- Email: [email protected]
Further evidence that the serotonin receptor in the rat stomach fundus is not 5HT1A or 5HT1B
L:l~ ,%~ICUCC%, \hi. ~8, ira. t-5 Prllltod ill t h e L.S.A.
Pergamoll Pru~%
FURTHER EVIDENCF THAT THE SEROTONIN RECFPTOR IN ]'HE RAT STOMACH FUNDI'S IS NOT 5HTIA OR 5HTIB Marlene I.. Cohen and Laura A. Wlttenauer ll]]y Research Laboratories Ell Lilly and Company Indianapolls, Indlana 46285 (Re~.clved in ftndJ
form October
14, ]083)
Summary The nature of the receptor medlating serotonin contraction in the rat stomach fundus has not been clearly associated with either 5HT 1 or 5FT 2 receptors. We have explored the possibility that such receptors :p the rat fundus may better correlate with 5HTI^. or 5HT.~ receptor subtypes as defined by radio]abeled ligand b~n~Ing studies with brain cortica] membranes. Meta chlorophenylpiperazine (C}'P) and meta trif]uoromethylphenylpiperazine (TFMPP), selective ]igands for the 5HT. B receptor and LY165163, a selective ligand for i the 5HTIA receptor, have been evaluated for their agonJst and antagonist activity at serotonin receptors in the rat stomach fundus. CPP and TFMPP were partial agonists in the rat stomach fundus whereas LY]65163 showed no ~gonist activity in this smooth muscle in concentrations up to 10--M. All three phenylpiperazines antagonized serotonin-induced contractions in the rat stomach fundus. The affinity for serotonin receptors ~n the rat fundus was similar for all three phenylplperazlnes in spite of the reported selectlvlty of MCPP and TFMPP for 5HII~_ and, of LY165163 for 5HTIA receptors. Furthermore, the affinity of these agents for serotonln receptors in the rat stomach fundus did not agree with thelr reported afflnity for either 5HT.. or 5HI.~ binding sites in rat cortical membranes. Thus, the slmllarlty In affinities of these phenvlpiperazlne derivatives for serotonin receptors mediating contraction in the rat fundus along with their different affinities for 5HTIA and 5HTIB blndlng sites argues against the possibl]ity that the serotonln receptor in the rat fundus is of the 5HT]A or 5HTIB subtype of serotonin receptor. The receptor that mediates serotonin-lnduced contractions in the rat fundus is dlst~nct from r 5}IT^ Y receptor (1,2,3), 5HT 1 (1,2,3) or trvptam~ne receptor (3). However, the possibility remains that %he receptor in the rat stomach fundus, although not correlatJpg with radlo]abeled l!gand binding data for the 5HTI receptor, may more close]v resemble either the 5HTIA. or 5HTIB receptor subtypes recently defined wlth radlolabeled ]Igand blndln~studles ~n rat cortical membranes (4). Two agent,,, meta chlorophenylp~perazlne (CPP) and mete trlfluoromuthvlphenylpiperazine (TFMPP) have been identlfied as selective liganas for the 5HTIB receptor subtype (5). These agents have previously been demonstrated to be antagonists of vascular 5H%. receptor q~tes in the rat ~ugular vein (6). Asarch et al. (7) have r e c e n c y identified LY165163 (para amino phenylethyl TFMPP) as showing relatlve]y high affinity at 5HT]A binding sites in rat cortlcal membranes (Table I). If the receptors that mediate serotonJn-lnduced stomach fundus were of the 5HTIB subtype, we would ~3.00 + .00 (c) 19~6 Pcr~$alnOn P r e ~
contraction% In the rat anticipate that CPP and
0024-3205/86
Copvrlght
J td.
?
Serotonin
Receptor
in Rat Stolna(i~
TABLE
Affinity
Subtypes
$8, ',o. I,
[~)g6
1
of Pheny]piperaztnes
5tlT1B R e c e p t o r
\~l.
in Brain
for 5HT
Cortical
1
and
~embranes
a
- L o g KB 5ttT1A
~'UI]1%
mTFMPP
6.44
8.06
LY165163
8.53
6.48
aData
transposed
from A~arch
et al.
