Activation Of NK1 Receptors Research Articles

Molecular Aspects Involved in the Mechanisms of Bothrops jararaca Venom-Induced Hyperalgesia: Participation of NK1 Receptor and Glial Cells.

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

The Neurokinin-1 Receptor: Structure Dynamics and Signaling

Substance P (SP), the first isolated neuropeptide, belongs to the family of tachykinin peptides and is the natural ligand of neurokinin-1 receptors (NK-1R), also named SP receptors. The undecapeptide activates the receptor after specifically binding to the protein and triggers intracellular signals leading to different biochemical events and subsequent physiological responses. This study reviews the main architectural features of this receptor, its interaction with natural and synthetic ligands, and the functional conformational states adopted after interacting with ligands and effector G proteins. The analysis of the main intracellular signaling pathways turned on by the activation of NK-1 receptors reveals the participation of different proteins supporting metabolic changes and genetic and epigenetic regulations. Furthermore, the analysis of receptor occupancy and receptor downregulation and internalization represents a complex and estimable field for basic and clinical research focused on the role of SP in physiopathology. Profound knowledge of the structural dynamics of NK-1R may help develop and assay new selective synthetic non-peptide antagonists as potential therapeutic agents applied to various pathologies and symptoms.

Ionic signalling mechanisms involved in neurokinin-3 receptor-mediated augmentation of fear-potentiated startle response in the basolateral amygdala.

The tachykinin peptides include substance P (SP), neurokinin A and neurokinin B, which interact with three G-protein-coupled neurokinin receptors, NK1Rs, NK2Rs and NK3Rs, respectively. Whereas high densities of NK3Rs have been detected in the basolateral amygdala (BLA), the functions of NK3Rs in this brain region have not been determined. We found that activation of NK3Rs by application of the selective agonist, senktide, persistently excited BLA principal neurons. NK3R-elicited excitation of BLA neurons was mediated by activation of a non-selective cation channel and depression of the inwardly rectifying K+ (Kir) channels. With selective channel blockers and knockout mice, we further showed that NK3R activation excited BLA neurons by depressing the G protein-activated inwardly rectifying K+ (GIRK) channels and activating TRPC4 and TRPC5 channels. The effects of NK3Rs required the functions of phospholipase Cβ (PLCβ), but were independent of intracellular Ca2+ release and protein kinase C. PLCβ-mediated depletion of phosphatidylinositol 4,5-bisphosphate was involved in NK3R-induced excitation of BLA neurons. Microinjection of senktide into the BLA of rats augmented fear-potentiated startle (FPS) and this effect was blocked by prior injection of the selective NK3R antagonist SB218795, suggesting that activation of NK3Rs in the BLA increased FPS. We further showed that TRPC4/5 and GIRK channels were involved in NK3R-elicited facilitation of FPS. Our results provide a cellular and molecular mechanism whereby NK3R activation excites BLA neurons and enhances FPS. KEY POINTS: Activation of NK3 receptors (NK3Rs) facilitates the excitability of principal neurons in rat basolateral amygdala (BLA). NK3R-induced excitation is mediated by inhibition of GIRK channels and activation of TRPC4/5 channels. Phospholipase Cβ and depletion of phosphatidylinositol 4,5-bisphosphate are necessary for NK3R-mediated excitation of BLA principal neurons. Activation of NK3Rs in the BLA facilitates fear-potentiated startle response. GIRK channels and TRPC4/5 channels are involved in NK3R-mediated augmentation of fear-potentiated startle.

Angiotensin (1-7) Attenuates the Nociceptive Behavior Induced by Substance P and NMDA via Spinal MAS1.

