NK3R signalling in the posterodorsal medial amygdala is involved in stress-induced suppression of pulsatile LH secretion in female mice.

Psychosocial stress negatively impacts reproductive function by inhibiting pulsatile luteinizing hormone (LH) secretion. The posterodorsal medial amygdala (MePD) is responsible in part for processing stress and modulating the reproductive axis. Activation of the neurokinin 3 receptor (NK3R) suppresses the gonadotropin-releasing hormone (GnRH) pulse generator, under hypoestrogenic conditions, and NK3R activity in the amygdala has been documented to play a role in stress and anxiety. We investigate whether NK3R activation in the MePD is involved in mediating the inhibitory effect of psychosocial stress on LH pulsatility in ovariectomised female mice. First, we administered senktide, an NK3R agonist, into the MePD and monitored the effect on pulsatile LH secretion. We then delivered SB222200, a selective NK3R antagonist, intra-MePD in the presence of predator odour, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Senktide administration into the MePD dose-dependently suppresses pulsatile LH secretion. Moreover, NK3R signalling in the MePD mediates TMT-induced suppression of the GnRH pulse generator, which we verified using a mathematical model. The model verifies our experimental findings: (i) predator odour exposure inhibits LH pulses, (ii) activation of NK3R in the MePD inhibits LH pulses and (iii) NK3R antagonism in the MePD blocks stressor-induced inhibition of LH pulse frequency in the absence of ovarian steroids. These results demonstrate for the first time that NK3R neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator.

Neurokinin B Administration Induces Dose Dependent Proliferation of Seminal Vesicles in Adult Rats.

BACKGROUND: Neurokinin B; an endogenous decapeptide, mediates its reproductive physiological actions through gonadotropin releasing hormone. Despite the potential role of Neurokinin B on seminal vesicles, its effects on seminal vesicles in adult male mammals remain elusive. We aimed to investigate the potentials of variable doses of Neurokinin B, its agonist and antagonist on histomorphology and expression of NK3R on seminal vesicles, and secretory activity of seminal vesicles in adult male rats. METHODS: Adult male Sprague Dawley rats (n=10 in each group) were administered intraperitoneally with Neurokinin B in three variable doses: 1 µg, 1 ηg and 10 ρg while, Senktide (Neurokinin B agonist) and SB222200 (Neurokinin B antagonist) in 1 µg doses consecutively for 12 days. After 12 days of peptide treatment, half of the animals (n=05) in each group were sacrificed while remaining half (n=05) were kept for another 12 days without any treatment to investigate treatment reversal. Seminal vesicles were dissected and excised tissue was processed for light microscopy, immunohistochemistry and estimation of seminal fructose levels. RESULTS: Treatment with Neurokinin B and Senktide significantly increased while SB222200 slightly decrease the seminal vesicles weight, epithelial height and seminal fructose levels as compared to control. Light microscopy revealed increased epithelial height and epithelial folding as compared to control in all Neurokinin B and Senktide treated groups while decreased in SB222200. Effects of various doses of Neurokinin B, Senktide and SB222200 on seminal vesicles weight, epithelial height, seminal fructose levels and histomorphology were reversed when rats were maintained without treatments. Immuno-expression of Neurokinin B shows no change in treatment and reversal groups. CONCLUSION: Continuous administration of Neurokinin B and Senktide effect positively while SB222200 have detrimental effects on cellular morphology, epithelial height and seminal fructose levels in seminal vesicles. Effects of peptide treatments depicted a reversal towards control group when rats were kept without any treatment.

Interplay between hippocampal TACR3 and systemic testosterone in regulating anxiety-associated synaptic plasticity.

