Eugenol and liposome-based nanocarriers loaded with eugenol protect against anxiolytic disorder via down regulation of neurokinin-1 receptors in mice.

Anxiety disorder is a psychiatric disorder characterized by extreme fear or worry. It is highly prevalent worldwide which affects daily life and is also an enormous health burden. Neurokinin 1 receptor (NK1R) is a G protein coupled receptor, expressed in both central and peripheral nervous system, involved in affective behaviors. NK1R has established role in anxiety and it is also an important target for pathogenesis of anxiety disorder. Therefore, it has been hypothesized in previous studies that the blockades of NK1R may have antidepressant and anxiolytic effects. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of NK1R was analyzed by real time RT-PCR. Moreover, the NK1R protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating NK1R protein expression in mice.

Emerging targets for cough therapies; NK1 receptor antagonists.

Cough is mediated by vagal afferent fibres innervating the larynx and proximal airways. Pre-clinical studies suggest that vagal C fibres produce Substance P, one of the tachykinin family of neuropeptides, which has been shown to enhance cough via the neurokinin-1 (NK-1) receptor and studies in animal models have also shown that NK-1 antagonists are effective at blocking induced cough. In the past, tachykinin receptor antagonists have yielded disappointing results in treating asthma and cough, however most of the activity of the agents tested was restricted to the peripheral nervous system and also the outcomes measures evaluating cough not optimal. More recently a small proof of concept study has suggest that aprepitant, an NK-1 antagonist licensed for the prevention of chemotherapy induced nausea and vomiting, might have beneficial effects on cough frequency in patients with lung cancer. In this review we investigate the current evidence for the anti-tussive effect of these therapies and the clinical trials in progress.

One shot NEPA plus dexamethasone to prevent multiple-day chemotherapy in sarcoma patients.

PURPOSE: Chemotherapy-induced nausea and vomiting (CINV) is one of the most feared and disturbing adverse events of cancer treatment associated with decreased adherence to effective chemotherapy regimens. For high-risk soft tissue sarcoma patients, receiving multiple-day chemotherapy (MD-CT), antiemetic guidelines recommend a combination of an NK1 receptor antagonist (NK1-RA), a 5-HT3 receptor antagonist (5HT3-RA), and dexamethasone on each day of the antineoplastic treatment. NEPA is the first oral fixed-dose combination of a highly selective NK1-RA, netupitant, and second-generation 5HT3-RA, palonosetron. So far, no data has been published in literature about the efficacy of a single dose of NEPA in MD-CT. METHODS: We performed a prospective, non-comparative study to assess the efficacy of one shot of NEPA plus dexamethasone in sarcoma patients receiving MD-CT. The primary efficacy endpoint was a complete response (CR: no emesis, no rescue medication) during the overall phase (0-120 h) in cycle 1. The main secondary endpoints were CR during the overall phase of cycles 2 and 3. RESULTS: The primary endpoint was reached in 88.9% of patients. Cycles 2 and 3 overall CR rates were 88.9% and 82.4%, respectively. The antiemetic regimen was well tolerated. CONCLUSIONS: This pilot study showed the benefit of one shot of NEPA to prevent CINV in sarcoma patients receiving MD-chemotherapy.

Substance P and dopamine interact to modulate the distribution of delta-opioid receptors on cholinergic interneurons in the striatum.

