Gly166 in the NK1 receptor regulates tachykinin selectivity and receptor conformation

We have studied the pharmacological properties of genetically engineered human NK1 tachykinin receptors in which residues at the extracellular surface of the fourth transmembranal domain were substituted with the corresponding amino acids from the NK2 receptor. We show that substitution of G166C:Y167F in the human NK1 receptor induces high affinity binding of a group of tachykinin ligands, known as 'septides' (i.e. neurokinin A, neurokinin B, [pGlu6,Pro9]-substance P6-11 and substance P-methylester). In contrast, binding of substance P and non-peptide antagonists is unaffected by these mutations. This effect parallels that found on the rat receptor and is therefore species specific. Second, we demonstrate that mutation of Gly166 to Cys alone is both necessary and sufficient to create this pan-reactive tachykinin receptor, whereas replacement of Tyr167 by Phe has no detectable effect on the pharmacological properties of the receptor. Furthermore, analysis of the effect of N-ethylmaleimide and dithiothreitol on binding of radiolabelled substance P documents differences in the mode in which this ligand interacts with wild-type and mutant receptors and supports the existence of a mutational induced change in the conformational status of the NK1 receptor.     

A tachykinin NK3 receptor antagonist, SR 142801 (osanetant), prevents substance P-induced bronchial hyperreactivity in guinea-pigs

Aerosolized substance P (0.1 M, for 30 min) induced airway hyperresponsiveness in guinea-pigs 24 h after they were pre-treated with salbutamol (8.7 mM by aerosol for 10 min) and phosphoramidon (0.1 mM by aerosol for 10 min). This was displayed by an exaggerated response to the bronchoconstrictor effect of acetylcholine. A microvascular leakage hypersensitivity also occurred and was demonstrated by a potentiation of the plasma protein extravasation from bronchial vessels induced by histamine. The aim of this study was to investigate the effects of the non-peptide and potent tachykinin NK3 receptor antagonist, SR 142801 (osanetant), in comparison with those of the tachykinin NK1 and NK2 receptor antagonists, SR 140333 (nolpitantium) and SR 48968 (saredutant) respectively, on substance P. When given once at 1 mg/kg i.p. 45 min before exposure to substance P, SR 142801 prevented both hyperresponsiveness to acetylcholine and the potentiation of histamine-induced increase in microvascular permeability. SR 142801 did not exhibit any tachykinin NK1 or NK2 antagonistic activity in experiments on guinea-pig isolated airways, in vitro or in vivo. The results suggest that tachykinin NK3 receptors might be involved in these substance P-induced effects on airways.     

Potencies of agonists acting at tachykinin receptors in the oestrogen-primed rat uterus: effects of peptidase inhibitors

The uterotonic potencies of the naturally occurring mammalian tachykinins and the synthetic subtype-selective agonist analogues of these agents [Lys5,MeLeu9,Nlel0]neurokinin A-(4-10) and [Nle10]neurokinin A-(4-10) (tachykinin NK2 receptor-selective), [Sar9,Met(O2)11]substance P (tachykinin NK1 receptor-selective) and senktide (tachykinin NK3 receptor-selective) were determined using preparations from oestradiol-treated rats. The endopeptidase 24.11 inhibitor, N-[N-[1-(S)-carboxyl-3-phenylpropyl]-(S)-phenyl-alanyl-(S)-isoserine+ ++ (SCH 39370), potentiated responses to neurokinin A, neurokinin B and substance P, but not to [Lys5,MeLeu9,Nle10)]neurokinin A-(4-10) or senktide. [Nle10]neurokinin A-(4-10) effects were potentiated by SCH 39370 with amastatin and those to [Sar9,Met(O2)11]substance P were potentiated by SCH 39370 and captopril in combination. In the presence of optimal concentrations of peptidase inhibitors the relative order of agonist potency was: neurokinin A > substance P > neurokinin B for the naturally occurring mammalian tachykinins and [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) > [Nle10]neurokinin A-(4-10) > [Sar9,Met(O2)11]substance P > senktide for the synthetic tachykinin analogues. Thus, while a tachykinin NK2 receptor predominates in the oestrogen-primed uterus, a tachykinin NK1 receptor may also be present. The non-peptide tachykinin NK3 receptor antagonist, SR 142801, did not antagonise the effects of senktide suggesting that tachykinin NK3 receptors do not mediate its relatively minor effect on the uterus of the oestrogen-primed rat.     

Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors

We compared the desensitization of neurokinin1 and neurokinin2 (NK1 and NK2) receptors expressed in Chinese hamster ovary cells to substance P and neurokinin A, respectively. Substance P and neurokinin A stimulated a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) for both receptors, which was due to release of Ca2+ from intracellular stores. This was followed by a plateau in [Ca2+]i, which was due to influx of extracellular Ca2+, and was more sustained for the NK2 receptor. When Ca2+ was present in the extracellular solution, the Ca2+ response of the NK1 receptor, but not the NK2 receptor, rapidly desensitized and slowly resensitized to two exposures to agonist. In contrast, the [Ca2+]i response, measured in Ca2+-free solution, and inositol triphosphate generation desensitized and resensitized similarly for the NK1 and NK2 receptors. Thus, differences in desensitization between the NK1 receptor and the NK2 receptor may be related to differences in entry of extracellular Ca2+. We compared endocytosis of the NK1 and NK2 receptors to determine whether disparities could account for differences in desensitization. Fluorescent and radiolabeled substance P and neurokinin A were internalized similarly by cells expressing NK1 and NK2 receptors. Thus, disparities in internalization cannot account for differences in desensitization. We used inhibitors to examine the contribution of endocytosis, recycling, and phosphatases to desensitization and resensitization of the NK1 receptor. Desensitization did not require endocytosis. However, resensitization required endocytosis, recycling, and phosphatase activity. This suggests that the NK1 receptor desensitizes by phosphorylation and resensitizes by dephosphorylation in endosomes and recycling.     

Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury

Substance P, acting via the neurokinin 1 receptor (NK1R), plays an important role in mediating a variety of inflammatory processes. However, its role in acute pancreatitis has not been previously described. We have found that, in normal mice, substance P levels in the pancreas and pancreatic acinar cell expression of NK1R are both increased during secretagogue-induced experimental pancreatitis. To evaluate the role of substance P, pancreatitis was induced in mice that genetically lack NK1R by administration of 12 hourly injections of a supramaximally stimulating dose of the secretagogue caerulein. During pancreatitis, the magnitude of hyperamylasemia, hyperlipasemia, neutrophil sequestration in the pancreas, and pancreatic acinar cell necrosis were significantly reduced in NK1R-/- mice when compared with wild-type NK1R+/+ animals. Similarly, pancreatitis-associated lung injury, as characterized by intrapulmonary sequestration of neutrophils and increased pulmonary microvascular permeability, was reduced in NK1R-/- animals. These effects of NK1R deletion indicate that substance P, acting via NK1R, plays an important proinflammatory role in regulating the severity of acute pancreatitis and pancreatitis-associated lung injury.     

Expression of a retrovirally transduced gene under control of an internal housekeeping gene promoter does not persist due to methylation and is restored partially by 5-azacytidine treatment

BACKGROUND: Tachykinins, such as substance P, might be involved in the development of airway hyperresponsiveness and airway inflammation. OBJECTIVE: This study was designed to investigate the effects of the tachykinin NK1 receptor antagonist SR 140333 (Nolpitantium) and the NK2 receptor antagonist SR 48968 (Saredutant) on the activation of alveolar macrophages in the guinea-pig. METHODS: Guinea-pigs sensitized and challenged by ovalbumin administered by aerosol or naive guinea-pigs were exposed by aerosol to the neutral endopeptidase, phosphoramidon and, 15 min later, to substance P. Twenty-four hours later, bronchoalveolar lavages were performed and the cell composition of bronchoalveolar lavage fluids and the arachidonate release from alveolar macrophages stimulated in vitro with fMLP were evaluated. RESULTS: Antigen challenge in sensitized guinea-pigs induced an increase in the total number of cells and granulocytes in the bronchoalveolar lavage fluids that was not reduced by pre-treatment of guinea-pigs with a single dose of SR 140333 or SR 48968 (1 mg/kg). Substance P exposure in phosphoramidon-pretreated guinea-pigs did not induce an increase in the total number of cells. In contrast, antigen or substance P exposure induced a significant increase in the in vitro fMLP-induced arachidonate release from alveolar macrophages. Pre-treatment of the guinea pigs with SR 140333 or SR 48968 did not reduce the increase in arachidonate release from fMLP-stimulated alveolar macrophages from sensitized and challenged guinea-pigs. Pre-treatment of the animals by SR 140333 and SR 48968 reduced the enhanced arachidonate release induced by fMLP from substance P-exposed guinea-pigs. CONCLUSION: The present data demonstrate the importance of NK1- and NK2-receptor stimulation in the development of substance P-induced increased reactivity of alveolar macrophages.     

