTD.,-induced
Drugs were added
(-lqs.
g/ml)
contractile
5 min heforc
responses in isoiuted stomach smooth muscle of
the addition
of LTD,.
Each point and vertical
rcPrcscnt!+ the mean and S.E.M. of four to five experiments. Each value in the figure represents the slope and pA, calculated Drug cone-ntratkn; I: ccm!r::!. G t!) ” g/m!. A it) en g/ml, A tt! ~’ g/ml, � 1[)~-”g/ml, 8 1()-y g/ml.
har
hy Schild analysis.
331
belong to the LT, type, but the type of cys-LT reccptors in rat skin is not clear. Since MCI-826 is not a hydro~acetophenone derivative, unlike the other three drugs used in this study, the inhibitory action on rat cys-LT-induced cutaneous reactions may be related to the difference in their chemical structures, In addition, the report of Metters et al. (1991) concerned with cys-LT receptors is very interesting. These authors indicated that cys-LTs and SK&Fl~4353 compete for [‘HILTD, binding in rat lung with the same potency as they compete for [3H]LTD, binding in guinea pig lung. MK571 and ICI198615, however, compete for the receptors in the rat with less potency than they compete for those of guinea pig. These results suggest that more multiple receptors for cys-LTs exist in these tissues. We need to study the effects of other characteristic cys-LT antagonists such as BAYu9773, lC1198615, SK &F104353 and MK571 on our experimental models. Secondly, in order to confirm whether the low efficacy of anti-cys-LT drugs as seen in rat skin was characteristic only for the tissue, we investigated the effect of these drugs on the contractile response induced by cys-LTs in rat smooth muscle. Since it is reported that rat stomach and colon, but not duodenum and ileum (Goldenberg and Subers, 19821, are contracted by LTCJ and LTD,, we selected stomach smooth muscfe for the present study. LTCj and LTRj caused the tissue to contract in a concentration-depcndent manner and found a submaximal contraction at a concentration of IO-’ g/ml 11.6 x 1Uw7M LTC, and 2 x 10e7 M LTD,). The contractile response was observed at con~ntrations of cys-LTs lower than those needed to Cause the increase in vascular permeability, suggesting that, in rats, stomach tissue may bc more sensitive to cys-LT than skin. Though LTE, also contracted stomach strips in a concentration-dependent manner, the activity was weaker than that of LTC, or LTD,. On the other hand, histamine produced little contraction of stomach tissue. It is known that histamine H , and H 2 receptors are present in rat stomach smooth muscle, and that histamine contracts them through H, receptors and relaxes them through H, receptors (Ercan and Tiirker, 19771. The four anti-cys-LT drugs all inhibited the contraction induced by LTC, and LTD, in a concentrationdependent manner. However, these drugs had no effect on acetylcholine-induced contractile response, suggesting that the inhibitory activity is cys-LT specific. The slopes of the four anti-cys-LT drugs, as dctermined by Schild analysis, were close to 1 in LTD,-induced contraction, and the inhibitory patterns were competitive. These results suggest strongly that the contraction induced by LTD, is activated through LTt receptors. In LTC,-induced contraction, the SIOPCSof LY 171883 and L-648051 were aiso close to 1. but those of FPL 55712 and MCI-826 varied considerably from 1,
suggesting that another type of cys-LT receptors may be concerned with the contraction induced by LTC,. In the present study, however, since we examined the effects of these drugs in the absence of an inhibitor of ~-g~uta~yl transferase, which metabolizes LTC, to LTD, (Orning and ~ammarstr~m, 19801, it is possible that LTC,-induced reactions may occur after a complete or partial change from LTC, to LTD+ This suggestion will require further investigation. Finally, these anti-cys-LT drugs did not affect passive cutaneous anapbyIaxis in either mice or rats. These findings correspond with previous finding with FPL 55712 in mice llnagaki et al., 1986) and AA-861, which is an inhibitor of Slipoxygenase, in mice (Inagaki et al.. 1985) and rats (Taira et al., 19881.We therefore believe that cys-LTs do not play a crucial role in passive cutaneous anaphylaxis in mice and rats. in conclusion, the present results indicate differences between cys-LT receptors in rat skin and those in mouse skin or rat stomach tissue, and a lack of involvement of cys-LT in passive cutaneous anaphylaxis in mice and rats.
Acknowledgements The authors are greatly indebted to Fisons Ph~rm~ceuti~i. Co. Ltd. (Leictstcrshirr. U.K.). Shitmngi Pharmaceutical Ca.Ltd. (Osaka. Japan), Mitsubishi-kuse~ Co. Ltd. (Tokyo, Japan) and Merck Frosst Cam&r, Inc. @&her. Carrada) for pnividing FfL 557l-7. LYIflXK.3. MCI-X26 and L-64X051.