It has been recently demonstrated that predictive learning induces a persistent accumulation of delta-opioid receptors (DOPrs) at the somatic membrane of cholinergic interneurons (CINs) in the nucleus accumbens shell (Nac-S). This accumulation is required for predictive learning to influence subsequent choice between goal-directed actions. The current experiments investigated the local neurochemical events responsible for this translocation. We found that (1) local administration of substance P into multiple striatal sub-territories induced DOPr translocation and (2) that this effect was mediated by the NK1 receptor, likely through its expression on CINs. Interestingly, whereas intrastriatal infusion of the D1 agonist chloro-APB reduced the DOPr translocation on CINs and infusion of the D2 agonist quinpirole had no effect, co-administration of both agonists again generated DOPr translocation, suggesting the effect of the D1 agonist alone was due to receptor internalisation. In support of this, local administration of cocaine was found to increase DOPr translocation as was chloro-APB when co-administered with the DOPr antagonist naltrindole. These studies provide the first evidence of delta-opioid receptor translocation in striatal cholinergic interneurons outside of the accumbens shell and suggest that, despite differences in local striatal neurochemical microenvironments, a similar molecular mechanism - involving an interaction between dopamine and SP signalling via NK1R - regulates DOPr translocation in multiple striatal regions. To our knowledge, this represents a novel mechanism by which DOPr distribution is regulated that may be particularly relevant to learning-induced DOPr trafficking.

Effects of NK1 receptors on gastric motor functions and satiation in healthy humans: results from a controlled trial with the NK1 antagonist aprepitant.

Aprepitant, an NK1 receptor antagonist, is approved for the treatment of chemotherapy-induced or postoperative emesis by blocking NK1 receptors in the brain stem vomiting center. The effects of NK1 receptors on gastric functions and postprandial symptoms in humans are unclear; a single, crossover study did not show a significant effect of aprepitant on gastrointestinal transit. Our aim was to compare, in a randomized, double-blind, placebo-controlled, parallel-group study (12 healthy volunteers per group), the effects of aprepitant vs. placebo on gastric emptying of solids (by scintigraphy) with a 320-kcal meal, gastric volumes (GVs; fasting and accommodation by single photon emission-computed tomography ), satiation [maximum tolerated volume (MTV)], and symptoms after a dyspeptogenic meal of Ensure. Aprepitant (125 mg on day 1, followed by 80 mg on days 2-5) or placebo, one tablet daily, was administered for 5 consecutive days. Statistical analysis was by unpaired rank sum test, adjusted for sex difference and body mass index. To assess treatment effects on symptoms, we incorporated MTV in the model. Aprepitant increased fasting, postprandial, and accommodation GV and tended to increase volume to fullness and MTV by ~200 kcal. However, aprepitant increased aggregate symptoms, nausea, and pain scores after ingestion the MTV of Ensure. There was no significant effect of aprepitant on gastric half-emptying time of solids. We conclude that NK1 receptors are involved in the control of GV and in determining postprandial satiation and symptoms. Further studies of the pharmacodynamics and therapeutic role of NK1 receptor antagonists in patients with gastroparesis and dyspepsia are warranted.NEW & NOTEWORTHY Aprepitant increases fasting, postprandial, and accommodation gastric volumes. Aprepitant increases volume to fullness and maximum tolerated volume during a nutrient drink test. NK1 receptors are involved in the control of gastric volume and in determining postprandial satiation and symptoms.

Antagonism of the Neurokinin-1 Receptor Improves Survival in a Mouse Model of Sepsis by Decreasing Inflammation and Increasing Early Cardiovascular Function.

OBJECTIVES: Sepsis remains a serious clinical problem despite intensive research efforts and numerous attempts to improve outcome by modifying the inflammatory response. Substance P, the principal ligand for the neurokinin-1 receptor, is a potent proinflammatory mediator that exacerbates inflammatory responses and cardiovascular variables in sepsis. DESIGN: The current study examined whether inhibition of the neurokinin-1 receptor with a specific antagonist (CJ-12,255) would improve survival in the cecal ligation and puncture model of sepsis in adult female outbred mice. SETTING: University basic science research laboratory. MEASUREMENTS AND MAIN RESULTS: Neurokinin-1 receptor treatment at the initiation of sepsis improved survival in cecal ligation and puncture sepsis (neurokinin-1 receptor antagonist survival = 79% vs vehicle = 54%). Delaying therapy for as little as 8 hours postcecal ligation and puncture failed to provide a survival benefit. Neurokinin-1 receptor antagonist treatment did not prevent the sepsis-induced decrease in circulating WBCs, augment the early (6 hr postcecal ligation and puncture) recruitment of inflammatory cells to the peritoneum, or improve phagocytic cell killing of pathogens. However, the neurokinin-1 receptor antagonist significantly reduced both circulating and peritoneal cytokine concentrations. In addition, the cardiovascular variable, pulse distension (a surrogate for stroke volume) was improved in the neurokinin-1 receptor antagonist group during the first 6 hours of sepsis, and there was a significant reduction in loss of fluid into the intestine. CONCLUSION: These data show that early activation of the neurokinin-1 receptor by substance P decreases sepsis survival through multiple mechanisms including depressing stroke volume, increasing fluid loss into the intestine, and increasing inflammatory cytokine production.

