Analysis of the Expression of Tachykinins and Tachykinin Receptors in the Rat Uterus During Early Pregnancy.

The peptides of the tachykinin family participate in the regulation of reproductive function acting at both central and peripheral levels. Our previous data showed that treatment of rats with a tachykinin NK3R antagonist caused a reduction of litter size. In the present study, we analyzed the expression of tachykinins and tachykinin receptors in the rat uterus during early pregnancy. Uterine samples were obtained from early pregnant rats (Days 1-9 of pregnancy) and from nonpregnant rats during the proestrus stage of the ovarian cycle, and real-time quantitative RT-PCR, immunohistochemistry, and Western blot studies were used to investigate the pattern of expression of tachykinins and tachykinin receptors. We found that all tachykinins and tachykinin receptors were locally synthesized in the uterus of early pregnant rats. The expression of substance P, neurokinin B, and the tachykinin receptors NK1R and NK3R mRNAs and proteins underwent major changes during the days around implantation and they were widely distributed in implantation sites, being particularly abundant in decidual cells. These findings support the involvement of the tachykinin system in the series of uterine events that occur around embryo implantation in the rat.

Neuropeptide signaling activates dendritic cell-mediated type 1 immune responses through neurokinin-2 receptor.

Neurokinin A (NKA), a neurotransmitter distributed in the central and peripheral nervous system, strictly controls vital responses, such as airway contraction, by intracellular signaling through neurokinin-2 receptor (NK2R). However, the function of NKA-NK2R signaling on involvement in immune responses is less-well defined. We demonstrate that NK2R-mediated neuropeptide signaling activates dendritic cell (DC)-mediated type 1 immune responses. IFN-γ stimulation significantly induced NK2R mRNA and remarkably enhanced surface protein expression levels of bone marrow-derived DCs. In addition, the DC-mediated NKA production level was significantly elevated after IFN-γ stimulation in vivo and in vitro. We found that NKA treatment induced type 1 IFN mRNA expressions in DCs. Transduction of NK2R into DCs augmented the expression level of surface MHC class II and promoted Ag-specific IL-2 production by CD4(+) T cells after NKA stimulation. Furthermore, blockade of NK2R by an antagonist significantly suppressed IFN-γ production by both CD4(+) T and CD8(+) T cells stimulated with the Ag-loaded DCs. Finally, we confirmed that stimulation with IFN-γ or TLR3 ligand (polyinosinic-polycytidylic acid) significantly induced both NK2R mRNA and surface protein expression of human PBMC-derived DCs, as well as enhanced human TAC1 mRNA, which encodes NKA and Substance P. Thus, these findings indicate that NK2R-dependent neuropeptide signaling regulates Ag-specific T cell responses via activation of DC function, suggesting that the NKA-NK2R cascade would be a promising target in chronic inflammation caused by excessive type 1-dominant immunity.

NK receptors, Substance P, Ano1 expression and ultrastructural features of the muscle coat in Cav-1(-/-) mouse ileum.

Caveolin (Cav)-1 is an integral membrane protein of caveolae playing a crucial role in various signal transduction pathways. Caveolae represent the sites for calcium entry and storage especially in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Cav-1(-/-) mice lack caveolae and show abnormalities in pacing and contractile activity of the small intestine. Presently, we investigated, by transmission electron microscopy (TEM) and immunohistochemistry, whether the absence of Cav-1 in Cav-1(-/-) mouse small intestine affects ICC, SMC and neuronal morphology, the expression of NK1 and NK2 receptors, and of Ano1 (also called Dog1 or TMEM16A), an essential molecule for slow wave activity in gastrointestinal muscles. ICC were also labelled with c-Kit and tachykinergic neurons with Substance P (SP). In Cav-1(-/-) mice: (i) ICC were Ano1-negative but maintained c-Kit expression, (ii) NK1 and NK2 receptor immunoreactivity was more intense and, in the SMC, mainly intracytoplasmatic, (iii) SP-immunoreactivity was significantly reduced. Under TEM: (i) ICC, SMC and telocytes lacked typical caveolae but had few and large flask-shaped vesicles we called large-sized caveolae; (ii) SMC and ICC contained an extraordinary high number of mitochondria, (iii) neurons were unchanged. To maintain intestinal motility, loss of caveolae and reduced calcium availability in Cav-1-knockout mice seem to be balanced by a highly increased number of mitochondria in ICC and SMC. Loss of Ano-1 expression, decrease of SP content and consequently overexpression of NK receptors suggest that all these molecules are Cav-1-associated proteins.

