Female infertility is associated with an altered expression of the neurokinin B/neurokinin B receptor and kisspeptin/kisspeptin receptor systems in ovarian granulosa and cumulus cells.

OBJECTIVE: To analyze and compare the expression profile of TAC3, TACR3, KISS1, and KISS1R in mural granulosa and cumulus cells from healthy oocyte donors and patients with different infertility etiologies, including advanced maternal age, endometriosis, and low ovarian response. DESIGN: Genetic association study. SETTING: Private fertility clinic and public research laboratory. PATIENT(S): Healthy oocyte donors and infertile women undergoing in vitro fertilization (IVF) treatment. INTERVENTION(S): IVF. MAIN OUTCOME MEASURE(S): Gene expression levels of KISS1, KISS1R, TAC3, and TACR3 in human mural granulosa and cumulus cells. RESULT(S): Infertile women showed statistically significantly altered expression levels of KISS1 (-2.57 ± 2.30 vs. -1.37 ± 2.11), TAC3 (-1.21 ± 1.40 vs. -1.49 ± 1.98), and TACR3 (-0.77 ± 1.36 vs. -0.03 ± 0.56) when compared with healthy oocyte donors. Advanced maternal age patients, endometriosis patients, and low responders showed specific and altered expression profiles in comparison with oocyte donors. CONCLUSION(S): Abnormal expression levels of KISS1/KISS1R and TAC3/TACR3 systems in granulosa cells might be involved in the decreased fertility associated to advanced maternal age, endometriosis, and low ovarian response.

Regulation of prepubertal dynorphin secretion in the medial basal hypothalamus of the female rat.

The onset of puberty is the result of an increase in secretion of hypothalamic gonadotrophin-releasing hormone (GnRH). This action is a result of not only the development of stimulatory inputs to its release, but also the gradual decrease in inhibitory inputs that restrain release of the peptide prior to pubertal onset. Dynorphin (DYN) is one of the inhibitory inputs produced in the medial basal hypothalamus (MBH); however, little is known about what substance(s) control its prepubertal synthesis and release. Because neurokinin B (NKB) increases in the hypothalamus as puberty approaches, we considered it a candidate for such a role. An initial study investigated the acute effects of an NKB agonist, senktide, on the secretion of DYN from MBH tissues incubated in vitro. In other experiments, central injections of senktide were administered to animals for 4 days then MBHs were collected for assessment of DYN synthesis or for the in vitro secretion of both DYN and GnRH. Because insulin-like growth factor (IGF)-1 has been shown to play an important role at puberty, additional animals received central injections of this peptide for 4 days to assess NKB and DYN synthesis or the in vitro secretion of NKB. The results obtained show that senktide administration up-regulates the NKB receptor protein, at the same time as suppressing the DYN and its receptor. Senktide consistently suppressed DYN and elevated GnRH secretion in the same tissue incubates from both the acute and chronic studies. IGF-1 administration caused an increase in NKB protein, at the same time as decreasing DYN protein. Furthermore, the central administration of IGF-1 caused an increase in NKB release, an action blocked by the IGF-1 receptor blocker, JB-1. These results indicate that the IGF-1/NKB pathway contributes to suppressing the DYN inhibitory tone on prepubertal GnRH secretion and thus facilitates the puberty-related increase in the release of GnRH to accelerate the onset of puberty.

Bronchoconstrictor effect of the tachykinin NK3-receptor agonists [MePhe7]-neurokinin B and senktide in the isolated guinea pig lung.

To determine whether bronchoconstriction can be mediated via the tachykinin NK3 receptors, isolated guinea pig lungs were challenged with the exogenous tachykinin NK3-receptor agonists [MePhe7]-neurokinin B ([MePhe7]-NKB) and senktide. [MePhe7]-NKB induced bronchoconstriction (EC50 = 11.8 ± 1.7 µM) that was significantly inhibited by the tachykinin NK3-receptor antagonist SB 223412 at 10 µM (EC50 = 24.4 ± 4.5 µM). Senktide also induced bronchoconstriction (EC50 = 96.2 ± 20.3 µM) and the bronchoconstriction was significantly reduced by SB 223412 at 1 and 10 µM (EC50 = 270.8 ± 78.9 µM and 388.3 ± 105.5 µM, respectively). Although the authors demonstrated that SB 223412, [MePhe7]-NKB, and senktide are potent and selective for the tachykinin NK3 receptors in binding and functional (Ca(2+) mobilization) assays, the tachykinin NK1-receptor antagonist CP 99,994 at 1 µM (EC50 = 32.7 ± 8.5 µM) produced inhibition of [MePhe7]-NKB-induced bronchoconstriction, whereas the tachykinin NK2-receptor antagonist SR 48968 at 0.1 µM (EC50 = 213.2 ± 42.9 µM) blocked senktide-induced bronchoconstriction. These data suggest that [MePhe7]-NKB and senktide caused bronchoconstriction in guinea pig through activation of the tachykinin NK3-receptors but the tachykinin NK1- and/or NK2-receptors are also involved in the response.