Substrate assistance in the mechanism of family 18 chitinases: theoretical studies of potential intermediates and inhibitors

Gastric adaptive relaxation is a vago-vagal reflex, probably involving the site of interface of vagal afferents and efferents in the dorsal vagal complex of the medulla. Previous studies have shown that both substance P and nitric oxide in the dorsal vagal complex decrease intragastric pressure. The purpose of this study is, firstly, to localize NK1 tachykinin receptor immunoreactive (ir) staining in the dorsal vagal complex and, secondly, to determine its anatomical relationship to nitrergic cells in the dorsal motor nucleus of the vagus. Sections were stained by avidin-biotin immunocytochemistry using antiserum to NK1 receptor alone or combined with NADPH-diaphorase histochemistry. In the nucleus tractus solitarius, NK1 receptor-ir varicosities were moderately dense in the medial subnucleus, but sparse in the centralis and gelatinosus subnuclei. In the dorsal motor nucleus of the vagus, NK1 receptor-ir staining in cell bodies and fibers was present throughout, with a markedly dense varicose fiber and cell body staining in a lateral column of the rostral portion of the nucleus. NADPH-diaphorase staining is most marked in cell bodies in the same region of the dorsal motor nucleus of the vagus. In dual-stained sections, there was complete overlap of NADPH-diaphorase and NK1 receptor-ir stain; however, the markers were very rarely colocalized within the same vagal motor neurons. Ipsilateral vagotomy almost completely abolished NK1r-ir staining in vagal motor neurons. We conclude that, in the dorsal motor nucleus of the vagus, NK1 receptor is synthesized by a population of vagal motor neurons which are in close anatomical proximity to, but separate from, nitrergic neurons. Based on these observations, substance P-mediated gastric relaxation in this region is unlikely to be via activation of nitrergic vagal preganglionic neurons. In the nucleus tractus solitarius, the NK1 receptor and NADPH-diaphorase stain are not codistributed in subnuclei mediating gastric and esophageal control. Therefore, substance P and nitric oxide may mediate their respective gastrointestinal effects via separate afferent pathways.     

In vitro and in vivo pharmacology of S 16474, a novel dual tachykinin NK1 and NK2 receptor antagonist

Since tachykinins released from lung sensory nerve endings are thought to play a role in inflammatory diseases of airways via NK1 and NK2 receptors, dual tachykinin NK1 and NK2 receptor antagonists may have a great therapeutic potential. In vitro, the cyclopeptide S 16474 (cyclo-[Abo-Asp(D-Trp(Suc0Na)-Phe-N-(Me)Bzl)]) bound to both human tachykinin NK1 and NK2 receptors expressed in two lines of transfected Chinese hamster ovary cells (IC50 values 85 nM and 129 nM, respectively), while showing a poor affinity for the rat tachykinin NK1 receptor. S 16474 inhibited the contractions induced by substance P in isolated rabbit vena cava (pA2 7.0) and by neurokinin A in rabbit pulmonary artery (pA2 5.6). In vivo in anaesthetized guinea-pigs, S 16474 was found to dose dependently inhibit the bronchoconstrictions induced by intravenously administered substance P, neurokinin A and capsaicin. Plasma extravasation evoked in bronchi by endogenously released tachykinins under vagus nerve stimulation was abolished by S 16474 (10 mu mol/kg i.v.). These results demonstrate clearly that S 16474 is a tachykinin receptor antagonist exhibiting, in vitro and in vivo, a dual inhibitory effect on NK1 and NK2 receptors.     