Multifaceted approach to determine the antagonist molecular mechanism and interaction of ibodutant ([1-(2-phenyl-1R-[[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl]-ethylcarbamoyl)-cyclopentyl]-amide) at the human tachykinin NK2 receptor.

Ibodutant (MEN15596, [1-(2-phenyl-1R-[[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl]-ethylcarbamoyl)-cyclopentyl]-amide) is a tachykinin NK(2) receptor (NK(2)R) antagonist currently under phase II clinical trials for irritable bowel syndrome. This study focuses on the ibodutant pharmacodynamic profile at the human NK(2)R and compares it with two other antagonists, nepadutant (MEN11420, (cyclo-[[Asn(beta-D-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2beta-5beta)]) and saredutant [SR48968, (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide]. In functional experiments (phosphatidylinositol accumulation) in Chinese hamster ovary cells expressing the human NK(2)R, ibodutant potency measured toward concentration-response curves to neurokinin A as pK(B) was 10.6, and its antagonism mechanism was surmountable and competitive. In the same assay, antagonism equilibration and reversibility experiments of receptor blockade indicated that ibodutant quickly attains equilibrium and that reverts from receptor compartment in a slower manner. Kinetic properties of ibodutant were assessed through competitive binding kinetics experiments performed at [(3)H]nepadutant and [(3)H]saredutant binding sites. Determined K(on) and K(off) values indicated a fast association and slow dissociation rate of ibodutant at the different antagonist binding sites. Last, by radioligand binding experiments at some mutated human tachykinin NK(2)Rs, the amino acidic determinants crucial for the high affinity of ibodutant were identified at the transmembrane (TM) level: Cys167 in TM4; Ile202 and Tyr206 in TM5; Phe270, Tyr266, and Trp263 in TM6; and Tyr289 in TM7. These results indicated an extended antagonist binding pocket in the TM portion of the receptor, which is conceived crucial for TM3 and 6 arrangement and leads to G protein-coupled receptor activation. By combining this information and molecular modeling, the docking mode of ibodutant-human NK(2)R complex is proposed.

Changes in the response to excitatory antagonists, agonists, and spasmolytic agents in circular colonic smooth muscle strips from patients with diverticulosis.

BACKGROUND: Colonic samples from asymptomatic diverticulosis (DS) patients presented enhanced electrical field stimulation (EFS)-contractions, in an earlier study of ours, suggesting increased endogenous responses. The aim of this study was to explore changes in excitatory neuromuscular transmission and to assess the pharmacodynamics of spasmolytic agents in DS. METHODS: Circular muscle strips from sigmoid colon of DS patients (n = 30; 69.5 ± 14.8 years) and controls (n = 32; 64.7 ± 16.2 years) were studied using organ baths to evaluate the direct effect of excitatory agonists (carbachol, neurokinin A [NKA] and substance P [SP]), and the effect of antagonists (atropine and NK2 antagonist GR94800) and spasmolytic drugs (otilonium bromide [OB] and N-butyl-hyoscine) on the contractions induced by EFS-stimulation of excitatory motorneurons. qRT-PCR was also performed to compare mRNA expression of M2 , M3 , NK2 receptors and L-type calcium channels. KEY RESULTS: Contractions to carbachol (Emax : 663.7 ± 305.6% control vs 2698.0 ± 439.5% DS; p < 0.0005) and NKA (Emax : 387.8 ± 35.6% vs 1102.0 ± 190.1%; p < 0.0005) were higher in DS group, without differences for SP. Higher potency for DS patients was observed in the concentration-response curves for atropine (pIC50  = 8.56 ± 0.15 control vs pIC50  = 9.95 ± 0.18 DS group; p < 0.005) and slightly higher for GR94800 (pIC50  = 7.21 ± 0.18 control vs pIC50  = 7.97 ± 0.32 group; p < 0.0001). Lower efficacy (Emax ) and potency (pIC50 ) was observed for spasmolytic drugs in DS, whereas no differences were found regarding the relative expression of the receptors evaluated between groups. CONCLUSIONS & INFERENCES: The greater response to cholinergic and tachykinergic agonists and greater potency for muscarinic and NK2 antagonists observed in DS might play a role in the spasticity found in diverticular disease.