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E~~r[~p~~r~Joi~r?lt~tof Ftiuri~l~~~~~fc~~,72
333
E3P 52662
Leukotriene receptors in the skin of rats differ from those of mouse skin or rat stomach strip Tom Miura, Naoki Inagaki, Shoichi Goto, Kenji Yoshida, Hiroichi Nagai and Akihide Koda Dcpartrrwt
t of Ptwrtnuccdogy~
Gift
Phannacchd
Ut~iwrsity. S-6- I Mimttorat~i~ast~i, Gifr 502. Japatr
Rt,eived 5 December 1991. revised MS received P,7 June 1992. accepted 30 June f9Y2
To compare the reccpfors for cystcinyl-leuk~~triene (cys-LT) in rat skin with those in other tissues. we investigated the effects and L-648051) on cys-f T-induced cutaneous reactions of specific cys-LT receptor antagonists (FPL 55712. LY 171883, MCI-826 in rats and mice, and on cys-LT-induced contractile responses in rat stomach smooth muscle. WC .~lso studied the effects of thcsc drugs irii h~molago~s pass& cutaneous anaphylaxis. The four cys-LT receptor antagonists dose dependently inhibited cys-LT-induced cutaneous reactions in mouse car, but failed to inhibit passive cutaneous anaphylaxis and the histamine-induced cutaneous reaction. In rats, only MCI-826 inhibited cys-LT-induced ~ut:~neous reactions although the other three drugs failed to inhibit these reactions. In contrast, the ~~-LT-induced contractile n_sjjcnses of rat stomach smooth muscle were inhibited by alf thcsc drugs in a concentration-dependent manner. Thcsc results suggest that cys-LT rcccptors in rat skin have an affinity diffcrcnt from that of rcccptors in mouse skin and rat stomach. They also suggest that cys-LTs arc not involved in passive cutaneous anaphylaxis in mice and rats. Cystcinyl-leukotricnc;
Cystcinyl-lcukotrienc
rcccptor antagonists; Cutaneous anaphylaxis (passive!; (Rat); tMousef
1. Introduction Cysteinyl-leukotrienes including (cys-LTs), leukotriene (LT) C,, LTD, and LTE, are chemical mediators that are metabolitcs of the 5-lipo~genase pathway of arachidonic acid and cause potent and long-lasting contraction of smooth muscles (Samuelsson, 1983). Cys-LTs are also known to cause an increase in vascular permeability (Ueno et al., 1981; Welton et al., 1981) and changes in blood pressure (SmedegHrd et al., 1982). Recently, it has been suggested that cys-LT might play very important roles in several kinds of diseases, especially allergy (Schwartz and Austen, 1982; Kaliner, 1985). Studies of specific antagonists against cys-LTs have therefore been actively undertaken. Sodium 7-[3-14-acetyl-3-hydroxy-2propylphenoxy~-2-hydroxypropyi]4-oxo-8-propyl-4H-lbenzopyran-2-carboxylacetate (FPL 55712) (Augsten et al., 1973) is one of the earliest discovered cys-LT receptor antagonists and has very often been used as a standard cbmpound in the development of anti-cys-LT drugs. it was reported that FPL 55712 inhibited LTC,-
Cifu Pharmaceutical University. S-6-1 Mitehor;~tlig;lshi,Cil’u 502, Japan. Tel. Xl.54X37-3931,fax 8 I.5X2-37-5979. Correspondence to: T. Miura, Department of Plwmacoltrgy,
and LTD.+-induced contractions of human airway (Buckner et al., 1986) and LTD,-induced contraction of guinea pig trachea (Snyder and Krcll. 1984). However, it was also reported that the agent inhibited neither LTC,-induced contraction of guinea pig trachea (Snyder and Krell, 1984) nor LTD~-induced contraction of guinea pig heart and cat gall bladder (Feniuk et al., 1984). This evidence suggests the cxistcnce of subtypes of receptors for cys-LTs in several mammalian tissues. Recently, we found that FPL 55712 did not inhibit the LTC~-induced cutaneous reaction in rats (Koda et al., 1990). In this study, we compared the response of rat skin against cys-LTs with responses of the mouse skin and rat stomach smooth muscle, and investigated the effects of the specific cys-LT antagonists, FPL 55712, 1-~2-hydroxy-3-propyl-4-~4-( I H-tctrazol-~-yl)butoxyl phenyl] ethanone (LYl7~~~3) (Flcisch et al., 19%). (E)-2,2-diethyl-3’-[2-[2-(4-isopropyl)thiazolyl]ethcnyl~ succinanilic acid (MCI-8261 (Takahashi et al., 1987) and sodium 4-[3-(4-acetyl-3-hydroxy-Zpropylphenoxy)propylsulfonyl-~-ox~-benzeneb~tanoaie IL-648051 1 (Jones ct al.. 19BG. on cys-LT-induced cutaneo~ls reactions in rats and mice and contractile responses in rat stomach smooth muscle. In addition, the effects of these drL!gd on homologous passive cutaneous anaphylaxis were iwxstigatcd. ‘i hc role of cys-LTs is also discussed.
passive cutaneous
anaphylaxis,
30-fold
diluted
mouse
antiserum was injected in a volume of 10 ~1 into both ears under cthcr anesthesia. After 48 h, the mice were challenged Female BALB/c mice weighing ahrut 2tt g and female Wistar rats weighing about 150 g were used for preparing antisera. In other experiments, male ddY mice weighing about 30 g and male Wistar rats weighing about 250 g were used throughout. All animals were purchased Japan 1.
from Japan
SLC.
Inc. (Hamamatsu.
with an intravenous
injection
of 0.25 ml of
0.5% Evans blue dye saline solution containing
0.25 mg
of dinitrophcnylatcd bovine serum albumin. In mediator-induced cutaneous reactions, 10 r.tl of mediator solution
(2 x lo-
LTC, or 5 x IO-” cars under ether
g/ml
histamine,
g/ml LTD,) anesthesia.
