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[125i]Neurokinin A labels pharmacologically distinct populations of NK2 binding sites in hamster and rabbit urinary bladder
European Journal of Pharmacology, 232 (1993) 287-290
287
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 21194 Short communication
[125I]Neurokinin A labels pharmacologically distinct populations of NK 2 binding sites in hamster and rabbit urinary bladder S t e v e n G u a r d , D e b a s i s h Pain, R u t h F r a n k s a n d K e i t h J. W a t l i n g Parke-Davis Neuroscience Research Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB, UK
Received 19 November 1992,revised MS received 6 January 1993,accepted 12 January 1993
The pharmacological profile of NK 2 binding sites has been characterised in homogenates of rabbit urinary bladder and compared with that present in homogenates of hamster bladder. In both species, [125I]neurokinin A-specific binding to urinary bladder membranes was displaced by neurokinin A and the NK 2 agonist [fl-AlaS]neurokinin A-(4-10) whilst the NK 1 ligands [Sar9,Met(O2)n]substance P and (+)-CP-96,345, and the NK 3 agonist, senktide, were only weak displacers or ineffective. At rabbit NK 2 sites, the rank order of affinity of NK 2 receptor-selective antagonists was; MEN 10,376 > MEN 10,207 > L-659,877 >> R 396. In contrast, the rank order of displacement of [x25I]neurokinin A-specific binding to hamster bladder membranes was: L-659,877 > R 396 > MEN 10,376 > MEN 10,207. These data demonstrate that [lZSI]neurokinin A binds to pharmacologically distinct NK2 binding sites in hamster and rabbit urinary bladder. Tachykinin; Neurokinin A; Neurokinin receptor subtypes; Tachykinin receptor antagonists; Bladder (rabbit, hamster)
1. Introduction
Substance P, neurokinin A and neurokinin B belong to the tachykinin family of peptides which are characterised by a common C-terminal amino acid sequence Phe-X-Gly-Leu-Met-NH 2. It is now well established that the biological effects of the tachykinins are mediated by at least three receptor types termed NK l, NK 2 and NK 3. The pharmacology of tachykinin receptors has progressed markedly in the last few years, due primarily to the development of both peptide and non-peptide receptor antagonists with high affinity and selectivity for a particular receptor type (for recent reviews see Guard and Watson, 1991; Watling, 1992). NK 2 receptors are widely distributed throughout the peripheral organs of several mammalian species and are characterised by their preferential affinity for neurokinin A and its N-terminally extended forms, neuropeptide K and neuropeptide Y (van Giersbergen et al., 1992). Most recently, the use of selective NK2 receptor antagonists (see table 1), specifically the cyclic peptide, L-659,877 (McKnight e t a . , 1991), a linear hexapeptide analogue, R 396 (Dion et al., 1990), and a heptapeptide derivative of neurokinin A-(4-10), M E N
Correspondence to: S. Guard, Parke-Davis Neuroscience Research Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB, UK. Tel. 44.223.210929, fax 44.223.249106.
10,376 (Maggi et al., 1991), has revealed the possible existence of NK 2 receptor subtypes. Thus, in rabbit tissue bioassays, the following rank order of affinity is observed; M E N 10,376 > L-659,877 > R 396; whereas in hamster tissue bioassays, the rank order is; L659,877 > R 396 > M E N 10,376 (Maggi et al., 1992a). In the present study, we have identified and characterised NK 2 receptor binding sites in the rabbit urinary bladder using [125I]neurokinin A. The pharmacological profile obtained with tachykinin receptor-selective agonists and the above NK 2 receptor antagonists has been compared with that observed in the hamster urinary bladder.