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

Presynaptic NK1 receptor activation by substance P suppresses EPSCs via nitric oxide synthesis in the rat insular cortex

Presynaptic NK1 receptor activation by substance P suppresses EPSCs via nitric oxide synthesis in the rat insular cortex

Presynaptic NK1 Receptor Activation by Substance P Suppresses EPSCs via Nitric Oxide Synthesis in the Rat Insular Cortex

Substance P (SP) regulates inhibitory synaptic transmission mediated by GABAA receptors in the cerebral cortex; however, SP-mediated regulation of excitatory synaptic transmission remains poorly understood. We performed whole-cell patch-clamp recordings from pyramidal neurons to examine the effects of SP on excitatory postsynaptic currents (EPSCs) mediated via AMPA receptors in the insular cortex (IC), which is involved in nociceptive information processing. First, EPSCs evoked by minimal electrical stimulation (eEPSCs) including stepwise EPSCs and failure events, were examined. SP dose-dependently suppressed mean eEPSC amplitude, partially due to an increase in the failure rate of eEPSCs. The SP-induced suppression of eEPSCs was accompanied by an increase in the paired-pulse ratio and was inhibited by the preapplication of SR140333, an NK1 receptor antagonist. [Sar9,Met(O2)11]-substance P, an NK1 receptor-selective agonist, mimicked the effects of SP on eEPSCs and decreased the frequency of miniature EPSCs (mEPSCs) without changing the average mEPSC amplitude. Considering that most NK1 receptors in the cerebral cortex are expressed in nitric oxide synthase (NOS)-positive GABAergic neurons, the SP-induced suppressive effect on EPSCs may be mediated by nitric oxide (NO) in this subtype of GABAergic neurons. NO imaging using the fluorescent probe DAX-J2 Red supports this hypothesis: SP increased the fluorescence intensity of DAX-J2 Red in some GABAergic neurons. Furthermore, both L-NAME, an NOS inhibitor, and PTIO, an NO scavenger, diminished the SP-induced suppression of eEPSCs. These results suggest that the activation of presynaptic NK1 receptors contributes to SP-induced eEPSC suppression by activating the NO synthesis pathway in GABAergic neurons. (246 words)

Effects of Electrical Stimulation of NAc Afferents on VP Neurons' Tonic Firing.

Afferents from the nucleus accumbens (NAc) are a major source of input into the ventral pallidum (VP). Research reveals that these afferents are GABAergic, however, stimulation of these afferents induces both excitatory and inhibitory responses within the VP. These are likely to be partially mediated by enkephalin and substance P (SP), which are also released by these afferents, and are known to modulate VP neurons. However, less is known about the potentially differential effects stimulation of these afferents has on subpopulations of neurons within the VP and the cellular mechanisms by which they exert their effects. The current study aimed to research this further using brain slices containing the VP, stimulation of the NAc afferents, and multi-electrode array (MEA) recordings of their VP targets. Stimulation of the NAc afferents induced a pause in the tonic firing in 58% of the neurons studied in the VP, while 42% were not affected. Measures used to reveal the electrophysiological difference between these groups found no significant differences in firing frequency, coefficient of variation, and spike half-width. There were however significant differences in the pause duration between neurons in the dorsal and ventral VP, with stimulation of NAc afferents producing a significantly longer pause (0.48 ± 0.06 s) in tonic firing in dorsal VP neurons, compared to neurons in the ventral VP (0.21 ± 0.09 s). Pauses in the tonic firing of VP neurons, as a result of NAc afferent stimulation, were found to be largely mediated by GABAA receptors, as the application of picrotoxin significantly reduced their duration. Opioid agonists and antagonists were found to have no significant effects on the pause in tonic activity induced by NAc afferent stimulation. However, NK-1 receptor antagonists caused significant decreases in the pause duration, suggesting that SP may contribute to the inhibitory effect of NAc afferent stimulation via activation of NK-1 receptors.

Gender-Related Differences of Tachykinin NK2 Receptor Expression and Activity in Human Colonic Smooth Muscle.