5 x lo-”
g/ml
was injected into both Immediately after the
injection, mice received an intravenous injection of 0.25 ml of 0.5% Evans blue dye saline solution.
mediator
Thirty min after the injection of dye solution, the mice Dinitrophenylated
Ascoris SIII~II~ extract and dinitro-
phenylated bovine serum albumin were used as antigens. Asruris SIII~III estract prepared according to the method of Strejan and Campbell (1968) and bovine serum albumin were dinitrophenylatcd according to the method of Eisen et al. (1953). Dinitrophenylated .-tst~ris SIIII~I extract was used for preparing antisera, and dinitrophcnylatcd bovine serum albumin served as the antigen for *a _liciting passive cutaneous anaphylaxis.
were killed by cervical dislocation removed. The pair of ears obtained
and their ears were
from
dissolved in 0.7 ml of I N KOH
each mouse was
solution at 37 “C
overnight and 9.3 ml of extraction solvent, a mixture of 0.6 N H3P0, After
solution and acetone (5: 13), was added.
vigorous shaking, the precipitates
off and the amount tally at 620 nm.
of dye was measured
were filtered colorimetri-
2.6. Cutarwous reactiorts it1 rats Asrtrris swat extract Antisera to dinitrophenylated in mouse and rat were prepared according to the methods of Levine and Vaz (1970) and of Tada and Okumura (1971). respectively. The titers of the both preparations measured by 48-h homologous passive cutaneous anaphylaxis were I : 5 12.
Six treatment points were marked on the backs of rats shaved I day before. In concentration-response studies of mediators, 0.1 ml of physiological saline was injected intradermally into one point as a control, and mediator solutions were injected in the order diluted into the other five points. Immediately after the mediator injections, I ml of 0.5% Evans blue dye saline solution was injected intravenously.
FPL 55712 (MW: 56s)). LY 1718X3 (MW: 318). MCIS26 (MW: 122.5) and L-64X051 (MW: 49X) were donated by Fisons Pharmaceutical Co. Ltd. (Leicestershire. U.K.). Shionogi Pharmaceutical Co. Ltd. iOsaka; Japan). Mitsubishi-kasei Co, Ltd. (Tokyo. Japan) and Merck Frosst Canada, Inc. (Quebec. Canada), respcctively. LY 171X83 was dissolved in saline containing 0.05 M NaHCO, in saline.
and the other compounds
were dissolved
into one of the six points as a control, and the same volume of chemical mediator solution (lo-’ g/ml histamine 3 x IO -” g/ml LTC, or 3 x IO-" g/ml LTD,) was injected intradermalty into the second point. The mediator solution containing an antagonist was injected into the other four points. Immediately after this 1 ml of 0.5% Evans blue saline solution was injected intravenously.
Histamine tdihydrochloride: Nacalai Tesque. Inc., Kyoto. Japan), scrotonin (creatininc sulfate: Merck, NJ. USA). LTC,. LTD,. LTE, IWako Pure Chemical Industries. Ltd.. Osaka, Japan) and acetylcholine tchloride: Nacalai Tesque. Inc.) were used as mediators for eliciting cutaneous reaction and smooth muscle contraction. All mediators were prepared in saline. 2.X Cutaticou~ rcactiorls iti momc
To examine the effects of locally administered antagonists, 0.1 ml of vehicle was injected intradermally
ear
When passive cutaneous anaphylaxis and cutaneous reactions by mediators were induced simultaneously in the same rats (Koda et al., 1990), 0.1 ml of saline was injected intradermally into one point as a control for passive cutaneous anaphylaxis, and 40-fold diluted rat antiserum was injected into the second point. After 48 h, 0.1 ml each of saline as a control for cutaneous reactions, IO-’ g/ml histamine, 3 x IO-” g/ml LTC, and 3 x IO-” g/ml dermally
Passive cutaneous anaphylaxis and mediator-induced cutaneous reactions were elicited in ddY mice as reported hy lnagaki et al. (1986). Briefiy. in the case of
LTDj
solutions were injected intra-
into the 3rd, 4th, 5th and 6th points, respec-
tively., Then, 0.5 ml of I% Evans blue dye solution containing 1 mg of dinitrophenylated bovine serum albumin was injected intravenously.
In all experiments, the rats were killed by bleeding 30 min after the reactions were induced, and the skin containing each reaction spot was excised. The amount of dye leaked within the reaction. sites was measured according to the same method as used in mice except that the volumes of IN KOH and extraction solvent were 1 ml and 8.3 ml, respectively. Each group consisted of six rats, and injection orders were rotated in consideration of biases in reactivity among the six spots.
dye
Amount of
(~g;
20 -
10 -
o-
2.7. Contractile response of smooth muscle strips of rat stomach Rat stomach smooth muscle strips were prepared according to the method of Offermeier and Ariens (1966). After killing the rat by bleeding, the stomach fundus was removed and carefully cleaned with Tyrode solution at 31°C. The fundus was halved by careful cutting in the direction of the longitudinal muscle fibres. Alongside this incision, two strips about 3 mm wide were taken, one from each half cf the fundus. Each strip was placed in a 5-ml Magnus bath and suspended in Tyrode solution. After a l-h equi!ibration period, responses were recorded isotonically with an initial load of 0.3 g. Cumulative concentration-response curves for each mediator were made for the tissue by successive increases in the bath concentration of the reagent. For testing the effects of antagonists on LTC,and LTD,-induced contractile responses, one of the two strips was used for LTC,, and the other for LTD,. The curve for IO- "'-lo-'g/ml LTC, or LTD, was generated twice, and the antagonist (or saline as a control) was added into the organ bath 5 min before the second curve was made. There was no tachyphylaxis observed against LTC, or LTD, in the second response. The data were expressed as a percentage of the maximum contraction obtained at the first response. pAz values were calculated according to the method of Arunlakshana and Schild (1959).
20 r
10 -
OI
I
9
Concentration
Fig. I. lncreasz LTD.,
in vascular
and LTE,
,
7
8
6
of mediator
permeabihy
in mice (upper)
tions were injected
5
4
(-log.
inducc~
3
LIJ
ti$ histamine.
and rats (lower).