Tachykinin receptor 3 (TACR3) is a member of the tachykinin receptor family and falls within the rhodopsin subfamily. As a G protein-coupled receptor, it responds to neurokinin B (NKB), its high-affinity ligand. Dysfunctional TACR3 has been associated with pubertal failure and anxiety, yet the mechanisms underlying this remain unclear. Hence, we have investigated the relationship between TACR3 expression, anxiety, sex hormones, and synaptic plasticity in a rat model, which indicated that severe anxiety is linked to dampened TACR3 expression in the ventral hippocampus. TACR3 expression in female rats fluctuates during the estrous cycle, reflecting sensitivity to sex hormones. Indeed, in males, sexual development is associated with a substantial increase in hippocampal TACR3 expression, coinciding with elevated serum testosterone and a significant reduction in anxiety. TACR3 is predominantly expressed in the cell membrane, including the presynaptic compartment, and its modulation significantly influences synaptic activity. Inhibition of TACR3 activity provokes hyperactivation of CaMKII and enhanced AMPA receptor phosphorylation, associated with an increase in spine density. Using a multielectrode array, stronger cross-correlation of firing was evident among neurons following TACR3 inhibition, indicating enhanced connectivity. Deficient TACR3 activity in rats led to lower serum testosterone levels, as well as increased spine density and impaired long-term potentiation (LTP) in the dentate gyrus. Remarkably, aberrant expression of functional TACR3 in spines results in spine shrinkage and pruning, while expression of defective TACR3 increases spine density, size, and the magnitude of cross-correlation. The firing pattern in response to LTP induction was inadequate in neurons expressing defective TACR3, which could be rectified by treatment with testosterone. In conclusion, our study provides valuable insights into the intricate interplay between TACR3, sex hormones, anxiety, and synaptic plasticity. These findings highlight potential targets for therapeutic interventions to alleviate anxiety in individuals with TACR3 dysfunction and the implications of TACR3 in anxiety-related neural changes provide an avenue for future research in the field.

A novel antidiuretic hormone governs tumour-induced renal dysfunction.

Maintenance of renal function and fluid transport are essential for vertebrates and invertebrates to adapt to physiological and pathological challenges. Human patients with malignant tumours frequently develop detrimental renal dysfunction and oliguria, and previous studies suggest the involvement of chemotherapeutic toxicity and tumour-associated inflammation1,2. However, how tumours might directly modulate renal functions remains largely unclear. Here, using conserved tumour models in Drosophila melanogaster3, we characterized isoform F of ion transport peptide (ITPF) as a fly antidiuretic hormone that is secreted by a subset of yki3SA gut tumour cells, impairs renal function and causes severe abdomen bloating and fluid accumulation. Mechanistically, tumour-derived ITPF targets the G-protein-coupled receptor TkR99D in stellate cells of Malpighian tubules-an excretory organ that is equivalent to renal tubules4-to activate nitric oxide synthase-cGMP signalling and inhibit fluid excretion. We further uncovered antidiuretic functions of mammalian neurokinin 3 receptor (NK3R), the homologue of fly TkR99D, as pharmaceutical blockade of NK3R efficiently alleviates renal tubular dysfunction in mice bearing different malignant tumours. Together, our results demonstrate a novel antidiuretic pathway mediating tumour-renal crosstalk across species and offer therapeutic opportunities for the treatment of cancer-associated renal dysfunction.

The overexpression of neurokinin B-neurokinin 3 receptor system exerts direct effects on the ovary under PCOS-like conditions to interfere with mitochondrial function.

PROBLEM: The increased hypothalamic neurokinin B (NKB) level may contribute to the hyperactive LH pulse secretion in Polycystic ovary syndrome (PCOS). However, the expression and role of the neurokinin B-neurokinin 3 receptor (NKB-NK3R) system in the local ovarian tissue of PCOS have not been clarified. We constructed in vivo and in vitro models to elucidate the mechanism of the NKB-NK3R pathway in reproductive endocrine disorders of PCOS. METHOD OF STUDY: The granulosa cell line-KGN cells were set in palmitic acid (PA) and dihydrotestosterone (DHT) to simulate the PCOS-like conditions. And we used the high-fat/high-glucose diet to build a PCOS-like mice model and neurokinin 3 receptor antagonist (NK3Ra) was administered to half of the mice. The expression of the NKB-NK3R system, mitochondrial functions, hormone levels, and inflammatory state was evaluated. RESULTS: The PCOS-like stimulations induced the NKB-NK3R system and MAPK-ERK pathway overexpression in KGN cells, in an approximate dose and time-dependent manner. The NKB-NK3R system overactivated the MAPK-ERK pathway to increase NNT overexpression, disturb NADH/NADPH pools, aggravate the oxidation state, and decrease ATP production. With overexpression of the NKB-NK3R system in the local ovarian tissue, ovulatory dysfunction, progesterone deficiency, and pro-inflammatory states were apparent in PCOS-like mice. Antagonizing the receptor, NK3R, reversed the adverse reproductive endocrine phenotypes via improving mitochondrial dysfunction. CONCLUSIONS: In addition to the central regulation, local ovarian overexpression of the NKB-NK3R system participated in the adverse reproductive endocrine phenotypes, supporting the therapeutic implications of NK3Ra for PCOS.