Altered circadian feeding behavior and improvement of metabolic syndrome in obese Tac1-deficient mice.

BACKGROUND: Metabolic function is regulated by the interplay of central and peripheral factors that ultimately regulate food intake (FI) and energy expenditure. The tachykinin substance P (SP) has been identified as a novel regulator of energy balance, however, the mechanisms underlying this effect are ill-defined and conflicting data regarding the role of SP on FI have been reported by different groups. OBJECTIVE: To further characterize the metabolic role of the Tac1 gene products (SP and neurokinin A) in mice through a series of genetic, metabolic and behavioral studies in Tac1-deficient mice. RESULTS: Tac1-/- mice are leaner than controls and display reduced FI and altered feeding circadian rhythm, supported by disrupted expression of the clock genes Cry1/2, Per1/2 in the suprachiasmatic nucleus, mediobasal hypothalamus (MBH) and liver, as well as increased proopiomelanocortin expression in the MBH. Tac1 ablation induced resistance to obesity, improved glucose tolerance, prevented insulin resistance under high-fat diet, increased activation of brown adipose tissue and improved hepatic steatosis. Moreover, deletion of Tac1 in ob/ob mice ameliorated body weight gain in females only but was sufficient to decrease fat and triglyceride content in the liver of males. CONCLUSIONS: These results provide further evidence that Tac1 controls circadian feeding behavior and metabolism in mice through mechanisms that involve the regulation of the melanocortin system. In addition, these studies suggest that the blockade of SP may offer a new method to treat metabolic syndrome.

Substance P promotes hepatic stellate cell proliferation and activation via the TGF-ß1/Smad-3 signaling pathway.

Prolonged activation and proliferation of hepatic stellate cells (HSCs) usually results in the initiation and progression of liver fibrosis following injury. Recent studies have shown that Substance P (SP) participates in the development of fibrosis. However, whether SP is involved in liver fibrosis, especially in the activation and proliferation of HSCs, is largely unknown. In the present study, we measured the effects of a series of concentrations of SP on the cell viability and activation of HSC-T6 cells and LX2 cells. The underlying mechanism was also investigated. We found that SP effectively increased cell viability, both in an MTT assay (p<0.05) and in a lactate dehydrogenase activity assay (LDH) (p<0.05). Moreover, SP upregulated the protein expression of α-SMA and Collagen I (both p<0.05) and decreased the release of lipid droplets (LDs) (p<0.05), all of which are associated with HSC activation. Apoptosis analysis revealed that SP can attenuate the increase of cell apoptosis induced by serum withdrawal (p<0.05). Furthermore, these effects were all blocked by an SP receptor antagonist, L732138. More importantly, L732138 decreased the activation of the TGF-ß1/Smad3 signaling pathway, which is highly associated with liver fibrosis. Taken together, our results demonstrate that SP can promote HSC proliferation and induce HSC activation via the TGF-ß1/Smad3 signaling pathway.

EGFR-TKI, erlotinib, causes hypomagnesemia, oxidative stress, and cardiac dysfunction: attenuation by NK-1 receptor blockade.