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.

Differential expression of Nk1 and NK3 neurokinin receptors in neurons of the nucleus tractus solitarius and the dorsal vagal motor nucleus of the rat and mouse.

Tachykinins (substance P, neurokinin A and neurokinin B) influence autonomic functions by modulating neuron activity in nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV) through activation of neurokinin receptors NK1 and NK3. Our purpose was to identify and define by neurochemical markers, the subpopulations of NK1 and NK3 expressing neurons in NTS and DMV of rat and mouse. Because the distribution of the NK1 and NK3 expressing neurons overlaps, co-expression for both receptors was tested. By double labeling, we show that NK1 and NK3 were not co-expressed in NTS neurons. In the DMV, most of neurons (87%) were immunoreactive for only one of the receptors and 34% of NK1 neurons, 7% of NK3 neurons and 12% of NK1-NK3 neurons were cholinergic neurons. None of the neurons immunoreactive for NK1 or NK3 were positive for tyrosine hydroxylase, suggesting that catecholaminergic cells of the NTS (A2 and C2 groups) did not express neurokinin receptors. The presence of NK1 and NK3 was examined in GABAergic interneurons of the NTS and DMV by using GAD65-EGFP transgenic mouse. Immunoreactivity for NK1 or NK3 was found in a subpopulation of GAD65-EGFP cells. A majority (60%) of NK3 cells, but only 11% of the NK1 cells, were GAD65-EGFP cells. In conclusion, tachykinins, through differential expression of neurokinin receptors, may influence the central regulation of vital functions by acting on separate neuron subpopulations in NTS and DMV. Of particular interest, tachykinins may be involved in inhibitory mechanisms by acting directly on local GABAergic interneurons. Our results support a larger contribution of NK3 compared with NK1 in mediating inhibition in NTS and DMV.

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.

Upregulation of tachykinin NK-1 and NK-3 receptor binding sites in the spinal cord of spontaneously hypertensive rat: impact on the autonomic control of blood pressure.

1 Effects of intrathecally (i.t.) injected tachykinin NK-1 and -3 receptor agonists and antagonists were measured on mean arterial blood pressure (MAP) and heart rate (HR) in awake unrestrained spontaneously hypertensive rats (SHR,15-week-old) and age-matched Wistar Kyoto rats (WKY). Quantitative in vitro autoradiography was also performed on the lower thoracic spinal cord of both strains and Wistar rats using specific radioligands for NK-1 receptor ([(125)I]HPP[Arg(3),Sar(9),Met(O(2))(11)]SP (3-11)) and NK-3 receptor ([(125)I]HPP-Asp-Asp-Phe-N-MePhe-Gly-Leu-Met-NH(2)). 2 The NK-1 agonist [Sar(9),Met(O(2))(11)]SP (650 and 6500 pmol) decreased MAP and increased HR in WKY. The fall in MAP was blunted in SHR and substituted by increases in MAP (65-6500 pmol) and more sustained tachycardia. The NK-3 agonist senktide (6.5-65 pmol) evoked marked increases in MAP and HR (SHR>>>WKY), yet this response was rapidly desensitized. Cardiovascular effects of [Sar(9),Met(O(2))(11)]SP (650 pmol) and senktide (6.5 pmol) were selectively blocked by the prior i.t. injection of LY303870 (NK-1 antagonist, 65 nmol) and SB235375 (NK-3 antagonist, 6.5 nmol), respectively. Antagonists had no direct effect on MAP and HR in both strains. 3 Densities of NK-1 and -3 receptor binding sites were significantly increased in all laminae of the spinal cord in SHR when compared to control WKY and Wistar rats. The dissociation constant was however not affected in SHR for both NK-1 (K(d)=2.5 nM) and NK-3 (K(d)=5 nM) receptors. 4 Data highlight an upregulation of NK-1 and -3 receptor binding sites in the thoracic spinal cord of SHR that may contribute to the hypersensitivity of the pressor response to agonists and to the greater sympathetic activity seen in this model of arterial hypertension.