(7).
TFMPP would show higher affinity than LY]65163 at qerotonln receptors in tbo rat fundus. Conversely, if the receptor~ that medlate ~erotun]n c~ntracttons in the rat stomach fundus are of the 511TIA ~ubtype, then IY165163 ~hould ~how higher affinity for these receptorq than the other phenylplperazine derlvarives. Materials
and Methods
Isolation of smooth muscle preparations. Male W1star rats (130-~75 g; Laboratory Supply, Indianapolis, IN) were sacrificed by cervical di~!ocatlon and longltudina] sections of the ,,tomach fundus were prepared for Jn vitro examination. Four preparat~on~ were obtained from one rat fundus. "lls~,ues were mounted in organ baths containing I0 ml of modified Krebs' bc~lutton of the fo]lowlng composition (m111imolar concentrations): Na(,l, 118.2; KCI, q.6; CaCI2.2H20, 1.6; KH2PO4, 1.2; b!gSO4, 1.2; dextrose, I0.0; and NaHCN,, 2~..8. Fissue bath solutions were maintained at ~7°(: and equlllbrated with 9~% O, ,-nd 57 CO 2. Tissues were placed under optimum restlng force (4F,) and were al~owed to equilibrate for approximately ] hr before exposure to
of A$onlst
contractile concentiation xncrease in concentration
Actlvity
Noncumulat ~ve 5HT w e r e o b t , l l n e d b) a s t e p w i s e preceding cc)neentratlonq every
of Phenylpi]~.erazines.
response curves after wa~hin%
for out
15-20 sin. Tissues were then challenged in o ~imllar noncumulative f,~hion with either LPP, TFMPP, er IY16516~. Only one phe~ylpiper,~.zine derivative was examined per tlsbue. Determinatlon of apparent anta$onist dissociation constants. Nuncumu~,~ tlve contractlJe concentration-response curves for 511'I were obtained by stepwise increase in concentration after washlng out the precedlng concontrattons every 15 to 20 m]n. Each agon~st concentration remained ]n contact with the tissue for approximately 2 mln before washout and maximum response to each agonlst concentration was measure(]. FD= wilueq were take~ a,, the concentration of ogonist that produce~ ha]f-max)iOmal contraction. After control response,~ were obtained, tl,q~;ne'~ were incubated wlth on apprnpr[ate concentratlon of buffer or antagonlst for I hr. Responses to 5HT wele then repeated in the presence of antagonist. ('oncentr
Vol.
38,
No.
1,
1986
Serotontn
Receptor
I n Rat
Stomach
3
Apparent antagonlst dissociation constants (KB) were determined for each concentration of antagonist according to the follo~ing equation:
(B) KB = ( d o ~ e
ratio
-
l)
where (B) i s the concentration of the antagonist and dose ratio is the ED. 0 of the agonist in the presence of the antagonlst dlvided" bv. the control F~.ju Generally, paralle] shifts in the concentration-response curves occurred zn the presence of antagonists. These results were then expressed as the negatlve logarithm of the K B (1:e:, -log KR). Calculations were performed wlth the aid of a computer and dzglta] plotter as descrzbed previously (8) for competitive antagonists. The d a t a w e r e a l s o a n a l y z e d according to the procedure of Arunlakshana and Schild (9). According to Arunlakshana and Schlld (9), if blockade is competitive under equiJibrium conditions, a plot of the logarithm of (dose ratio - 1) a g a i n s t the negative logarithm of the molar concentration of antagonist should yield a straight line whose ~lope is ] and intercept along the abscissa i s t h e pA 2 w h i c h ~s e q u a l t o - l o g KB. Results CPP and TI~IPP were partial agonJsts in the rat stomach fundus producing a maximum contractile response that was approximately 40% of the maximum response to serotonin (Figure i). In contrast, compound~l,Y165i63 did not contract the rat stomach fundus in concentrations up to I0 M.
