DTIC ADA250745: Evidence of NK1 and NK2 Tachykinin Receptors and their Involvement in Histamine Release in a Murine Mast Cell Line

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I Form Apoved
AD-A250'745 JMENTATION PAGE OiS o o.raB0
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2 REPORTD ATE 3. REPORT TYPE AND DATES COVERED
1992 Open literature publication
4 TITLE AND SUBTITLE S. FUNDING NUMBERS
(U)Evidence of NKI and NK2 tachykinin receptors and their
involvement in histamine release in a murine mast cell line 61102A
3M161 102BSI AA
6. AUTHOR(S)
Krumins, SA and Broomfield, CA
7. PERFORMINGO RGANIZATION NAME(S) AND AODRESS(ES) B. PERFORMING ORGANIZATION
US Army Medical Research Institute of Chemical Defense REPORT NUMBER
ATTN: SGRD-UV- PB
Aberdeen Proving Ground, MD 21010-5425
(
9 SPONSORING/MONITORING AGENCY NAME(S) AND .r,UY IES|ES|] 10 SAPGOENNSCOY RRINEPGO/MROTN NIUTMORBIENRG
ELECTE
USAMRICD
MAY2 9 1992
ATTN: SGRD-UV-RC
APG, MD 21010-5425 USAMRICD-P91-017
11. SUPPLEMENTARY NOTES
Appeared in Neuropeptides, vol 21, 65-72, 1992
12a DISTRIBUTION/AVAILABILITY STATEMENT 12b DISTRIBUTION CODE
Approved for public release; distribution unlimited
13. ABSTRACT (Maximum 200 words)
Abstract-Binding of )kHs ubstance P (SP) and histamine release were examined using a
cloned mouse mast cell line SP binding was saturable and specific in the presence of 30mM
Nanad2S 4OOdn5MO.m HMig hT-raisff ibnuitfyfe Sr.P SbPin idnitnegra cwteasd bwloitchk etwd ob tyy tphees ionfc lbuisnidoinn go fs i0te 5su Mwi tho f Kth5 ev aNluKeTs of 0 3
receptor selective lgand septide in the binding mixture Neurokinin A (NKA) evoked
concentration dependent histamine release At concentrations in the nanomolar range, the
NKI preferring agonists SP, SP methylester and physalaemm evoked !0% net release of
histamine, which was substantially less than the maximum effect of NKA (+37%) in the
mNKIcAro-imndoulacre dra hnigseta mPirneetr eraetlemaesnet oIDf .tAherg c',eDll.sP hweit'h,D t-hTer pN0K32.L aeuntta)ngsounbisst tapnecpeti dPe, aA pruetdauticveed SP
antagonist. also elicited histamine release in the micromotar range, apparently acting as an
agonist at the NK2 site Compound 48/80, N-terminal SP fragments, neurokinin B and the two
selective NK2 receptor antagonists cyclo(Gln-Trp Phe (R)ANC-2ILeu.Met) (peptide A) and
cyclo(GIn.Tro.Phe-Gly Leu Met) Ipeotide 8) were ineffective Although the results suggest the
coexistence of functional NKI and NK2 receptors, it appears that in this mast cell line
neuroliminin.duced histamine release is primarily mediated by the NK2 receptor,
characterized biochemically as a low affinity binding site with a K5 value of 40nM for SP
I4 SUBJECTT ERMS 15 NUMBER OF PAGES
histamine release, substance P, tachykinint receptors 8
16 PRICEC ODE
17 SECURiTY CLASSIFiCATiON IS SECURITYC LASSIFICATION t9 SECURITYC LASSIFICATION 20 LIMITATION OF ABSTRACT
OF REPORT OF THIS PAGI OF ABSTRACT
UNCLASSIFIED UNCLASSIFIED UNCLASSIFIED None
NSN 754O0' 280 5500 Statdad Form 298 iRe, 2 89)
192-40
A'europeptidrs (1592)2 1. 6.5-72
©DLu ngnvn Group UK Lid I992
Evidence of NK1 and NK2 Tachykinin Receptors and
their Involvement in Histamine Release in a Murine
Mast Cell Line
S. A. KRUMINS and C. A. BROOMFIELD
Biochemical Pharmacology Branch, United States Army Medical Research Institute of Chemical Defense,
Aberdeen Pro ving Ground, Maryland 21010, USA, fReprint request to CAB)
Abstract-Binding of [3Hjsubstance P (SP) and histamine release were examined using a
cloned mouse mast cell line. SP binding was saturable and specific. In the presence of 30mM
Na SO4S50mM Tris buffer, SP interacted with two types of binding sites with K values of 0.3
2 0
and 40nM. High-affinity SP binding was blocked by the inclusion of 0.5uM of the NKI
receptor selective ligand septidle in the binding mixture. Neurokinin A (NKA) evoked
conce nt ration-dlependent histamine release. At concentrations in the nanomolar range, the
NK1 preferring agonists SIDS, P methylester and physalaemnin evoked _<5%n et release of
histamine, which was substantiallyless than the maximum effect of NKA (+37%) in the
micromolar range. Pretreatment of the cells with the NK2 antagonist peptidle A reduced
NKA-induced histamine release. ID.Arg',D-Phe5 DTp 9,Leu'I ]substance P. a putative SP
antagonist, also elicited histamine release in the micromolar range, apparently acting as an
agonist at the NK2 site. Compound 48/80, N-terminal SP fragments, neurokinin 8 and the two
selective NK2 receptor antagonists cyclo(Gln-Trp-Phe-(R)-[AMC-2Leu-Met (peptidle A) and
cyclo(Gln-Trp-Phe-Gly-Leu-Met) (peptide B) were ineffective. . Ithough the results suggest the
coexistence of functional NKI and NK2 receptors, it appea, s tiat in this mast cell line
neurokinin-induced histamine release is primarily mediated Iny the NK2 receptor,
characterized biochemically as a low affinity binding site wi4 ..i K(I value of 40nM for SP.