To determine whether the epidermal growth factor receptor tyrosine kinase inhibitor, erlotinib may cause hypomagnesemia, inflammation, and cardiac stress, erlotinib was administered to rats (10 mg · kg(-1)· d(-1)) for 9 weeks. Plasma magnesium decreased progressively between 3 and 9 weeks (-9% to -26%). Modest increases in plasma substance P (SP) occurred at 3 (27%) and 9 (25%) weeks. Neutrophil superoxide-generating activity increased 3-fold, and plasma 8-isoprostane rose 210%, along with noticeable appearance of cardiac perivascular nitrotyrosine. The neurokinin-1 (NK-1) receptor antagonist, aprepitant (2 mg · kg(-1) · d(-1)), attenuated erlotinib-induced hypomagnesemia up to 42%, reduced circulating SP, suppressed neutrophil superoxide activity and 8-isoprostane elevations; cardiac nitrotyrosine was diminished. Echocardiography revealed mild to moderately decreased left ventricular ejection fraction (-11%) and % fractional shortening (-17%) by 7 weeks of erlotinib treatment and significant reduction (-17.5%) in mitral valve E/A ratio at week 9 indicative of systolic and early diastolic dysfunction. Mild thinning of the left ventricular posterior wall suggested early dilated cardiomyopathy. Aprepitant completely prevented the erlotinib-induced systolic and diastolic dysfunction and partially attenuated the anatomical changes. Thus, chronic erlotinib treatment does induce moderate hypomagnesemia, triggering SP-mediated oxidative/inflammation stress and mild-to-moderate cardiac dysfunction, which can largely be corrected by the administration of the SP receptor blocker.

Meta-analysis of adjunctive non-NK1 receptor antagonist medications for the control of acute and delayed chemotherapy-induced nausea and vomiting.

PURPOSE: Chemotherapy-induced nausea and vomiting (CINV) is a distressing chemotherapy-induced symptom that may adversely impact the quality of life of cancer patients. METHODS: We conducted a systematic search of the Pubmed, Bireme, and Cochrane databases for randomized clinical trials that were published in English and that evaluated the combination of adjunctive non-neurokinin 1 (NK1) antagonist drugs (i.e., neuroleptics, anticonvulsants, benzodiazepines, and cannabinoids) with 5-hydroxytryptamine 3 (5-HT3) antagonists for adult cancer patients who were scheduled to receive moderate or highly emetogenic chemotherapy. We employed the Review Manager (RevMan) Computer program Version 5.2 for statistical calculations. RESULTS: We included 13 studies with a total of 1,669 patients. We observed a higher complete protection for acute CINV with adjunctive medications (risk ratio (RR) = 0.55; 95% confidence interval (CI) 0.30-1.01; p = 0.05; I2 = 47%), which was not the case for the delayed period (RR = 0.89; 95% CI 0.73-1.10, p = 0.29, I2 = 15%). We also observed that these adjunctive medications significantly increased the complete control of nausea (RR = 0.72; 95% CI 0.55-0.95; p = 0.02, I2 = 83%) and vomiting (RR = 0.61; 95% CI 0.50-0.75; p < 0.00001; I2 = 60%). There was no subgroup analysis evidence of the superiority of any single group of adjunctive medications. CONCLUSIONS: We conclude that adjunctive non-NK1 antagonist medications may be useful for CINV control. Prospective randomized studies incorporating these low-cost medications into new regimens combining 5-HT3 and NK1 antagonists may be warranted.

Substance P is essential for maintaining gut muscle contractility: a novel role for coneurotransmission revealed by botulinum toxin.

