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.

Probiotic potential of Lacticaseibacillus rhamnosus VHProbi M15 on sucralfate-induced constipation in mice.

The main objective of this study was to investigate the potential probiotic properties of Lacticaseibacillus rhamnosus VHProbi®M15 (M15). This study examined the effects of M15 on sucralfate-induced constipation in a mouse model. The BALB/c mice were randomly divided into four groups: the normal group (NOR) was without any treatment, while the constipation (CON), phenolphthalein (PHE), and probiotic (PRO) treatment groups were fed with sucralfate until the appearance of constipation symptoms. Afterward, the NOR and CON groups were given 1 ml saline orally every day until the end of the experiment; the PHE and PRO groups were given phenolphthalein or M15 suspension in 1 ml orally, respectively. Compared with the CON group, the fecal water content and intestinal peristalsis improved in the PRO group. Here, intake of M15 effectively attenuated sucralfate-induced constipation, recuperated colonic epithelial integrity, and increased serum levels of gastrointestinal excitatory neurotransmitters (motilin, gastrin, substance P). Analysis of the intestinal microbiota of mice by 16S rRNA metagenomic revealed an increase in the relative abundance of Bacteroides and a decrease in Sclerotinia, Verrucosa and Proteus in the PRO group. Compared with the CON group, the constipation-induced intestinal microecological changes were partially recovered in the PHE and PRO groups. These results demonstrate that M15 enhanced gastrointestinal transit and alleviated in mice with sucralfate-induced constipation.

Senktide blocks aberrant RTN3 interactome to retard memory decline and tau pathology in social isolated Alzheimer's disease mice.

Sporadic or late-onset Alzheimer's disease (LOAD) accounts for more than 95% of Alzheimer's disease (AD) cases without any family history. Although genome-wide association studies have identified associated risk genes and loci for LOAD, numerous studies suggest that many adverse environmental factors, such as social isolation, are associated with an increased risk of dementia. However, the underlying mechanisms of social isolation in AD progression remain elusive. In the current study, we found that 7 days of social isolation could trigger pattern separation impairments and presynaptic abnormalities of the mossy fibre-CA3 circuit in AD mice. We also revealed that social isolation disrupted histone acetylation and resulted in the downregulation of 2 dentate gyrus (DG)-enriched miRNAs, which simultaneously target reticulon 3 (RTN3), an endoplasmic reticulum protein that aggregates in presynaptic regions to disturb the formation of functional mossy fibre boutons (MFBs) by recruiting multiple mitochondrial and vesicle-related proteins. Interestingly, the aggregation of RTN3 also recruits the PP2A B subunits to suppress PP2A activity and induce tau hyperphosphorylation, which, in turn, further elevates RTN3 and forms a vicious cycle. Finally, using an artificial intelligence-assisted molecular docking approach, we determined that senktide, a selective agonist of neurokinin3 receptors (NK3R), could reduce the binding of RTN3 with its partners. Moreover, application of senktide in vivo effectively restored DG circuit disorders in socially isolated AD mice. Taken together, our findings not only demonstrate the epigenetic regulatory mechanism underlying mossy fibre synaptic disorders orchestrated by social isolation and tau pathology but also reveal a novel potential therapeutic strategy for AD.

The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep†.

The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65 days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9 days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn't display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.

Occurrence of blaNDM-7 and association with blaKPC-2, blaCTX-M15, aac, aph, mph(A), catB3 and virulence genes in a clinical isolate of Klebsiella pneumoniae with different plasmids in Brazil.

To characterize phenotypically and genotypically an isolate of multidrug-resistant (MDR) K. pneumoniae from a patient with septicemia in a hospital in Recife-PE, Brazil, resistance and virulence genes were investigated using PCR and sequencing the amplicons, and the plasmid DNA was also sequenced. The K74-A3 isolate was resistant to all ß-lactams, including carbapenems, as well as to aminoglycosides and quinolones. By conducting a PCR analysis and sequencing, the variants blaNDM-7 associated with blaKPC-2 and the cps, wabG, fim-H, mrkD and entB virulence genes were identified. The analysis of plasmid revealed the presence of blaCTX-M15, aac(3)-IVa, aph(3')-Ia, aph(4)-Ia, aac(6')ib-cr, mph(A) and catB3, and also the plasmids IncX3, IncFIB, IncQ1, ColRNAI and ColpVC. To our knowledge, this is the first report of the blaNDM-7 gene in Recife-PE and we suggest that this variant is located in IncX3. These results alert us to the risk of spreading an isolate with a vast genetic arsenal of resistance, in addition to which several plasmids are present that favor the horizontal transfer of these genes.