Neurokinin 3 receptor antagonist-induced adipocyte activation improves obesity and metabolism in PCOS-like mice.

AIMS: Neurokinin-B (NKB)-Neurokinin-3-receptor (NK3R) pathway is remarkably sensitive to energy equilibrium; however, its role in metabolic regulation remains unexplored in polycystic ovary syndrome (PCOS). Therefore, this work aimed to investigate the role of NK3R antagonists (NK3Ra) on metabolic dysfunction and obesity in in vitro and in vivo PCOS models. MAIN METHODS: First, an observational study using serum samples collected from 19 PCOS patients was performed. Second, prospective case-control experimental studies where NK3Ra (SB222200) was used to treat PCOS-like mice (BALB/c mice), ovariectomized+estrogen implanted obese mice (C57BL/6J mice) and 3T3-L1 murine preadipocytes were carried out to investigate its effect on metabolism in vivo and in vitro. The fat volumes, serum biochemical indexes, adipokines and inflammatory cytokines, metabolism-related gene expression and the concentrations of ATP, NAD+, NADPH…etc. were studied. KEY FINDINGS: We found a positive correlation between serum NKB and lipid metabolism indicators in PCOS women. Using the mouse models, we demonstrated that administration of NK3Ra regulates serum adipokines, inhibits weight gain with a marked decrease in fat volume, adipocyte size, and inflammatory cytokines, and promotes oxidative metabolism and energy consumption. NK3Ra reduces lipid accumulation in mature murine adipocytes by inhibiting the expression of peroxisome proliferator- activated receptor gamma (PPAR-γ) and fatty acid binding protein 4 (FABP4) genes. NK3Ras also enhances oxidative metabolism and energy consumption by maintaining intracellular redox homeostasis. SIGNIFICANCE: This study backs the use of NK3Ras as a potential therapeutic for PCOS since it ameliorates both reproductive and metabolic aberrations.

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 Cß (PLCß), but were independent of intracellular Ca2+ release and protein kinase C. PLCß-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 SB 218795, 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 Cß 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.

Nk3R blockade has sex-divergent effects on memory in mice.

BACKGROUND: Memory consolidation is a process required for the formation of long-term memories. The G-protein-coupled receptor (GPCR) neurokinin-3-receptor (Nk3R) and its interactions with sex hormones seem important for the modulation of fear memory consolidation: Nk3R antagonism in male mice impairs fear memory, but enhances it in females. However, the involvement of the Nk3R as a modulator of other memories in both sexes remains unexplored. METHODS: We use the novel object recognition paradigm to test the effect of a systemic blockade of Nk3R during memory consolidation. Further, we assess the expression of estrogen receptor α, estrogen receptor ß, and androgen receptor and heterodimerization with Nk3R in the medial prefrontal cortex (mPFC) and dorsal hippocampus (DH) of mice. RESULTS: Nk3R systemic antagonism elicited decreased memory consolidation in males while it enhanced it in females during proestrus. Nk3R analysis in the different subregions of the mPFC and the DH showed a higher expression in males than females. Moreover, females presented upregulation of the androgen receptor in the CA1 and the estrogen receptor beta in the cingulate cortex, CA1, and dentate gyrus. Overall, males presented an upregulation of the estrogen receptor alpha. We also explored the heterodimerization of GCPR membrane sex hormone receptors with the Nk3R. We found a higher percentage of Nk3R-membrane G-protein estrogen receptors heterodimers in the prelimbic cortex of the mPFC in females, suggesting an interaction of estradiol with Nk3R in memory consolidation. However, males presented a higher percentage of Nk3R-membrane G-protein androgen receptors heterodimers compared to females, pointing to an interaction of testosterone with Nk3R in memory consolidation. CONCLUSION: These data propose novel ideas on functional interactions between Nk3R, sex hormones, estrogen receptors, and androgen receptors in memory consolidation.