Co-expression of the neurokinin NK2 receptor and G-protein components in the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has proven useful for studying molecular interactions between a range of signal transduction components. We now report the first co-expression of a mammalian seven-transmembrane receptor and G-protein components in S. pombe. We selected the human neurokinin NK2 receptor together with its G-protein-signalling partner Gq for this study. Yeast membrane fractions showed high levels of NK2 receptor-binding activity (1159 +/- 534 (n = 3) fmol/mg protein) although initial experiments with intact cells revealed an absence of receptors at the cell surface. Using a construct comprising the NK2 coding sequence fused with the signal sequence from an endogenous phosphatase (phoI), we detected approximately 400 NK2 receptors/cell in unbroken yeast. Successful co-expression of the NK2 receptor with the G-protein subunits G alpha q, beta 1 or beta 2 and gamma 3 failed to modulate agonist binding, suggesting the absence of functional interaction between these components. As an alternative test of G alpha q function, we next expressed its downstream effector target phospholipase C-beta 1 (PLC beta 1) in S. pombe. Although PLC beta 1 undergoes powerful in vitro activation by G alpha q derived from baculovirus-infected Sf9 cells and mammalian cells, G alpha q expressed in S. pombe is totally ineffective. Similar results were also achieved with the G-protein subunit G alpha 16. Together, these data suggest that seven-transmembrane receptors can be expressed in S. pombe at high levels and directed to the cell surface although their interaction with co-expressed G-proteins in undetectable. Production of inactive G alpha-chains in S. pombe may account for these observations.

The relationship between the agonist-induced activation and desensitization of the human tachykinin NK2 receptor expressed in Xenopus oocytes.

1. Repeated applications of neurokinin A (NKA) to oocytes injected with 25 ng wild-type hNK2 receptor cRNA caused complete attenuation of second and subsequent NKA-induced responses while analogous experiments using repeated applications of GR64349 and [Nle10]NKA(4-10) resulted in no such desensitization. This behaviour has been previously attributed to the ability of the different ligands to stabilize different active conformations of the receptor that have differing susceptibilities to receptor kinases (Nemeth & Chollet. 1995). 2. However, for Xenopus oocytes injected (into the nucleus) with 10 ng wild-type hNK2 receptor cDNA, a single 100 nM concentration of any of the three ligands resulted in complete desensitization to further concentrations. 3. On the other hand, none of the ligands caused any desensitization in oocytes injected with 0.25 ng wild-type hNK2 receptor cRNA. even at concentrations up to 10 microM. 4. The two N-terminally truncated analogues of neurokinin A have a lower efficacy than NKA and it is likely that it is this property which causes the observed differences in desensitization, rather than the formation of alternative active states of the receptor. 5. The peak calcium-dependent chloride current is not a reliable measure of maximal receptor stimulation and efficacy is better measured in this system by studying agonist-induced desensitization. 6. The specific adenylyl cyclase inhibitor SQ22536 can enhance NKA and GR64349-mediated desensitization which suggests that agonist-induced desensitization involves the inhibition of adenylyl cyclase and the subsequent down-regulation of the cyclic AMP-dependent protein kinase, possibly by cross-talk to a second signalling pathway.