Semi-mechanistic population pharmacokinetics analysis reveals distinct CYP2C19 dependency in the bioactivation of vicagrel and clopidogrel to active metabolite M15-2.

AIMS: Vicagrel is a novel antiplatelet drug used to mitigate clopidogrel resistance due to CYP2C19 polymorphism. This study aimed to develop a semi-mechanistic population pharmacokinetic (PopPK) model to characterize the pharmacokinetic (PK) profile of M15-2, the active metabolite of vicagrel and clopidogrel, and to evaluate the influence of CYP2C19 polymorphisms and other covariates in healthy subjects and patients with acute coronary syndrome (ACS) after oral administration. METHODS: The analysis utilized data from 213 subjects, including 178 healthy subjects and 35 patients, from five clinical trials. PopPK modeling and simulation were used to estimate PopPK parameters and evaluate the impact of covariates. RESULTS: The M15-2 PK profiles were well characterized by a model incorporating transit compartments, two-compartment parent models and two-compartment M15-2 models for both vicagrel and clopidogrel. The parameter estimates indicated the dose fraction of vicagrel that formed M15-2 was approximately 20-fold that of clopidogrel. Covariate analysis identified a significant effect of CYP2C19 on M15-2 apparent clearance (CL/F) and apparent volume of distribution (V3/F) for clopidogrel but only CL/F for vicagrel. The analysis suggested that the nonlinear PK of M15-2 for clopidogrel was due the first-step bioactivation of clopidogrel to 2-oxoclopidogrel. CONCLUSION: The model illustrated the bioactivation of vicagrel is more efficient and less dependent on CYP2C19 than that of clopidogrel. M15-2 is formed in a linear process from vicagrel, versus a nonlinear and less predictable process from clopidogrel. Advantages in both PK and pharmacogenetics suggest that vicagrel may reduce the complexity of currently recommended CYP2C19-based dosage adjustment for clopidogrel.

Ceftriaxone treatment failure in two cases of Enteric Fever caused by CTX-M15 positive Salmonella Typhi and its gastrointestinal carriage.

We report two cases of culture positive typhoid fever caused by ceftriaxone resistant Salmonella Typhi. Bacterial isolates from both the cases were positive for ESBL by phenotypic methods. Both patients didn't respond to ceftriaxone and were finally treated with meropenem. Screening of family members of one patient isolated a similar strain from a healthy carrier with the same antibiogram pattern. All isolates were subjected to PCR, which confirmed the presence of blaCTX-M15 ESBL gene. These two cases confirm emergence of ESBL-producing Salmonella Typhi causing Enteric Fever in India and also their presence in the gut flora of healthy carriers.

Sex-specific pubertal and metabolic regulation of Kiss1 neurons via Nhlh2.

Hypothalamic Kiss1 neurons control gonadotropin-releasing hormone release through the secretion of kisspeptin. Kiss1 neurons serve as a nodal center that conveys essential regulatory cues for the attainment and maintenance of reproductive function. Despite this critical role, the mechanisms that control kisspeptin synthesis and release remain largely unknown. Using Drop-Seq data from the arcuate nucleus of adult mice and in situ hybridization, we identified Nescient Helix-Loop-Helix 2 (Nhlh2), a transcription factor of the basic helix-loop-helix family, to be enriched in Kiss1 neurons. JASPAR analysis revealed several binding sites for NHLH2 in the Kiss1 and Tac2 (neurokinin B) 5' regulatory regions. In vitro luciferase assays evidenced a robust stimulatory action of NHLH2 on human KISS1 and TAC3 promoters. The recruitment of NHLH2 to the KISS1 and TAC3 promoters was further confirmed through chromatin immunoprecipitation. In vivo conditional ablation of Nhlh2 from Kiss1 neurons using Kiss1Cre:Nhlh2fl/fl mice induced a male-specific delay in puberty onset, in line with a decrease in arcuate Kiss1 expression. Females retained normal reproductive function albeit with irregular estrous cycles. Further analysis of male Kiss1Cre:Nhlh2fl/fl mice revealed higher susceptibility to metabolic challenges in the release of luteinizing hormone and impaired response to leptin. Overall, in Kiss1 neurons, Nhlh2 contributes to the metabolic regulation of kisspeptin and NKB synthesis and release, with implications for the timing of puberty onset and regulation of fertility in male mice.