Immunocytochemical distribution of NK-1 and NK-3 tachykinin receptors in isolated pancreatic acini of guinea pigs and rats.

In this study, we investigated the immunocytochemical distribution of NK-1 and NK-3 tachykinin receptors in guinea pig and rat isolated pancreatic acini. In dispersed acinar cells from guinea pig, immunofluorescence staining detected similar densities of NK-1 and NK-3 receptors; conversely, rat acinar cells expressed NK-1 receptors more strongly than NK-3 receptors. In line with previous functional studies, these immunocytochemical findings suggest that guinea pig NK-1 and NK-3 receptors and rat NK-1 receptors alone play a direct stimulatory role in the basal pancreatic acinar amylase release.

Characterization of subpopulations of neurons producing melanin-concentrating hormone in the rat ventral diencephalon.

Neurons producing melanin-concentrating hormone (MCH) are involved in a large array of functions. Some of these functions may be mediated by specific subpopulations. One such subpopulation was characterized by the expression of the neurokinin 3 receptor and the 'cocaine- and amphetamine-regulated transcript' (CART) peptide, while another expresses neither one of these two molecules. MCH+/CART+ axons were traced throughout the brain and showed a strikingly different pattern of distribution than that of MCH+/CART- axons. Particularly, many MCH+/CART+ axons are observed in the telencephalon, while MCH+/CART- projections are mostly directed toward the brainstem. Calbindin, a protein involved in calcium homeostasis, has been largely used in many structures of the brain for the identification of neuronal phenotypes. However, few MCH neurons were labeled for this protein. On the other hand, neurons producing the peptides hypocretins (Hcrt), and codistributed with the MCH neurons, were all labeled for calbindin. Thus, at least two subpopulations of MCH neurons can be distinguished on the basis of neuronal phenotypes and connections. These neurons may be involved in distinct circuitry and in distinct functions.

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.

Expression of connexin36 in cone pedicles and OFF-cone bipolar cells of the mouse retina.

Transgenic technology, immunocytochemistry, electrophysiology, intracellular injection techniques, and reverse transcription PCR were combined to study the expression of neuronal connexin36 (Cx36) in the outer plexiform layer of the mouse retina. Transgenic animals expressed either a fusion protein of full-length Cx36 with enhanced green fluorescent protein (EGFP) attached at the C terminus or exon 2 of Cx36 was replaced bybeta-galactosidase (beta-gal). In the outer nuclear layer,beta-gal-positive cell bodies, which were confined to the most distal region close to the outer limiting membrane, displayed immunoreactivity against S-cone opsin. Cx36-EGFP puncta colocalized with cone pedicles, which were visualized by intracellular injection. In reverse transcriptase PCR experiments, Cx36 mRNA was never detected in samples of rods harvested from the outer nuclear layer. These results strongly suggest expression of Cx36 in cones but not in rods. In vertical sections, Cx36 expression in the vitreal part of the outer plexiform layer was characterized by a patchy distribution. Immunocytochemistry with antibodies against the neurokinin-3 receptor and the potassium channel HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel) displayed clusters of the Cx36 label on the dendrites of OFF-cone bipolar cells. In horizontal sections, these clusters of Cx36 appeared as round or oval-shaped groups of individual puncta, and they were always aligned with the base of cone pedicles. Double-labeling experiments and single-cell reverse transcriptase PCR ruled out expression of Cx36 in horizontal cells and rod bipolar cells. At light microscopic resolution, we found close association of Cx36-EGFP with the AMPA-type glutamate receptor subunit GluR1 but not with GluR2-GluR4, the kainate receptor subunit GluR5, or the metabotropic glutamate receptor mGluR6.

Immunocytochemical description of five bipolar cell types of the mouse retina.