RAT STOMACH FUNDUS 100
TON IN (n=38) 60
~
FMPP ( n = 4 )
I
40
O 10
9
8 -LOG
I 7
--
T6
--
= 5
--
4
AGONIST (M)
Fig. 1 Coptra( tile concentration response curves to serotonin, meta chlorophenylplperazine (CPP), meta tr]fJuorometh~]phenylpzperazzne (TFMPP) and I,Y165163, para am~nophenylethy] TFMPP in the rst stnmach fundus. Point~ are mean values ± SE for the number of tissues ind]cated in parentheses.
qt, r , > t O l i i i l
i~,( ¢ , p t o l
ill
'~, } t
Vn] .
%to,n,i~ ~l
However, CPP, TFMPP and I,Y16~16~ were all induced contractions in the rat stomach fundus. soc~ation constants for these phen>ipiperazines All three phenylp~peraz~ne~ qhnwed roughly s~m~lar receptor In the rat stomach fundus in contrast to ( ' F a b l e l~ f e r 5HTIA ( L Y l b 5 1 6 ~ ) and 5HTIB (CPP,
)~,
~,o.
] ,
}c)~G
antagon~t~ of ~erotontnTable > ~nd~cateq the d~sin the ~,~.t stomach fundus. affinity f(~r ti~e ,,erntontn their reported ~pec~f~c~tv '!'F~.IPP) r e c e p t o r subtypes.
TABLE 2 Afi~nitv of PbEny]plperaz~nes for Serotonzn Receptors Mediating Contraction :n the Rat Funduq
Compound
Receptor Selectiv±ty
m'IFMPP
(5HT1B)
7.32
+- 0 . 1 4
(9)
7.36
O.q7
m('PP
(5H'IIB)
7.84
÷ 0.09
(1',)
7.84
1.00
IY16S163
(SHTIA)
7.4~ ± 0.~ ~ (]0)
7.16
].f0
-l,og
k B *_ qF
(n) a
PA2 b
-Slnpe b
a v a t u e s a r e m e a n s i S . t . . l o r KB = ( B ) / ' ( d n ~ e r a t i o - ]) w h e r e (P,~ -: e o n c e n t r , ~ tlon of antagonist and d o s e r a t t o = FD of the Jgonzst after the antagont~t 5 divided bv t h e El).() ~li t h e a g u n L q t b e ~ o r e t h e a n t a g o n i s t . The n u m b e r o f bti.~sues examined f~r each antagonist is indicated ~n p a r e n t h e s e s , . pA 2 i s t h e a h s c t s s a ~ntercept o f t h e l i n e f o r m e d f~om a S c h t l d p l o t o f lo~ ( d o s e r a t t o - 1) v s . - l o g (B) ( q ) , Sch~]d plots were constructed from three concentrations (~f t h e a n t a g o n i s t u~,ing t h e n . m h e r oI t i s s u e q ~ n d ~ c a t e d ±n parenthese,~. The slope of the Schild plots d~d 'nnt differ from unit\.
I)rscuss
ton
Although serotonln ~s a p o t e n t a g o n l ~ t zn t h e r . t , , t o m a c h f u n d u s ( 1 , 2 , ~ ) qpectfic antagoniqtq o f th~.~ r e c e p t o r have not been tdentlfLed. Recent data from our laboratory and o t h e r s h a v e ± n d l c a t e d t h a t t h e r e c e p t o r ~ responsible for serotonin-ipduced (ontractlonq in the rat stom¢Ich fllrlduq are ni)t similar to 51112, SHT. or trvptamlne recept¢~rs as deflned by ra(lio]igand l~ipdlng in rat cortical m e m ~ r a n ~ "(].2,),). Although not ,i~] compounds wlth h~gh alf~nltv at 51ITI bra~n binding s~tes showed high sfl~n~ty for ~er(~tontn rec~.ptors in the rat fun(lu¢ (~), the possib~ Lit> existed that ~eroton~n recept~.~, in the rat ~tomach fundus were more specifically of the 511TI. of 5H'II~ subtype. With the recent ident~fleation ~f agents showing high a~ltn~tv ,']~t 51!T md 5HT • ,¢ I'~ 1t, receptor s u b t y p e . s , t h l ~ p o ~ l h ~ l t t v wa~ ~, . p l o r e d lp t h e p r e ~ e n t . ~ u d v . The selective 5If'liB receptor a~;ents, CPP nnd IFMPP were partlal rig,mists In the rat stomach fund.~ whereas the sel~,cttve SH'FI~ agentg IY16~]6~, did ,~et contract the rat qtnm,lch fun(hls in concentrations u~"to i0 -~v. These dnt,l may sugge.