Detection, Structural Elucidation, and Biological Effects of Diverse N-Acyl-homoserine Lactone Signaling Molecules in the Plant-Promoting Endophytic Bacterium Rhizobium oryzihabitans M15.

Quorum sensing (QS), usually performed by N-acyl-homoserine lactones (AHLs) in Gram-staining-negative bacteria, plays an important role in plant-bacteria interactions. Rhizobium oryzihabitans M15 is a plant-growth-promoting rhizobacterium (PGPR) isolated from rice roots. In this study, we found a QS system in the endogenous plasmid of R. oryzihabitans M15 and detected the activity of AHLs by a bioassay method. We identified five AHL analogues in R. oryzihabitans M15 using liquid chromatography-tandem mass spectrometry (LC-MS). The most dominant AHL analogue was N-(3R-hydroxy-7-cis-tetradecanoyl)-l-homoserine lactone according to nuclear magnetic resonance (NMR) and Mosher's reactions. Furthermore, the rosI mutant abolished AHL production and significantly decreased growth, exopolysaccharide (EPS) production, biofilm formation, and motility compared to the wild-type strain. These results lay the foundation for further investigating the QS regulation mechanism and signal pathway of R. oryzihabitans M15 and its interactions with the host plant.

A single nucleotide mutation drastically increases the expression of tumor-homing NGR-TNFα in the E. coli M15-pQE30 system by improving gene transcription.

Due to their potent immune stimulation, tumor necrosis factor alpha (TNFα) variants with tumor-homing activity are attractive as novel antitumor drugs. The promising antitumor effect of NGR-TNFα in clinical trials triggered extensive interest in developing novel tumor-homing TNFα variants in recent years. Owing to its promising antitumor effect, NGR-TNFα is usually used as a control for newly developed tumor-homing TNFα variants. In our previous works, we produced a pericyte-targeting Z-TNFα at high levels using the Escherichia coli (E. coli) M15-pQE30 system. To further compare Z-TNFα and NGR-TNFα, we attempted to express NGR-TNFα using the same system. Surprisingly, native NGR-TNFα was expressed at a low (~ 0.2 mg/L) level in E. coli M15 containing the pQE30 plasmid. However, a single nucleotide mutation of C to G, resulting in a substitution of leucine (L) with valine (V) at the start of TNFα, increased the expression of NGR-TNFα by ~ 100 times through improving transcription. In addition, the amino acid substitution showed a little impact on the receptor binding, in vitro cytotoxicity, and in vivo antitumor effect of NGR-TNFα. As fusing NGR to the N-terminus of TNFα with a valine substitution did not reduce the protein yield, the TNFα gene with a C > G mutation might be used to prepare novel tumor-homing TNFα when the native TNFα-based variant is expressed at an extremely low level in E. coli. Notably, in addition to the mutated valine, the impact of N-terminal additional amino acids provided by pQE30 vector on the function of TNFα variant must be carefully evaluated. KEY POINTS : • A single nucleotide mutation increased the expression of NGR-TNFα by two orders. • Nucleotide mutation-induced amino acid substitution did not reduce NGR-TNFα activity.

A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes.

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of ß-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease.

UVR-B-induced NKR-1 Expression in Ocular Tissues is blocked by Substance P Receptor Antagonist Fosaprepitant in the Exposed as well as Unexposed Partner Eye.