Substance P (SP) is commonly coexpressed with ACh in enteric motor neurons, and, according to the classical paradigm, both these neurotransmitters excite smooth muscle via parallel pathways. We hypothesized that, in addition, SP was responsible for maintaining the muscular responsiveness to ACh. We tested this hypothesis by using botulinum toxin (BoNT/A), a known blocker of vesicular release of neurotransmitters including ACh and neuropeptides. BoNT/A was injected into rat pyloric sphincter in different doses; as control we used boiled BoNT/A. At the desired time point, pylorus was dissected out and pyloric contractility was measured ex vivo in an organ bath and by measuring phosphorylation of myosin light chain 20 (MLC20). BoNT/A (10 IU) significantly reduced the response of pyloric muscle to exogenous ACh, an effect that was accompanied by reduced MLC20 phosphorylation in the muscle. Both effects were reversed by exogenous SP. CP-96345, a NK1 receptor antagonist, blocked the ability of exogenous SP to reverse the cholinergic hyporesponsiveness as well as the reduction in MLC20 phosphorylation induced by BoNT/A. In conclusion, we have identified a novel role for SP as a coneurotransmitter that appears to be important for the maintenance of muscular responsiveness to the principal excitatory neurotransmitter, ACh. These results also provide new insight into the effects of botulinum toxin on the enteric nervous system and gastrointestinal smooth muscle.

The function of P2X3 receptor and NK1 receptor antagonists on cyclophosphamide-induced cystitis in rats.

PURPOSE: The purpose of the study is to explore the function of P2X3 and NK1 receptors antagonists on cyclophosphamide (CYP)-induced cystitis in rats. METHODS: Sixty female Sprague-Dawley (SD) rats were randomly divided into three groups. The rats in the control group were intraperitoneally (i.p.) injected with 0.9% saline (4 ml/kg); the rats in the model group were i.p. injected with CYP (150 mg/kg); and the rats in the intervention group were i.p. injected with CYP with subsequently perfusion of bladder with P2X3 and NK1 receptors' antagonists, Suramin and GR 82334. Spontaneous pain behaviors following the administration of CYP were observed. Urodynamic parameters, bladder pressure-volume curve, maximum voiding pressure (MVP), and maximum cystometric capacity (MCC), were recorded. Pathological changes in bladder tissue were observed. Immunofluorescence was used to detect the expression of P2X3 and NK1 receptors in bladder. RESULTS: Cyclophosphamide treatment increased the spontaneous pain behaviors scores. The incidence of bladder instability during urine storage period of model group was significantly higher than intervention group (χ(2) = 7.619, P = 0.007) and control group (χ(2) = 13.755, P = 0.000). MCC in the model group was lower than the control and intervention groups (P < 0.01). Histological changes evident in model and intervention groups rats' bladder included edema, vasodilation, and infiltration of inflammatory cells. In model group, the expression of P2X3 receptor increased in urothelium and suburothelium, and NK1 receptor increased in suburothelium, while the expression of them in intervention group was lower. CONCLUSIONS: In CYP-induced cystitis, the expression of P2X3 and NK1 receptors increased in urothelium and/or suburothelium. Perfusion of bladder with P2X3 and NK1 receptors antagonists ameliorated the bladder function.

Efficacy and safety of repeated dosing of netupitant, a neurokinin-1 receptor antagonist, in treating overactive bladder.

AIM: NK-1 receptors in sensory nerves, the spinal cord and bladder smooth muscle participate in complex sensory mechanisms that regulate bladder activity. This study was designed to assess the efficacy and safety of a new NK-1 receptor antagonist, netupitant, in patients with OAB. METHODS: This was a phase II, multicenter, double-blind study in which adults with OAB symptoms >6 months were randomized to receive 1 of 3 doses of netupitant (50, 100, 200 mg) or placebo once daily for 8 weeks. The primary efficacy endpoint was percentage change from baseline in average number of daily micturitions at week 8. Urinary incontinence, urge urinary incontinence (UUI), and urgency episodes were also assessed. RESULTS: The primary efficacy endpoint was similar in the treatment groups (-13.85 for placebo to -16.17 in the netupitant 200 mg group) with no statistically significant differences between netupitant and placebo. The same was true for most secondary endpoints although a significant difference for improvement in UUI episodes and a trend for the greatest decrease in urgency episodes were seen in the netupitant 100 mg group. Netupitant was well tolerated with most treatment emergent adverse events (AEs) being mild. While the overall incidence of AEs increased with netupitant dose, there was no evidence for this dose dependency based on relationship to treatment, intensity, or time to onset. CONCLUSIONS: The study failed to demonstrate superiority of netupitant versus placebo in decreasing OAB symptoms, despite a trend favoring netupitant 100 mg. There were no safety concerns with daily administration of netupitant over 8 weeks.