Introduction belongs to ill peptide family known as tachyki-
The undecapeptide substance 11( SPt). considered nins. characiv ited by a common C-tetminal
to be a nicurot ransinitter involved in sensorys' eqlenice. Phc-X-GIv-I~uNlet-N I.,2( Table 0). In
processes, is nowv thought to cause a wide range of addition to SP,. t%o othier inammialian tachvkinins.
peripheral effects. e.g.. vasodilation. plasmia namned neutokinmas. are known.- the decapeptides
extravasation and iiniminoiodulation (1). Sp rieurokiiiin A I NKA) and neurokinin BI( N K13)
_________________________________________(2). the biological effects of SPt, NKA and[ NKII
t),ue rmeecd 28N la t991 are mediated through their respective tachykinin
I),lie cccPIC(I3 Ociotc 1991 reepLltors. NKI. NK2 and NK3 (2-4). Among the
65
92 5 2I Z Y'S 2
66 SLUtRoi'Fi'tiDF.S
Table I Amino acid sequences of the mammalian tachykinins (ncurokinins),
SulmtancPe (S P) Arg*Pw*-Ly- -Pro-Gtn-Gtn-t'hc.Phc*Gt-Lu.Mic't-I
Ncurotkinin A (INKA) Itis-L~s.Ttir.Asp.Scr-I~'tiea.Otv.Lcu.M\c.Nl t-
Neurokinn IB( NKIt) Asp-.Mietisi-AMp-Pi-Val.Gly.tlcu.Ntci-Nit.
threc neurokinins. only SI1 is known to induce of SP-induced histamine release have been per-
histamine release from some types of mast cells, formed on heterogeneous but enriched popu-
such as rat peritoneal (S. 6) and human skin mast lations of rat peritoncal mast cells or blood
cells (7. 8). SIPa ction on mast cells is not fully basophils. not well suited for biochemical assays.
understood. Whereas most of the biological effects Autoradiographic studies of SP binding have
of neurokinins depend on the interaction of the failed to show specific binding of Sp to rat peri-
common C-termninal sequence (4). the histanmine toneal mast cells (21).
releasing activity of SI1. in contrast, requires the In pilot experiments. NKA was found to release
basic N-terminal domain of the SP' molecule (9. histamine from MCI9 cells, while SI'. N-terminal
10(-i .e.. Arg-l'ro-Lys-l'ro. However, interactions SP' fragments, compound 48180 and the mast cell
of the C-termninal portion arc needed for maximumn degranulating peptide (MCD), another potent
histamine release (11). Because receptors onl mast histamine releasing compound isolated from wasp
cells recognizing the N-termninal domain of the SP' venom, caused- little or no histamine relese.
molecule have not yet been identified (12). media- Moreover. specific bindingof SI to MNI9 cells was
tion of SP' action via such receptors is presently observL ' These experiments were expanded and
being questioned. Other mechaniqms for SP'action are described in detail here. A part of this investi-
onl mast cells have been suggested. For example. a gation has previously been presented in an abstract
receptor-independent pathway involving GTI'- (22).
binding regulatory proteins (G proteins) has been
suggested as a common mechanisnm for the hista-
mtine releasing action of SP' and the poly-basic Mlaterials and Methods
molecules mastoparan andl compound 48/801
(12-14). Mastoparan. atp eptide toxin from wasp 'iaterhils
venom. aitd compouitd 480(a re potent histamine Radioligand used was J2-prolyI-3 4-al IIsubstance
releasers in rat peritoneal mast cells (11. 13) but IP( [al IS11. 36.3 and 42.6Cilmittol. New England
not in guintea pig tmascte lls (IS). htuman pulmio. Nuclear, MA). Unlabeled peptide!s wvere pur-
nary mast cells (16) or humait basophtilic leuko. chased from P'eninsula Laboratories. CA, or Cam-
evtes (17, 18). SIPd isplays similar variation in bridge Research Biochemicals, NY. and mast cell
evoking histamine release from mast cells of degrantilating peptide (MCI)) was from IBachein
dlifferettt tissue,; antd species (5-11. 13, 19). These CA. Bovine albumin. F~ractionVm. bacitracin.
variations it response remain inexplicable. bestatimt. compouind 48/80 andl histamine diphos-
In thtis report. tlte actiont of netirokinlins ottit pliate salt were purcltised front Signta Chemical
cloned nlouse moait cell lite.- MC19. dlerivedl from Company, MO. Fltioraldehyde o-Ilhtlalaldehyde
fetal liver cells fronma ( 136X A/J )[- mtouse (20). is reagent solution was purchasedl from l'ierce
describedl. 'I lie aint of this study wast;o exaiie Chienmical Compatny. II.. 'Il ie MC19 nmascet ll lite
biochemtically S, bindintg, usintg a homlogeneous" was pureltaed front Aitterican 'lype Cuiltumre Col-
1
cell population. aind correlate the bindimng patterit lectioti (ATICC, CR1. 8306), Nit).