Purpose: To investigate the effect of NKR-1 antagonists in an established UVR-B-induced cataract mouse model. Furthermore, to examine the expression of pro-inflammatory cytokines/chemokines in mouse eyes following unilateral UVR-B exposure.Methods: Mice received intraperitoneally injections of Fosaprepitant and Spantide I, before and after unilateral exposure to UVR-B. After day 3 and 7 post-exposure, ocular tissues were extracted for the detection of NKR-1 protein level by ELISA.Results: Pretreatment with Fosaprepitant decreases NKR-1 expression in exposed ocular tissues as well as in the unexposed lens epithelium compared to the saline group. Spantide I treatment showed a tendency of NKR-1 overexpression in ocular tissues.Conclusion: The clinically approved NKR-1 receptor antagonist Fosaprepitant decreases NKR-1 protein expression effectively not only in the exposed but also in the unexposed partner eye in a UVR-B irradiation mouse model. No effect was seen on the protein concentration of pro-inflammatory cytokines/chemokines in either eye.

Neurokinin 3 receptor-selective agonist, senktide, decreases core temperature in Japanese Black cattle.

Heat stress disrupts reproductive function in cattle. In summer, high ambient temperature and humidity elevate core body temperature, which is considered to be detrimental to reproductive abilities in cattle. Neurokinin B (NKB) is a factor that generates pulsatile GnRH and subsequent LH secretion in mammals. Recent studies have reported that NKB-neurokinin 3 receptor (NK3R) signaling is associated with heat-defense responses in rodents. The present study aimed to clarify the role of NKB-NK3R signaling in thermoregulation in cattle. We examined the effects of an NK3R-selective agonist, senktide, on vaginal temperature as an indicator of core body temperature in winter and summer. In both seasons, continuous infusion of senktide for 4 h immediately decreased vaginal temperature, and the mean temperature change in the senktide-treated group was significantly lower than that of both vehicle- and GnRH-treated groups. Administration of GnRH induced LH elevation, but there was no significant difference in vaginal temperature change between GnRH- and vehicle-treated groups. Moreover, we investigated the effects of senktide on ovarian temperature. Senktide treatment seemed to suppress the increase in ovarian temperature from 2 h after the beginning of administration, although the difference between groups was not statistically significant. Taken together, these results suggest that senktide infusion caused a decline in the vaginal temperature of cattle, in both winter and summer seasons, and this effect was not due to the gonadotropin-releasing action of senktide. These findings provide new therapeutic options for senktide to support both heat-defense responses and GnRH/LH pulse generation.

Antiproliferative effects of [D-Pro2, D-Trp7,9]-Substance P and aprepitant on several cancer cell lines and their selectivity in comparison to normal cells.

A neuropeptide, Substance P (SP), has mitogenic action in many types of cancer cells mediated via the neurokinin-1 receptor (NK1R). Small molecular NK1R antagonists have been frequently shown to possess anticancer activity both in vivo and in vitro, but there are only a few papers on such activity regarding peptide antagonists. In order to extend the data on this class of compounds, we have compared the effects of a peptide antagonist, [D-Pro2, D-Trp7,9]-Substance P, and a small molecular antagonist, aprepitant on the proliferation of five cancer and three normal cell lines. The comparison was based on three assays: cell proliferation test, MTT test and assay for colony formation. Consistently with earlier reports, aprepitant potently reduced cell proliferation in cancer cell lines in all assays, but in contrast to previous works, the compound was not selective and it affected normal cell lines to a similar degree. The studied peptide antagonist, [D-Pro2, D-Trp7,9]-Substance P, was able to decrease proliferation only in a few cell lines, and only in the highest concentration (100 µM). In a lower concentration, a slight pro-proliferative effect was observed in a few cell lines. No statistically significant effects on colony formation were found for this compound.

225Ac- and 213Bi-Substance P Analogues for Glioma Therapy.