Mediator
!.TC;.
solu-
into the mouse ear and the rat dorsal skin just
before injecting Evans blue dye solution. Each point and vertical bar represents
the
mean
and S.E.M.
Histamine.
�
LTC,.
of S-10 A LTD,.
mice �
and
12 rats.
o
LTE,.
After confirmation of the significance of differences (P < 0.05) by Kruskal-Wallis’ analysis, the differences between individual pairs of means were analyzed using Williams-Wilcoxon’s analysis.
3. Results 2.8. Statistical arlalysis 3.1. Increase k lqascular permeability induced by cys-LTs The data are reported as means and S E.M. Results for cutaneous reactions were calculated after subtraction of the corresponding control value from the individual value. Multiple comparisons were made for statistical analysis. When uniformity of variances in the data was identified by Bartrett’s analysis (P < 0.051, I-way analysis of variance was used to test for statistical differences. Significant differences (P < 0.05) were identified, after which the data were further analyzed for significant dtfferences between individual pairs of means using Duncan’s multiple range test. If uniformity was not demonstrated in the variances of the data, multiple comparisons were made non-parametrically.
The activities of three kinds of cys-LTs to increase vascular permeability in mice and rats were examined and compared with that of histamine (fig. 1). In mice, the cutaneous reactions induced by histamine, LTC, and LTD, increased concentration dependently and their intensity was in the order: LTC, > LTD, > histamine, but LTE, failed to increase vascular pcrmeability even at a concentration of 2.28 X lo-’ M. In rats, LTC, and LTD, also caused a concentration-dependent increase in vascular permeability and were stronger than histamine. The cutaneous reaction to LTE., was observed only at a concentration of 2.28 X
33fi TABLE I Effects of FPL 55712. LYI71!!!!3. MCI-!!2fi and L-M8051 on cutaneous reactions induced by antigen. histamine. LTC 4 and LTD4 in mice. Drug"
Control FPL 55712
Dose mg/kg
1 2 5
Control LYI7IS!!3 3
10 Control MCI-S2fi
IWI 0.1 I
Control L-MS051
Amount n of dye J.Lg (% inhibition) Passive cutaneous anaphylaxis
Ilistamine
LTC 4
2!!.M±2.99 29.29±3.47 (-2.3) 24.1l5 ± 2.5fi (13.2) 21.fiH 1.!!7 (24.3)
12'<)( ±0.43
\3.59± 1.7!! S.O!!± 1.39 c 7.33±0.77 c 4.fi3±0.51 d
(40.5) (4fi.1) (65.9)
14.40± 1.84 13.38± 1.30 (7.0 1O.72±O.70 (25.6) 7.94 ± 0.95 d (44.9)
22.40± 2.44 23.9fi ± :1.3S ( -7.0) (S.fi) 20.4!! ± 2.21 (1.5) 22.0fi±4.94
7.5S± 1.44
17.22±2.lfi 15.M± 1.3fi \3.43±2.45 fi.32±0.fiO
(9.2) (22.0) (63.3)
13.50±0.40 13.IH 1.42 (2.4) 11.37 ± 0.88 (I5.8) 8.31 ±0.71 d (38.4)
4.2HO.70 5.21 ± I.fi9 ( -22.0) (15.0) 3.fi3±0.84 1.5!!±0.2!! ,. (63.0)
6.16±0.96 4.1 t± 0.52 (33.3) 5.3HO.79 (12.8) 2.3HO.71 d (fiI.5)
10
ROfi ± 1.23 ( - 6.3)
2!!.M±2.99 (9.0) 2fi.07 ± 3.3fi 34.fiS ± 3.57 ( - 21.1) 23.3S±4.23 (1S.4) 2S.M±2.99 24.21 ± 1.74 24.13 ± 2.01 23.3fi±3.23
3
10.74 ± 1.09 ( 10.6)
!!.06± 1.47
7.90±0.98
(2.3)
12.01 ±0.43 (15.5) (t5.7) (l8.4)
14.06± 1.07 14.06± 1.33 13.91 ± 1.19 9.64± 1.26
1O.2HO.77 (14.5)
Drugs were administered intravenously just before the reactions were elicited. mice. ,. P < 0.05. d P < lUll. II
10- 5 M. the intensity was thus weaker than that of the other mediators.
3.2. Effects of cys-L T antagonists on cutaneous reactions in mice The effects of FPL 55712, LY171883, MCI-826 and L-648051 on passive cutaneous anaphylaxis and cuta neous reactions induced by histamine, LTC 4 and LTD4
I>
LTD4
d
(0)
(1.1) c
(31.4)
12.16±0.S5 11.21 ± 1.60 1O.27± 1.60 6.32 ± 1.06
d
(7.S) (15.5) (4!!.0)
Each value represents the mean and S.E.M. for seven to nine
in mouse ear were investigated. Table 1 shows the results of these drugs administered intravenously im mediately before eliciting the reaction. All drugs signif icantly inhibited LTC 4- and LTD4-induced cutaneous reactions in a dose-dependent manner. MCI-826 proved to be the most potent of the four drugs. However, these drugs had no effect on passive cutaneous anaphy laxis and histamine-induced cutaneous reaction. The effects of local administration of these drugs on
TABLE 2 Effects (If FPL 55712. LYI7IS!!3. MCI-!!2fi and L-MS051 on cutaneous reactions induced by histamine. LTC 4 and LTD4 in mice. Drug
II
Control FPL 55712
Control LYI71!!!!3
Cone. -Ing. glml
Amount
I>
of dye J.Lg (% inhihition)
Ilistamine
LTC 4
13.59±2.M fi 5 4
IS.96±2.73 (-39.5) 13.59± 2.64
fi
5 4 Control MCI-!!2fi
Control L-M!!051
15.49 ± 2.97 ( - 14.0) 13.59±2.M
fi 5 4
11.54 ± 2.48
(l5.])