The tachykinin NK2 receptor plays a key role in gastrointestinal motor function. Enteric neurons release neurokinin A (NKA), which activates NK2 receptors on gastrointestinal smooth muscle, leading to contraction and increased motility. In patients with diarrhea-predominant irritable bowel syndrome, the NK2 receptor antagonist ibodutant had a greater therapeutic effect in females than males. The present study aimed to determine whether gender influences the expression and activity of NK2 receptors in human colonic smooth muscle. In vitro functional studies were performed to examine the contractile responses of colonic muscle strips to NKA and the selective NK2 receptor agonist [Lys5,MeLeu9,Nle10]NKA(4-10). Contractions were also measured in the presence of ibodutant to determine its antagonistic potency. The signal transduction pathways coupled to NK2 receptor activation were investigated using second messenger inhibitors. Western blot and fluorescent immunohistochemistry were conducted to determine the protein expression and localization of NK2 receptors. NK2 receptor-mediated contractility was greater in females compared with males. When against NKA, ibodutant was more potent in females. NK2 receptor expression increased with age in females, but not in males. Phospholipase C-mediated signaling was less prominent in females compared with males, whereas Ca2+ sensitization via Rho kinase and protein kinase C appeared to be the dominant pathway in both genders. The distribution of NK2 receptors in the human colon did not differ between the genders. Overall, gender differences exist in the expression and activity of NK2 receptors in colonic smooth muscle. These gender distinctions should be considered in the therapeutic development of NK2 receptor agents. SIGNIFICANCE STATEMENT: The tachykinin NK2 receptor has been identified as a therapeutic target for the treatment of bowel and bladder dysfunctions. The present study has revealed gender-related variations in NK2 receptor activity, signaling transduction pathways, antagonist potency, and changes in expression with age. These factors may underlie the gender differences in the treatment of diarrhea-predominant irritable bowel syndrome with NK2 receptor antagonists. Our findings highlight that gender differences should be considered in the therapeutic development of NK2 receptor agents.

Endokinin A/B stimulates rat gastric motility through myogenic NK1 receptors located in the fundus.

Endokinin A/B (EKA/B), the common C-terminal decapeptide in endokinins A and B, is a preferred ligand of the NK1 receptor and regulates pain and itch. The study focused on the effects of EKA/B on rat gastric motility in vivo and in vitro. Gastric emptying was measured to evaluate gastric motility in vivo. Intragastric pressure and the contraction of gastric muscle strips were measured to evaluate gastric motility in vitro. Moreover, various neural blocking agents and neurokinin receptor antagonists were applied to explore the mechanisms. TAC4 and TACR1 mRNAs were expressed throughout rat stomach. EKA/B promoted gastric emptying by intraperitoneal injection in vivo. Correspondingly, EKA/B also increased intragastric pressure in vitro. Additionally, EKA/B contracted the gastric muscle strips from the fundus but not from the corpus or antrum. Further studies revealed that the contraction induced by EKA/B on muscle strips from the fundus could be significantly reduced by NK1 receptor antagonist SR140333 but not by NK2 receptor antagonist, NK3 receptor antagonist, or the neural blocking agents used. Our results suggested that EKA/B might stimulate gastric motility mainly through the direct activation of myogenic NK1 receptors located in the fundus.

Role of Neuro‐immune Interactions in a Cigarette Smoke‐induced Chronic Airway Inflammation Mouse Model