Functional Rescue of Inactivating Mutations of the Human Neurokinin 3 Receptor Using Pharmacological Chaperones.

G protein-coupled receptors (GPCRs) facilitate the majority of signal transductions across cell membranes in humans, with numerous diseases attributed to inactivating GPCR mutations. Many of these mutations result in misfolding during nascent receptor synthesis in the endoplasmic reticulum (ER), resulting in intracellular retention and degradation. Pharmacological chaperones (PCs) are cell-permeant small molecules that can interact with misfolded receptors in the ER and stabilise/rescue their folding to promote ER exit and trafficking to the cell membrane. The neurokinin 3 receptor (NK3R) plays a pivotal role in the hypothalamic-pituitary-gonadal reproductive axis. We sought to determine whether NK3R missense mutations result in a loss of cell surface receptor expression and, if so, whether a cell-permeant small molecule NK3R antagonist could be repurposed as a PC to restore function to these mutants. Quantitation of cell surface expression levels of seven mutant NK3Rs identified in hypogonadal patients indicated that five had severely impaired cell surface expression. A small molecule NK3R antagonist, M8, increased cell surface expression in four of these five and resulted in post-translational receptor processing in a manner analogous to the wild type. Importantly, there was a significant improvement in receptor activation in response to neurokinin B (NKB) for all four receptors following their rescue with M8. This demonstrates that M8 may have potential for therapeutic development in the treatment of hypogonadal patients harbouring NK3R mutations. The repurposing of existing small molecule GPCR modulators as PCs represents a novel and therapeutically viable option for the treatment of disorders attributed to mutations in GPCRs that cause intracellular retention.

Different pituitary action of NK3Ra and NK3Rb in grass carp.

In mammals, NK3R is the specific receptor for NKB, which played an important role in reproduction. Recently, two NK3R isoforms, namely NK3Ra and NK3Rb, have been identified in fish. However, little is known about the pituitary actions of the two NK3R isoforms in fish. In this study, both NK3Ra and NK3Rb were isolated from grass carp pituitary. Although their sequence similarity was only 61.6%, the two NK3R isoforms displayed similar ligand selectivity and binding affinity to TAC3 gene products (NKBa, NKBRPa and NKBRPb). In addition, both NK3Ra and NK3Rb displayed similar signaling pathways, including PKA, PKC, MAPK and Ca2+ cascades. Tissue distribution indicated that both NK3Ra and NK3Rb were highly detected in grass carp pituitary. Further study found that NK3Ra was mainly located in pituitary LHß cells, while NK3Rb was only detected in pituitary SLα cells. Furthermore, NK3Ra and NK3Rb activation could induce LHß and SLα promoter activity, respectively. These results suggested that the two NK3R isoforms displayed different pituitary actions in fish. Using grass carp pituitary cells as model, we found that PACAP could significantly reduce NK3Ra, but induce NK3Rb mRNA expression coupled with cAMP/PKA and PLC/PKC pathways. Interestingly, PACAP could also significantly inhibit LHß, but stimulate SLα mRNA expression in grass carp pituitary cells. Furthermore, NK3R antagonist could not only inhibit LHß mRNA expression, but also block PACAP-induced SLα mRNA expression in grass carp pituitary cells. These results suggested that NK3Ra and NK3Rb could mediate PACAP-reduced LHß and -induced SLα mRNA expression in grass carp pituitary, respectively.

Localization of kisspeptin, NKB, and NK3R in the hypothalamus of gilts treated with the progestin altrenogest.

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirms they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB.

Characterization of the Action of Tachykinin Signaling on Pulsatile LH Secretion in Male Mice.

