On the presence of NK2 receptor subtypes in peripheral and central tissues

Regulatory Peptzdes, 46 (1993) 311-313

311

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On the presence of NK 2 receptor subtypes in peripheral and central tissues K . J . W a t l i n g a, S. G u a r d a, J . E . K r a u s e b, Y. T a k e d a b, R. Q u i r i o n c, R. Z a r n e g a r a, D. P a i n a a n d R. F r a n k s

a

aParke-Daws Neurosctence Research Centre, Addenbrookes Hospital Stte. Cambridge (UK), t,Department of Anatomy and Neurobiology, Washmgton Umverslty School of Medtc,ne, St Louts, MO (USA) and ~Douglas Hospttal Research Centre and Department of Psychiatry. McGdl Umverszty, Verdun (Canada) K e y words" Tachykinin; N K 2 receptor subtype; [ t25I]Iodohistidyl neurokmm A; Hamster bladder; Rabbit

bladder; [12SI]Neuropeptide 7; Rat brain

Whilst the presence o f N K 2 receptors throughout the peripheral organs of several mammalian species is n o w well established, questions remain concerning the existence of N K 2 receptor subtypes and also whether or not N K 2 receptors are present m the C N S . However, with the recent development of several selective N K 2 receptor antagonists, speclficaUy the cyclic peptide, L-659,877 [1 ], a linear hexapeptide analogue, R 396 [2] and a heptapeptide derivative of N K A ( 4 - 1 0 ) , M E N 10,376 [3], it is n o w possible to attempt to answer these questions. Accordingly, the use of these antagomsts has revealed the existence of species-dependent N K 2 receptor subtypes. Thus, at NK2A receptors present in the rabbit pulmonary artery, contractions ehcited by the N K 2 selective agonist [fl-AlaS]NKA(4-10) are blocked by the above antagonists with a rank order of affinities o f M E N 10,376 > L-659,877 > R 396. In Correspondence to K J Wathng, Parke-Davls Neurosclence Re-

search Centre, Addenbrookes Hospital Site, Hdls Road, Cambndge, CB2 2QB, UK

contrast, at NK2B receptors in hamster trachea, the rank order of affinities for these c o m p o u n d s versus [B-Ala 8 ] N K A ( 4 - 1 0 ) - i n d u c e d contractions is L-659,877> R 3 9 6 > M E N 10,376 [4].

TABLE I AffimUes of tachykanm receptor agonlsts and antagomsts for [ ~2SI]NKA binding sites m rabbit and hamster bladder membranes Data are gwen as mean pK, values + S.E.M Compound

Rabbit bladder

Hamster bladder

NKA [fl-Ala8]NKA(4-10) [Sarg,Met(O2)ll ]SP SenkUde ( _+)CP-96,345 MEN 10,207 MEN 10,376 L-659,877 R 396

8 89 + 0 06 741+003 5.54+0_11 < 5.00 < 5 00 7.03 + 0 04 7 45 + 0 04 6.46 + 0 06 <5 00

03 766+012 <5_00 < 5.00 < 5 00 5_28+ 0.08 5 88 + 0 05 8 09 + 0 04 7 82+0 07 8.85 + 0

312 In the present study, we have identified N K 2 bindmg sites in rabbit and hamster urinary bladder, tissues known to possess N K 2 receptors, and assessed their N K 2 subtype profile using the above antagonists Binding studies were performed on crude m e m b r a n e suspensions using [ 125I]iodohistidylNKA (0 1 nM for hamster unnary bladder and 0.5 n M for rabbit urinary bladder), essentially as prewously described [5]. N K A and the selective N K 2 receptor agonist [fl-AIaS]NKA(4-10) inhibited binding in both tissues with high affinity, whereas the N K 1- and NK3-selective agonlsts, [Sar9,Met(02)il]sP and senktide, respectively, together with the selective

non-peptlde NKl-selective antagonist, ( + ) C P 96,345 were essentially inactive at displacing binding in either tissue (Table I) The relative affinities of the above NK2 selectwe antagonists for the above two Ussues, expressed as mean pKI values ( - logK,) _+ S E . M where n = 3 - 6 (see Table I), confirm the presence of NK2A and NK2n binding sites m rabbit and hamster urinary bladder, respectwely. These data thus support earlier in vitro p h a r m a c o logical data which indicate the existence of species dependent N K 2 receptor subtypes. To investigate the presence of NK2 receptors in rat CNS, we have examined the autoradiograph~c dis-

i

Fig 1_ Autoradlographlc distnbutaon of [ Iz~I]NP ? (25 pM) binding sites to coronal rat brmn sections at the level of the dorsal htppocampus Total binding (A), [12SI]NP ), binding m the presence of 1 nM [Sarg,Met(O,.)n ]SP (B), 30 nM L-659,877 (C) and 100 nM senkUde (D)

313 tribution of binding sites for [IZSI]neuropeptide 7 ([ tzsI]NP ~), a radioligand previously shown to label both N K 1 and N K 2 binding sites [6]. When brain sections were incubated with [ 125I]NP ? (25pM) in the presence of the N K t selective agontst [Sar9,Met(O2) It ]SP (1 nM), binding was displaced markedly from all brain regaons with the notable exceptions of the CA1, CA2 and CA3 subfields of the A m m o n ' s horn of the hlppocampus and various thalamic nuclei (Ftg. 1). In contrast, [ L25I]NP y binding to these [ Sar9,Met(O2)li ]SP-InsensitIve regions was displaced by the NKEB selecttve antagonist L-659,877 (30 nM) suggesting that in these brain regions [t25I] N P 7 is labelling NKEB binding sites. In contrast, senktlde (100 nM) appeared to have very little effect on [125I]NP ? specific binding indicating an absence of binding to N K 3 sites (Fig. l) The presence of central N K 2 binding sites was further investigated by performing competition experiments for [ t25I] N P 7 binding to rat hippocampal membranes. Approx. 20~o of [a25I]NP 7 specific binding to rat hippocampal membranes was dtsplaced wtth high affinity by the NKEB selective antagonist L-659,877 (IC50 0.54 n M ) In contrast, the NKEA selective antagonist M E N 10,207 displayed a significantly lower affinity for these sites (IC50 9 82 nM). In summary, these data confirm the existence of species dependent N K 2 receptor subtypes, namely NK2A receptors present in the rabbit whtch display high affinity for M E N 10,207, and NKEBreceptors

present in the hamster which display high affinity for L-659,877 and R 396. Furthermore, the high affinity of L-659,877 for [ 125I]NP 7 binding sites in rat hlppocampus suggests that the N K z receptor present in this tissue is of the NK2B subtype.

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