Within the last decades, there has been no major improvement in treatment of patients with glioma, especially with glioblastoma multiforme (GBM) which is related to specific features of this tumor type, such as heterogeneity at the macroscopic, microscopic and genetic level, the infiltrative nature of tumors and the obstacle of the brain-blood barrier which limits the accessability of most drugs. The current standard of care is surgical resection, followed by radio- and chemotherapy. After first-line treatment of the primary lesion, tumor recurrence is diagnosed in virtually all GBM patients. Treatment of tumor recurrence represents a challenging clinical task. Surgical resection to relief symptoms of mass effect and/or salvage chemotherapy are often considered as last therapeutic option. A new treatment option is urgently needed. Targeted alpha therapy with an intratumoral injection of 213Bi-DOTA-Substance P (SP) or 225Ac-DOTAGA-Substance P has been introduced into the therapeutic armamentarium of recurrent GBM. There are many advantages of using SP such as very high prevalence of increased NK-1 expression in GBM cells, regardless of the degree of malignancy, and expression of the NK-1 receptor system not only on the membrane of cancer cells but also strong expression of NK1 receptors within the tumor neovasculature suggesting concomitant targeting of vascular and neoplastic structures. Radioisotopes with different physical properties, mainly beta-emitting metallic radionuclides, were implemented for brain tumor treatment. Based on their radiophysical properties, however, alpha emitters exhibit more promising properties. In investigator-initiated phase I and II studies, targeted alpha therapy using Bi-213/Ac-225 radiolabeled Substance P for malignant gliomas compare favorably with standard therapy, with the limitation that no large controlled series have so far been generated. Further development should focus on the improvement of the biological and chemical properties of the compound and the application by dedicated catheter systems to improve the intratumoral distribution of the radiopharmaceutical within growth and infiltrative zone of these glial neoplasms.

Role for Kisspeptin and Neurokinin B in Regulation of Luteinizing Hormone and Testosterone Secretion in the Fetal Sheep.

Evidence suggests that the hypothalamic-pituitary-gonadal (HPG) axis is active during the critical period for sexual differentiation of the ovine sexually dimorphic nucleus, which occurs between gestational day (GD) 60 and 90. Two possible neuropeptides that could activate the fetal HPG axis are kisspeptin and neurokinin B (NKB). We used GD85 fetal lambs to determine whether intravenous administration of kisspeptin-10 (KP-10) or senktide (NKB agonist) could elicit luteinizing hormone (LH) release. Immunohistochemistry and fluorescent in situ hybridization (FISH) were employed to localize these peptides in brains of GD60 and GD85 lamb fetuses. In anesthetized fetuses, KP-10 elicited robust release of LH that was accompanied by a delayed rise in serum testosterone in males. Pretreatment with the GnRH receptor antagonist (acyline) abolished the LH response to KP-10, confirming a hypothalamic site of action. In unanesthetized fetuses, senktide, as well as KP-10, elicited LH release. The senktide response of females was greater than that of males, indicating a difference in NKB sensitivity between sexes. Gonadotropin-releasing hormone also induced a greater LH discharge in females than in males, indicating that testosterone negative feedback is mediated through pituitary gonadotrophs. Kisspeptin and NKB immunoreactive cells in the arcuate nucleus were more abundant in females than in males. Greater than 85% of arcuate kisspeptin cells costained for NKB. FISH revealed that the majority of these were kisspeptin/NKB/dynorphin (KNDy) neurons. These results support the hypothesis that kisspeptin-GnRH signaling regulates the reproductive axis of the ovine fetus during the prenatal critical period acting to maintain a stable androgen milieu necessary for brain masculinization.

The pivotal role of glycogen synthase kinase 3 (GSK-3) in vomiting evoked by specific emetogens in the least shrew (Cryptotis parva).