\%ilt%s elective iettrokiii-imtdced histamiite rel-
ease to I1)id enttify the tacmykinimt receptors preseit
onl mast cells and2) assess their role it neurokinimt- Cll'1
iinduced histaitte release. Biochemtical studies of 'I lie NIC19 cells wsere Incuutated its suspmtsiomt
SI1b imtdimtg to ttast cells are lackimtg. Most studies cothtires in lDulbecco's modified Eagle's ttediumt
07
,1K,I ,\ANDN Q,+1+A(,IIM INRI.(,I+I ORS
(1)ME) containing L-mrginine IIC(II 1 6ml). L- Binding data were analyzed using the LIGAND
•al isapl araanlglmuneo ( 36acmidgs/ I). (0fo.1limcaMci)d. (6smodgiul)m. nopny-reusvsaetne- capopmapruentet r peqrougilriabmriu m(2 4) dtioss oobctiaatinio ne stcimonastetasn fto. r tKh,e.
il .0mnM) and fetal bovine serum (10%), supplied and maximum binding capacity. B,,,.,. Models
by ATCC. Actively growing cultures. seeded at involving one or two independent classes of
105cells/ml in Corning 25cm2 polystyrene tissue binding sites were evaluated for each experiment.
culture flasks and supplemented with fresh culture The most appropriate model was shown on the
medium every 2-3 days. were allowed to multiply basis of the 'extra suin-of-squares" F-test. The
at 37°C in a 7% C0 -air mixture for 5 to 6 days. protein concentration was determined by Peter-
2
The cultures were harvested at ac ell density of 1.5 son's modification of the micro-l.owry method
x 10cellshml. Aliquots of the cultured cells were (Sigma Protein Assay Kit) using bovine serum
prepared for histamine release studies, while albumin as a standard.
remaining cells were pelleted and stored at -70°C
or in liquid nitrogen. Cell viability was assessed by
the Trypan blue exclusion test. Cultured cells of
viability ->75%w ere used in the experiments. 'Histamine release studies. Cultured cells were
pelleted. washed twice and resuspended in Tyrode
solution of the follwing composition (mM): NaCI
Binding studies. A modification of the method 137. KCI 2.7. Nal12PO4 0.4. MgCI2, I,g lucose 5.6,
described by Maruyama (23) was used. The frozen H EPES (4.(2-hydroxyethyl)- I-piperazine-ethane-
cell pellets were thawed and diluted with 50 sulphonic acid) 10.a nd with or without CaCI.
volumes of ice-cold 50mM Tris'HCI buffer (pH I.8mM (p't 7.42,0 'C). Aliquots (0.9n1) of cell
7.4). vortexed vigorously, then centrifuged at suspensions (10'eells/rl). subsequent to teni-
40000g for 15amn at 40C and washed one more perature equilibration (5min, 370C). were incu-
time. The resulting pellet was resuspended inTris bated with the indicated concentrations of a
buffer (600mg protein/iol), now containing baci- possible releasing agent or the vehicle (spon-
tracin (final concentration 40ug/ml), and distri- tancous release) in a volume of 0.1 11. Tilere ac-
buted in 0.25 ml aliquots to a set of polypropylene tion was stopped after 15miu incubation by
tubes containing 40uM bestatin, 0.4mg bovine chilling the tubes in an ice bath. The cells were
avolbluummein aI nmdl )w. itFho orr wbiinthdoinugt 3s0amtuMra Ntioan2S Oan4a (ltyostiasl, s1e0pmairna teadt fr3o(m0g .t he4 0mCe. diRuemle absye cde nhtisritfaumgaintieo nw foars
increasing concentrations of 131tISP were added in measured in the supernatants. Residual histamine
the presence and absence of I uM of unlabeled SP. in the cells was measured after lysing the cells by
In competitive inhibition studies, increasing con- addition of distilled water to the pellets followed
centrations (0.1 nM-1buM) of unlabeled SP were by protein precipitation with 20% tricholoroacetic
added in the presence of a fixed concentration of acid and centrifugation at 10(0)g. listamine was
I1IlISP t0.2-2nM). After 2(mm incubation on ice, assayed fluorimetrically by the method of Shore et
the labeled membranes were collected by filtration al. (25) using an excitation wavelength of 348iut
over Whatnan GF/B filters under vacuum. fol- and emission wavelength of 439 nm, Ilistamine
lowed by three rapid washes with ice-cold Tris standard ranging from 25nighrl to 250ug/nl were
buffer using the M-24R Cell-Ilarvester (Brandel used for calibration of the assay. I listamiie rel-
Instruments). Both the tubes and the filters were eased in the supernatant was expressed as a
soaked with (1.1% polyethylenimine for 24h percentage of total histamine presem in the cells
before use. The binding of [I lISP to crude MCt9 plu supernatant. Spontaneous release in the
membranes was determined from the difference absence of a releasing agent was subtracted from
between parallel experiments with membranes or the stimulated release to estimate net induced
the vehicle for each combined concentration of release. The releasing agents \%ere examined for
labeled and unlabeled SP to rule out contributions posible interference with histamine determin-
o1l I IISP binding to nonl-biological materials. ation. and corrections were made when required
68 NEt Ei nIDEs
-C7 s rsehsouwltns) i.n inco wnchaicvhe dKod wvnawriaersd wSictha tcahmaordu nptl obtos u(nnodt.