Inhibitors of endopeptidase and angiotensin-converting enzyme lead to an amplification of the morphological changes and an upregulation of the substance P system in a muscle overuse model.

BACKGROUND: We have previously observed, in studies on an experimental overuse model, that the tachykinin system may be involved in the processes of muscle inflammation (myositis) and other muscle tissue alterations. To further evaluate the significance of tachykinins in these processes, we have used inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), substances which are known to terminate the activity of various endogenously produced substances, including tachykinins. METHODS: Injections of inhibitors of NEP and ACE, as well as the tachykinin substance P (SP), were given locally outside the tendon of the triceps surae muscle of rabbits subjected to marked overuse of this muscle. A control group was given NaCl injections. Evaluations were made at 1 week, a timepoint of overuse when only mild inflammation and limited changes in the muscle structure are noted in animals not treated with inhibitors. Both the soleus and gastrocnemius muscles were examined morphologically and with immunohistochemistry and enzyme immunoassay (EIA). RESULTS: A pronounced inflammation (myositis) and changes in the muscle fiber morphology, including muscle fiber necrosis, occurred in the overused muscles of animals given NEP and ACE inhibitors. The morphological changes were clearly more prominent than for animals subjected to overuse and NaCl injections (NaCl group). A marked SP-like expression, as well as a marked expression of the neurokinin-1 receptor (NK-1R) was found in the affected muscle tissue in response to injections of NEP and ACE inhibitors. The concentration of SP in the muscles was also higher than that for the NaCl group. CONCLUSIONS: The observations show that the local injections of NEP and ACE inhibitors led to marked SP-like and NK-1R immunoreactions, increased SP concentrations, and an amplification of the morphological changes in the tissue. The injections of the inhibitors thus led to a more marked myositis process and an upregulation of the SP system. Endogenously produced substances, out of which the tachykinins conform to one substance family, may play a role in mediating effects in the tissue in a muscle that is subjected to pronounced overuse.

Substance P reduces TNF-α-induced apoptosis in human tenocytes through NK-1 receptor stimulation.

BACKGROUND: It has been hypothesised that an upregulation of the neuropeptide substance P (SP) and its preferred receptor, the neurokinin-1 receptor (NK-1 R), is a causative factor in inducing tenocyte hypercellularity, a characteristic of tendinosis, through both proliferative and antiapoptotic stimuli. We have demonstrated earlier that SP stimulates proliferation of human tenocytes in culture. AIM: The aim of this study was to investigate whether SP can mediate an antiapoptotic effect in tumour necrosis factor-α (TNF-α)-induced apoptosis of human tenocytes in vitro. RESULTS: A majority (approximately 75%) of tenocytes in culture were immunopositive for TNF Receptor-1 and TNF Receptor-2. Exposure of the cells to TNF-α significantly decreased cell viability, as shown with crystal violet staining. TNF-α furthermore significantly increased the amount of caspase-10 and caspase-3 mRNA, as well as both BID and cleaved-poly ADP ribosome polymerase (c-PARP) protein. Incubation of SP together with TNF-α resulted in a decreased amount of BID and c-PARP, and in a reduced lactate dehydrogenase release, as compared to incubation with TNF-α alone. The SP effect was blocked with a NK-1 R inhibitor. DISCUSSION: This study shows that SP, through stimulation of the NK-1 R, has the ability to reduce TNF-α-induced apoptosis of human tenocytes. Considering that SP has previously been shown to stimulate tenocyte proliferation, the study confirms SP as a potent regulator of cell-turnover in tendon tissue, capable of stimulating hypercellularity through different mechanisms. This gives further support for the theory that the upregulated amount of SP seen in tendinosis could contribute to hypercellularity.

