Autoradiographic localization of tachykinin and calcitonin gene-related peptide receptors in adult urinary bladder.

PURPOSE: In bladder, sensory afferent nerve fibers contain the "sensory neuropeptides" substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), which interact with tachykinin NK-1 and NK-2 receptors and CGRP receptors, respectively. The purpose of this study was to examine the autoradiographic distribution of these three receptor types in the human bladder, to determine whether the anatomic location of the receptors was consistent with their known functional roles. MATERIALS AND METHODS: Specimens of urinary bladder from 9 patients (58-74 years) were obtained at cystectomy. Frozen sections of dome were labeled with [125I]-Bolton-Hunter [Sar9,Met(O2)11]-SP (NK-1 receptors), [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10) (NK-2 receptors) and [125I]-rat CGRP-I. Binding sites were visualized using emulsion autoradiography. RESULTS: NK-1 receptors were found over the endothelium of arterial blood vessels within the detrusor muscle and lamina propria, and over small vessels in the subepithelium. NK-2 receptors were seen over the detrusor muscle and very sparsely over blood vessels, whereas CGRP receptors were expressed densely over the smooth muscle layer of arteries and arterioles, and weakly over collecting venules. NK-1 and CGRP receptors were not observed over the detrusor muscle. CONCLUSIONS: Although the afferent nerves contain all three peptides, not all cell types express receptors for each peptide. The general distribution of receptors is in good agreement with the location of nerves, and with the known actions of SP and CGRP as vasodilator agents, and of NKA (but not SP or CGRP) in contracting the detrusor muscle.

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.

Autoradiographic localization of tachykinin NK2 and NK1 receptors in the guinea-pig lung, using selective radioligands.

The distribution of tachykinin receptors in guinea-pig airways was studied using newly developed selective radioligands, [125I][Lys5,Tyr(I2)7,MeLeu9,Nle10]neurokinin A-(4-10) for NK2 receptors and [125I]Bolton-Hunter-[Sar9,Met(O2)11]substance P for NK1 receptors. Optimum incubation times were 60 and 45 min for tachykinin NK2 and NK1 sites, respectively, in slide-mounted sections of guinea-pig lung. Bacitracin (40 micrograms/ml) greatly reduced specific binding of [125I][Lys5,Tyr(I2)7,MeLeu9,Nle10]neurokinin A-(4-10), whereas phosphoramidon (1 and 10 microM) and bacitracin (40 micrograms/ml) significantly increased the specific binding of [125I]Bolton-Hunter-[Sar9,Met(O2)11]substance P. Dense specific binding of [125I]Bolton-Hunter-[Sar9,Met(O2)11]substance P occurred over bronchial smooth muscle of large and small airways, with moderate binding on bronchial epithelium and over pulmonary arterial smooth muscle. Moderate specific binding of [125I][Lys5,Tyr(I2)7, MeLeu9,Nle10]neurokinin A-(4-10) was associated with bronchial smooth muscle of mainly large airways but not with other histological regions. This is the first autoradiographic report of (a low density of) tachykinin NK2 binding sites on airway smooth muscle and supports the potent actions of NK2 receptor ligands as contractile agents in guinea-pig isolated bronchi.

Sensory nerves play an efferent role in the function of the arterioles, but not the dilator muscle, of the rat iris.

We have studied the expression, distribution and function of receptors for the sensory neurotransmitters, substance P (SP) and calcitonin gene-related peptide (CGRP) in the dilator muscle and arterioles of the rat iris. Immunohistochemical studies showed that the sensory fibres containing these peptides are distributed throughout the connective tissue stroma of the iris and in association with the larger arterioles, but do not come into close association with the dilator muscle cells. Using reverse-transcription polymerase chain reaction, we have shown that both NK1 and NK3 receptor message is expressed by iris tissue, comprising both dilator muscle and stromal tissue. Binding sites for the NK1 agonist, [Sar9, Met(O2)11]-substance P (SarSP), and for CGRP are confined to the stromal layer and to the larger arterioles within that layer and do not appear to be associated with the dilator muscle itself. Application of either SarSP or CGRP produced both a vasodilatation and an inhibition of sympathetic nerve-induced vasoconstriction of the larger arterioles. Neither SarSP nor CGRP altered the resting tone of the dilator nor were they capable of modulating the contractions due to sympathetic nervous activity. These results suggest that the sensory fibres perform an efferent role in the larger irideal arterioles while their presence in the irideal stroma appears not to modulate the activity of the dilator muscle.

Tachykinin receptors in guinea-pig airways: characterization using selective ligands.

Tachykinin receptors in guinea-pig airways were examined using radioligand binding techniques in lung homogenates, and using isolated bronchial segments. Binding of the NK1 selective radioligand 125I-labelled Bolton-Hunter [Sar9,Met(O2)11]substance P ([125I]BHSarSP) was saturable and of high affinity (KD, 0.26 nM). The rank potency order of competitors for [125I]BHSarSP binding was [Pro9]SP > CP 96345 >> septide > [pGlu6]SP(6-11) > RP 67580 > or = [DPro9,t beta Pro10(phi),Trp11]SP > [DPro9,t beta Pro10(CH2 phi),Trp11]physalaemin > or = GR82334 > or = 127I Bolton-Hunter neurokinin A (BHNKA). Septide had higher affinity than expected, and it was the only ligand to bind to two sites. Agonists interacting with NK2 receptors were more potent contractile agents than NK1 receptor agonists. Responses to BHNKA (pD2 8.4) were antagonized by MDL 29913 and MEN 10207, with pKB values 6.42 and 6.79, and also by SR 48968 and GR 94800, although this was not dose dependent. This agonist was also weakly inhibited by CP 96345 and RP 67580. These data demonstrate that BHNKA can interact with both NK1 and NK2 receptors. There was no relationship between the binding affinity of NK1 ligands in lung homogenates, with GR 82334 being notably weak, and their agonist or antagonist potency in bronchial smooth muscle.