13.59±2.M fi 5 4
12.9fi±2.69
(4.6)
II Drugs were mixed with mediator and injected intradcrmally. " P < (1.01.
I>
LTD4
5.06± 1.50 1.74± 0.22 3.32± 1.26 2.37±0.79
(65.6) (34.4) (53.2)
10.11 ±2.34 8.06± 1.55 fi.32±2.02 3.32± 1.01
(20.3) 07.5) (67.2)
11.21 ± 1.53 9.4!!± 1.39 6.95 ± 1.12 3.95 ± 1.14
(15.4) (3S.O) (64.S)
16.59±2.54 15.1fi±2.21 12.95 ± 1.52 9.4!!±0.68
(S.6) (21.9) (42.9)
6.16±0.77 8.8Si 1.04 ( - 43.7) 5.06± 1.56 (17.9) 1.2H 0.95 d (79.4)
S.37±O.96 9.00 ± 1.98 6.47± 1.39 2.84 ± 0.93
( -7.5) (22.7) (66.1)
5.06± 1.50 3.32±0.71 3.16± I.M l.74±0.70
C
d
(34.4) 07.5) (65.6)
10.11 ±2.34 9.64 ±2.53 6.oo± 1.26 4.42 ± 1.56
C
(4.6) (40.7) (56.3)
Each value represents the mean and S.E.M. for six or seven mice.
C
P < 0.05.
337
TABLE 3 Effects of FPL 55712, LYi7iX83, MCI-826 and L+64iWl on cutaneous reactions induced by antigen. histamine. LTC, and LTD, in rats. Drug a
Control FPL 55712
Dose me/k
Arn~~unt* of dye pg <% inhibition)
0.3
1 3
Chmrl LY1718X3
Control MCI-826
I 3 10 O.lil 0.1
1 Control L-h4HOSI
1 3 10
Passive cutaneous ana~hyiaxis
histamine
LTC,
LTD,
26.30 & 6.82 28.44_t3.36 (-8.1) 25.93 & I .94 (1.4) 12.5Xf 4.40 (52.23
iS.25t I.54 15.93Lt:il.92 (-45) i3.WXfti.91 (9.0) il.81 f 1.62 (22.6)
13.9s rf:I .xs 14.X7kO.69 (-6.6) 15.59~0.98(-11.X) 15.~~~0.~3 (-7.71
itl.hc)* I.23 13.142tO.67 (- 22.9) 11.03~0.83 i-3.2) 12.04&0.73 I- 12.61
29.92 -r_4.67 31.19*s.37 (-4.2) 31.32&2.?6 (-4.7) 25.11 k4.49 (ih.i)
19.962 1.70 20.74 f 1.hS t - 3.Y) 25.00 *I .OS( - 25.3) 22.14k3.07 i - 10.9)
l&69* 1.47 17.2xlt 1.39 f-3.5) 17.82-f 1*OS t - 6.8) ih.22I 1.73 (2.X)
II.271 I.22 1X62& 1.30 i - 12.0) i3.34 &fl.X(i f - 1X.4) 13.181 I.55 i - 16.9)
19.09-r_3.30 17.45 * 3.37 W) 23.13 e ?.tlOf - 21.2) 17.04 i 4.29 f lfl.7i
24.70+ I.12 22.81 * 1.68 (7.71 23.57 f 2.70 (4&f 24.02 & 3.73 i -0.9)
l&54* 1.3s 13.63rt i.tui (17.6) 13.67a 1.77 (17.4) 7.91+ 11.76c 62.2)
13.54f 0.96 lZ.lhlt: I.16 ( 10.2I ff.llh* 1.15 fiX.3l 7.91 *ti.fiJ E (41.61
26.ti8 $z3.21 23.87 jl4.07 I x.79 & 3.37
28.94 & I A:! 34.45 f 2.52 ( - 19.0) 30.13k2.79 (-4.1) 28.73 f I .73 in.71
20.41+ 2.22 22.36 & 1.25 ( - 9.6) 2l.hOk 1.22 (-S.8) i9.ssz5:2.17 (4.2)
18.14*0.94 10.09~ I.44 ( - i0.h) IX.25& 1.29 ( -0.h) 17.17* I.61 (5.3)
It%3 ,+ 3.15
’ Drugs were administered c P < 0.01.
( 10.5) (29.h) (37.7)
int~ven{3usiy just before the reactions were elicited. ’ Each value represents the mean and S.E.M. fitr six rats.