Hemokinin-1 stimulates prostaglandin E2 production in human colon through activation of cyclooxygenase-2 and inhibition of 15-hydroxyprostaglandin dehydrogenase.

Hemokinin-1 (HK-1) is a newly identified tachykinin, originating from the immune system rather than neurons, and may participate in the immune and inflammatory response. In colonic mucosa of patients with inflammatory bowel disease (IBD), up-regulation of the TAC4 gene encoding HK-1 and increased production of prostaglandin E2 (PGE2) occur. Our aim was to examine the mechanistic link between human HK-1 and PGE2 production in normal human colon. Exogenous HK-1 (0.1 µM) for 4 h evoked an increased PGE2 release from colonic mucosal and muscle explants by 10- and 3.5-fold, respectively, compared with unstimulated time controls. The HK-1-stimulated PGE2 release was inhibited by the tachykinin receptor antagonists (S)1-2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl-4-phenyl-l azonia-bicyclo[2.2.2]octane (SR140333) [neurokinin-1 (NK1)] and N-[(2S)-4-(4-acetamido-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)butyl]-N-methylbenzamide (SR48968) [neurokinin-2 (NK2)] and was also inhibited by the cyclooxygenase (COX)-2 inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide) (NS-398) but not by the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560). A parallel study with substance P showed similar results. Molecular studies with HK-1-treated explants demonstrated a stimulatory effect on COX-2 expression at both transcription and protein levels. It is noteworthy that this was coupled with HK-1-induced down-regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) mRNA and protein expression. Immunoreactivity for 15-PGDH occurred on inflammatory cells, epithelial cells, platelets, and ganglia. This finding provides an additional mechanism for HK-1-evoked PGE2 increase, in which HK-1 may interfere with the downstream metabolism of PGE2 by suppressing 15-PGDH expression. In conclusion, our results uncover a novel inflammatory role for HK-1, which signals via NK1 and NK2 receptors to regulate PGE2 release from human colonic tissue, and may further explain a pathological role for HK-1 in IBD when abnormal levels of PGE2 occur.

Substance P receptor activation induces downregulation of the AMPA receptor functionality in cortical neurons from a genetic model of Amyotrophic Lateral Sclerosis.

Substance P (SP), a neuropeptide member of the tachykinin (TK) family, has a functional role both in physiological and pathological conditions, including Amyotrophic Lateral Sclerosis disease. One hypothesis of the selective motor neuron death in ALS involves the excitatory neurotransmitter glutamate, because these neurons are extremely susceptible to excessive stimulation of AMPA receptors. It has been reported that SP exerts its action against a variety of insults including excitotoxicity, and that altered levels of SP have been observed in the cerebrospinal fluid (CSF) of patients with ALS. Here we have analyzed the interaction between SP and AMPA receptor functionality, both in Control cortical neurons in culture and in those obtained from a genetic mouse model of ALS (G93A). Our studies demonstrate that SP reduces the kainate-activated currents in Control and G93A neurons and that this reduction is significantly higher in the mutated neurons. SP effect is mediated by its receptor NK1 because GR 82334 (5 µM), a NK1 competitive antagonist, is able to suppress the current reduction. Analysis of miniature excitatory postsynaptic currents (mEPSCs) in Control and G93A neurons indicates that SP (200 nM) is able to significantly decrease the mEPSC amplitudes in G93A neurons, whereas it is ineffective on Control mEPSCs. Western blotting experiments in cultures and cortical tissues show a higher NK1 expression level in G93A mice compared to that of Control. This is also confirmed by immunocytochemistry experiments in cultured neurons. In addition, the amount of GluR1 subunit AMPA receptors is not modified following SP exposure, indicating a non internalization of the AMPA receptors. Finally, toxicity experiments have revealed that SP is able to rescue G93A cortical cells whereas it is ineffective on those of Control. These findings provide the first evidence of SP having a physiological and protective role in the G93A mouse model of ALS, and may suggest the possible use of SP as a clinical therapeutic treatment.