With the ever-growing number of transgenic mice being used in vision research, a precise knowledge of the cellular organization of the mouse retina is required. As with the cat, rabbit, rat, and primate retinae, as many as 10 cone bipolar types and one rod bipolar type can be expected to exist in the mouse retina; however, they still have to be defined. In the current study, several immunocytochemical markers were applied to sections of mouse retina, and the labeling of bipolar cells was studied using confocal microscopy and electron microscopy. By using antibodies against the neurokinin-3 receptor NK3R; the plasma membrane calcium ATPase1 (PMCA1); and the calcium (Ca)-binding proteins CaB1, CaB5, caldendrin, and recoverin, three different OFF-cone bipolar cells could be identified. One type of ON-cone bipolar cell was identified through its immunoreactivity for CaB5 and PMCA1. Rod bipolar cells, comparable in morphology to those of other mammalian retinae, expressed protein kinase Calpha and CaB5. It was also shown that putative OFF-cone bipolar cells receive light signals through flat contacts at the cone pedicle base, whereas ON-cone bipolar signaling involves invaginating contacts. The distribution of the kainate receptor subunit GluR5 was studied by confocal and electron microscopy. GluR5 was expressed at flat bipolar cell contacts; however, it appears to be involved with only certain types of OFF-cone bipolar cells. This suggests that different bipolar cell types receive their light signals through different sets of glutamate receptors.

Decreases in neurokinin-3 tachykinin receptor-immunoreactive and -mRNA levels are associated with salt appetite in the deoxycorticosterone-treated rat.

Tachykinins are a family of neuropeptides that inhibit salt appetite. Although decreased tachykinin-mRNA levels are observed in natriorexic sodium-deplete rats, no decrease is seen in natriorexic sodium-replete rats that are administered the aldosterone-mimetic deoxycorticosterone acetate (DOCA). Since reduced synthesis of tachykinins could not account for increased appetite, we hypothesized that increased salt appetite was due to a downregulation of tachykinin receptors. Thus, we injected rats with DOCA once daily for 11 days and analyzed tachykinin receptor subtype, neurokinin 3 (NK3r)-immunoreactivity by Western blot analysis since intracerebroventricular injection of senktide (NK3r agonist) attenuates salt intake in DOCA-treated animals. We examined NK3r-immunoreactivity in several brain regions thought to be associated with the control of water and electrolyte balance including the bed nucleus of the stria terminalis, central nucleus of the amygdala, diagonal band of Broca, hippocampus, nucleus tractus solitarius, parabrachial nucleus, paraventricular nucleus of the hypothalamus, and supraoptic nucleus. Consistent with our hypothesis, we found decreased NK3r-immunoreactivity in all brain regions analyzed except for increases in the amygdala and no changes in the paraventricular nucleus of the hypothalamus. To examine whether DOCA's effects on NK3r synthesis are direct, we used differentiated N1E-115 neuroblastoma cells that express NK3r and treated them with a range of concentrations of DOCA and found a dose-dependent decrease in NK3r-mRNA abundance via Northern blotting. The present results suggest that the tachykinin receptors are downregulated after subchronic DOCA treatment and this finding is consistent with the hypothesis that suppressed inhibition of salt appetite as mediated through the tachykininergic system.

SR 142801, a tachykinin NK(3) receptor antagonist, prevents beta(2)-adrenoceptor agonist-induced hyperresponsiveness to neurokinin A in guinea-pig isolated trachea.

We investigated whether fenoterol was able to enhance contractile responsiveness to neurokinin A (NKA) on the guinea-pig isolated trachea. We then studied the effects of two inhibitors of nuclear factor kappa B (NFkappaB), gliotoxin and pyrrolidine dithiocarbamate, and of the tachykinin NK(1), NK(2) and NK(3) receptor antagonists, SR 140333, SR 48968 and SR 142801 and determined whether tachykinin receptor gene expression was up-regulated in the trachea after exposure to fenoterol. Fenoterol (0.1 microM, 15 h, 21 degrees C) induced an increased contractile response to NKA (mean of difference in maximal tension between control and fenoterol +/- S.E.M; +0.47 +/- 0.14 g, n = 26, P < 0.01). This hyperresponsiveness was strongly reduced by co-incubation with gliotoxin (0.1 microg/ml) or pyrrolidine dithiocarbamate (0.1 mM) and abolished by SR 140333 (0.1 microM) and SR 142801 (0.1 microM). SR 48968 (0.1 microM) diminished the tracheal contractility to NKA but failed to reduce the hyperreactivity induced by fenoterol. Tachykinin NK(1) receptor (NK(1)R), NK(2) receptor (NK(2)R) and NK(3) receptor (NK(3)R) gene expression was analyzed by semiquantitative RT-PCR. Compared to control tissues, NK(1)R and NK(2)R mRNA expression was increased by about 1.6-fold and 1.4-fold, respectively, in tissues treated with fenoterol. We were unable to detect the presence of NK(3)R mRNA in the guinea-pig trachea. In conclusion, fenoterol induces tracheal hyperresponsiveness to NKA and an up-regulation of NK(1)R and NK(2)R gene expression. The hyperresponsiveness implicates the NFkappaB pathway and is abolished by tachykinin NK(1) (SR 140333) and NK(3) (SR 142801) receptor antagonists.