~t t h a t i nteraction ~ith a receptor r e s e m b l i n g t h e 5PT 1. b i n d i n g s i t e rnav be associated w±th contr,~ct I le respnn,,eq in the rat stomach ~imdus. All three phenylpiptr,3~zne derlvattve~ ~t.dled in the pre.sent r e p o r t antagontzed ',erotontn-znduced contractions in the r a t f . n d u s . Although these c o m p o u n d s ~how m a r k e d l y d i f f e r e n t affinities f o r 5ttT 1. and 5PT] r e c e p t o r .~ubt y p e s ( 7 , s u m m a r i z e d in T a b l e l ) , a f f l n r t l e ~ f o r s e r o ~ o n i n r e c e pBt o r ~ m e d l a t a v ~ contraction in t h e rat fundus were similar amon~ t h e s e ohenylplperazlnt
Vol. 38, No. I, 1986
Serotonin Re('eptor in R~it Stomach
5
derivatives (Table 2). If the serotonln receptors in the rat fundus were of the 5 H T I A O r 5HTIB subtype, then we would have anticipated higher affinity for one of k~ese pHenylpzperazines than for the other, based on the receptor subtype specificity previously documented from binding studies. Since the affinltles of all three phenylplperazznes were similar in the rat fundus, we must conclude that the receptors that mediate the contractile response to serotonln are neither 5HT]A nor 5HTIB. Further confirmation of this concluslon might be derived from an evaluation of the affinities of TFMPP and LY165163 based on binding studies in brain cortical membranes (7). This information as summarized in Table I indicates that TFMPP and LY165163 show dissociation constants of approximately i0 M at 5HT._ and 5HT~. receptor subtypes, respectively. Lzkewlse, TFMPP and LY165163 I~ IA . " -f show affinities of approximately 3.5 x i0 at 5HT . and 5HT._ receptor IA Ib subtypes, respectlve~y. Affinity for the serotonin receptor in the rat fundus approximated J x 10--M for these phenylplperazine derivatives, a value different from their affinlties at either 5HTIA or 5HTIB binding sites. In summary, using these 5HT 1 rat stomach fundus, we further mediating serotonin contractions identified with a 5HT 1 subtype of
receptor subtype specific antagonists in the support the contention that the receptor in the rat fundus is not 5HT] nor can it be receptor.
Acknowledsements The authors wish to express their appreciation to Ms. Gretchen Beckhelm for the computer graphics and to Ms. Elaine Gardner for the expert typing of this manuscript. References I. 2. 3. 4. 5. 6. 7. 8. 9.
J. E. LEYSEN AND J. P. TOLLANAERE, Ann. Rep. Med. Chem. 17 i-i0 (1982). M. L. COHEN, K. W. SCHENCK, W. COLBERT AND L. WITTENAUER, J. Pharmacol. Exp. Ther. 232 770-774 (1985). M. I.. COHEN AND L. A. WITTENAUER, J. Pharmacol. Exp. Ther. 233 75-79 (1985). N . W . PEDIGO, H. I. YAMAMURA AND D. L. NELSON, .I. Neurochem. 36 220-226 (1981). M. A. SILLS, B. B. WOLFE AND A. FRAZER, J. Pharmacol. Exp. Ther. 23] 480-487 (1984). M . L . COHEN AND R. W. FULLER, Life Sci. 32 711-718 (1983). K . B . ASARCH, R. W. RANSOM AND J. C. SFIH, Life Sci. 36 1265-]273 (1985). B. R. ZABOROWSKY, W. C. MCMAHON, W. A. GRIFFIN, F. H. NORRIS AND R. R. RUFFOLO, JR., J. Pharmacol. Methods. 4 4165-4178 (1980). O. ARUNLAKSHANA AND H. O. SCHILD, B~. J. Pharmacol. 14 48-58 (1959).