Chronic obstructive pulmonary disease characterized by progressive persistent airway inflammation & airway resistance is caused mainly by cigarette smoke‐triggered inflammatory cascades & tissue damage. The airways are densely innervated by capsaicin‐sensitive sensory nerves expressing Transient Receptor Potential Vanilloid 1 (TRPV1) ion channels, which play an important regulatory role in inflammation through the release of sensory neuropeptides. Pro‐inflammatory peptides, such as tachykinins (substance P, neurokinin A) encoded by the TAC1 gene induce neurogenic inflammation via NK1 & NK2 receptor activation in the innervated area. The overall significance of these mediators depends on the pathophysiological mechanisms involved in certain inflammatory processes. Therefore, we established a predictive mouse model of cigarette smoke‐induced chronic bronchitis & consequent airway hyperresponsiveness to investigate the roles of TRPV1 channel & tachikinins.We exposed TRPV1−/− & TAC1−/− gene‐deleted mice and their C57Bl/6 wildtype counterparts to cigarette smoke in a whole body smoke exposure chamber for 3 months. Bronchial responsiveness & enhanced pause (Penh) correlating with airway resistance to inhalation of 22 mM carbachol were measured with unrestrained whole body plethysmography weekly. At the end of each month inflammatory cell profile and count from the bronchoalveolar lavage fluid (BALF), semiquantitative histopathological scoring of the lung, myeloperoxidase (MPO) activity with spectrophotometry & IL‐1β concentrations with ELISA were performed.In wildtype mice mild bronchial hyperresponsiveness developed in the 1st month, then it decreased & started to increase after 10 weeks. The inflammatory cell peak in BALF was observed in the 2nd month, correlating with the histology. In the 1st month significant perivascular/peribronchial edema, in the 2nd month remarkable inflammatory cell accumulation, then increasing atelectasy, emphysema, mucus production & irregular bronchiolar mucosa were observed. Lung IL‐1β concentration gradually increased, but MPO activity only transiently elevated in the 2nd month. Hyperreactivity occured earlier & increased significantly in TRPV1−/−, but did not change in TAC1−/− mice. Remarkably more severe inflammatory histopathological parameters, higher BALF cell counts, MPO activity a& IL‐1β production were observed in TRPV1‐deficient mice. Meanwhile, some inflammatory alterations in the 1st month, & BALF cell counts in the 2nd month were significantly decreased in the TAC1 gene‐deleted mice.Neurogenic inflammatory mechanisms are not crucial in chronic cigarette smoke‐induced inflammation & hyperresponsiveness. The overall role of TRPV1 receptor is protective in smoking‐induced chronic bronchitis.Support or Funding InformationÚNKP‐18‐3 New National Excellence Program of the Ministry of Human CapacitiesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hemokinin-1 is an important mediator of pain in mouse models of neuropathic and inflammatory mechanisms

The Tac4 gene-derived hemokinin-1 (HK-1) is present in pain-related regions and activates the tachykinin NK1 receptor, but with binding site and signaling pathways different from Substance P (SP). NK1 receptor is involved in nociception, but our earlier data showed that it has no role in chronic neuropathic hyperalgesia, similarly to SP. Furthermore, NK1 antagonists failed in clinical trials as analgesics due to still unknown reasons. Therefore, we investigated the role of HK-1 in pain conditions of distinct mechanisms using genetically modified mice.Chronic neuropathic mechanical and cold hyperalgesia after partial sciatic nerve ligation (PSL) were determined by dynamic plantar aesthesiometry and withdrawal latency from icy water, motor coordination on the accelerating Rotarod. Peripheral nerve growth factor (NGF) production was measured by ELISA, neuronal and glia cell activation by immunohistochemistry in pain-related regions. Acute somatic and visceral chemonocifensive behaviors were assessed after intraplantar formalin or intraperitoneal acetic-acid injection, respectively. Resiniferatoxin-induced inflammatory mechanical and thermal hyperalgesia by aesthesiometry and increasing temperature hot plate.Chronic neuropathic mechanical and cold hypersensitivity were significantly decreased in HK-1 deficient mice. NGF level in the paw homogenates of intact mice were significantly lower in case of HK-1 deletion. However, it significantly increased under neuropathic condition in contrast to wildtype mice, where the higher basal concentration did not show any changes. Microglia, but not astrocyte activation was observed 14 days after PSL in the ipsilateral spinal dorsal horn of wildtype, but not HK-1-deficient mice. However, under neuropathic conditions, the number of GFAP-positive astrocytes was significantly smaller in case of HK-1 deletion. Acute visceral, but not somatic nocifensive behavior, as well as neurogenic inflammatory mechanical and thermal hypersensitivity were significantly reduced by HK-1 deficiency similarly to NK1, but not to SP deletion.We provide evidence for pro-nociceptive role of HK-1, via NK1 receptor activation in acute inflammation models, but differently from SP-mediated actions. Identification of its targets and signaling can open new directions in pain research.