2. Materials and methods 2.1. Radioligand binding assays
For radioligand binding assays in rabbit and hamster urinary bladder, preparation of crude membrane suspensions and binding assays were performed essentially as previously described (Lee et al., 1986). Briefly, tissues from New Zealand White rabbits stored at - 70°C, or fresh tissues from Golden Syrian hamsters were homogenised by Polytron in 50 mM Tris-HC1 buffer (pH 7.4) containing 100 mM NaC1 and 5 mM KCI and centrifuged at 48 000 x g at 4°C for 30 min. The pellet was resuspended in 50 mM Tris-HC1 (pH 7.4) contain-
288
ing 100 mM NaC1, 10 mM EDTA and 300 mM KC1 and incubated for 30 min at 4°C. The membranes were centrifuged at 48000 x g for 30 min and subsequently washed twice with 50 mM Tris-HCl (pH 7.4). The final pellet was resuspended in a small volume of 50 mM Tris-HC1 (pH 7.4) and protein content determined by the method of Bradford (1976) using bovine serum albumin (BSA) as a standard. Binding assays were performed in polypropylene tubes in a final volume of 300/xl Tris-HC1 buffer (pH 7.4) containing 3 mM MnC12, 0.02% BSA, and 2 ~ g / m l chymostatin, 4 / z g / m l leupeptin, 40 ~ g / m l bacitracin, 2 /zM phosphoramidon to inhibit peptidase activity. Crude membranes (150 /xg protein for rabbit bladder or 20 /~g protein for hamster bladder) were incubated at room temperature for 90 min with 0.5 nM (rabbit assays) or 0.1 nM (hamster assays) [125I]neurokinin A in the presence or absence of test compounds. Nonspecific binding was defined using 1 /xM neurokinin A or 10 /zM of the NK 2 receptor-selective agonist [/3AlaS]neurokinin A-(4-10). Incubations were terminated by rapid filtration through Whatman G F / C filters presoaked in 0.3% polyethylenimine/0.5% Triton X-100. Radioactivity bound to filters was measured in a Hewlett Packard Gamma counter at 74% efficiency.
2.2. Data analysis The ability of natural and synthetic tachykinins to inhibit the binding of [~25I]neurokinin A to rabbit or hamster urinary bladder membranes was determined using 6-10 test concentrations (0.! nM-10 /zM) assayed in duplicate. Competition data were analysed by non-linear regression using an iterative curve-fitting program implemented on RS1 (Bolt, Beranek and Newman Inc.). IC50 values were calculated for each curve and converted to K i values using the ChengPrussoff equation (Cheng and Prussoff, 1973). 2.3. Materials The peptides, neurokinin A, senktide, and L-659,877 were o b t a i n e d from Bachem Ltd., U.S.A. [Sar9,Met(Oz)ll]substance P and MEN 10,376 were purchased from Peninsula Laboratories, CA, U.S.A and [/3-AlaS]neurokinin A-(4-10) was obtained from Novabiochem, Switzerland. MEN 10,207 and R 396 were custom synthesised by Biomac, Glasgow, Scotland. (_+)-CP-96,345 ((2S/R,3S/R)-[cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1-azabicyclo[2.2.2]octan-3-amine]) was synthesised by the Medicinal
TABLE 1 Primary sequences of tachykinin peptide agonists and antagonists used. Peptide
Sequence
Neurokinin A [/3-AlaS]neurokinin A-(4-10) [Sarg,Met(O2)U ]substance P Senktide MEN 10,376 MEN 10,207 L-659,877 R 396
His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 Asp-Ser-Phe-Val-/3-Ala-Leu-Met-NH 2 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Sar-Leu-Met(O2)-NH 2 Succinyl-Asp-Phe-MePhe-Gly-Leu-Met-NH 2 Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Lys-NH z Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Met-NH 2 Cyclo(Leu-Met-Gln-Trp-Phe-Gly) Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH 2
TABLE 2 Affinities of tachykinin receptor agonists and antagonists for [1251]neurokinin A binding sites in rabbit and hamster urinary bladder membranes. Values are mean PKis ( - l o g K i ) + S.E.M. with the number of experiments (n) shown in parentheses. IC50 values were converted to K i values using the Cheng and Prussoff relationship and K D values for [1251]neurokinin A of 0.85 nM and 0.64 nM for rabbit and hamster bladder NK 2 binding sites, respectively. Compound
Neurokinin A [/3-AlaS]neurokinin A-(4-10) [Sar 9,Met(O2)11]substance P Senktide ( + )-CP-96,345 MEN 10,376 MEN 10,207 L-659,877 R 396
Rabbit bladder
Hamster bladder
pK i
n
pK i
n
8.95 + 0.06 7.47 +_0.03 5.60 _+0.11 < 5.00 < 5.00 7.51 + 0.03 7.08 _+0.04 6.52 + 0.06 < 5.00
(3) (3) (5) (3) (4) (4) (4) .(4) (4)
8.85 + 0.03 7.66 _+0.12 < 5.00 < 5.00 < 5.00 5.88 +_0.05 5.28 + 0.08 8.09 + 0.04 7.82 + 0.07
(3) (4) (3) (3) (3) (3) (6) (3) (4)
289 Chemistry D e p a r t m e n t , Parke-Davis Neuroscience Research Centre, Cambridge, U.K. (2-[lZ~I]iodohistidyll) neurokinin A ([125I]neurokinin A; specific activity 2 000 C i / m m o l ) was purchased from A m e r s h a m International, U.K.