Pergamoll Pru~%
FURTHER EVIDENCF THAT THE SEROTONIN RECFPTOR IN ]'HE RAT STOMACH FUNDI'S IS NOT 5HTIA OR 5HTIB Marlene I.. Cohen and Laura A. Wlttenauer ll]]y Research Laboratories Ell Lilly and Company Indianapolls, Indlana 46285 (Re~.clved in ftndJ
form October
14, ]083)
Summary The nature of the receptor medlating serotonin contraction in the rat stomach fundus has not been clearly associated with either 5HT 1 or 5FT 2 receptors. We have explored the possibility that such receptors :p the rat fundus may better correlate with 5HTI^. or 5HT.~ receptor subtypes as defined by radio]abeled ligand b~n~Ing studies with brain cortica] membranes. Meta chlorophenylpiperazine (C}'P) and meta trif]uoromethylphenylpiperazine (TFMPP), selective ]igands for the 5HT. B receptor and LY165163, a selective ligand for i the 5HTIA receptor, have been evaluated for their agonJst and antagonist activity at serotonin receptors in the rat stomach fundus. CPP and TFMPP were partial agonists in the rat stomach fundus whereas LY]65163 showed no ~gonist activity in this smooth muscle in concentrations up to 10--M. All three phenylpiperazines antagonized serotonin-induced contractions in the rat stomach fundus. The affinity for serotonin receptors ~n the rat fundus was similar for all three phenylplperazlnes in spite of the reported selectlvlty of MCPP and TFMPP for 5HII~_ and, of LY165163 for 5HTIA receptors. Furthermore, the affinity of these agents for serotonln receptors in the rat stomach fundus did not agree with thelr reported afflnity for either 5HT.. or 5HI.~ binding sites in rat cortical membranes. Thus, the slmllarlty In affinities of these phenvlpiperazlne derivatives for serotonin receptors mediating contraction in the rat fundus along with their different affinities for 5HTIA and 5HTIB blndlng sites argues against the possibl]ity that the serotonln receptor in the rat fundus is of the 5HT]A or 5HTIB subtype of serotonin receptor. The receptor that mediates serotonin-lnduced contractions in the rat fundus is dlst~nct from r 5}IT^ Y receptor (1,2,3), 5HT 1 (1,2,3) or trvptam~ne receptor (3). However, the possibility remains that %he receptor in the rat stomach fundus, although not correlatJpg with radlo]abeled l!gand binding data for the 5HTI receptor, may more close]v resemble either the 5HTIA. or 5HTIB receptor subtypes recently defined wlth radlolabeled ]Igand blndln~studles ~n rat cortical membranes (4). Two agent,,, meta chlorophenylp~perazlne (CPP) and mete trlfluoromuthvlphenylpiperazine (TFMPP) have been identlfied as selective liganas for the 5HTIB receptor subtype (5). These agents have previously been demonstrated to be antagonists of vascular 5H%. receptor q~tes in the rat ~ugular vein (6). Asarch et al. (7) have r e c e n c y identified LY165163 (para amino phenylethyl TFMPP) as showing relatlve]y high affinity at 5HT]A binding sites in rat cortlcal membranes (Table I). If the receptors that mediate serotonJn-lnduced stomach fundus were of the 5HTIB subtype, we would ~3.00 + .00 (c) 19~6 Pcr~$alnOn P r e ~
contraction% In the rat anticipate that CPP and
0024-3205/86
Copvrlght
J td.
?
Serotonin
Receptor
in Rat Stolna(i~
TABLE
Affinity
Subtypes
$8, ',o. I,
[~)g6
1
of Pheny]piperaztnes
5tlT1B R e c e p t o r
\~l.
in Brain
for 5HT
Cortical
1
and
~embranes
a
- L o g KB 5ttT1A
~'UI]1%
mTFMPP
6.44
8.06
LY165163
8.53
6.48
aData
transposed
from A~arch
et al.
(7).