Cholinergic neurons expressing neuromedin K receptor (NK3) in the basal forebrain of the rat: a double immunofluorescence study.

By using a double immunofluorescence method we have examined the distribution of cholinergic neurons expressing neuromedin K receptor (NK3) in the rat brain and spinal cord. The distribution of neuromedin K receptor-like immunoreactive neurons completely overlapped with that of choline acetyltransferase-positive neurons in certain regions of the basal forebrain, e.g. the medial septal nucleus, nucleus of the diagonal band of Broca, magnocellular preoptic nucleus and substantia innominata. Partially overlapping distributions of neuromedin K receptor-like immunoreactive and choline acetyltransferase-positive neurons were found in the basal nucleus of Meynert, globus pallidus, ventral pallidum of the forebrain, tegmental nuclei of the pons and dorsal motor nucleus of the vagus. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities, however, were found predominantly in the medial septal nucleus, nucleus of the diagonal band of Broca and magnocellular preoptic nucleus of the basal forebrain: 66-80% of these choline acetyltransferase-positive neurons displayed neuromedin K receptor-like immunoreactivity. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities were hardly detected in other aforementioned regions of the forebrain, brainstem and spinal cord. The present study has provided morphological evidence for direct physiological modulation or regulation of cholinergic neurons by tachykinins through the neuromedin K receptor in the basal forebrain of rats.

Retinal dopaminergic neurons (A17) expressing neuromedin K receptor (NK(3)): a double immunocytochemical study in the rat.

By using a double immunofluorescence method we examined the distribution of dopaminergic neurons (A17) expressing neuromedin K receptor (NKR, NK(3)) in the rat retina. The distribution of NKR-like immunoreactive (-LI) neurons partially overlapped that of tyrosine hydroxylase (TH)-LI neurons in the inner retina of section and flat-mount preparation. Neurons showing both TH- and NKR-like immunoreactivities were found in the retina (A17): 100% of these TH-LI neurons displayed NKR-like immunoreactivity, and they constituted about 3.5% of total NKR-LI neurons. The majority of double-labeled neurons with TH- and NKR-like immunoreactivities were distributed in the proximal inner nuclear layer and the upper part of inner plexiform layer of the retina, and characterized with appearance of amacrine cells. The present study has provided morphological evidence for direct physiological modulation of dopaminergic neurons by tachykinins through NKR in the rat retina (A17).

Central and peripheral expression of neurokinin-1 and neurokinin-3 receptor and substance P-encoding messenger RNAs: peripheral regulation during formalin-induced inflammation and lack of neurokinin receptor expression in primary afferent sensory neurons.

The neurokinin-1 receptor and its tachykinin neuropeptide ligand substance P are associated with the mediation of nociception. Substance P released from primary afferent sensory neurons activates neurokinin receptors on both central and peripheral targets that mediate specific aspects of central sensitization and inflammatory function; however, an autoreceptor function for the neurokinin-1 receptor remains highly controversial. Activation of the neurokinin-1 receptor by substance P during chronic nociception increases neurokinin-1 receptor gene expression in the spinal cord. Similarly, neurokinin-3 receptors on peripheral or target tissues or neurons could play an important role in the sensitization of sensory neurons. Therefore, this study (i) mapped the steady-state levels of substance P-encoding preprotachykinin, neurokinin-1 and neurokinin-3 receptor messenger RNAs in central and peripheral tissues including sensory ganglia, and (ii) investigated whether formalin-evoked nociception altered the quantity or location of neurokinin-1 or neurokinin-3 receptor messenger RNAs in the sensory ganglia or inflamed peripheral targets for substance P. Solution hybridization-nuclease protection assays quantified neurokinin receptor messenger RNA levels in central and peripheral tissues from normal and formalin-inflamed rats. High concentrations of the neurokinin-1 receptor were found in whole brain, spinal cord, and peripheral target organs innervated by substance P-containing neurons. Measurable levels of neurokinin-3 receptor messenger RNA were found only in brain, spinal cord and urinary bladder. Results also show that neither neurokinin-1 nor neurokinin-3 receptor messenger RNAs were detectable in primary afferent sensory neurons in the dorsal root ganglia of normal or formalin-inflamed rats. Neurokinin-1 receptor messenger RNA levels were, however, significantly increased in hindpaw tissues inflamed by formalin for 6 h. These results indicate that the plasticity of neurokinin-1 receptor gene expression in non-neuronal peripheral cells could regulate sensitivity to substance P in a manner similar to that in the spinal cord dorsal horn. Altered neurokinin-1 receptor gene expression provides a useful marker of long-term nociceptive activation and may mediate peripheral mechanisms of hyperalgesia and cellular sensitization during inflammation. Importantly, inflammation does not induce a phenotypic change in afferent sensory neurons providing neurokinin receptor targets for the direct sensitization of these neurons by substance P.

