Xiang-Ping Zeng

Characterization of Tachykinin NK sub 2 Receptors in Human Urinary Bladder

AbstractFunctional and radioligand binding studies with selective agonists and antagonists were used to investigate tachykinin receptors in the human bladder. Strips of detrusor muscle were contracted by the tachykinins neurokinin A and neuropeptide gamma, and by the NK2 receptor selective agonists [Lys5, MeLeu9, Nle10]-NKA(4-10) and [Lys5, Tyr(I sub 2)7, MeLeu9, Nle10]-NKA(4-10), with pD2 values 8.2, 8.0, 8.1 and 7.1. [Sar9, Met(O2)11]-SP and senktide were ineffective agonists, indicating an absence of NK1 and NK3 receptors. The contractile responses to [Lys5, MeLeu9, Nle10]-NKA(4-10) were inhibited competitively by the NK2 receptor selective antagonists SR 48968, GR 94800 and MDL 29913, with pA2 values 9.1, 8.6 and 7.0. Specific binding of the new NK2 receptor selective radioligand [sup 125 I]-[Lys5, Tyr(I2)7, MeLeu9, Nle10]-NKA(4-10) was saturable to a high affinity site (KD 2.3 nM.). Specific binding was inhibited by NK2 receptor agonists and antagonists, but not by NK1 and NK3 analogues, showing bind...

Mammalian tachykinins stimulate rat uterus by activating NK-2 receptors

Neurokinin A (NKA), substance P (SP), and neurokinin B (NKB) enhanced the contractile force of uterine preparations from estrogen-treated rats. Neurokinin A was more and NKB less potent than SP. The actions of SP were enhanced by phosphoramidon (1 microM) but were unaffected by captopril (10 microM) or bestatin (10 microM). The actions of the peptides were enhanced in the combined presence of phosphoramidon, captopril, and bestatin; the potency order remained NKA > SP > NKB. Atropine inhibited responses to NKB but not to NKA, and slightly reduced those to SP. Specific binding of [125I]-iodohistidyl-neurokinin A (INKA) to uterine membranes was displaced by the tachykinins with a potency order of NKA > SP > NKB. These findings indicate that in the rat uterus 1) tachykinins act at an NK-2 receptor, and that another tachykinin receptor on cholinergic nerves may also be present; and 2) endopeptidase-24.11 participates in the inactivation of the tachykinins.

Characterisation of a novel, selective radioligand. [125I]Lys5, Tyr(I2)7,MeLeu9,Nle10]neurokinin A-(4-10), for the tachykinin NK2 receptor in rat fundus

The tyrosyl derivative of the tachykinin NK2 selective agonist [Lys5,MeLeu9,Nle10]NKA-(4-10) was iodinated and the product [125I][Lys5,Tyr(I2)2,MeLeu9,Nle10]NKA-(4-10) purified using reverse phase HPLC. The binding characteristics of this novel radioligand were investigated in homogenates of rat gastric fundus. Binding was saturable, reversible and to a single population of high affinity sites of KD 1.3 +/- 0.2 nM (n = 4). Specific binding of [125I][Lys5,Tyr(I2)7,MeLeu9,Nle10]NKA-(4-10) was inhibited by neuropeptide gamma SR 48968 > or = neurokinin A (NKA) > or = [Lys5,MeLeu9,Nle10]NKA-(4-10) > [Lys5,Tyr7,MeLeu9,Nle10] NKA-(4-10) > neuropeptide K > [Lys5,Tyr(I2)7,MeLeu9,Nle10]NKA-(4-10) > MDL 29,913 > [127I]- Bolton-Hunter-NKA > neurokinin B > substance P (SP) >> MEN 10207 > [Sar9,Met(O2)11]SP >> senktide, indicating binding to NK2 receptors. NKA, [Lys5,MeLeu9,Nle10]NKA-(4-10) and [Lys5,Tyr(I2)7,MeLeu9,Nle10]NKA-(4-10) contracted the isolated fundus strip, with pD2 values 7.9, 7.7 and 7.4, respectively. This novel, highly selective radioligand should prove useful in characterisation studies in peripheral tissues.

Evidence for tachykinin NK-2 receptors in guinea-pig airways from binding and functional studies, using [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10).

The potent contractile responses of guinea-pig airways to neurokinin A (NKA) and neuropeptide gamma (NP gamma) are thought to be mediated by NK-2 receptors. However, NK-2 binding sites are not detectable using the radioligand [125I]-iodohistidyl-NKA. Here, a novel, highly selective iodinated radioligand, [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10), and a number of related peptides have been used to characterize NK-2 receptors on guinea-pig airways, using binding and functional studies. Specific binding of [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10), was saturable and to a single high affinity site, with KD 1.29 +/- 0.36 nM (n = 4). The rank order of potency for tachykinins and analogues as competitors for the binding was: [Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10) > or = NP gamma > or = [Lys5,MeLeu9,Nle10]-NKA(4-10) > NKA > or = SR 48968 >> MDL 29913 > or = substance P (SP) = [127I]-Bolton-Hunter NKA (BHNKA) > or = MEN 10207 > neurokinin B (NKB). Septide, [DPro9,Pro10,Trp11]-SP, the NK-1 selective ligands [Sar9,Met(O2)11]-SP, [Pro9]-SP and CP 96345, the NK-3 selective senktide, and calcitonin gene-related peptide (CGRP) were weak or ineffective. On guinea-pig isolated bronchi, the potency order of contractile agonists was: [Lys5,MeLeu9,Nle10]-NKA(4-10) > NKA > or = NP gamma > or = [Lys5,Tyr7,MeLeu9, Nle10]-NKA(4-10) > or = septide = BHNKA > or = [Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10) > or = [Sar9,Met(O2)11]-SP > or = NKB = [Pro9]-SP > or = SP >> senktide.(ABSTRACT TRUNCATED AT 250 WORDS)

An investigation of tachykinin NK2 receptor subtypes in the rat.