TFMPP would show higher affinity than LY]65163 at qerotonln receptors in tbo rat fundus. Conversely, if the receptor~ that medlate ~erotun]n c~ntracttons in the rat stomach fundus are of the 511TIA ~ubtype, then IY165163 ~hould ~how higher affinity for these receptorq than the other phenylplperazine derlvarives. Materials
and Methods
Isolation of smooth muscle preparations. Male W1star rats (130-~75 g; Laboratory Supply, Indianapolis, IN) were sacrificed by cervical di~!ocatlon and longltudina] sections of the ,,tomach fundus were prepared for Jn vitro examination. Four preparat~on~ were obtained from one rat fundus. "lls~,ues were mounted in organ baths containing I0 ml of modified Krebs' bc~lutton of the fo]lowlng composition (m111imolar concentrations): Na(,l, 118.2; KCI, q.6; CaCI2.2H20, 1.6; KH2PO4, 1.2; b!gSO4, 1.2; dextrose, I0.0; and NaHCN,, 2~..8. Fissue bath solutions were maintained at ~7°(: and equlllbrated with 9~% O, ,-nd 57 CO 2. Tissues were placed under optimum restlng force (4F,) and were al~owed to equilibrate for approximately ] hr before exposure to
of A$onlst
contractile concentiation xncrease in concentration
Actlvity
Noncumulat ~ve 5HT w e r e o b t , l l n e d b) a s t e p w i s e preceding cc)neentratlonq every
of Phenylpi]~.erazines.
response curves after wa~hin%
for out
15-20 sin. Tissues were then challenged in o ~imllar noncumulative f,~hion with either LPP, TFMPP, er IY16516~. Only one phe~ylpiper,~.zine derivative was examined per tlsbue. Determinatlon of apparent anta$onist dissociation constants. Nuncumu~,~ tlve contractlJe concentration-response curves for 511'I were obtained by stepwise increase in concentration after washlng out the precedlng concontrattons every 15 to 20 m]n. Each agon~st concentration remained ]n contact with the tissue for approximately 2 mln before washout and maximum response to each agonlst concentration was measure(]. FD= wilueq were take~ a,, the concentration of ogonist that produce~ ha]f-max)iOmal contraction. After control response,~ were obtained, tl,q~;ne'~ were incubated wlth on apprnpr[ate concentratlon of buffer or antagonlst for I hr. Responses to 5HT wele then repeated in the presence of antagonist. ('oncentr
Vol.
38,
No.
1,
1986
Serotontn
Receptor
I n Rat
Stomach
3
Apparent antagonlst dissociation constants (KB) were determined for each concentration of antagonist according to the follo~ing equation:
(B) KB = ( d o ~ e
ratio
-
l)
where (B) i s the concentration of the antagonist and dose ratio is the ED. 0 of the agonist in the presence of the antagonlst dlvided" bv. the control F~.ju Generally, paralle] shifts in the concentration-response curves occurred zn the presence of antagonists. These results were then expressed as the negatlve logarithm of the K B (1:e:, -log KR). Calculations were performed wlth the aid of a computer and dzglta] plotter as descrzbed previously (8) for competitive antagonists. The d a t a w e r e a l s o a n a l y z e d according to the procedure of Arunlakshana and Schild (9). According to Arunlakshana and Schlld (9), if blockade is competitive under equiJibrium conditions, a plot of the logarithm of (dose ratio - 1) a g a i n s t the negative logarithm of the molar concentration of antagonist should yield a straight line whose ~lope is ] and intercept along the abscissa i s t h e pA 2 w h i c h ~s e q u a l t o - l o g KB. Results CPP and TI~IPP were partial agonJsts in the rat stomach fundus producing a maximum contractile response that was approximately 40% of the maximum response to serotonin (Figure i). In contrast, compound~l,Y165i63 did not contract the rat stomach fundus in concentrations up to I0 M.