Regulation of spinal substance p release by intrathecal calcium channel blockade.

BACKGROUND: The authors investigated the role of different voltage-sensitive calcium channels expressed at presynaptic afferent terminals in substance P release and on nociceptive behavior evoked by intraplantar formalin by examining the effects of intrathecally delivered N- (ziconotide), T- (mibefradil), and L-type voltage-sensitive calcium channel blockers (diltiazem and verapamil). METHODS: Rats received intrathecal pretreatment with saline or doses of morphine, ziconotide, mibefradil, diltiazem, or verapamil. The effect of these injections upon flinching evoked by intraplantar formalin (5%, 50 µl) was quantified. To assess substance P release, the incidence of neurokinin-1 receptor internalization in the ipsilateral and contralateral lamina I was determined in immunofluorescent-stained tissues. RESULTS: Intrathecal morphine (20 µg), ziconotide (0.3, 0.6, and 1 µg), mibefradil (100 µg, but not 50 µg), diltiazem (500 µg, but not 300 µg), and verapamil (200 µg, but not 50 and 100 µg) reduced paw flinching in phase 2 compared with vehicle control (P < 0.05), with no effect on phase 1. Ziconotide (0.3, 0.6, and 1 µg) and morphine (20 µg) significantly inhibited neurokinin-1 receptor internalization (P < 0.05), but mibefradil, diltiazem, and verapamil at the highest doses had no effect. CONCLUSION: These results emphasize the role in vivo of N-type but not T- and L-type voltage-sensitive calcium channel blockers in mediating the stimulus-evoked substance P release from small primary afferents and suggest that T- and L-type voltage-sensitive calcium channel blockers exert antihyperalgesic effects by an action on other populations of afferents or mechanisms involving postsynaptic excitability.

In vivo assessment of antiemetic drugs and mechanism of lycorine-induced nausea and emesis.

Lycorine is the main alkaloid of many Amaryllidaceae and known to cause poisoning with still unknown mechanisms. Longer lasting toxicological core symptoms of nausea and emesis may become a burden for human and animal patients and may result in substantial loss of water and electrolytes. To optimise the only empirical symptomatic antiemetic drug treatment at present, it is important to elucidate the causative involved targets of lycorine-induced emesis. Therefore, in the current study, we have tested the actions of a various antiemetic drugs with selective receptor affinities on lycorine-induced nausea and emesis in vivo in dogs. Beagle dogs were pre-treated in a saline vehicle-controlled crossover and random design with diphenhydramine, maropitant, metoclopramide, ondansetron or scopolamine prior lycorine administration (2 mg/kg subcutaneously). In vivo effects were assessed by a scoring system for nausea and emesis as well as by the number and lag time of emetic events for at least 3 h. Moreover, plasma pharmacokinetic analysis was carried out for ondansetron before and after lycorine injection. The data show that histaminergic (H1), muscarinic and dopaminergic (D2) receptors are presumably not involved in lycorine-induced emetic effects. While ondansetron significantly reduced the number of emetic events, lycorine-induced emesis was completely blocked by maropitant. Only ondansetron also significantly decreased the level of nausea and was able to prolong the lag time until onset of emesis suggesting a preferential participation of 5-HT3 receptors in lycorine-induced nausea. Thus, it is the first in vivo report evidencing that predominantly neurokinin-1 (NK1) and to a lesser extent 5-hydroxytryptamine 3 (5-HT3) receptors are involved in lycorine-induced emesis facilitating a target-oriented therapy.