Therefore, the parameters for the binding of
E ['HISP were not calculated based on these binding
50 data.
3
9Competitive inhibition studies of [HISP
abinding by increasing concentrations of unlabeled
25 SP were performed in high ionic strength solutions
by the inclusion of NaSOa in the binding mixtures,
2
which according to Bahouth and Masacchio (27)
acts mainly by reducing nonspecific SP binding.
The lowering of nonspecific SP binding was con-
0L(Mo)g '.e mS 1A DDED 2 firWmbeidn.d iTnmghi xet uared dpitrioodnu coefd 3n0omnMsp eNciafi2cS bOin4 dtion gt haes
Fig. I Concentration dependence of the specific binding of low as 35% of total binding and was routinely used
IIIISP to mastc ell membranes in 50mM Tris buffer. 4C. All in the competition experiments. The binding para-
values are the mean of quadruple samples The differenc meters were estimated from the displacement f
between samples %ere less than IS%. 2nM [3H]SP binding (88000cpm) by increasing
concentrations of unlabeled SP. The radioligand
was used at the lowest practical concentration for
detection of binding to sites of disparate character-
Results istics. This concentration provided about 1200cpm
Binding of fHlsubstance P to mast cell ,nent- of total binding and about 800cpm of specific
branes. The binding of [3HISP to mast cell mem- binding after corrections for [3H]SP binding to
branes was rapid. Time course studies of 0.2nM non-biological material. The displacement curve
(5HISP binding revealed that maximum specific with the 95% confidence limit curves (Fig. 2)
bindingwas reached after 20min incubation at 4C represents a conglomerate plot of binding data
(data not shown). After I h incubation, 87% of from four separate experiments. A two-site model
maximum binding remained bound, while after 3h was found to be a best fit for (3HISP binding to
incubation, 72% remained bound. Hence, 20min mast cell membranes. The estimated binding para-
incubation was routinely used. Figure 1 demon- meters are shown in Table 2.
strates that specific binding of I[HISP is saturable. Blocking studies with the highly selective NKI
Artifactual non-receptor interactions were presu- receptor ligand [pGlu6,Progjsubstance P (septide)
mably absent since background counts were (28) were performed to further examine the heter-
subtracted from each assay point (see Methods). ogeneity ofSP binding sites, Fig, 3 shows the effect
The specific binding, obtained by deducting non- ofO.5uM septide on the displacement curve in the
specific binding in the presence of I uM unlabeled
SP from that of total binding without SP. was only
10 to 15% of total binding. For example, the Table 2 Parameters for substatce P binding to ntast
addition of l8nM (3H]SP (797300cpm) resulted in cell (MC79) membrates m the presette of 30m
total j3tHjSP binding of 11 154cpm and specific NaZSO,
L'HISP binding of 1267cpm (2mL total volume of
binding mixture) after binding to nonbiological ApparouKeBnt,
substances had been accounted for. (lAl) fnolesI,gProtein
The shape of the saturation curve is indicative of tIgh.affiniis ite 03 67
cooperative binding. For positive cooperativity. Low.affmitysite 40.0 2710)
the ascending portion of the curve is almost Binding parameters "ere detised fromthe ombined anal)sis
parallel with the y-axis (26). here indicated by offour displacement epCeriments uing the LtGAND program
13111S P bound. Facilitated or cooperative binding (24). Data are shon inI lgure 2.
7NKAI NI)NK2TAClY~% HCl'l,Rll-'ORS 69
002- Table 3 IHistamine release froin mist Cell% (NIC19)
induced by iachykinin receptor ligatids
001 a Liand 1C xo n0ea-1il (, n) %r ocfhus-aiusf)
Substance P' (StI' 0 010 (3) 5-1
0.110 (I) 4
10t (I) 3
0..1001 (I) 3
-710001 (2) 4!±0,7
LOGM I
SI'niineh~tester 00( (W3) 31 1
Fig.2 Displacements of the Linding of 2n~tl 'IIjSI' by 0.1100 (2) 4 140
unlabeled SP from mast cell membranes in the presenceo f
30mM Na-SO4. 'The displacement curse ssilh9 5% confidence Ph)salaeininl 0 010I (5) 4- (17.
limit curNc s is a conglomerate plot bascdo n four comlpetition Neuroinin A (NIKA) 0Onto( (I) 0
experiments. indicating binding to (l ites( Table 2). o. 1I (2) 2 1 () 7
Blrr bound divided by tot it adjoact wit) Iog('l)- loga rilthnm I (IIo) 4!±06
of the total ligand concentration. Anil (5) 7 ± I
20 (2) 101! 0.7
parfefisneilcye d iospf l3a0c emmMe nNt ao2fS I3O4II.ISTPh eb inadbsinegn.c es hoofw hnitg hb-y [0 1N(0O001 1 (((3S) )) .13374 1± S17
an initial plateau in (he plot, is indicative ofseptide 030( (1) 4)
blocking of NK I sites. ((1 (1) .1t
Release ofh ismntine froin inast cells. The amnount 11r 7 .~ul 101 (2) 4 : 2
of histamine in the MC/9 cells varied with number substance P' 50 (2) 03 0,(7
of days in culture. A maximum amount up 10 (pcptideM 1001(S (2) 41
0,608ug/106 Cells was measured, which was more
than 10-fold that of mast ells derived from murine I ties .Ituese,s prcssed.is.te.In .4S tUt in a- 5)a ndMea±inS D)
bone marrow cultures (29), 0,053ug/10' cells, but (n 2). represent Induced rcteascs.s bichi st igind siulated
releasem inuss pontaneous reteise.
much lower than that of either human p~ulmo(nary,. Delectaible in thea bsencoef estrmcclular CaCI'. i'Potent
4.23ug/105 cells, or rat peritoneal mast cells. bousbesin antagoni (30ta)i ndl utiie SlI. nigonm'I 311
30ug/106 cells, (16).