3. Results Specific binding of [t25I]neurokinin A (0.5 nM) to crude m e m b r a n e s of rabbit urinary bladder represented approximately 76% of total binding ( ~ 2600 specific cpm). In comparison, [125I]neurokinin A binding (0.1 nM) to hamster bladder m e m b r a n e s was routinely 98% specific or greater ( ~ 7140 specific cpm). In competition experiments, neurokinin A and [/3AlaS]neurokinin A-(4-1 0) inhibited [125I]neurokinin A-specific binding to hamster and rabbit bladders with high affinity (table 2). In contrast, the NK~ receptorselective agonist [Sar9,Met(O2)H]substance P inhibited specific binding at micromolar concentrations, while the N K 1 receptor-selective non-peptide antagonist (+)-CP-96,345 was virtually inactive at 10 ~ M . Similarly, the N K 3 receptor-selective agonist, senktide, was virtually inactive in displacing [t25I]neurokinin A binding from either tissue (table 2). With regard to the N K 2 receptor-selective antagonists, a different rank order of affinities was obtained in the two tissues. Thus, in the rabbit urinary bladder, [125I]neurokinin A specific binding was displaced with a rank order of: M E N 10,376> M E N 10,207> L659,877 >> R 396. In the hamster urinary bladder, the rank order of affinities was: L-659,877 > R 396 > M E N 10,376 > M E N 10,207 (see table 2).
4. Discussion Previous studies have shown that [t25I]neurokinin A labels a population of N K 2 binding sites in the hamster urinary bladder (Buck and Shatzer, 1988). In the present study, the N K 2 binding sites labelled by [125I]neurokinin A in rabbit and hamster urinary bladder have been compared. The presence of N K 2 binding sites is evident from the high affinity of unlabelled neurokinin A and the NK2 receptor-selective agonist [/3-Ala8]neu rokinin A-(4-10) for [t25I]neurokinin A binding sites and the weak, or negligible, affinity of the N K 1 receptor agonist [Sar9,Met(O2)11]substance P, the selective non-peptide N K 1 receptor antagonist (__+)-CP-96,345 and the N K 3 receptor-selective agonist, senktide. The results of the present study also confirm previous findings from functional studies which suggest that N K 2 receptors present in rabbit tissues, tentatively referred to as NK2A, are pharmacologically distinct from those of the putative NK2B subtype present in
hamster tissues (Maggi et al., 1992a,b). Thus, while both rabbit and hamster N K 2 binding sites appear to be equally sensitive to the agonists neurokinin A and [3-Ala8]neurokinin A-(4-10), [lzSI]neurokinin A binding sites in the two species a p p e a r to show a differential affinity for several N K 2 receptor-selective antagonists. In rabbit tissues, as with smooth muscle bioassays, the NKA-(4-10)-derived peptide antagonists M E N 10,376 and M E N 10,207 displayed significantly higher affinity for [125I]neurokinin A binding sites as compared with the cyclic peptide antagonist L-659,877 or its linear analogue, R 396. In comparison, in the hamster bladder binding assay, L-659,877 and R 396 were significantly more active than M E N 10,376 at displacing binding to N K 2 sites, consistent with previous findings using functional assays (Maggi et al., 1992a,b). In summary, we have shown that the rabbit urinary bladder contains a population of N K 2 binding sites which display preferential affinity for the N K 2 receptor antagonists, M E N 10,376 and M E N 10,207, with L-659,877 and R 396 being weakly active or inactive, respectively. These data have provided further evidence for the existence of species-dependent N K 2 receptors in rabbit and hamster urinary bladder. It remains to be established whether these pharmacologically distinct receptors are true receptor subtypes or species-dependent variations of the same receptor.