mediator-induced cutaneous reactions are shown in table 2. The drugs were mixed with mediator solutions and injected ~ntradermaliy. All drugs ~~h~bjted LTC4and LT~~-induced cutaneous reactions in a ~oncentration-dependent manner. The inhibition was significant at concentrations of lo-’ and lo-’ g/mi in LY1718X3
on LTC,-induced cutaneous reaction and at a concentration of NJ-” g/ml in ~Cl-82~ on both reactions. The percent i~b~b~tion~of IO-’ g/ml FI?L 55712 and L-6~051 on LTC, anti LT~~-induced reactions were greater than 50% although the inhibition was not statistically significant. However, these four drugs did not
TABLE 4 Effects of WC 55712. LYl71tW. MCi-~2~ and t_-~4~ilSl on cutane~~us re~tcti~~nsinduced by his~mine, LTCJ and LTI), in r&s. Drug ”
Control FPL 55712
Cont. -log. s/ml
Amn:mt ” of dye pg (‘;j. inhibition) -_ LTC, Histamine
7 6 s 4
is.23 f I .Y2 17.hOt 1.80 t - iS.hf IS.98 * ff*?h f - 4.9) 14.21,* ii.96 fh.lf i3.2Xk I.70 (12.X)
LTD,
is.ss* 1x.47* I.61 (-25.7) lfi.S2& i.S2(- iZ.Sf
i2.42+ I.11 12.1nte.4s 12.74&0.71) i2.xs *fl.Xtl ii.77i 1.03
(2.h) t - 2.6) t ,:;: . ._
19.76_+157 ih.OYf I .3J 16.42 + 2. i I 17.39+0.X7 211.2t(1* Il.67
(it&% i ih.Yf f i3.Of ( - 2.2)
14.6Yk I.37 ih.lf*
I.44 f- 10.3) I.13 f -5%
Control LY 171883
24.52 f I .49 23.44 f I .43 23.54+ Lib 22.36 f I .4Y 28.62 & I .s7 f -
(4.4) (4.0) tS.81 16.7)
2l.hO~2.91 (2.5) 2 1.Oh* 1hh 21.0,,~2.‘33 1- i.S) 23.54 f 2.hl ( - 9.0) X&14+2.21 (-2l.fii
Control MCI-WY
19.12+ I.11 i7.60+ 2.08 18.14&“O.h9 19.01_t(1,9s 1x.79* 1.36
(7.9) (5.1) (O.hf (1.7)
is.23 + 0.9S Ui.7) 15.12*0.x4 (2.X) 14J!o*ti.91 ls.hh+o.3X f -2.81 3.78* f).SXrt (75.2)
Wh _t0.39 Y.hl+l.?X (--KS) 9.50& 1.33 (- 7.2) IX.hl X.iOji*li 2.4x + (3.48(’ (72.0)
13.39*:0.93 iX.i4f2.37~-35.5f 18.58+.2.47 ( - 3X.X) 20.20 rt i.76 ( - 50‘9)
12.85t WI4 13.2X~rO.69 (-3.3) i3.39* 1.no (-4.2) i4,yo* 1.62 t - lh.0)
17.42&il.82 12.53f 1.07 I-0.9) ( - 0.9) 12.53_t0.9I lS.SSf. I.21 c t-25.2)
Control L-h4WS I
h 5 4
” Drugs were mixed with medi~tl)r and injroted intmd~r~l~y.
” E;&
v&g
r~~~sents the mean and S.E.M. for six rats,
’ P < tt.OS,’ P < tl.tii.
IOO-
80 -
AS shown in table 3, MCI-826 significantly inhibited LTC,- and LTD,-induced cutaneous reactions at a dose of I mg/kg when administered intravenously immed~tely before eliciting of the reactions, but did not affect passive cutaneous anaphylaxic and histamine-induced cutaneous reaction elicited simultaneously in the ume rats. The other three drugs did not yield any significant inhibition in any of the cutaneous reactions at doses sufficient to inhibit cys-LT-induced cutaneous reactions in mouse ear. Table 4 shows the effects of loeat administration of these drugs on histamine-. LTC,- and LTD,-induced cutaneous reactions in rats. FPL 55712 and LYl71883 did not inhibit any of these cutaneous reactions. MCI-826 significantly inhibited LTC,- and LTD,-induced cutaneous reactions at a ~n~entration of IO-’ g/ml. but did not affect histamine-induced cutaneous reaction. L-648U51 dose dependently enhanced all reactions tested.
A> the first step in evaluating the effects of cys-LT receptor antagonists on .cys-LT-induced contractile responses in rat stomach, WC studied the relationships between the concentration of various mediators and the intenstty of the contractile responses. LTC, and LTD, caused concentration-dependent contractions and their potencies at lower concentrations were greater than those of ?.cetylcholine and serotonin. AIthough LTE, caused contraction in a concentrationdependent manner, the intensity was weaker than those of LTCJ and LTD,, histamine also caused cont~ction at high concentrations (over 9 x lo-’ M), but the contraction induced by 9 x IO-’ M histamine was only about 20% of that produced by acetylcholine (fig. 2). Figure 3 shows the results of FPL 55712, LY171883, MCI-826 and L-6480,51 on LTC,-induced contractile response in rat stomach. All drugs inhibited LTC,-induced contraction in a concentration-dependent manner. The intensities were in the following order: MCI826 (PA, 7.81) > FPL 55712f6.86) > L-648051 (6.38) > LY I71883 (6.28). The slopes calculated by Schild analysis for LY 171883 and L-64805 1 were close to 1 (LY 17 18% 0.96 and L-648OSl: 0.84) and showed competitive antagonism against LTCJ. FPL 55712 and MCI-826 however, antagonized the contraction non-competitive!y as the slopes were not close to 1 (FPL 55712: 0.59 and MCI-826 0.65). All these drugs also inhibited LTI),-induced contraction in a concentration-depen-
(10 -
40 -
20 -
oIO
a
7
Gmccntr8tion
of
9
6
mediator
5
t-log.