Split-receptors in the tachykinin neurokinin-1 system--mutational analysis of intracellular loop 3

Several membrane proteins have been functionally expressed from non-covalently coupled, contiguous segments especially with the split-site located between natural domains. Experiments using such 'split-proteins' were here performed in the tachykinin neurokinin-1 (NK1) receptor with co-expression of contiguous segments with split-sites positioned in various intracellular and extracellular loops. The construct where the split-site was located in intracellular loop 3 gave a reasonable expression level of substance-P-binding sites, i.e. 12% of wild-type expression. Of the other split-receptors tested, only the one with the split-site located just outside transmembrane (TM) segment-V gave any detectable substance P binding, which however only was 1% of the wild-type expression level. The construct with the split-site located in intracellular loop 3 bound all of the tested peptide agonists and non-peptide antagonists with normal affinity and was able to stimulate inositol phosphate turnover with a normal EC50 for substance P and an Emax according to the expression level. When intracellular loop 3 was either extended with 112 amino acid residues derived from the muscarine M2 receptor or, when major parts of the loop were deleted in the non-split NK1 receptor, the affinity for neither substance P nor for the prototype nonpeptide antagonist, CP96,345 was affected, yet an increase in EC50 for substance P was observed. Also in the split-receptor, most of intracellular loop 3 could be substituted or even deleted without affecting ligand affinity, although a decreased expression level was observed in constructs having major deletions. It is concluded, that the NK1 receptor is preferentially reconstituted by co-expression of a putative A-domain including TM-I-V and a B-domain including TM-VI and -VII. It is suggested that a number of rhodopsin-like 7TM receptors may function as two-domain structures based on the finding that a network of short loops has been highly conserved within each of the putative domains and, that these domains are separated by a relatively long and in respect of length poorly conserved loop, i.e. intracellular loop 3.     

Tachykinin NK1 receptor antagonists enhance stress-induced c-fos in rat locus coeruleus

These experiments tested the hypothesis that substance P neurotransmission at tachykinin NK1 receptors in the locus coeruleus is involved in stress-induced activation of the locus coeruleus, using c-fos as an index of activation. Selective tachykinin NK1 receptor antagonists administered systemically did not result in substantial locus coeruleus c-fos expression. Restraint stress resulted in a large number of locus coeruleus c-fos expressing cells. Administration of two selective tachykinin NK1 receptor antagonists prior to restraint resulted in an increase in the number of locus coeruleus c-fos expressing cells, compared to restraint alone. These results suggest that the enhanced c-fos expression observed in response to tachykinin NK1 receptor antagonists combined with stress, could be due to the blockade of tachykinin NK1 receptor-mediated activity at sites other than the locus coeruleus, resulting in an overall activation of the locus coeruleus.     

Are inner or outer hair cells the source of summating potentials recorded from the round window?

Previous studies indicated that antidromic stimulation of capsaicin-sensitive vagal afferent fibers activated, via peripheral release of tachykinins, nonadrenergic, noncholinergic parasympathetic ganglion neurons that mediate relaxations of guinea pig trachealis. On the basis of the effects of selective agonists and inhibition with a nonselective receptor antagonist (SR 48968), we speculated that tachykinin-mediated activation of neurokinin3 (NK3) receptors might be involved. Using the recently developed NK3-selective receptor antagonist SR 142801, we further assessed the role of NK3 receptors in these relaxant responses. Relaxations of the guinea pig trachea elicited by antidromic stimulation of capsaicin-sensitive vagal afferent nerves were markedly inhibited by 0.3 microM SR 142801 and were abolished by a combination of SR 142801 and either of the NK1-selective receptor antagonists SR 140333 and CP 99994 (0.3 microM each). The NK3 receptor antagonist had similar effects on the relaxant responses elicited by capsaicin and substance P, but it had no effect on relaxations of the trachealis elicited by electrical field stimulation of the postganglionic nerves that innervate the trachealis or by stimulation of the preganglionic parasympathetic vagal nerves that innervate the trachea. These results and the observation that the ganglion neurons that mediate these responses are densely innervated by substance P-containing nerve fibers lead us conclude that stimulation of capsaicin-sensitive visceral afferent fibers activates, upon peripheral release of tachykinins, nonadrenergic, noncholinergic inhibitory neurons innervating guinea pig trachealis via activation of both NK3 and NK1 receptors.     