00t istamine was released fronm slispenioi((t of
NlC/9 cells by rticroinolar contcentrationts of the
~NK2
00 preferring agottist. NKA. attclilily toa minior
degree by nautotiolar conicenttratiotns of the INKI
z 00n0 selective agottists. SP. Sli meulylestcr and the
amphibian tachlykinlin. pllysalaeintt (Table 3).
Inctreasinig conlcenttratiotns of te NK I preferrintg
itgonists till to ](XuNl did not produltce an increase
Of________ in histamtinte release. Septide appeared to htave no
.5 itminle releasing Capability. To detect itnduced
LOGIT ) re leas e for SPI a td pltvsal ae ti n. i t was ntecesa ry to
Fig.-' Reprcsentattse cutsco f the distulacemnotsf l the o(mit extra-cellular COCI2 frotleIlt ledlujoin. Tis Is.
binding of 201 ['I lSt' by untlateled SI1f rom mastc ell a condition Ithe 1s109 cells %harev ,'i th rat peritotical
menmbralneisn the presenceo ftb oth1NitNt NSa mndu li SuM latcls(0,O ilng al,
smeipctaisien.c iiAlerlntmt,.s antodft sh bein edsinpge mdaimtae nssts saas%br e apeoa tedd0 uop1nliccesa uteit Y.itesld edc eal lt (1tv0e) rOl 1m stIting llowe-rc aCvv a~la\%ue flolira tlte
simlar xvsuls. sponttanteous release. 16 i I",, (t =10). as
70 .1-LUROi'LflhtS
compared to 18± I% (nt= 32) in the presence of SI1 bainds to two types of sitcs of vastly different
I.SaMl CaCd. lbut had littlc or no effect on binding propertics. As SI1 interacts with all three
2
ind~uced histamine release except when SI' or tachykinin reccptors. but preferentially with the
pliysalaciii were the releasers. Extending the NKI type, the high-affinity binding is compatible
incubation period with the releasing agents front with that of SP binding to NK I receptors. While
lin tiptlo~fmin did not change the valuesofthc blocking of high-affinity SP binding using the
.spontneous releaise. Induced histamine release selective NK I ligand septide (Fig. 3) confirms the
reachied a mtaximnumat i 7.5miit incubation and presence of NK I receptors, the functional studies
remained coistat tiup to(Ah0mtmhe longest period support the presence of NK2 receptors.
exalnc(. [l).ArgIJ.i-he.DTrp .Leu~i We have demonstrated here that none of the
subsance IP (peptide X). a potent boibesin well-known histamine releasers, compound 48/80.
antagoiit (30))a nd putatilse SIPa ntagonist (31) MCD and N-terminal SP fragments evoked hista-
induced the release of histamine iii a similar mine release from the MC/9 cells. On the other
concentration range as (lid NKA. while two hand, the results have revealed the novel observa-
selective antagonists for the NK2 receptor (32). tion that NKA acts as a histamine secretagogue in
cc(GnrP.Ihe-(R)Glv.I ANC-21 [.ell.Met) this cell type. Moreover, the reduction in NKA-
(peptide A) and cyclo(Gln.-rrp-l'he-GIy-.eu. induced histamine release observed after pretreat-
Met) (peptide 13). were without effect tip to ment with a NK2 antagonist but not with a NKI
lIN)uN. Compound 48/80). examined from 1.00 antagonist (spantide) suggests the involvement of
to l(XhighmnL.a range kntown to cause releamins era t NK2 receptors in this process and thereby the
p)eritoniea[l mast cells (1011. 1. 13). NICI at I uMt. existence of NK2 receptors on this cell line. The
the N-terminal SIP fragments. SPI'(-4) (0.01- weak activity of the selective NKI agonists sug-
lO~nNI) and SI'(l.6). SPI'(-7) or SPI'(l9) at gests the presence of a nminor population of
IfiuM. as well as NKI3. a preferential NK3 functional NKI sites. However, the involvement
receptor agonist, in thmrean ge 0.0l-35tiM. evoked of NK I receptors in NKA-induced histamine rel-
little or no histamine release (data not shtown). ease was dismissed onl the following grounds: (1)
The two NK2 selective amtagoimists. peptide A and NKA is shown to interact preferentially with NK2
11( ,ee above), were examined for their ability to receptors; (2) pretreamtment of the cells with the SP
inhibit NKA-induced histamitte release. P'eptide (N KI ) antagonist spantide led to no loss in NKA-
A appeared to blem ost effective. l'ricbation of induced histantine release, while (3) pretreatment
the cells with either I10or lOQnNI of peptide A for with a NK2 antagonist did. A lack of effect by the
10m ma t 371~pC~ rior to the expostire to e(Itiinolar NK 3-preferring ligand NK13 precluded the pos-
concentrations of NK A redutced the histamine sible involvement of NK3 receptors in NKA-
release by 25 and STY,- respectivelyv. I'reimcu- induced histamnine release.