BDNF-overexpression regulates the reactivity of small pulmonary arteries to neurokinin A.

Brain-derived neurotrophic factor (BDNF) is a key modulator during the development of jugular and nodose ganglia neurons, which represent the origin of a large proportion of the sensory innervation of the lung. It belongs to the family of neurotrophins, which have been shown to induce the expression of tachykinins. To assess the interactions of BDNF and the tachykinin neurokinin A (NKA) in small pulmonary vessels, BDNF-transgenic mice were examined for tachykinin contents in the airways, heart, trigeminal ganglion and jugular and nodose ganglion complex (JNC) using reverse phase HPLC (rpHPLC) and radioimmunoassay. BDNF-overexpression led to increased NKA levels in the heart and the JNC, whereas only slightly enhanced levels in the trigeminal ganglion were detected. Lower NKA levels were found in the lung. To assess vasoreactivity in small arteries, precision cut lung slices were subjected to videomorphometry and the response to NKA was examined, which showed significantly stronger effects in the BDNF-transgenic mice, while NK-2 receptor mRNA expression, assayed by real-time RT-PCR, was reduced. In conclusion, BDNF-overexpression results in decreased levels of NKA in the lung with subsequently increased NKA-sensitivity of small arteries. These findings point to a modulatory role of neurotrophins in small respiratory vessel tone regulation.

mRNA expression of tachykinins and tachykinin receptors in different human tissues.

The tachykinins substance P, neurokinin A and neurokinin B are involved in many pathophysiological processes. A reverse transcription-polymerase chain reaction (RT-PCR) assay was used to analyse the expression of TAC1 and TAC3, the genes that encode substance P/neurokinin A and neurokinin B, respectively, and the genes encoding the tachykinin NK(1), NK(2) and NK(3) receptors in different human tissues. The data show that tachykinins and their receptors mRNAs are broadly distributed in different human tissues being present in neuronal and non-neuronal types of cells. The presence of TAC3 and the tachykinin NK(3) receptor (TACR3) in a wide variety of peripheral tissues argue for a still unexplored role of this ligand-receptor pair in mediating visceral effects of tachykinins. We found, for the first time, that TAC3 and TACR3 mRNAs are expressed in human airways and pulmonary arteries and veins, providing further evidence for the involvement of this system in lung physiopathology.

Identification of a tachykinin NK(2) receptor splice variant and its expression in human and rat tissues.

The tachykinins substance P, neurokinin A and neurokinin B are implicated in different diseases and play an important role in neuroimmunomodulation. These peptides interact with three distinct types of tachykinin receptors termed NK(1), NK(2) and NK(3). While most mammalian genes encoding G protein-coupling cell membrane receptors are intron-less, the three tachykinin receptors contain introns in their genomic structures. In the present study, we have identified a splice variant of the tachykinin NK(2) receptor that results from skipping of exon 2 in the processing of the tachykinin NK(2) receptor mRNA. By using reverse transcription-polymerase chain reaction analysis, we observed that the tachykinin NK(2) receptor splice variant, that we named NK(2)beta, appeared in different human and rat tissues that also express the wild type, tachykinin NK(2)alpha isoform. Compared to tachykinin receptor NK(2)alpha isoform mRNA levels, the NK(2)beta isoform was strongly expressed in human and rat uteri, expressed in a moderate degree in the rat urinary bladder, colon, duodenum and stomach and unexpressed in the rat cerebral cortex, kidney, thoracic aorta, skeletal muscle and heart. These data describe the first known tachykinin receptor splice variant and suggest that the variety of tachykinin receptors may be further expanded through the generation of splicing isoforms. The presence of the truncated isoform may have a physiological significance in the regulation of tachykinin NK(2) receptor protein levels.

Characterization of tachykinin NK2 receptor in the anterior pituitary gland.