Modulation by substance P of synaptic transmission in the mouse hippocampal slice

The modulatory action of substance P on synaptic transmission of CA1 neurons was studied using intra- or extracellular recording from the mouse hippocampal slice preparation. Bath-applied substance P (2-4 microM) or the selective NK1 receptor agonist substance P methylester (SPME, 10 nM-5 microM) depressed field potentials (recorded from stratum pyramidale) evoked by focal stimulation of Schaffer collaterals. This effect was apparently mediated via NK1 receptors since it was completely blocked by the selective NK1 antagonist SR 140333. The field potential depression by SPME was significantly reduced in the presence of bicuculline. Intracellular recording from CA1 pyramidal neurons showed that evoked excitatory postsynaptic potentials (EPSPs) and evoked inhibitory postsynaptic potentials (IPSPs) were similarly depressed by SPME, which at the same time increased the frequency of spontaneous GABAergic events and reduced that of spontaneous glutamatergic events. The effects of SPME on spontaneous and evoked IPSPs were prevented by the ionotropic glutamate receptor blocker kynurenic acid. In tetrodotoxin (TTX) solution, no change in either the frequency of spontaneous GABAergic and glutamatergic events or in the amplitude of responses of pyramidal neurons to 4 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or 10 microM N-methyl-D-aspartate (NMDA) was observed. On the same cells, SPME produced minimal changes in passive membrane properties unable to account for the main effects on synaptic transmission. The present data indicate that SPME exerted its action on CA1 pyramidal neurons via a complex network mechanism, which is hypothesized to involve facilitation of a subset of GABAergic neurons with widely distributed connections to excitatory and inhibitory cells in the CA1 area.     

Using chromosomal lacIQ1 to control expression of genes on high-copy-number plasmids in Escherichia coli

Tachykinin NK1 receptor antagonists injected into the medulla oblongata are known to abolish vomiting induced by vagal afferent stimulation. Emetic vagal afferents have been shown to synapse with neurons in the medial solitary nucleus (mNTS), which suggests that substance P is a transmitter in the synapse. To examine this possibility, the effects of GR205171, an NK1 receptor antagonist, on retching and mNTS neuronal responses to the stimulation of abdominal vagal afferents were investigated in decerebrate dogs. GR205171 (0.05-0.7 mg kg-1, i.v.) abolished retching induced by either vagal or mNTS stimulation within 5 min. Firing of mNTS neurons in response to pulse-train and sustained vagal stimulation did not change even after the abolition of retching. Similarly, GR205171 did not have any effects on mNTS evoked potentials induced by pulse-train vagal stimulation. In about 20% of mNTS neurons, the peak firing frequency was facilitated to about 150% with repetitive pulse-train vagal stimulation. This facilitation remained even after the abolition of retching. Administration of GR205171 (1 mg ml-1, 30 microliters) into the 4th ventricle abolished retching, with latencies in excess of 120 min These results suggest that substance P does not participate in synaptic transmission between emetic vagal afferents and mNTS neurons in dogs.     