Inhibition of substance P-induced defensive behavior via neurokinin-1 receptor antagonism in the central and medial but not basolateral nuclei of the amygdala in male Wistar rats

RationaleThe production of unconditioned defensive behaviors has been related to the amygdala, a key component of the encephalic aversion system. Microinjection of the neuropeptide substance P (SP) in the amygdala elicits defensive behaviors via the activation of type 1 neurokinin (NK-1) receptors. However, no studies have investigated whether intra-amygdala SP/NK-1 mechanisms can elicit other types of defensive responses, such as antinociception and ultrasonic vocalizations (USVs). MethodsThe present study investigated the effects of SP-induced activation of the neurokininergic system in three main nuclei of the amygdala—basolateral (BLA), central (CeA), and medial (MeA) nuclei—in rats that were subjected to the elevated plus maze (EPM), tail-flick test, and USV recording. The effects of SP in these amygdaloid nuclei were challenged with combined injections of the NK-1 receptor antagonist spantide. ResultsThe present study showed that SP injections in the CeA and MeA but not BLA exerted anxiogenic-like effects. In contrast to the CeA, the anxiogenic-like effects of SP in the MeA were not dependent on NK-1 mechanisms. In the tail-flick test, SP microinjections produced antinociceptive effects only in the MeA through NK-1 receptor activation. No USV emissions were detected after the SP microinjections. ConclusionsThe present study showed that NK-1 receptors in the CeA and MeA but not BLA are involved in defensive reactions to conditions of fear. The present results may provide a better understanding of the neurochemical mediation of fear states.

モルヒネの低用量化を可能にする鎮痛補助薬の探索研究

Morphine with its potent analgesic property has been widely used for the treatment of various types of pain. However, the intrathecal (i.t.) administration of morphine at doses far higher than those required for antinociception exhibited nociceptive-related behaviors consisting of scratching, biting and licking, hyperalgesia, and allodynia in mice. Morphine-3-glucuronide (M3G), one of the major metabolites of morphine, has been found to evoke nociceptive behaviors similar to those after high-dose i.t. morphine. It is plausible that M3G may be responsible for nociception seen after high-dose i.t. morphine treatment. This article reviews the potential mechanism of spinally mediated nociceptive behaviors evoked by i.t. M3G in mice. We discuss the possible presynaptic release of nociceptive neurotransmitters/neuromodulators such as substance P, glutamate, dynorphin, and Leu-enkephalin in the primary afferent fibers following i.t. M3G administration. It is possible to speculate that i.t. M3G could indirectly activate NK1, NMDA, and δ2-opioid receptors that lead to the release of nitric oxide (NO) in the dorsal spinal cord. The major function of NO is the production of cGMP and the activation of protein kinase G (PKG). The NO-cGMP-PKG pathway plays an important role in M3G-induced nociceptive behavior. The phosphorylation of extracellular signal-related kinase (ERK) in the dorsal spinal cord was also evoked via the NO-cGMP-PKG pathway through the activation of δ2-opioid, NK1, and NMDA receptors, contributing to M3G-induced nociceptive behaviors. The demonstration of a neural mechanism underlying M3G-induced nociception provides a pharmacological basis for improved pain management with morphine at high doses.

Induction and antagonism of pica induced by teriparatide in rats

Intermittent subcutaneous injection of teriparatide, an active fragment of human parathyroid hormone, is clinically used for the treatment of osteoporosis. Patients suffer from nausea, which is one of the side effects teriparatide induces; however, the etiology of teriparatide-induced nausea remains unknown. We have reported pica, kaolin ingestion behavior, can be used as an assessment of nausea-related response in rats. In this study, we investigated the characteristics of teriparatide-induced pica and the abilities of anti-emetic drugs to inhibit teriparatide-induced pica. Male and female adolescent (4-week-old), young (8-week-old), and adult (30-week-old) naive rats, and ovariectomized (OVX: 17-week-old) and sham-operated (17-week-old) rats subcutaneously received teriparatide (0.4mg/kg, n=4), and their kaolin and food intakes were monitored for 24h after the injection. Among the tested rats, we found that OVX rats, rather than male, female, and sham-operated rats, showed marked teriparatide-induced pica (0mg/kg: 0.17±0.07g, 0.4mg/kg: 6.18±0.91g). Teriparatide-induced pica in OVX rats was inhibited by intraperitoneal pretreatment with serotonin 5-HT3 (granisetron 0.5mg/kg), dopamine D2 (prochlorperazine 0.5mg/kg), neurokinin NK1 (fosaprepitant 1mg/kg), and histamine H1 (diphenhydramine 10mg/kg) receptor antagonists to 70%, 11%, 19%, and 59% of that in vehicle-treated control, respectively. These results suggest that teriparatide-induced pica in OVX rats has the potential to reflect teriparatide-induced nausea; 5-HT3, D2, NK1, and H1 receptor activation is involved in the development of this behavior; antagonists of these receptors have the potential to be medical candidates used as treatments for teriparatide-induced nausea in human patients.