Glycogen synthase kinase 3 (GSK-3) is a constitutively active multifunctional serine-threonine kinase which is involved in diverse physiological processes. GSK-3 has been implicated in a wide range of diseases including neurodegeneration, inflammation, diabetes and cancer. GSK-3 is a downstream target for protein kinase B (Akt) which phosphorylates GSK-3 and suppresses its activity. Based upon our preliminary findings, we postulated Akt's involvement in emesis. The aim of this study was to investigate the participation of GSK-3 and the antiemetic potential of two GSK-3 inhibitors (AR-A014418 and SB216763) in the least shrew model of vomiting against fully-effective emetic doses of diverse emetogens, including the nonselective and/or selective agonists of serotonin type 3 (e.g. 5-HT or 2-Methyl-5-HT)-, neurokinin type 1 receptor (e.g. GR73632), dopamine D2 (e.g. apomorphine or quinpirole)-, and muscarinic 1 (e.g. pilocarpine or McN-A-343) receptors, as well as the L-type Ca2+ channel agonist (FPL64176), the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, and the chemotherapeutic agent, cisplatin. We first determined if these emetogens could regulate the phosphorylation level of GSK-3 in the brainstem emetic loci of least shrews and then investigated whether AR-A014418 and SB216763 could protect against the evoked emesis. Phospho-GSK-3α/ß Ser21/9 levels in the brainstem and the enteric nerves of jejunum in the small intestine were upregulated following intraperitoneal (i.p.) administration of all the tested emetogens. Furthermore, administration of AR-A014418 (2.5-20 mg/kg, i.p.) dose-dependently attenuated both the frequency and percentage of shrews vomiting in response to i.p. administration of 5-HT (5 mg/kg), 2-Methyl-5-HT (5 mg/kg), GR73632 (5 mg/kg), apomorphine (2 mg/kg), quinpirole (2 mg/kg), pilocarpine (2 mg/kg), McN-A-343 (2 mg/kg), FPL64176 (10 mg/kg), or thapsigargin (0.5 mg/kg). Relatively lower doses of SB216763 exerted antiemetic efficacy, but both inhibitors barely affected cisplatin (10 mg/kg)-induced vomiting. Collectively, these results support the notion that vomiting is accompanied by a downregulation of GSK-3 activity and pharmacological inhibition of GSK-3 protects against pharmacologically evoked vomiting.

SPA: a peptide antagonist that acts as a cell-penetrating peptide for drug delivery.

Although cell-penetrating peptides (CPPs) has been proven to be efficient transporter for drug delivery, ideal peptide vectors for tumor therapy are still being urgently sought. Peptide antagonists have attracted substantial attention as targeting molecules because of their high tumor accumulation and antitumor activity compared with agonists. SPA, a derivative of substance P, is a potent antagonist that exhibits antitumor activity. Based on the amino acid composition of SPA, we speculate that it can translocate across cell membranes as CPPs do. In this study, our results demonstrated that SPA could enter cells similarly to a CPP. As a vector, SPA could efficiently deliver camptothecin and plasmids into cells. In addition, our results showed that SPA exhibited low toxicity to normal cells and high enzymatic stability. Taken together, our results validated the ability of SPA for efficient drug delivery. More importantly, our study opens a new avenue for designing ideal CPPs based on peptide antagonists.

Δ9-THC and related cannabinoids suppress substance P- induced neurokinin NK1-receptor-mediated vomiting via activation of cannabinoid CB1 receptor.

Δ9-THC suppresses cisplatin-induced vomiting through activation of cannabinoid CB1 receptors. Cisplatin-evoked emesis is predominantly due to release of serotonin and substance P (SP) in the gut and the brainstem which subsequently stimulate their corresponding 5-HT3-and neurokinin NK1-receptors to induce vomiting. Δ9-THC can inhibit vomiting caused either by the serotonin precursor 5-HTP, or the 5-HT3 receptor selective agonist, 2-methyserotonin. In the current study, we explored whether Δ9-THC and related CB1/CB2 receptor agonists (WIN55,212-2 and CP55,940) inhibit vomiting evoked by SP (50 mg/kg, i.p.) or the NK1 receptor selective agonist GR73632 (5 mg/kg, i.p.). Behavioral methods were employed to determine the antiemetic efficacy of cannabinoids in least shrews. Our results showed that administration of varying doses of Δ9-THC (i.p. or s.c.), WIN55,212-2 (i.p.), or CP55,940 (i.p.) caused significant suppression of SP-evoked vomiting in a dose-dependent manner. When tested against GR73632, Δ9-THC also dose-dependently reduced the evoked emesis. The antiemetic effect of Δ9-THC against SP-induced vomiting was prevented by low non-emetic doses of the CB1 receptor inverse-agonist/antagonist SR141716A (<10 mg/kg). We also found that the NK1 receptor antagonist netupitant can significantly suppress vomiting caused by a large emetic dose of SR141716A (20 mg/kg). In sum, Δ9-THC and related cannabinoids suppress vomiting evoked by the nonselective (SP) and selective (GR73632) neurokinin NK1 receptor agonists via stimulation of cannabinoid CB1 receptors.

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

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