Electrophysiological characterisation of tachykinin receptors in the rat nucleus of the solitary tract and dorsal motor nucleus of the vagus in vitro

1. Recent studies have shown antagonists at the NK1 subtype of receptor for tachykinins are antiemetics and suggested that this may result from blockade of tachykinin-mediated synaptic transmission at a central site in the emetic reflex. 2. We have used intracellular recording in vitro to study the pharmacology of tachykinins in the nucleus of the solitary tract (NST) and dorsal motor nucleus of the vagus (DMNV). 3. Neurones in the NST were depolarized by substance P (SP), the presumed endogenous ligand for the NK1 receptor and these effects were mimicked by the NK1 agonists, SP-O-methylester (SPOMe), GR73632 and septide; however, SP was nearly an order of magnitude less potent than the latter two agonists. 4. In the DMNV, SP and NK1 receptor agonists evoked similar depolarising responses but SP appeared to be more potent than in the NST and was closer in potency to the other agonists. 5. NK1-receptor antagonists blocked responses to septide and GR73632 in the NST but had little effect on responses to SP and SPOMe. In contrast, in the DMNV the NK1-receptor antagonists blocked responses to septide and GR73632 but also reduced responses to SP and SPOMe. 6. Neurokinin A (NKA) was almost equipotent with septide and GR73632 in depolarizing both NST and DMNV neurones but these effects were not mimicked by a specific NK2-receptor agonist. Responses to NKA were unaffected by an NK2-receptor antagonist; however, the depolarizing effects of NKA were blocked by NK1-receptor antagonists. 7. Neurones in both DMNV and NST were unaffected by the endogenous NK3-receptor ligand, neurokinin B and by a specific agonist for this site, senktide. 8. The results with NK1 receptor agonists and antagonists suggest that the septide-sensitive NK1 site is involved in the excitation of both NST and DMNV neurones. The 'classical' NK1 receptor may play more of a role in the DMNV and a third unknown site may be responsible for the depolarizing response to SP in the NST. The effects of NKA are best interpreted as an action at the septide-sensitive NK1 site. This raises the possibility that anti-emetic action of the NK1 antagonists may be due to blockade of NKA transmission at the septide-sensitive site.     

Involvement of tachykinin NK1 and NK2 receptors in substance P-induced microvascular leakage hypersensitivity and airway hyperresponsiveness in guinea-pigs

Tachykinins, such as substance P, might be involved in the development of airway hyperresponsiveness (AHR) and airway inflammation. However, it is unknown which tachykinin receptors mediate these biological activities. The effects of two antagonists of tachykinin neurokinin-1 (NK1) and tachykinin neurokinin-2 (NK2) receptors, SR 140333 and SR 48968, respectively, were investigated on substance P (SP)-induced airway hyperresponsiveness and potentiation of the histamine-induced increase in microvascular leakage, in phosphoramidon-pretreated guinea-pigs. Guinea-pigs were pretreated with phosphoramidon (0.1 mM aerosol for 15 min) and exposed 15 min later to saline solution alone or to saline solution containing SP (0.1 mg.mL-1 for 30 min). Twenty four hours later, the animals were anaesthetized and prepared for the recording of the pulmonary inflation pressure (PIP) to acetylcholine or for the investigation of microvascular leakage to histamine. Pretreatment of the guinea-pigs with a single dose of SR 48968 (1 mg.kg-1, i.p.) 30 min before SP exposure, significantly prevented the development of AHR, whereas SR 140333 (1 mg.kg-1, i.p.) did not. In a second set of experiments, phosphoramidon-pretreated guinea-pigs exposed to SP presented a significant potentiation of the histamine-induced increase in microvascular leakage in pulmonary airways. When the guinea-pigs were pretreated with SR 140333, an inhibition of the increased microvascular leakage to histamine was observed. In contrast, no significant inhibitory activity was noted when the guinea-pigs were pretreated with SR 48968. The present data demonstrate the importance of tachykinin NK2 receptor stimulation in the development of airway hyperresponsiveness and that of tachykinin NK1 receptor stimulation in microvascular leakage hypersensitivity in phosphoramidon-pretreated and substance P-exposed guinea-pigs. The results also suggest a dissociation between the presence of microvascular leakage and the occurrence of airway hyperresponsiveness.     