f
bation with the SI' antagonist (33). I)Arg .1)- Recenitly. pharmacological and biochemical
'lrp7 1..em I s11bstince I) (spamitide). onl the other evidence hlas suggested that there are multiple
htand, had Ito redtucing effect. subtypes of NK2 receptors (35). A series of linear
The N109 cells, like rat peritoneal mast cells and cyclic NK2 anttagonist,. were fotind to have
(34), released histamnine with concanas aImA (conmv arying potency in different NK2 containing
A). an Igl*-type secretagogite. Incubation for tisstues.lihe finding that thmsee lective NK 2 recep-
I51m1m isi itli 2.5 and Sugtilm of con A without time tor antagonist -peptide A.- inhibited only pariallI'
potentiator plmosplmatidylerime evoked I P/ and NKA-induced histamine release mighmt inidicate
91"i,n duced release, respctively, thmpere sence (ifa hecterogenons pool of NK2 sites
onl MC/9 cells.
lDisclmsimmi The present sttudies have provided evidence for
tie cistemee of N KI and NK 2 receptors onl thme
Tlhe resti demnonstrate th thecN C'09 iiias cell MC/9 cell line. Tlhe coexistencee of phmysmologically
line poCse Specific binldimgsites for SIP.M lore- relevanit NK I and NK2 receptors has recently been
os er anahmsisl of thle bi in , damli&tas resecaled th at describmed for different functions muotmhe r types of
NKIANNIsK r5CtYKININIZI OXIIORS 71"
tissues IFor exsample. both SI1a nd NKA were shown lhere that in *t mast cell type. unresponsive
found to stimulate thle release of i-hydrosytrypiti- to known basic sccretagaoguces suich as compound
mine via NKI and NK2 receptors in di le rat 4880and N-terminal SIPfr agments. a NK2 recep.
cerebral cortex. (33) B~oth receptor types have also tor-dependent pathway might ex.ist for the release
b)en implicated in SP-indticed superoxide anion of histaine.
production inl guineca-pig alveolar macrophages
(36).
cn~ldeet
It has lbecii argued that. because some SIP~ cn~ldert
antagonists. such as (D-Arg1.-D-l'ro2.D-T'rp'- t1osoksssdn itj11wauhr S ,Knin1 .tcd
"'Lell"Istibsiancc P1e.l icit histamiine release (37). National ReseaircCho uncil (USANIRICI)) Senior Researchi
the iniductioii of histainiie release by SIPis not Assiiiieup I tieo piniiiiisor asetlionsoniaineit hervin are
tachkinn reeptr-nediaed.Becuse eveal tic ivaic lins of the anihors init aren ot to lie conisirued as
tachkinn rcepor-ediaed.Becusesevraloffic,1t or asr eflecting ihe \~ivS of %he Atii or the D~epart-
an tagonists of SI')w ithI two or more D-Trp residuies menio f tDefense.
of liiiited selectivity for all three tachykiuin reed-
torg have considerable agonist properties for these
sites (38, 39). it isp ossible that we deal with a class Referenuces
of comtpounids with imixed agonlist-auttagottist I tLeiiieck. ri (t988). tile 19mU,l f \oii [.ier Itcr.
properties. l'eptide X (lTble 3) is apparently such Substniice 1'. Froni emrtr~to eseirent. ,\eia Phl,t
aIc oimpounid. *'bis peptide described ittitially as it Seand1,3 34: 35-454.
SP' anutagonist that blocks the hyoscitie-resistait 2 egiili.) .ta~peau. C6 oS ntl)ieisjse '
olliate withidrawval contractutre Ii gititia pig ileutim kls$7) m~siin'nidre siew. phtarmiacological receptor% for
sibsi.iiie neiirol~inins. t.ife Sc. 41) 101-117
(32). was later fouind to be a potent aittagonist of 3I Ilele. C J-. Krause. J C.. Manih. t, \V.. Couturc. It
the amuphibian let radecapept ide bomlbesin and aod tiii. M . t'Wo IDilersiq in niannntaian
structurally related mammalian peptides. includ- iactkini ii jidergic neurons. mitltiple peptide.. retell.
iiig gasirtin-releasitig peptide (GRIP), (31 ). l'eptde tor.. nu regulatoty iiehannins I-ASLii J 4. IldiS-1615
X has also been lounrd to inhibit the action of tile Ifegoli, 1) , tDraipeanC. AD ion, S., iid Coiiiure. Rt(1988$)
nneetruohrphohyhoryproisoianileih uvyaessp ersls i' :tpresiit (44)~. ) NcoelOt. gsiccalle tcouoilss afogro rnecisepifstoo rr neccilalrroa5ic.iieurniz areiiocne pto'Irrs e ispl haPrnliiaa-r-
Sinucvca sopressiti anidth lep eplidles of thle bomlbe- iiacot. sci. 9:2, 1M9.52.