Tachykinins are a family of bioactive peptides that interact with three subtypes of receptors: NK1, NK2 and NK3. Substance P has greater affinity for NK1, and neurokinin A (NKA) for NK2 receptor subtype. Although only NK1 receptor has been characterized in the anterior pituitary gland, some evidence suggests the existence of NK2 receptors in this gland. Therefore, we investigated the presence of NK2 receptors in the anterior pituitary gland of male rats by radioligand binding studies using labeled SR48968, a non peptidic specific antagonist. [3H]SR48968 specific binding to cultured anterior pituitary cells was time-dependent and saturable, but with a lower affinity than previously reported values for cells expressing NK2 receptors. Unlabeled NKA inhibited only partially [(3)H]SR48968 specific binding to whole anterior pituitary cells. Since SR48968 is a non polar molecule, we performed experiments to discriminate surface from intracellular binding sites. SR48968 exhibited both surface and intracellular specific binding. Analysis of the surface-bound ligand indicated that [3H]SR48968 binds to one class of receptor with high affinity. Neurokinin A completely displaced [3H]SR48968 surface specific binding fitting to a two-site/two-state model with high and low affinity. Additionally, immunocytochemical studies showed that the NK2 receptor is expressed at least in a subset of lactotropes. These results demonstrate the presence of NK2 receptors in the anterior pituitary gland and suggest that NKA actions in this gland are mediated, at least in part, by the NK2 receptor subtype.

Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells.

Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [beta-Ala8]-neurokinin A(4-10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase.

Tachykinin receptor expression and function in human esophageal smooth muscle.

Tachykinins are present in enteric nerves of the gastrointestinal tract and cause contraction of esophageal smooth muscle; however, the mechanisms involved are not understood. Our aim was to characterize tachykinin signaling in human esophageal smooth muscle. We investigated functional effects of tachykinins on human esophageal smooth muscle using tension recordings and isolated cells, receptor expression with reverse transcription (RT)-polymerase chain reaction (PCR) and immunoblotting, intracellular Ca2+ responses using fluorescent indicator dyes, and membrane currents with patch-clamp electrophysiology. The mammalian tachykinins [substance P and neurokinin (NK) A and NKB] elicited concentration-dependent contractions of human esophageal smooth muscle. These responses were not affected by muscarinic receptor or neuronal blockade indicating a direct effect on smooth muscle cells (SMCs). Immunofluorescence and RT-PCR identified tachykinin receptors (NK1, NK2, and NK3) on SMCs. Contraction was mediated through a combination of Ca2+ release from intracellular stores and influx through L-type Ca2+ channels. NK2 receptor blockade inhibited the largest proportion of tachykinin-evoked responses. NKA evoked a nonselective cation current (I(NSC)) with properties similar to that elicited by muscarinic stimulation. The following paradigm is suggested: tachykinin receptor binding to SMCs releases Ca2+ from stores along with activation of I(NSC), which in turn results in membrane depolarization, L-type Ca2+ channel opening, rise of Ca2+ concentration, and contraction. These studies reveal new aspects of tachykinin signaling in human esophageal SMCs. Excitatory tachykinin pathways may represent targets for pharmacological intervention in disorders of esophageal dysmotility.

Regions of the human neurokinin A receptor involved in the generation of second messengers and in receptor desensitization.

Deletion analysis was used to study sites of human Neurokinin A receptor (HNKAR) necessary for signal transduction in CHO cells. Deletion of 62 and 81 amino acids from the c-terminus of HNKAR forms mutant receptors HNKAR delta 62 and HNKAR delta 81, which bind neurokinin A with high affinity but are functionally different. Wild type HNKAR and HNKAR delta 62 are functionally active whereas HNKAR delta 81 is functionally inactive. In addition, HNKAR and HNKAR delta 62 were both desensitized to the neurokinin A signal within 5 minutes. The data indicates: 1) an intact cytoplasmic tail of the HNKAR is not critical for signal transduction, but the n-terminal amino acids of the cytoplasmic tail are necessary for signaling and 2) the c-terminal 62 amino acids are not necessary for desensitization.

Expression of rat NK-2 (neurokinin A) receptor in E. coli.