The heterogeneity of tachykinin NK2 receptor subtypes was examined in five tissues from the rat, using binding and functional techniques. Initial experiments with the selective radioligand [125I][Lys5,Tyr(I2)7,MeLeu9,Nle10]neurokinin A-(4-10) showed no specific binding to rat spinal cord membranes or sections. However, this radioligand exhibited high specific binding (80-95% of total) in membranes from the rat fundus, colon, bladder and vas deferens. Dissociation constants (KD) were lower in bladder and colon (0.4 nM) than in fundus (1.9 nM) or vas deferens (1.4 nM). Neurokinin A, neuropeptide gamma, [Lys5,MeLeu9,Nle10]NK(4-10), SR 48968 [(S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophen yl)butyl]benzamine], GR 94800 [PhCO-Ala-Ala-DTrp-Phe-DPro-Pro-Nle-NH2] and MEN 10627 [cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2beta-5beta)] displayed high affinity (pIC50 8.4-9.5) as competitors, with no significant difference in potency between these four tissues. [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) contracted the isolated fundus (EC50 117 nM) and bladder (EC50 10 nM) and these responses were similarly inhibited by the tachykinin NK2 receptor antagonists, SR 48968 and MEN 10627 (pA2 values 7.6-8.2). In spite of differences in KD seen in some tissues, these results do not provide compelling evidence for tachykinin NK2 receptor heterogeneity in smooth muscle-containing tissues in the rat. The absence of detectable binding in rat spinal cord may be due to very low expression of tachykinin NK2 receptors, or to existence of a different receptor subtype.

Tachykinin NK-2 Receptors in Child Urinary Bladder*

PURPOSE Although NK-2 receptors mediate contractions to tachykinins in adult detrusor muscle, little is known about the functions of tachykinins in child urinary bladder. Here we have used highly selective agonists and antagonists to examine NK-2 receptors in child detrusor muscle. MATERIALS AND METHODS Specimens of urinary bladder from 23 children (0 to 10 years0 were obtained at operation for vesicoureteric reflux. Strips of detrusor muscle were mounted in organ baths in Krebs solution containing phosphoramidon (10 microM.), and isometric tension was recorded. Contractile responses were elicited by tachykinins and selective agonists in the presence and absence of autonomic inhibitors and of tachykinin NK-2 receptor antagonists. RESULTS The NK-2 receptor agonists neurokinin A (NKA), neuropeptide gamma and [Lys5, MeLeu9, Nle10]-NKA(4-10) contracted the isolated child detrusor, with pD2 values of 7.7, 7.2 and 7.3. The maximum response to NKA was greater than that to the other 2 agonists. No age-related differences were seen. Selective agonists for NK-1 receptors ([Sar9, Met(O2)11]-SP and septide) and NK-3 receptors (senktide) were ineffective contractile agents. Responses to NKA were unaffected by phentolamine (5 microM.), propranolol (3 microM.), tetrodotoxin (1 microM.) and indomethacin (1 microM.), indicating a direct action on smooth muscle. The tachykinin NK-2 receptor antagonists SR 48968 and MEN 10627 caused a concentration-dependent antagonism of responses to NKA, with apparent pKB values of 9.4 and 8.1. CONCLUSIONS Neurokinin A appears to act directly on NK-2 receptors on detrusor muscle of infant and child urinary bladder, without involvement of neural or indirect contractile mechanisms. Potency of antagonists was similar to that seen in other tissues. However, agonist potency was significantly lower in the isolated detrusor from children, compared with our previous study in adult detrusor. This discrepancy may be related to age-related differences in NK-2 receptors or in contractile mechanisms; alternatively it may be a result of the reflux condition.

A novel, selective radioligand, [125I]-[Lys5, Tyr(I2)7, Meleu9, Nle10]-NKA(4–10), for the tachykinin NK-2 receptor

A new radioligand, [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10), based on the selective agonist [Lys5,MeLeu9,Nle10]-NKA(4-10) has been developed. Binding in rat fundus membranes was displaced by NP gamma > NKA > or = [Lys5,MeLeu9,Nle10]-NK(4-10) > neuropeptide K > [Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10) > SP > [Sar9,Met(O2)11]-SP >> senktide, indicating binding to NK-2 receptors. Preliminary studies demonstrated high specific binding in membranes from rat urinary bladder, duodenum and colon. Specific binding in rat brain and lung was negligible, and binding in a range of guinea-pig tissues was no more than 35% specific. These data may indicate species differences in NK-2 receptors.