Sill family interact withi distincet anid Specific hligh- Johnson. A R. aind ErdosI.! . G, (197.1)R. ecleasAe li
affinuity recep~tors, it appears that peptide X inter- liisiaiiiin roiii m011taei1lss, t by' oisepeptides Proc.
acts asa blockitng agent with several itndependent No \1 BtiltN fcii1 421;2 32-1256
receptors. I Iolvcver. preset stud~ies Indicate that Ecr~ias oc1n. rrF ' .tLenlibacli., I.-. .I itdoIrtilme.e.(- irnian. t9I 8..t S),k olfeitsaeste.l~
peplide X,-w hich Ims muiniiial structural Imiti1ology iit~tniei. ns~ncI rh 'irnio.376-
with NKA (only Leu ..).. elicits a biological 7 lDemulici, P-. Itegoli. D-. Asserif. A.. tDcscour..I i,
respaonse. [hus. peptide X funictionls appareintly as Nla,iis,Ji .a iid Retious.M 0i9 8o)1. tttimi releasaen d
a muixed agoitst-anitagoist, expresing -gils losil esponsssosf rat aiid tiiijiskiil toi iilmaine 11a nd
characteristics a hNK2rcpo ndatagontist 'S Chiirch-.\tK.. Beton, It, C .Lossoan-MN. A,, litnisj
characteristics at various inidepenident recepator-,. :1 A. andI lolgaic-. . I195)Aligrmnluiiaiii
itncluditgilie NK I tacltykiniii receptor. Ini cont- 'roumt, ropspuis stimnulatioiin h iiin i llt mastte lls it.
elusion. this. ttid) hits demnitstfated the presetie .iiidiss oiiO Ii.u iueslu A1u usi t 1,i1 1 tsi lKiu tssponss.
of both NI(I and N (2r eceptorsott aIi tiirite liver Agetls, ind Actions 26, 11-).
ddeerireilsv~ui eetii asad~s~ai~ i~cd~~e ~li s~~t hie~emf rati ttrc sl to hhuwiwtat ha lO19'et 9.)t ilRuoe1le1r - O Iwftp ilreaNp -utieZi iu-,u Rueuanoirugani.nt iuNue.in a ntiiliedRh isetagmoinlniIe.). .
histamine is released by NK A via NK12 lacltykittin releasing aiusui ol mnlistanee 1'.t urainitamn aiid retmed
receptor activation,. Wlterewts septide appeafs to tpupdis. Lur. J Phtlluanmu1l6~8 53.W
have no histatiiie releasinig activity Init his cell In' te , Mi.rSl oicumiJ) ( .JomdanC. C -Oeluue,
typ.le other NK I preferring agottists. e.g., SIP P .Reuunet It anse'Nait. MN.0 t982) I ticefetiisol
methl lester. SIPa nd piysalactiti. exert a tltiitr 1nd ie rat J Phl\suI3tI3 )1W WII4
stimltilatittgz effect ottl michl release. suggetitig thle NImt uieL.N -teei.I- I eulavurg, I ani tiuniut'rg. S
possible ituso lvelieit of N 1( receptors. We have (lISSQt Ilis iols ,IiI h, XNImiinal mistiutse ti tile
72
NLUROI''IDES
istamnie releasing action of substanceP . Neuropharm. 28. Wormsr. U.. Laufer. R.. Hlart. Y,.Chorev. M..Gilon.C.
acologs2 0: 1025-1027. and Selinger. Z. (1980). Highly selective agonists for
12 NMou~liM. , Bauch, J -L., Bronner. C.. Rouot. B and substanceP r eceptor subl~pcs. EMNBOJ . 5: 2805280S
Landry. Y. (2990). G protein: a rcceptor-indeNrdent 29. Wodnar.Filipouiez. A.. Ilcusser. C 12 and Moroni. C.
urodeofactioflfor cationic aphiphilic reuropeptidcs and (1989). Production of [he hacntopoicie growuthfa ctors
senom peptides.1Irends Pharmacol. Sca,2 2:3 58-362, GMlCSF and interleukin-3 bym ast cells in responseto Igli
23. Muli.M.. Btrunner. C.. 2.andrv. Y.. Boekacrt. J. and recptor-mediated activation. Nature 339:1 50-152.
Rouot. 13 (19905)D. ~irecat ctivation of G1 P-binding regu. 30. Woll. P.1Ja. nd Rozengurt. E3(. 1988).I D-Arg'.D.Phc5.D.
latory proteins (G-protcius) bys ubstance Pand compound 'I rp? 9.Leuilsubstance P, a potent bombesin antagonist in
48/0 lEBS Letter, 259:2(4).202 murine Sw~is3sT 3 cells, inhibits the growth of human smtall
14. Repke. 12.a nd Itienet. NI. (2987).M ast cella ctivation -a cell lung cancer cells in vitro. Proc. Natl. Acad. Sc:.U SA
receptoir-independent miodeo f substanceP action. FCltS 85: 1859-1$63.
1 eitters2 21:2 .36-240. 31. Tson. K., Wut.S ..X.. Lu. Y.-A. and Way. 23.L . (1985)
15 2eldbergW. Va. nd Niongar, J. 1. (2954). Comparison of Block of the hioscine-resistant opiate wuithdrawal contrac. a
histamine releaseb y compound 4811$a0n do ct)lamine in ture of ilenin bya in ew substanc P antagonist (P.Argi.D.
perfused tissues.2 r.J. liharniacol. 9.2197-201. PheS.D-Trps.Lcuuijsubsancc P. Cur. J. Pharmacol. 220:
16i Ennis. NI. (1982).1 istarme releasef ront human pulmo- 155.156,
nat5r mastc ells. Agents and Actiiins 22: W-.63. 32. Williams. B. J.. Curtis. N. R.. McKnight. A. T., Maguire,
17 Piearce. F. L. (1986).O n the hetcrogeneity of mastc ells. J.. Foster. A. and Tridgelt. R. (1988).D evelopment of
Pliarnacology 32: 61.71. NK-2 selective antagonists, Reg. Pep.2 2: 289.