3
4
I41
Fig. 1. Dose-response curves of contractile responses induced by acetylcholine, LTC,, LTD4. LTE,. histamine and serotonin in isolated stomach smooth muscle of rats. Each point represents a percentage of the maximum contraction obtained with acetylchoiine. 0 Acrtylcholine. 0 LTC,. A LTD.,. A LTE,, 0 histamine. � serotonin.
dent manner (fig. 4). The intensities wcrc in the following order: MCI-826 (pA2: 7.61) > LY 171883 (6.36) > L-648051 (6.20) > FPL 55712t6.14). The slopes for all drugs were close to 1 (FPL 55712: 1.06, LY171883: 0.94, MCI-826: 1.03 and L-648051: 1.09) and contractions of rat stomach induced by LTDj were inhibited competitively, None of the drugs had any effects on contractile response induced by lo-’ g/ml acetylcholine at the highest concentrations studied here (MCI-826: IO-’ g/ml and FPL 55712, LYl71883 and L-648051: IO-” g/ml).
4. Discussion In the present study we first compared the sensitivity of mouse ear and rat dosal skin against cys-LTs. LTC, and LTD, caused dose-dependent increases in vascular permeability to almost the same extent. These cys-LTs were more potent than histamine in both species. LTE,. however, had little or no effect on vascular permeability in either mice or rats. Although it is generally known that LTE, causes an increase in vascular permeability as well as contraction of smooth muscle (Welton et al., 1981j, the activity in mice and rats might be less than that of LTC, or LTD,. In this
339
study, therefore, the effects of four anti-cys-LT drugs on cutaneous reactions in mice and rats induced by cys-LTs except LTE, were investigated. All anti-cys-LT drugs dose dep~~dentiy inhibited LTC,- and LTR~-induced cutaneous reactions in mouse ear when administered intravenously immediately before the reaction. In rat dorsal skin, although MCI-826 inhibited LTC,- and LTD,-induced cutaneous reactions, the other three drugs had no effect on these reactions at doses sufficient for inhibiting them in mice. Therefore, we investigated the effects of these drugs injected intradermally with mediator since it was possible that sufficient drugs may not have reached the reaction sites in rats. However, FPL 55712, LY171883 and L-648051 showed no effect on cys-LT-induced reactions in rat dorsal skin when administered locally as well as systemically. These three drugs did not affect LTC,-induced cutaneous reaction in rat ear at a con-
FI’L 100
centration of 10-j g/ml (data not shown), suggesting that the low efficacy of these drugs in rats was independent of the site on the skin. In addition, none of the drugs inhibited histamine-induced cutaneous reaction in either mice or rats. These results indicate that the inhibitory action of anti-cys-LT drugs on cys-LT-induced cutaneous reactions in mice and rats is quite different, suggesting that cys-LTs cause a vascular permeability increase in mice and rats through different types of cys-LT receptors. Cys-LT receptors of guinea pig skin may be similar to those of mice and different from those of rats since it is reported that FPL 55712 and LY 171883 inhibit LTD,-induced cutaneous reactions in guinea pigs (Fleisch et al., 1985; Krell et al., 1989). Recently, Gardiner et al. (1991) have proposed that cys-LT receptors can be classified into two large groups, LT, and LT,, depending on the action of some cys-LT antagonists LT, receptors are predominantly
LY171883
55712
Schild
analysis
Schild
analysis
Slope:
0.59
Slope:
0.96
P
SD
c :
0
.-I Y :: :
l-648051
MCI-826
z u 100
Schild
analysis
Srhild
aoalyeis
Slope:
0.65
Slope:
0.84
Pk.2
:
7.81
50
0
Concentration
of LTG
(-log.
g/ml1
Fig 7 Effects of FPL 55712, LY171RS3. MCI-826 and L-64X051 on LTC,-induced contructile responsc~ in isnlated stomach smooth muscle of r&.‘i..TC, was added IO the organ bath cumulatively. Drugs were added 5 min hefore the addition of LTCJ. Each point and vertical bar , calculated hy Schild analysis. Drug represents the mczn 2nd S.E.M, of 4-!tt experiments. Each value in the figure represents lhe slope and pA_ IO ’ gfmi. concentration; 0 control, 0 10 -” g/ml, d IO-’ g/ml. A IO- ’ g/ml. � 10 ” g/ml.
both LTD,
and LTE,
on guinea pig ileum
ated
responses
(Garuiner
et al..
1991).
FPL
55712,
al.. IWO) and trachea (Hand et al., 1989) c cys-LTs on guinea pig gall bladder (Fa;Lllexmcand Krell. I991 ) and human bronchial muscle
LY 171883 and L-648051 may belong to the LT, type antagonists because these drugs antagonize LTD.,-, but not LTC,-. induced contraction in guinea pig trachea
(Buckner et al.. IWhI. and are antagonized by most of the existing cys-LT antagonists (e.g. lCll986lS (Snyder
(Hand
et al.. 198> !Krcll et al.. 1987). SK&Flo4!3S3 (Hay et al.. 19S7) and MK.571 (Jones et al., 1989). which are
tion of guinea pig trachea induced by LTDJ (Takahashi et al., 1987) and of human bronchial muscle induced by
novel. wtent and selective cys-LT antagonists). LT, receptors, however. are predominantly activated by
three
LTC,
on guinea pig ileum (Gardincr
ct al.. 1990) and
et al..
1989).
responses through
cys-LTs
LT,
(Habe
MCI-826
also antagonizes
the
receptors, such as the contrac-
et al..
1987).
Furthermore,
we
found that the contraction of guinea pig ileum induced by IO-’ g/ml LTD, was almost completely inhibited by
trachea (Hand et al.. I9S9) and by three cys-LTs on ferret spleen. ferret trachea. sheep ainvay (Cuthbert et
IO-’
g/ml
Iti-’
g/ml LTC,
at.. WI) and human pulmonary vein (Brink and Gardiner. KIWI). but are not antagonized by the above mentioned antagonists. BAYu9773 is the first compound to antagonize both LT, and LT, receptor-medi-
MCI-826 in the presence of L-serine borate (data not shown), suggesting that MCI-826 had no effect on LT,
FPL 55712
LY171883
Schild
analysis
Schild
analysis
Slopr:
I.06
Slope:
0.94
nnalysis
Schild
analysis
Slope:
I.03 -
Slope:
1.09
L
,
9
a
Conccntraiion
ml%. LTDJ
of FPL Wl2.