With the goal of obtaining sufficient functional protein for structural analysis, rat neurokinin-2 receptor was produced in Escherichia coli by linking it to the periplasmic maltose-binding protein. As a first step, we present a biochemical and pharmacological investigation of the recombinant receptor. Western-blots showed that the fusion protein was associated with the membranes. The agonist [4,5-3H-Leu9]neurokinin A and the NK-2 antagonist [3H]SR48,968 bound to the receptor in a highly specific manner. Saturation binding of the [3H]agonist demonstrated a single class of receptors (KD = 10.5 nM, Bmax = 2.5 pmol/mg protein). The [3H]antagonist bound with higher affinity to a larger receptor population (KD = 0.2 nM, Bmax = 7.2 pmol/mg protein). Competition of [3H]agonist binding with other agonists demonstrated a potency order of: neurokinin A > [Nle10]NKA(4-10) = [beta-Ala8]NKA(4-10) >> substance P >>> senktide Against the [3H]antagonist, agonists were only partially inhibitory. Selective NK-2 antagonists inhibited binding of both [3H]ligands with an identical order of potency: SR48,968 >> R396 > MEN10,376, which is consistent with NK-2 receptor pharmacology in rat tissue.

Serotonin unmasks functional NK-2 receptors in vagal sensory neurones of the guinea-pig.

1. The regulation of substance P (SP) responsiveness in acutely isolated nodose neurones from adult guinea-pigs was investigated using standard intracellular recording techniques. 2. In control neurones, SP produced no measurable electrophysiological effects. However, following incubation with serotonin (5-HT, 10 microM), 64% of neurones were depolarized by 10 +/- 0.6 mV (n = 84 of 132 neurones) by SP (100 nM). 5-HT-induced SP responses were inhibited by SR48968 (100 nM, n = 6), a neurokinin 2 (NK-2) receptor antagonist, but were unaffected by CP99,994 and SR142801, NK-1 and NK-3 receptor antagonists (n = 3 each), respectively. 3. 5-HT-induced unmasking of SP responses was maximal within 5 min. Increasing the 5-HT incubation time up to 120 min did not increase the mean response amplitude or the percentage of SP responsive neurones (P = 0.611 and 0.867, respectively). 4. 5-HT-induced unmasking of SP responses was dose dependent (EC50 = 14 nM). A 5-HT3 receptor agonist CPBG (1 microM), mimicked the unmasking effects of 5-HT (n = 10 of 19 neurones), while 5-CT (10 microM), a non-selective 5-HT agonist devoid of action at 5-HT3 receptors, did not (n = 18). ICS205-930 (1 microM), a 5-HT3 receptor antagonist, completely blocked the 5-HT-induced unmasking of SP responses (n = 10 of 10 neurones). 5. In 68% of the neurones tested, bath-applied 5-HT (10 microM) evoked a 178 +/- 29.5 nM increase in [Ca2+]i (n = 16), which was blocked by nominally zero [Ca2+]o (n = 4) or by ICS205-930 (1 microM, n = 4). Nodose neurones incubated with 5-HT in the presence of nominally zero [Ca2+]o did not respond to SP (n = 12 of 13 neurones) in Locke solution containing normal [Ca2+]o, indicating that the 5-HT-mediated elevation of [Ca2+]i is required for unmasking of SP responses. Calmidazolium (100 nM), a calmodulin inhibitor, inhibited the unmasking effects of 5-HT (n = 5 of 5 neurones). 6. Incubating neurones with the nitric oxide (NO) donors papaNONOate (1 mM, 15-30 min) or SNAP (50 microM, 30-60 min) unmasked depolarizing SP responses in 71% and 45% of the neurones studied, respectively. L-NMMA (30 microM), a NO synthase inhibitor, blocked 5-HT-induced unmasking of SP responses (n = 10 of 10 neurones). 7. In sum, these results suggest that stimulation of 5-HT3 receptors activates an intracellular signalling cascade that couples calcium-calmodulin and NO activation to NK-2 receptor unmasking in sensory neurones.