The alternation of the stimulatory action of the tachykinin neurokinin B (NKB) and the inhibitory action of dynorphin within arcuate (ARH) Kiss1 neurons has been proposed as the mechanism behind the generation of gonadotropin-releasing hormone (GnRH) pulses through the pulsatile release of kisspeptin. However, we have recently documented that GnRH pulses still exist in gonadectomized mice in the absence of tachykinin signaling. Here, we document an increase in basal frequency and amplitude of luteinizing hormone (LH) pulses in intact male mice deficient in substance P, neurokinin A (NKA) signaling (Tac1KO), and NKB signaling (Tac2KO and Tacr3KO). Moreover, we offer evidence that a single bolus of the NKB receptor agonist senktide to gonad-intact wild-type males increases the basal release of LH without changing its frequency. Altogether, these data support the dispensable role of the individual tachykinin systems in the generation of LH pulses. Moreover, the increased activity of the GnRH pulse generator in intact KO male mice suggests the existence of compensation by additional mechanisms in the generation of kisspeptin/GnRH pulses.

Fezolinetant in the treatment of vasomotor symptoms associated with menopause.

Introduction: Although international clinical practice guidelines recognize a continued role for menopausal hormone therapy (HT), particularly for symptomatic women <60 years of age or within 10 years of menopause, safety and tolerability concerns have discouraged HT use due to potential links with a perceived increased risk of hormone-dependent cancers, and an established risk of stroke and venous thromboembolism. There is therefore a need for safe, effective non-hormonal therapy for relief of menopausal vasomotor symptoms (VMS).Areas covered: This narrative review summarizes the dataset accrued for fezolinetant, a neurokinin-3 receptor (NK3R) antagonist in clinical development for menopause-associated VMS.Expert opinion: Altered signaling in neuroendocrine circuits at menopause leads to VMS wherein NK3R activity plays a key role to modulate the thermoregulatory center in a manner conducive to triggering the 'hot flash' response. Thus, a new generation of NK3R antagonists has entered clinical development to specifically target the mechanistic basis of VMS. Fezolinetant is the most advanced NK3R antagonist in terms of stage of clinical development. Results to date have demonstrated rapid and substantial reduction in VMS frequency and severity and associated improvements in health-related quality of life. NK3R antagonists offer a non-hormonal alternative to HT for the treatment of menopause-related VMS.

Neurokinin 3 Receptor Antagonism Ameliorates Key Metabolic Features in a Hyperandrogenic PCOS Mouse Model.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine condition characterized by a range of endocrine, reproductive, and metabolic abnormalities. At present, management of women with PCOS is suboptimal as treatment is only symptomatic. Clinical and experimental advances in our understanding of PCOS etiology support a pivotal role for androgen neuroendocrine actions in PCOS pathogenesis. Hyperandrogenism is a key PCOS trait and androgen actions play a role in regulating the kisspeptin-/neurokinin B-/dynorphin (KNDy) system. This study aimed to investigate if targeted antagonism of neurokinin B signaling through the neurokinin 3 receptor (NK3R) would reverse PCOS traits in a dihydrotestosterone (DHT)-induced mouse model of PCOS. After 3 months, DHT exposure induced key reproductive PCOS traits of cycle irregularity and ovulatory dysfunction, and PCOS-like metabolic traits including increased body weight; white and brown fat pad weights; fasting serum triglyceride and glucose levels, and blood glucose incremental area under the curve. Treatment with a NK3R antagonist (MLE4901) did not impact the observed reproductive defects. In contrast, following NK3R antagonist treatment, PCOS-like females displayed decreased total body weight, adiposity, and adipocyte hypertrophy, but increased respiratory exchange ratio, suggesting NK3R antagonism altered the metabolic status of the PCOS-like females. NK3R antagonism did not improve circulating serum triglyceride or fasted glucose levels. Collectively, these findings demonstrate that NK3R antagonism may be beneficial in the treatment of adverse metabolic features associated with PCOS and support neuroendocrine targeting in the development of novel therapeutic strategies for PCOS.

Neurokinin receptor antagonism: a patent review (2014-present).