RAT STOMACH FUNDUS 100
TON IN (n=38) 60
~
FMPP ( n = 4 )
I
40
O 10
9
8 -LOG
I 7
--
T6
--
= 5
--
4
AGONIST (M)
Fig. 1 Coptra( tile concentration response curves to serotonin, meta chlorophenylplperazine (CPP), meta tr]fJuorometh~]phenylpzperazzne (TFMPP) and I,Y165163, para am~nophenylethy] TFMPP in the rst stnmach fundus. Point~ are mean values ± SE for the number of tissues ind]cated in parentheses.
qt, r , > t O l i i i l
i~,( ¢ , p t o l
ill
'~, } t
Vn] .
%to,n,i~ ~l
However, CPP, TFMPP and I,Y16~16~ were all induced contractions in the rat stomach fundus. soc~ation constants for these phen>ipiperazines All three phenylp~peraz~ne~ qhnwed roughly s~m~lar receptor In the rat stomach fundus in contrast to ( ' F a b l e l~ f e r 5HTIA ( L Y l b 5 1 6 ~ ) and 5HTIB (CPP,
)~,
~,o.
] ,
}c)~G
antagon~t~ of ~erotontnTable > ~nd~cateq the d~sin the ~,~.t stomach fundus. affinity f(~r ti~e ,,erntontn their reported ~pec~f~c~tv '!'F~.IPP) r e c e p t o r subtypes.
TABLE 2 Afi~nitv of PbEny]plperaz~nes for Serotonzn Receptors Mediating Contraction :n the Rat Funduq
Compound
Receptor Selectiv±ty
m'IFMPP
(5HT1B)
7.32
+- 0 . 1 4
(9)
7.36
O.q7
m('PP
(5H'IIB)
7.84
÷ 0.09
(1',)
7.84
1.00
IY16S163
(SHTIA)
7.4~ ± 0.~ ~ (]0)
7.16
].f0
-l,og
k B *_ qF
(n) a
PA2 b
-Slnpe b
a v a t u e s a r e m e a n s i S . t . . l o r KB = ( B ) / ' ( d n ~ e r a t i o - ]) w h e r e (P,~ -: e o n c e n t r , ~ tlon of antagonist and d o s e r a t t o = FD of the Jgonzst after the antagont~t 5 divided bv t h e El).() ~li t h e a g u n L q t b e ~ o r e t h e a n t a g o n i s t . The n u m b e r o f bti.~sues examined f~r each antagonist is indicated ~n p a r e n t h e s e s , . pA 2 i s t h e a h s c t s s a ~ntercept o f t h e l i n e f o r m e d f~om a S c h t l d p l o t o f lo~ ( d o s e r a t t o - 1) v s . - l o g (B) ( q ) , Sch~]d plots were constructed from three concentrations (~f t h e a n t a g o n i s t u~,ing t h e n . m h e r oI t i s s u e q ~ n d ~ c a t e d ±n parenthese,~. The slope of the Schild plots d~d 'nnt differ from unit\.
I)rscuss
ton
Although serotonln ~s a p o t e n t a g o n l ~ t zn t h e r . t , , t o m a c h f u n d u s ( 1 , 2 , ~ ) qpectfic antagoniqtq o f th~.~ r e c e p t o r have not been tdentlfLed. Recent data from our laboratory and o t h e r s h a v e ± n d l c a t e d t h a t t h e r e c e p t o r ~ responsible for serotonin-ipduced (ontractlonq in the rat stom¢Ich fllrlduq are ni)t similar to 51112, SHT. or trvptamlne recept¢~rs as deflned by ra(lio]igand l~ipdlng in rat cortical m e m ~ r a n ~ "(].2,),). Although not ,i~] compounds wlth h~gh alf~nltv at 51ITI bra~n binding s~tes showed high sfl~n~ty for ~er(~tontn rec~.ptors in the rat fun(lu¢ (~), the possib~ Lit> existed that ~eroton~n recept~.~, in the rat ~tomach fundus were more specifically of the 511TI. of 5H'II~ subtype. With the recent ident~fleation ~f agents showing high a~ltn~tv ,']~t 51!T md 5HT • ,¢ I'~ 1t, receptor s u b t y p e . s , t h l ~ p o ~ l h ~ l t t v wa~ ~, . p l o r e d lp t h e p r e ~ e n t . ~ u d v . The selective 5If'liB receptor a~;ents, CPP nnd IFMPP were partlal rig,mists In the rat stomach fund.