References Bradford, M.M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248. Buck, S.H. and S.A. Shatzer, 1988, Agonist and antagonist binding to tachykinin NK-2 receptors, Life Sci. 42, 2701. Cheng, Y.C. and W.H. Prussoff, 1973, Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50% inhibition (150) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099. Dion, S., N. Rouissi, F. Nantel, D. Jukic, N.E. Rhaleb, C. Tousignant, S. T616maque, G. Drapeau, D. Regoli, E. Naline, C. Advenier, P. Rovero and C.A. Maggi, 1990, Structure activity of neurokinins: antagonists for the neurokinin 2 receptor, Pharmacology 41, 184. Guard, S. and S.P. Watson, 1991, Tachykinin receptor types: classification and transmembrane signalling mechanisms, Neurochem. Int. 18 (2), 149. Lee, C.M., N.J. Campbell, B.J. Williams and L.L. Iversen, 1986, Multiple tachykinin binding sites in peripheral tissues and brain, Eur. J. Pharmacol. 130, 209. Maggi, C.A., S. Giuliani, L. Ballati, A. Lecci, S. Manzini, R. Patacchini, A.R. Renzetti, P. Rovero, L. Quartara and A. Giachetti, 1991, In vivo evidence for tachykininergic transmission using a new NK-2 receptor selective antagonist MEN 10,376, J. Pharmacol. Exp. Ther. 257, 1172. Maggi, C.A., A. Eglezos, L. Quartara, R. Patacchini and A. Giachetti, 1992a, Heterogeneity of NK-2 tachykinin receptors in hamster and rabbit smooth muscles, Reg. Pept. 37, 85.
290 Maggi, C.A., R. Patacchini, M. Astolfi, P. Rovero, A. Giachetti and P.L.M. Van Giersbergen, 1992b, Affinity of R 396 and NK-2 tachykinin receptor antagonist, for NK-2 receptors in preparations from different species, Neuropeptides 22, 93. McKnight, A.T., J.J. Maguire, N.J. Elliot, A.E. Fletcher, A.C. Foster, R. Tridgett, B.J. Williams, J. Longmore and L.L. Iversen, 1991, Pharmacological specificity of novel, synthetic, cyclic peptides as antagonists at tachykinin receptors, Br. J. Pharmacol. 104, 355.
Van Giersbergen, P.L.M., S.A. Shatzer, E. Burcher and S.H. Buck, 1992, Comparison of the effects of neuropeptide K and neuropeptide Y with neurokinin A at NK 2 receptors in the hamster urinary bladder, Naunyn-Schmiedeb. Arch. Pharmacol. 345, 51. Watling, K.J., 1992, Non-peptide antagonists herald new era in tachykinin research, Trends Pharmacol. Sci. 13, 266.