18 torcitian. J. C and Lichtenstein, L. St. (080). Induction 33. lscrfeldt. K.. Sol:i. M. and Bartfal. T. Y. (2990).
of histamine secretion bi) pol~cations Biochim. Bioph~s Substance Pa ndN enrokinin A. Issoc oenistiug tach))kinius.
Acta 629:5 87-603. stimulating the release of 1'1115-11Tf rom rat cerebral
29. Ali. I L, Leung. K. B. '.. PearrceF, . L.. Iiacs. N. A.a nd cortical slices. Brain Res. 506-.335-338.
Foreitian. J. C. (2986). Csimparison of the histamine. 34. Carnuccio. R.. Di Rosa. NI., Waenti. A.. lussine. T. and
releasingactioii of substance1 Po n mastc ells andb asophils Santebin. L. (1989).S electise inhibition by sasocorttn of
front different speciesa ndt issues.I nt. Arch. Allergy App histamine releasein duced by dentran andc oncanavalin.A
Intitntiol. 79:4 13-428 from rat peritoneal cells. Br. J. Pharmacol. 98:3 2.36
210N ibel. G., Fresnii..Chessman. A. and Cantiir, 11, 35. Bucd. S, 2H. and %an Giersbergen. P. L.. NI. (2990).
(1981).U seo f cloned pospulations of niouse lynmphocytetos Phartnacological and biochemical esidenc fur multiple
analyie cellulair differentiation. Cell2 3:219.28. types of lach))kinn NKl receptors. Abstract and oral
22. Butrcher. t...'iussap. C.J ., Geraghty.DP1.a .n dSlcClurc. presentation at the Substance P and Related Peptides:
Shasrp1.. M. (1299XC5o)n.c epts in characterization of Cellular and~lecular Ph)siology. Ns'w York Academyof
tlich~kiiul receptis. Abstract ando ral prestmation at the ScienceCso nference. Worcester. Mass. IS8t022 July 2990
SubstancPe as ndR elated Pcptides: Cellular and Noteen. 36. Brunellesehi. S.. Vanni. L.. Ledda. F.. Giotlt. A.. Nlaggi,
2.sPr lhsislogy. NessY ork Academy of ScienceCso nfer. C. A. and Fa'ntozzRi. . (2990). Tachylkinins aclisate
circe. WVorcester.MINS aitso s2.2 J uly 1990. guinica-pigalseolar macrophages-, insosenient of NK. and
22 Kruuiins.S A. (1"9)) Iindingof ['I 2ilsubstance 1it o mast NKI receptors. Br. J. Pharniacol. 2HM427.420.
cells. Abstraict andp oisteprr esentation at ibid. 37. fla1.anson. R.. Ilorig. J. and Leander. S. (2982). The
23 MIaru).,i. NI. (2986)S.e lectise solubilization of physa. mcchanisni of action of a substanceP aitagonist
lacntin-type substancPe binding sites froimr at biain (D-Plo.'DTrp'').SP. lit. J. Pharmacol. 77:6 97.7580.
tiinilbtanics by gl~csidcox)chstait and NaCI. Brain Res. 38. Regoli. D.. Nlizrahi. J., D'Orlean-Justc. 1P..D ion. S,
31751:8 6.1(A). Drapean. G. andC schcr. 2E(.2 985). SubstancPe a ntago.
24 Mlunsiin, 11 J. .ind Rodbard. D. (1980).2 .2GAND: A rusts showsisnogn iceselectiy for different rceptorqtpes.
sersatile cuniputetized approach for characterization of Cor. J. Plirmacol. IM81:2 2*25.
liganiiidniing )NltcnisA nal. Itiochein. 45:5 30.556. 39, Buad,.S . 11. and Shatner. S A. (2988)A. gonist and t
253S huireP. . A.. BunrkhailtAe.r .a ndC ohn. V, It, (2959). A asntagonist binding to tach~lkiniii peptide NK-2 receptors.,
tuetlrirsfot tile flul oiilicassasyol histantinetn tissuesJ. . Life Sci.4 2:2 7122708. F.I
Pliniacil. Etnp' Ih er.2 27:1 82-186 40. Zachary, I., and Rozengut. 13. (2986)A, substancPe
26 I)e2 e.rn.A..indRo dbard.1.(1979I Kinetics ofc oopetri. antagonist also Inhibits specific biniding and ttogenic
to isIning. In: I he Receptors. VOL 1, (O'Brien. R. 1). effects of sasopressin and iomnbesin-relpaetpetidde s in
ed.) PlenuimPutl iishiing Cotporation. p. 143.192. Sswis3s2 13ce lls Iliocheni, Ihiaphys. Res. Comni.17. touatod
27 Baliuth. S. W, and ushrijchlJin. .M I(.2 985), specific 135-141. 4.1tia~
iiig of Ij'IIsnssttiucc p)t o ther asst ubmiasitry gland.
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C 1:.' her2, 314:.126,136
!nsDis ri Spec,