~ whereas the sel~,cttve SH'FI~ agentg IY16~]6~, did ,~et contract the rat qtnm,lch fun(hls in concentrations u~"to i0 -~v. These dnt,l may sugge.~t t h a t i nteraction ~ith a receptor r e s e m b l i n g t h e 5PT 1. b i n d i n g s i t e rnav be associated w±th contr,~ct I le respnn,,eq in the rat stomach ~imdus. All three phenylpiptr,3~zne derlvattve~ ~t.dled in the pre.sent r e p o r t antagontzed ',erotontn-znduced contractions in the r a t f . n d u s . Although these c o m p o u n d s ~how m a r k e d l y d i f f e r e n t affinities f o r 5ttT 1. and 5PT] r e c e p t o r .~ubt y p e s ( 7 , s u m m a r i z e d in T a b l e l ) , a f f l n r t l e ~ f o r s e r o ~ o n i n r e c e pBt o r ~ m e d l a t a v ~ contraction in t h e rat fundus were similar amon~ t h e s e ohenylplperazlnt
Vol. 38, No. I, 1986
Serotonin Re('eptor in R~it Stomach
5
derivatives (Table 2). If the serotonln receptors in the rat fundus were of the 5 H T I A O r 5HTIB subtype, then we would have anticipated higher affinity for one of k~ese pHenylpzperazines than for the other, based on the receptor subtype specificity previously documented from binding studies. Since the affinltles of all three phenylplperazznes were similar in the rat fundus, we must conclude that the receptors that mediate the contractile response to serotonln are neither 5HT]A nor 5HTIB. Further confirmation of this concluslon might be derived from an evaluation of the affinities of TFMPP and LY165163 based on binding studies in brain cortical membranes (7). This information as summarized in Table I indicates that TFMPP and LY165163 show dissociation constants of approximately i0 M at 5HT._ and 5HT~. receptor subtypes, respectively. Lzkewlse, TFMPP and LY165163 I~ IA . " -f show affinities of approximately 3.5 x i0 at 5HT . and 5HT._ receptor IA Ib subtypes, respectlve~y. Affinity for the serotonin receptor in the rat fundus approximated J x 10--M for these phenylplperazine derivatives, a value different from their affinlties at either 5HTIA or 5HTIB binding sites. In summary, using these 5HT 1 rat stomach fundus, we further mediating serotonin contractions identified with a 5HT 1 subtype of
receptor subtype specific antagonists in the support the contention that the receptor in the rat fundus is not 5HT] nor can it be receptor.
Acknowledsements The authors wish to express their appreciation to Ms. Gretchen Beckhelm for the computer graphics and to Ms. Elaine Gardner for the expert typing of this manuscript. References I. 2. 3. 4. 5. 6. 7. 8. 9.
J. E. LEYSEN AND J. P. TOLLANAERE, Ann. Rep. Med. Chem. 17 i-i0 (1982). M. L. COHEN, K. W. SCHENCK, W. COLBERT AND L. WITTENAUER, J. Pharmacol. Exp. Ther. 232 770-774 (1985). M. I.. COHEN AND L. A. WITTENAUER, J. Pharmacol. Exp. Ther. 233 75-79 (1985). N . W . PEDIGO, H. I. YAMAMURA AND D. L. NELSON, .I. Neurochem. 36 220-226 (1981). M. A. SILLS, B. B. WOLFE AND A. FRAZER, J. Pharmacol. Exp. Ther. 23] 480-487 (1984). M . L . COHEN AND R. W. FULLER, Life Sci. 32 711-718 (1983). K . B . ASARCH, R. W. RANSOM AND J. C. SFIH, Life Sci. 36 1265-]273 (1985). B. R. ZABOROWSKY, W. C. MCMAHON, W. A. GRIFFIN, F. H. NORRIS AND R. R. RUFFOLO, JR., J. Pharmacol. Methods. 4 4165-4178 (1980). O. ARUNLAKSHANA AND H. O. SCHILD, B~. J. Pharmacol. 14 48-58 (1959).