LYl71XX3.
MCI-X%
by
g/ml
: 7.61
IO
4. Effect*
induced
by even IO-’
L-648051
Schild
PA2
but the contraction
was not inhibited
receptors. These findings lead us to believe that cys-LT receptors in mouse skin (probably guinea pig skin)
MCI -826
Fk
MCI-826
and L-h48tlSi
was added m the organ bath cumulatively.
7
oi
LTD.
on LTD.,-induced
Drugs were added
(-lqs.
g/ml)
contractile
5 min heforc
responses in isoiuted stomach smooth muscle of
the addition
of LTD,.
Each point and vertical
rcPrcscnt!+ the mean and S.E.M. of four to five experiments. Each value in the figure represents the slope and pA, calculated Drug cone-ntratkn; I: ccm!r::!. G t!) ” g/m!. A it) en g/ml, A tt! ~’ g/ml, � 1[)~-”g/ml, 8 1()-y g/ml.
har
hy Schild analysis.
331
belong to the LT, type, but the type of cys-LT reccptors in rat skin is not clear. Since MCI-826 is not a hydro~acetophenone derivative, unlike the other three drugs used in this study, the inhibitory action on rat cys-LT-induced cutaneous reactions may be related to the difference in their chemical structures, In addition, the report of Metters et al. (1991) concerned with cys-LT receptors is very interesting. These authors indicated that cys-LTs and SK&Fl~4353 compete for [‘HILTD, binding in rat lung with the same potency as they compete for [3H]LTD, binding in guinea pig lung. MK571 and ICI198615, however, compete for the receptors in the rat with less potency than they compete for those of guinea pig. These results suggest that more multiple receptors for cys-LTs exist in these tissues. We need to study the effects of other characteristic cys-LT antagonists such as BAYu9773, lC1198615, SK &F104353 and MK571 on our experimental models. Secondly, in order to confirm whether the low efficacy of anti-cys-LT drugs as seen in rat skin was characteristic only for the tissue, we investigated the effect of these drugs on the contractile response induced by cys-LTs in rat smooth muscle. Since it is reported that rat stomach and colon, but not duodenum and ileum (Goldenberg and Subers, 19821, are contracted by LTCJ and LTD,, we selected stomach smooth muscfe for the present study. LTCj and LTRj caused the tissue to contract in a concentration-depcndent manner and found a submaximal contraction at a concentration of IO-’ g/ml 11.6 x 1Uw7M LTC, and 2 x 10e7 M LTD,). The contractile response was observed at con~ntrations of cys-LTs lower than those needed to Cause the increase in vascular permeability, suggesting that, in rats, stomach tissue may bc more sensitive to cys-LT than skin. Though LTE, also contracted stomach strips in a concentration-dependent manner, the activity was weaker than that of LTC, or LTD,. On the other hand, histamine produced little contraction of stomach tissue. It is known that histamine H , and H 2 receptors are present in rat stomach smooth muscle, and that histamine contracts them through H, receptors and relaxes them through H, receptors (Ercan and Tiirker, 19771. The four anti-cys-LT drugs all inhibited the contraction induced by LTC, and LTD, in a concentrationdependent manner. However, these drugs had no effect on acetylcholine-induced contractile response, suggesting that the inhibitory activity is cys-LT specific. The slopes of the four anti-cys-LT drugs, as dctermined by Schild analysis, were close to 1 in LTD,-induced contraction, and the inhibitory patterns were competitive. These results suggest strongly that the contraction induced by LTD, is activated through LTt receptors. In LTC,-induced contraction, the SIOPCSof LY 171883 and L-648051 were aiso close to 1. but those of FPL 55712 and MCI-826 varied considerably from 1,
suggesting that another type of cys-LT receptors may be concerned with the contraction induced by LTC,. In the present study, however, since we examined the effects of these drugs in the absence of an inhibitor of ~-g~uta~yl transferase, which metabolizes LTC, to LTD, (Orning and ~ammarstr~m, 19801, it is possible that LTC,-induced reactions may occur after a complete or partial change from LTC, to LTD+ This suggestion will require further investigation. Finally, these anti-cys-LT drugs did not affect passive cutaneous anapbyIaxis in either mice or rats. These findings correspond with previous finding with FPL 55712 in mice llnagaki et al., 1986) and AA-861, which is an inhibitor of Slipoxygenase, in mice (Inagaki et al.. 1985) and rats (Taira et al., 19881.We therefore believe that cys-LTs do not play a crucial role in passive cutaneous anaphylaxis in mice and rats. in conclusion, the present results indicate differences between cys-LT receptors in rat skin and those in mouse skin or rat stomach tissue, and a lack of involvement of cys-LT in passive cutaneous anaphylaxis in mice and rats.
Acknowledgements The authors are greatly indebted to Fisons Ph~rm~ceuti~i. Co. Ltd. (Leictstcrshirr. U.K.). Shitmngi Pharmaceutical Ca.Ltd. (Osaka. Japan), Mitsubishi-kuse~ Co. Ltd. (Tokyo, Japan) and Merck Frosst Cam&r, Inc. @&her. Carrada) for pnividing FfL 557l-7. LYIflXK.3. MCI-X26 and L-64X051.
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