The distribution of tachykinin binding sites in the brain of an electric fish (Apteronotus leptorhynchus)

We mapped the distribution of tachykinin binding sites utilizing quantitative autoradiography of iodinated substance P and eledoisin as prototypic ligands for neurokinin-1 (NK1) and neurokinin-3 (NK3) receptors, respectively. The two ligands produced highly heterogenous and quantitatively different patterns of specific binding, suggesting that they revealed different tachykinin receptor subtypes. Although [125I]substance P and [125I]eledoisin binding were correlated in most brain regions, the binding of substance P was usually denser. [125I]substance P binding and substance P-like immunoreactivity were reasonably correlated in most brain areas, although discrepancies were found in some nuclei. Dense [125I]substance P binding was found in most areas of the subpallium and in parts of the pallium related to the olfactory system, as well as in the glomerular layer of the olfactory bulb. Moderate to dense binding of both ligands was observed in preoptic area, hypothalamus, habenula, parts of the thalamus and preglomerular complex. Especially noteworthy was the presence of [125I] substance P binding in the diencephalic prepacemaker nucleus, a region involved in the control of electroncommuncatory behavior. Substance P-like immunoreactivity is sexually dimorphic in certain diencephalic nuclei, including the prepacemaker nucleus (Weld and Maler, 1992); no obvious difference was seen between [125I]substance P or [125I]eledoisin binding in the brains of male versus female fish. In the mesencephalon striking laminar patterns of binding were seen in the torus semicircularis dorsalis and the optic tectum. Dense binding was also noted in the raphé nuclei, the locus ceruleus and the sensory nucleus of the vagus. Although binding of substance P in the electrosensory lateral line lobe and nucleus preeminentialis was light, it was distributed in a discrete fashion, suggesting a role of substance P in electrosensory processing.     

Desensitization of the neurokinin-1 receptor (NK1-R) in neurons: effects of substance P on the distribution of NK1-R, Galphaq/11, G-protein receptor kinase-2/3, and beta-arrestin-1/2

Observations in reconstituted systems and transfected cells indicate that G-protein receptor kinases (GRKs) and beta-arrestins mediate desensitization and endocytosis of G-protein-coupled receptors. Little is known about receptor regulation in neurons. Therefore, we examined the effects of the neurotransmitter substance P (SP) on desensitization of the neurokinin-1 receptor (NK1-R) and on the subcellular distribution of NK1-R, Galphaq/11, GRK-2 and -3, and beta-arrestin-1 and -2 in cultured myenteric neurons. NK1-R was coexpressed with immunoreactive Galphaq/11, GRK-2 and -3, and beta-arrestin-1 and -2 in a subpopulation of neurons. SP caused 1) rapid NK1-R-mediated increase in [Ca2+]i, which was transient and desensitized to repeated stimulation; 2) internalization of the NK1-R into early endosomes containing SP; and 3) rapid and transient redistribution of beta-arrestin-1 and -2 from the cytosol to the plasma membrane, followed by a striking redistribution of beta-arrestin-1 and -2 to endosomes containing the NK1-R and SP. In SP-treated neurons Galphaq/11 remained at the plasma membrane, and GRK-2 and -3 remained in centrally located and superficial vesicles. Thus, SP induces desensitization and endocytosis of the NK1-R in neurons that may be mediated by GRK-2 and -3 and beta-arrestin-1 and -2. This regulation will determine whether NK1-R-expressing neurons participate in functionally important reflexes.     

Activation of the cloned human NK3 receptor in Chinese Hamster Ovary cells characterized by the cellular acidification response using the Cytosensor microphysiometer