Discovery of a novel class of selective non-peptide antagonists for the human neurokinin-3 receptor. 2. Identification of (S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (SB 223412).

Optimization of the previously reported 2-phenyl-4-quinolinecarboxamide NK-3 receptor antagonist 14, with regard to potential metabolic instability of the ester moiety and affinity and selectivity for the human neurokinin-3 (hNK-3) receptor, is described. The ester functionality could be successfully replaced by the ketone (31) or by lower alkyl groups (Et, 21, or n-Pr, 24). Investigation of the substitution pattern of the quinoline ring resulted in the identification of position 3 as a key position to enhance hNK-3 binding affinity and selectivity for the hNK-3 versus the hNK-2 receptor. All of the chemical groups introduced at this position, with the exception of halogens, increased the hNK-3 binding affinity, and compounds 53 (3-OH, SB 223412, hNK-3-CHO binding Ki = 1.4 nM) and 55 (3-NH2, hNK-3-CHO binding Ki = 1.2 nM) were the most potent compounds of this series. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 53 is about 100-fold selective for the hNK-3 versus hNK-2 receptor, with no affinity for the hNK-1 at concentrations up to 100 microM. In vitro studies demonstrated that 53 is a potent functional antagonist of the hNK-3 receptor (reversal of senktide-induced contractions in rabbit isolated iris sphincter muscles and reversal of NKB-induced Ca2+ mobilization in CHO cells stably expressing the hNK-3 receptor), while in vivo this compound showed oral and intravenous activity in NK-3 receptor-driven models (senktide-induced behavioral responses in mice and senktide-induced miosis in rabbits). Overall, the biological data indicate that (S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (53, SB 223412) may serve as a pharmacological tool in animal models of disease to assess the functional and pathophysiological role of the NK-3 receptor and to establish therapeutic indications for non-peptide NK-3 receptor antagonists.

Extensive proteolysis inhibits high-level production of eukaryal G protein-coupled receptors in the archaeon Haloferax volcanii.

In this study the usage of the halophilic archaeon Haloferax volcanii as a production system for eukaryal G protein-coupled receptors (GPCRs) was characterized. The genes of four GPCRs were fused to the dihydrofolate reductase gene of H. volcanii. In Northern blots both 5' fragments and full-length fusion transcripts were found. In contrast, only C-terminal fusion protein fragments could be detected in Western blot analyses. Ligand binding experiments revealed that a minor amount of correctly folded human beta 2 adrenergic receptor was inserted into the membrane. The introduction of different modifications at the 5' and the 3' end of the receptor genes did not significantly increase the production level. Determination of the subcellular localization showed that fusion protein fragments containing one or more receptor helices were located in the membrane. The results indicate that neither transcription, translation nor membrane translocation but the activity of one or more proteases limits the level of GPCR production in H. volcanii.

The non-peptide tachykinin NK2 receptor antagonist SR 48968 interacts with human, but not rat, cloned tachykinin NK3 receptors.

SR 48968, a non-peptide tachykinin NK2 receptor antagonist, has been shown to possess sub-micromolar affinity for NK3 receptors present in the guinea pig. In the present study, we have compared the binding affinities of SR 48968 to the cloned human and rat NK3 receptors expressed in CHO cells. Using [125I]-[MePhe7]-neurokinin B as the radioligand, SR48968 displayed an IC50 value of 350 nM for the human NK3 receptor as compared with a value of greater than 10 microM for the rat NK3 receptor. Exposure of cells transfected with human NK3 receptor cDNA to [Pro7]-neurokinin B increased inositol phospholipid turnover in a concentration-dependent manner and this response was blocked competitively by SR 48968. Our results demonstrate that SR 48968 is an antagonist at the human NK3 receptor and may be a useful tool for elucidating the species-dependent variations in the non-peptide antagonist binding site(s) on the NK3 receptor.