287
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 21194 Short communication
[125I]Neurokinin A labels pharmacologically distinct populations of NK 2 binding sites in hamster and rabbit urinary bladder S t e v e n G u a r d , D e b a s i s h Pain, R u t h F r a n k s a n d K e i t h J. W a t l i n g Parke-Davis Neuroscience Research Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB, UK
Received 19 November 1992,revised MS received 6 January 1993,accepted 12 January 1993
The pharmacological profile of NK 2 binding sites has been characterised in homogenates of rabbit urinary bladder and compared with that present in homogenates of hamster bladder. In both species, [125I]neurokinin A-specific binding to urinary bladder membranes was displaced by neurokinin A and the NK 2 agonist [fl-AlaS]neurokinin A-(4-10) whilst the NK 1 ligands [Sar9,Met(O2)n]substance P and (+)-CP-96,345, and the NK 3 agonist, senktide, were only weak displacers or ineffective. At rabbit NK 2 sites, the rank order of affinity of NK 2 receptor-selective antagonists was; MEN 10,376 > MEN 10,207 > L-659,877 >> R 396. In contrast, the rank order of displacement of [x25I]neurokinin A-specific binding to hamster bladder membranes was: L-659,877 > R 396 > MEN 10,376 > MEN 10,207. These data demonstrate that [lZSI]neurokinin A binds to pharmacologically distinct NK2 binding sites in hamster and rabbit urinary bladder. Tachykinin; Neurokinin A; Neurokinin receptor subtypes; Tachykinin receptor antagonists; Bladder (rabbit, hamster)
1. Introduction
Substance P, neurokinin A and neurokinin B belong to the tachykinin family of peptides which are characterised by a common C-terminal amino acid sequence Phe-X-Gly-Leu-Met-NH 2. It is now well established that the biological effects of the tachykinins are mediated by at least three receptor types termed NK l, NK 2 and NK 3. The pharmacology of tachykinin receptors has progressed markedly in the last few years, due primarily to the development of both peptide and non-peptide receptor antagonists with high affinity and selectivity for a particular receptor type (for recent reviews see Guard and Watson, 1991; Watling, 1992). NK 2 receptors are widely distributed throughout the peripheral organs of several mammalian species and are characterised by their preferential affinity for neurokinin A and its N-terminally extended forms, neuropeptide K and neuropeptide Y (van Giersbergen et al., 1992). Most recently, the use of selective NK2 receptor antagonists (see table 1), specifically the cyclic peptide, L-659,877 (McKnight e t a . , 1991), a linear hexapeptide analogue, R 396 (Dion et al., 1990), and a heptapeptide derivative of neurokinin A-(4-10), M E N
Correspondence to: S. Guard, Parke-Davis Neuroscience Research Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB, UK. Tel. 44.223.210929, fax 44.223.249106.
10,376 (Maggi et al., 1991), has revealed the possible existence of NK 2 receptor subtypes. Thus, in rabbit tissue bioassays, the following rank order of affinity is observed; M E N 10,376 > L-659,877 > R 396; whereas in hamster tissue bioassays, the rank order is; L659,877 > R 396 > M E N 10,376 (Maggi et al., 1992a). In the present study, we have identified and characterised NK 2 receptor binding sites in the rabbit urinary bladder using [125I]neurokinin A. The pharmacological profile obtained with tachykinin receptor-selective agonists and the above NK 2 receptor antagonists has been compared with that observed in the hamster urinary bladder.
2. Materials and methods 2.1. Radioligand binding assays
For radioligand binding assays in rabbit and hamster urinary bladder, preparation of crude membrane suspensions and binding assays were performed essentially as previously described (Lee et al., 1986). Briefly, tissues from New Zealand White rabbits stored at - 70°C, or fresh tissues from Golden Syrian hamsters were homogenised by Polytron in 50 mM Tris-HC1 buffer (pH 7.4) containing 100 mM NaC1 and 5 mM KCI and centrifuged at 48 000 x g at 4°C for 30 min. The pellet was resuspended in 50 mM Tris-HC1 (pH 7.4) contain-
288
ing 100 mM NaC1, 10 mM EDTA and 300 mM KC1 and incubated for 30 min at 4°C. The membranes were centrifuged at 48000 x g for 30 min and subsequently washed twice with 50 mM Tris-HCl (pH 7.4). The final pellet was resuspended in a small volume of 50 mM Tris-HC1 (pH 7.4) and protein content determined by the method of Bradford (1976) using bovine serum albumin (BSA) as a standard. Binding assays were performed in polypropylene tubes in a final volume of 300/xl Tris-HC1 buffer (pH 7.4) containing 3 mM MnC12, 0.02% BSA, and 2 ~ g / m l chymostatin, 4 / z g / m l leupeptin, 40 ~ g / m l bacitracin, 2 /zM phosphoramidon to inhibit peptidase activity. Crude membranes (150 /xg protein for rabbit bladder or 20 /~g protein for hamster bladder) were incubated at room temperature for 90 min with 0.5 nM (rabbit assays) or 0.1 nM (hamster assays) [125I]neurokinin A in the presence or absence of test compounds. Nonspecific binding was defined using 1 /xM neurokinin A or 10 /zM of the NK 2 receptor-selective agonist [/3AlaS]neurokinin A-(4-10). Incubations were terminated by rapid filtration through Whatman G F / C filters presoaked in 0.3% polyethylenimine/0.5% Triton X-100. Radioactivity bound to filters was measured in a Hewlett Packard Gamma counter at 74% efficiency.