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening.

Involvement of substance P in the development of cisplatin‐induced acute and delayed pica in rats

Although substance P (SP) and neurokinin NK1 receptors have been reported to be involved in cisplatin-induced acute and delayed emesis, their precise roles remain unclear. Pica, the consumption of non-nutrient materials such as kaolin in rats, can be used as a model of nausea in humans. We investigated the time-dependent changes in cisplatin-induced pica and the involvement of SP and NK1 receptors in this behaviour. Rats were administered cisplatin with or without a daily injection of a 5-HT3 receptor antagonist (granisetron) or an NK1 receptor antagonist (aprepitant), and kaolin intake was then monitored for 5 days. The effects of granisetron on the cisplatin-induced expression of preprotachykinin-A (PPT-A) mRNA, which encodes mainly for SP, and on SP release in the medulla, measured by in vivo brain microdialysis, were also investigated. Cisplatin induced pica within 8 h of its administration that continued for 5 days. Granisetron inhibited the acute phase (day 1), but not the delayed phase (days 2-5), of pica, whereas aprepitant abolished both phases. Within 24 h of the injection of cisplatin, PPT-A mRNA expression and SP release in the medulla were significantly increased; these findings lasted during the observation period and were inhibited by granisetron for up to 24 h. The profiles of cisplatin-induced pica in rats are similar to clinical findings for cisplatin-induced emesis in humans, and we showed that SP production in the medulla and activation of NK1 receptors are involved in this cisplatin-induced pica.

Effects of substance P and Sar-Met-SP, a NK1 agonist, in distinct amygdaloid nuclei on anxiety-like behavior in rats

The amygdala, together with the dorsal periaqueductal gray (dPAG), medial hypothalamus, and deep layers of the superior and inferior colliculi, constitutes the encephalic aversion system, which has been considered the main neural substrate for the organization of fear and anxiety. The basolateral nucleus of the amygdala (BLA) acts as a filter for aversive stimuli to higher structures while the central (CeA) and the medial (MeA) nuclei constitute the output for the autonomic and somatic components of the emotional reaction through major projections to the limbic and brainstem regions. Although some findings point to the distinct participation of the substance P (SP) and the NK1 receptors system in the different nuclei of the amygdala on the expression of emotional behaviors, it is not clear if this system modulates anxiety-like responses in the distinct nuclei of the amygdala as well as the dPAG. Thus, it was investigated if the injection of SP into the BLA, CeA, or MeA affects the expression of anxiety-like responses of rats submitted to the elevated plus-maze (EPM) test and, if the effects are mediated by NK1 receptors. The results showed that SP and Sar-Met-SP (NK1 receptor selective agonist) injected into the CeA and MeA, but not into the BLA, caused anxiogenic-like effects in the EPM. Altogether, the data indicates that the SP may mimic the effects of anxiogenic stimuli via NK1 receptor activation only in the CeA and MeA (amygdala's nuclei output) and may activate the neural mechanisms involved in the defensive reaction genesis. The SP/NK1 receptors system activation may be phasically involved in very specific aspects of anxiety behaviors.