INTRODUCTION: The tachykinin family of peptides (substance P, neurokinin A) via the neurokinin-1 (NK-1), NK-2, and NK-3 receptors is involved in many physiological/physiopathological actions. Antagonists of these receptors may be used to treat many human pathologies. AREAS COVERED: This review offers an overview (from 2014 to present) of the actions exerted by NK receptor (NK-R) antagonists on emesis, pruritus, cardiomyopathy, respiratory tract diseases, bacterial infection, cancer, ocular pain, corneal neovascularization, excess of body fat/weight, conditioned fear, social isolation stress, hot flush, melanogenesis, follicle development, fish reproduction, and sex-hormone-dependent diseases. EXPERT OPINION: From 2014, no invention has been published using NK-2R antagonists. Although the tachykinin/NK receptor system is involved in a great number of mechanisms, to date, the use of only five NK-1R antagonists have been approved in humans but no NK-2R or NK-3R antagonist. NK receptor antagonists are safe in human trials and are potential therapeutic agents, but this potential is currently minimized. In humans, more studies on molecules acting as NK receptor antagonists and exerting a potential therapeutic action must be carried out. The antipruritic or antitumor action of NK-1R antagonists must be explored in greater depth: the highest safe dose and the time of administration (for a long period of time) of these antagonists must be well established.

Neurokinin receptors in drug and alcohol addiction.

The neurokinins are a class of peptide signaling molecules that mediate a range of central and peripheral functions including pain processing, gastrointestinal function, stress responses, and anxiety. Recent data have linked these neuropeptides with drug-related behaviors. Specifically, substance P (SP) and neurokinin B (NKB), have been shown to influence responses to alcohol, cocaine, and/or opiate drugs. SP and NKB preferentially bind to the neurokinin-1 receptor (NK1R) and neurokinin-3 receptor (NK3R), respectively, but do have some affinity for all classes of neurokinin receptor at high concentrations. NK1R activity has been shown to influence reward and reinforcement for opiate drugs, stimulatory and neurochemical responses to cocaine, and escalated and stress-induced alcohol seeking. In reinstatement models of relapse-like behavior, NK1R antagonism attenuates stress-induced reinstatement for all classes of drugs tested to date. The NK3R also influences alcohol intake and behavioral/neurochemical responses to cocaine, but less research has been performed in regard to this particular receptor in preclinical models of addiction. Clinically, agents targeting these receptors have shown some promise, but have produced mixed results. Here, the preclinical findings for the NK1R and NK3R are reviewed, and discussion is provided to interpret clinical findings. Additionally, important factors to consider in regards to future clinical work are suggested.

Endocytic recycling prevents copper accumulation in astrocytoma cells stimulated with copper-bound neurokinin B.

Neurokinin B (NKB) is a key neuropeptide in reproductive endocrinology where it contributes to the generation of pulses of gonadotropin-releasing hormone. NKB is a copper-binding peptide; in the absence of metal NKB rapidly adopts an amyloid structure, but copper binding inhibits amyloid formation and generates a structure that can activate the neurokinin 3 receptor. The fate of copper once it binds NKB and activates the neurokinin 3 receptor is not understood, but endocytosis of NKB occurs even when the peptide is coordinated to copper. Using astrocytoma cells that express endogenous neurokinin 3 receptor, this work shows that endocytosis of apo- and copper-bound NKB occurs in concert with the receptor via a trafficking pathway that includes the early endosome. When cells are stimulated with copper-bound NKB the cellular copper concentration does not significantly increase, however when the cells are pre-treated with the recycling inhibitor, brefeldin A, they are capable of accumulating copper. This data shows that copper-bound NKB can activate the neurokinin 3 receptor then endocytosis abstracts metal, peptide and receptor from the cell surface. The cell does not accumulate the copper but instead it enters recycling pathways that ultimately leads to metal release from the cell. The work reveals a novel receptor-mediated copper trafficking pathway that retains metal in membrane bound organelles until it is exported from the cell.

A single-nucleotide polymorphism induced alternative splicing in Tacr3 involves in hypoxic-ischemic brain damage.