2.2. Data analysis The ability of natural and synthetic tachykinins to inhibit the binding of [~25I]neurokinin A to rabbit or hamster urinary bladder membranes was determined using 6-10 test concentrations (0.! nM-10 /zM) assayed in duplicate. Competition data were analysed by non-linear regression using an iterative curve-fitting program implemented on RS1 (Bolt, Beranek and Newman Inc.). IC50 values were calculated for each curve and converted to K i values using the ChengPrussoff equation (Cheng and Prussoff, 1973). 2.3. Materials The peptides, neurokinin A, senktide, and L-659,877 were o b t a i n e d from Bachem Ltd., U.S.A. [Sar9,Met(Oz)ll]substance P and MEN 10,376 were purchased from Peninsula Laboratories, CA, U.S.A and [/3-AlaS]neurokinin A-(4-10) was obtained from Novabiochem, Switzerland. MEN 10,207 and R 396 were custom synthesised by Biomac, Glasgow, Scotland. (_+)-CP-96,345 ((2S/R,3S/R)-[cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1-azabicyclo[2.2.2]octan-3-amine]) was synthesised by the Medicinal
TABLE 1 Primary sequences of tachykinin peptide agonists and antagonists used. Peptide
Sequence
Neurokinin A [/3-AlaS]neurokinin A-(4-10) [Sarg,Met(O2)U ]substance P Senktide MEN 10,376 MEN 10,207 L-659,877 R 396
His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 Asp-Ser-Phe-Val-/3-Ala-Leu-Met-NH 2 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Sar-Leu-Met(O2)-NH 2 Succinyl-Asp-Phe-MePhe-Gly-Leu-Met-NH 2 Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Lys-NH z Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Met-NH 2 Cyclo(Leu-Met-Gln-Trp-Phe-Gly) Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH 2
TABLE 2 Affinities of tachykinin receptor agonists and antagonists for [1251]neurokinin A binding sites in rabbit and hamster urinary bladder membranes. Values are mean PKis ( - l o g K i ) + S.E.M. with the number of experiments (n) shown in parentheses. IC50 values were converted to K i values using the Cheng and Prussoff relationship and K D values for [1251]neurokinin A of 0.85 nM and 0.64 nM for rabbit and hamster bladder NK 2 binding sites, respectively. Compound
Neurokinin A [/3-AlaS]neurokinin A-(4-10) [Sar 9,Met(O2)11]substance P Senktide ( + )-CP-96,345 MEN 10,376 MEN 10,207 L-659,877 R 396
Rabbit bladder
Hamster bladder
pK i
n
pK i
n
8.95 + 0.06 7.47 +_0.03 5.60 _+0.11 < 5.00 < 5.00 7.51 + 0.03 7.08 _+0.04 6.52 + 0.06 < 5.00
(3) (3) (5) (3) (4) (4) (4) .(4) (4)
8.85 + 0.03 7.66 _+0.12 < 5.00 < 5.00 < 5.00 5.88 +_0.05 5.28 + 0.08 8.09 + 0.04 7.82 + 0.07
(3) (4) (3) (3) (3) (3) (6) (3) (4)
289 Chemistry D e p a r t m e n t , Parke-Davis Neuroscience Research Centre, Cambridge, U.K. (2-[lZ~I]iodohistidyll) neurokinin A ([125I]neurokinin A; specific activity 2 000 C i / m m o l ) was purchased from A m e r s h a m International, U.K.