Activation of the TRPV1 channel attenuates N-methyl-d-aspartic acid-induced neuronal injury in the rat retina

Capsaicin, a transient receptor potential vanilloid type1 (TRPV1) agonist, has been reported to protect against ischemia-reperfusion injury in various organs, including the brain, heart, and kidney, whereas activation of TRPV1 was also reported to contribute to neurodegeneration, including pressure-induced retinal ganglion cell death in vitro. We histologically investigated the effects of capsaicin and SA13353, TRPV1 agonists, on retinal injury induced by intravitreal N-methyl-d-aspartic acid (NMDA; 200nmol/eye) in rats in vivo. Under ketamine/xylazine anesthesia, male Sprague–Dawley rats were subjected to intravitreal NMDA injection. Capsaicin (5.0nmol/eye) was intravitreally admianeously with NMDA injection. SA13353 (10mg/kg) was intraperitoneally administered 15min before NMDA injection. Morphometric evaluation at 7 days after NMDA injection showed that intravitreal NMDA injection resulted in ganglion cell loss. Capsaicin and SA13353 almost completely prevented this damage. Treatment with capsazepine (TRPV1 antagonist, 0.5nmol/eye), CGRP (8–37) (calcitonin gene-related peptide (CGRP) receptor antagonist, 0.5pmol/eye), or RP67580 (tachykinin NK1 receptor antagonist, 0.5nmol/eye) almost completely negated the protective effect of capsaicin in the NMDA-injected rats. Seven days after intravitreal NMDA injection, the cell number of retinal ganglion cell was significantly smaller than in the eye that had received capsaicin in B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice that express the enhanced cyan fluorescent protein in retinal ganglion cells in the retina. These results suggested that activation of TRPV1 protects retinal neurons from the injury induced by intravitreal NMDA in rats in vivo. Activation of CGRP and tachykinin NK1 receptors is possibly involved in underlying protective mechanisms.

Cholinergic control of the murine trachealis muscle via non-vesicular acetylcholine release involving NK1 receptor activation

In addition to quantal, vesicular release of acetylcholine (ACh), there is also non-quantal release at the motor endplate which is insufficient to evoke postsynaptic responses unless acetylcholinesterase (AChE) is inhibited. We here addressed potential non-quantal release in the mouse trachea by organ bath experiments and (immuno)histochemical methods. Electrical field stimulation (EFS) of nerve terminals elicited tracheal constriction that is largely due to ACh release (atropine-sensitive) and is not significantly affected by AChE or butyrylcholinesterase (BChE) gene-deficiency.

Nerve growth factor enhances cough via a central mechanism of action

The mechanisms involved in enhanced cough induced by central and inhaled NGF in guinea pigs were investigated. Cough and airway function were assessed by plethysmography following inhaled or intracerebroventricular (i.c.v.) NGF treatment. Expression of TrkA and/or TRPV1 was determined in bronchi and/or brainstem by real-time PCR and immunoblotting. I.c.v. and inhaled NGF enhanced citric acid induced-cough and airway obstruction. Pretreatment (i.c.v.) with antagonists of TrkA (K252a) or TRPV1 (IRTX) significantly reduced both the NGF (i.c.v.) enhanced cough and airway obstruction whereas the NK1 antagonist (FK888) inhibited only cough. The H1 antagonist (cetirizine) did not affect either. Inhaled NGF increased phosphorylation of TrkA receptors in the bronchi but not the brainstem at 0.5h post-treatment. TrkA mRNA was elevated at 0.5h in the bronchi and at 24h in the brainstem while TRPV1 mRNA was elevated from 0.5h to 24h in brainstem and at 24h in the bronchi. Pretreatment (i.c.v.) with IRTX, but not K252a, significantly inhibited the inhaled NGF-enhanced cough. Central NGF administration enhances cough and airway obstruction by mechanisms dependent on central activation of TrkA, TRPV1 and NK1 receptors while inhaled NGF enhances cough via a mechanism dependent on central TRPV1 and not TrkA receptors. These data show that NGF, in addition to its effects on the airways, has an important central mechanism of action in the enhancement of cough. Therefore, therapeutic strategies targeting NGF signaling in both the airways and CNS may be more effective in the management of cough.