Single-nucleotide polymorphism (SNP) and Alternative splicing (AS) were found to be implicated in certain diseases, nevertheless, the contributions of mRNA SNPs and AS to pathogenesis in developing rat brains with hypoxic-ischemic encephalopathy (HIE) remained largely vague. Additionally, the disease associated with Tacr3 was normosmic congenital hypogonadotropic hypogonadism, while the relationship between HIE and Tacr3 remained largely elusive. The current study was designed to investigate the differentially expressed mRNAs and related SNPs as well as AS in neonatal rats subjected to HIE to identify if the exhibition of AS was associated with SNPs under pathological condition. Firstly, we used postnatal day 7 Sprague-Dawley rats to construct neonatal HIE model, and analyzed the expression profiles of SNP mRNA in hypoxic-ischemic (HI) and sham brains by using RNA sequencing. Then four genes, including Mdfic, Lpp, Bag3 and Tacr3, connecting with HIE and exhibiting SNPs and AS were identified by bioinformatics analysis. Moreover, combined with exonic splicing enhancer (ESE) and alternative splice site predictor (ASSP) analysis, we found that Tacr3 is associated specifically with HIE through 258547789 G > A SNP in inside the Alt First Exon and 258548573 G > A SNP in outside the Alt First Exon. Taken together, our study provides new evidence to understand the role of Tacr3 in HIE and it is possibly a potential target for the treatment of HIE in future clinic trial.

IGFs Potentiate TAC3-induced SLα Expression via Upregulation of TACR3 Expression in Grass Carp Pituitary Cells.

In mammals, the tachykinin 3 (TAC3)/tachykinin receptor 3 (TACR3) systems have been confirmed to play an important role in the regulation of puberty onset. Using grass carp pituitary cells as the model, our recent study found that the TAC3 gene products could significantly induce somatolactin α (SLα) synthesis and secretion via TACR3 activation. In the present study, we seek to examine if pituitary TACR3 can serve as a regulatory target and contribute to TAC3 interactions with other SLα regulators. Firstly, grass carp TACR3 was cloned and tissue distribution showed that it could be highly detected in grass carp pituitary. Using HEK293 cells as the model, functional expression also revealed that grass carp TACR3 exhibited ligand binding selectivity and post-receptor signaling highly comparable to its mammalian counterpart. Using grass carp pituitary cells as the model, TACR3 mRNA expression could be stimulated by insulin-like growth factor (IGF)-I and -II via the IGF-I receptor coupled to phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways. Interestingly, IGF-I/-II cotreatment could also significantly enhance TAC3-induced SLα mRNA expression and the potentiating effect was dependent on TACR3 expression and activation of adenylate cyclase (AC)/cAMP/protein kinase A (PKA), phospholipase C (PLC)/inositol 1,4,5-triphosphate (IP3)/protein kinase C (PKC), and Ca2+/calmodulin (CaM)/calmodulin-dependent protein kinase II (CaMK-II) cascades. Besides, IGF-I-induced Akt phosphorylation but not MEK, extracellular signal-regulated kinase (ERK1/2), and P38MAPK phosphorylation was notably enhanced by TACR3 activation. These results, as a whole, suggest that the potentiating effect of IGFs on TAC3 gene products-induced SLα mRNA expression was mediated by TACR3 upregulation and functional crosstalk of post-receptor signaling in the pituitary.

NKB/NK3 system negatively regulates the reproductive axis in sexually immature goldfish (Carassius auratus).

To ascertain the significance of the Neurokinin B/Tachykinin 3 receptor (NKB/NK3) system in goldfish reproduction, two cDNAs encoding tachykinin 3 receptors, namely tacr3a and tacr3b, were cloned. Subsequent studies revealed that the downstream signalling of both Tac3rs can be activated by different NKB peptides, suggesting that the cloned receptors are biologically functional in goldfish. RT-PCR analysis showed that tacr3s are widely expressed in brain regions. During the gonadal development, tacr3a and tacr3b exhibited different expression patterns in the hypothalamus and pituitary. The actions of NKB peptides on reproductive axis was further investigated in vivo. Intraperitoneal injections of NKB peptides significantly reduced the expression of kiss2 and gonadotropin releasing hormone 3 (gnrh3) in the hypothalamus, and the expression of luteinizing hormone beta subunit (lhb) and follicle stimulating hormone beta subunit (fshb) in the pituitary in sexually immature goldfish. Taken together, our findings revealed that NKB/NK3 system plays a negative role in the reproductive axis of immature goldfish.