1. The aim of the present study was to validate the Cytosensor microphysiometer, a novel system that measures the extracellular acidification rate as a reliable index of the integrated functional response to receptor activation, as a method for studying NK3 receptor pharmacology, and then to use this system to assess the functional activity of novel compounds at this receptor. 2. The selective NK3 agonist senktide caused reproducible, concentration-related increases in acidification ratein CHO-NK3 cells, with a pEC50 value of 8.72+/-0.11 (n=15). [Beta-Ala8]NKA(4-10), the selective NK2 agonist, elicited a much weaker response (pEC50=6.68+/-0.08, n=4), while the NK1-selective agonist substance P methylester only caused a very weak response at concentrations > or =3 microM (n=2). The rank order of potency for the endogenous tachykinins NKB>NKA>substance P (n=3) confirmed the response was mediated by the NK3 receptor. Moreover, the actual potencies obtained were consistent with affinities measured in radioligand binding studies. 3. The novel compounds PD156319-121 (0.3-1 microM), PD161182 (10-300 nM), PD168001 (10-100 nM) and PD168073 (10-100 nM) all acted as surmountable antagonists of the senktide-induced acidification response, with pA2 values of 7.49, 8.67, 9.17 and 9.25 respectively (n=3-5). In comparison the known NK3 antagonist SR142801 (10-100 nM) had a pA2 value of 8.83 (n=8) for the interaction with senktide. Again, these values are consistent with the radioligand binding data. 4. Amiloride (1 mM) inhibited the senktide-induced acidification response by 68.3+/-3.3 (n=4), indicating that the Na+/H+ antiporter plays an important role in this response, and this is consistent with the importance of this antiporter in other acidification responses. 5. Inhibition of protein kinase C with staurosporine (0.1 microM), or depletion of the intracellular Ca2+ stores with thapsigargin (1 microM), both resulted in a reduction in the maximum response to senktide (63.3+/-1.7 and 68.9+/-3.2% respectively, n=3-5), and co-application of these inhibitors abolished the response (n=3). This strongly suggested that the NK3 receptor was coupling via phospholipase C (PLC), as would be expected, although this could not be confirmed by the use of the putative PLC/PLA2 inhibitor U73122. 6. In conclusion, we have demonstrated the utility of the Cytosensor in the characterization of functional responses to agonists, and assessment of the affinities of antagonists in CHO cells expressing the human NK3, and have shown that our series of novel compounds are non-peptide NK3 antagonists of high affinity, as exemplified by PD168073.     

Impaired IL-1beta-induced neutrophil accumulation in tachykinin NK1 receptor knockout mice

Tachykinin NK1 receptors play an important role in the development of neurogenic inflammatory responses. We have used the murine air-pouch model to investigate whether the neurogenic component of the cellular inflammatory response to interleukin-1beta (IL-1beta, 10 ng into the air-pouch) is altered in NK1 receptor knockout mice compared to wild type controls. Air-pouches were washed following a 4 h IL-1beta treatment, the wash collected and neutrophil number estimated using a Neubauer haemocytometer. The response to IL-1beta was significantly attenuated in NK1 receptor +/- (40% reduction) and -/- mice (62% reduction) compared to wild type controls (+/+), whilst the response to cytokine-induced neutrophil chemoattractant (CINC, 0.3 microg) was unaffected. The response to substance P (7.5 nmol) was attenuated by approximately 50% in both NK1 receptor +/- and -/- mice compared to wild type controls. In conclusion NK1 receptors play a significant role in the cellular response to IL-1beta in a model of inflammation.     

The cytoplasmic tails of protease-activated receptor-1 and substance P receptor specify sorting to lysosomes versus recycling

The G protein-coupled receptor (GPCR) for thrombin, protease-activated receptor-1 (PAR1), is activated when thrombin cleaves its amino-terminal exodomain. The irreversibility of this proteolytic mechanism raises the question of how desensitization and resensitization are accomplished for thrombin signaling. PAR1 is phosphorylated, uncoupled from signaling, and internalized after activation like classic GPCRs. However, unlike classic GPCRs, which internalize and recycle, activated PAR1 is sorted to lysosomes. To identify the signals that specify the distinct sorting of PAR1, we constructed chimeras between PAR1 and the substance P receptor. Wild-type substance P receptor internalized and recycled after activation; PAR1 bearing the cytoplasmic tail of the substance P receptor (P/S) behaved similarly. By contrast, wild-type PAR1 and a substance P receptor bearing the cytoplasmic tail of PAR1 (S/P) sorted to lysosomes after activation. Consistent with these observations, PAR1 and the S/P chimera were effectively down-regulated by their respective agonists as assessed by both receptor protein levels and signaling. Substance P receptor and the P/S chimera showed little down-regulation. These data suggest that the cytoplasmic tails of PAR1 and substance P receptor specify their distinct intracellular sorting patterns after activation and internalization. Moreover, by altering the trafficking fates of PAR1 and substance P receptor, one can dictate the efficiency with which a cell maintains responsiveness to PAR1 or substance P receptor agonists over time.