3. Results Specific binding of [t25I]neurokinin A (0.5 nM) to crude m e m b r a n e s of rabbit urinary bladder represented approximately 76% of total binding ( ~ 2600 specific cpm). In comparison, [125I]neurokinin A binding (0.1 nM) to hamster bladder m e m b r a n e s was routinely 98% specific or greater ( ~ 7140 specific cpm). In competition experiments, neurokinin A and [/3AlaS]neurokinin A-(4-1 0) inhibited [125I]neurokinin A-specific binding to hamster and rabbit bladders with high affinity (table 2). In contrast, the NK~ receptorselective agonist [Sar9,Met(O2)H]substance P inhibited specific binding at micromolar concentrations, while the N K 1 receptor-selective non-peptide antagonist (+)-CP-96,345 was virtually inactive at 10 ~ M . Similarly, the N K 3 receptor-selective agonist, senktide, was virtually inactive in displacing [t25I]neurokinin A binding from either tissue (table 2). With regard to the N K 2 receptor-selective antagonists, a different rank order of affinities was obtained in the two tissues. Thus, in the rabbit urinary bladder, [125I]neurokinin A specific binding was displaced with a rank order of: M E N 10,376> M E N 10,207> L659,877 >> R 396. In the hamster urinary bladder, the rank order of affinities was: L-659,877 > R 396 > M E N 10,376 > M E N 10,207 (see table 2).
4. Discussion Previous studies have shown that [t25I]neurokinin A labels a population of N K 2 binding sites in the hamster urinary bladder (Buck and Shatzer, 1988). In the present study, the N K 2 binding sites labelled by [125I]neurokinin A in rabbit and hamster urinary bladder have been compared. The presence of N K 2 binding sites is evident from the high affinity of unlabelled neurokinin A and the NK2 receptor-selective agonist [/3-Ala8]neu rokinin A-(4-10) for [t25I]neurokinin A binding sites and the weak, or negligible, affinity of the N K 1 receptor agonist [Sar9,Met(O2)11]substance P, the selective non-peptide N K 1 receptor antagonist (__+)-CP-96,345 and the N K 3 receptor-selective agonist, senktide. The results of the present study also confirm previous findings from functional studies which suggest that N K 2 receptors present in rabbit tissues, tentatively referred to as NK2A, are pharmacologically distinct from those of the putative NK2B subtype present in
hamster tissues (Maggi et al., 1992a,b). Thus, while both rabbit and hamster N K 2 binding sites appear to be equally sensitive to the agonists neurokinin A and [3-Ala8]neurokinin A-(4-10), [lzSI]neurokinin A binding sites in the two species a p p e a r to show a differential affinity for several N K 2 receptor-selective antagonists. In rabbit tissues, as with smooth muscle bioassays, the NKA-(4-10)-derived peptide antagonists M E N 10,376 and M E N 10,207 displayed significantly higher affinity for [125I]neurokinin A binding sites as compared with the cyclic peptide antagonist L-659,877 or its linear analogue, R 396. In comparison, in the hamster bladder binding assay, L-659,877 and R 396 were significantly more active than M E N 10,376 at displacing binding to N K 2 sites, consistent with previous findings using functional assays (Maggi et al., 1992a,b). In summary, we have shown that the rabbit urinary bladder contains a population of N K 2 binding sites which display preferential affinity for the N K 2 receptor antagonists, M E N 10,376 and M E N 10,207, with L-659,877 and R 396 being weakly active or inactive, respectively. These data have provided further evidence for the existence of species-dependent N K 2 receptors in rabbit and hamster urinary bladder. It remains to be established whether these pharmacologically distinct receptors are true receptor subtypes or species-dependent variations of the same receptor.
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