Modification of the phenyl ring B of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates by pyridinyl moiety leads to novel antimitotics targeting the colchicine-binding site.

A series of 8 novel pyridinyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PYRIB-SOs) were designed, prepared and evaluated for their mechanism of action. PYRIB-SOs were found to have antiproliferative activity in the nanomolar to submicromolar range on several breast cancer cell lines. Moreover, subsequent biofunctional assays indicated that the most potent PYRIB-SOs 1-3 act as antimitotics binding to the colchicine-binding site (C-BS) of α, ß-tubulin and that they arrest the cell cycle progression in the G2/M phase. Microtubule immunofluorescence and tubulin polymerisation assay confirm that they disrupt the cytoskeleton through inhibition of tubulin polymerisation as observed with microtubule-destabilising agents. They also show good overall theoretical physicochemical, pharmacokinetic and druglike properties. Overall, these results show that PYRIB-SOs is a new family of promising antimitotics to be further studied in vivo for biopharmaceutical and pharmacodynamic evaluations.

Assessing the Impact of Chemical Algae Management Strategies on Anurans and Aquatic Communities.

Pond management with chemical and biological agents that reduce overgrowth of algae is an important means of maintaining water quality in residential ponds, yet the effects on nontarget species are not fully understood. We assessed the impact of Aquashade (a common nontoxic pond dye) and copper sulfate (a toxic algaecide) on American toad (Anaxyrus americanus), northern leopard frog (Lithobates pipiens), and Cope's gray treefrog (Hyla chrysoscelis) metamorphosis in outdoor mesocosm experiments. We also evaluated the relative impact of tadpole grazing versus chemical treatment on phytoplankton and periphyton abundance. We found no significant effects of pond management treatment on anuran metamorphosis, suggesting that addition of Aquashade and copper sulfate at tested concentrations does not significantly impact anurans under these experimental conditions. Interestingly, we found that the presence of tadpoles more strongly reduced algal abundance than Aquashade or copper sulfate by significantly decreasing phytoplankton and periphyton abundance over time. The present study suggests that anuran tadpoles may be effective at maintaining water quality, and that Aquashade and copper sulfate may have minimal effects on amphibian metamorphosis. Environ Toxicol Chem 2023;42:213-224. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

New 2-[(4-Amino-6-N-substituted-1,3,5-triazin-2-yl)methylthio]-N-(imidazolidin-2-ylidene)-4-chloro-5-methylbenzenesulfonamide Derivatives, Design, Synthesis and Anticancer Evaluation.

In the search for new compounds with antitumor activity, new potential anticancer agents were designed as molecular hybrids containing the structures of a triazine ring and a sulfonamide fragment. Applying the synthesis in solution, a base of new sulfonamide derivatives 20-162 was obtained by the reaction of the corresponding esters 11-19 with appropriate biguanide hydrochlorides. The structures of the compounds were confirmed by spectroscopy (IR, NMR), mass spectrometry (HRMS or MALDI-TOF/TOF), elemental analysis (C,H,N) and X-ray crystallography. The cytotoxic activity of the obtained compounds toward three tumor cell lines, HCT-116, MCF-7 and HeLa, was examined. The results showed that some of the most active compounds belonged to the R1 = 4-trifluoromethylbenzyl and R1 = 3,5-bis(trifluoromethyl)benzyl series and exhibited IC50 values ranging from 3.6 µM to 11.0 µM. The SAR relationships were described, indicating the key role of the R2 = 4-phenylpiperazin-1-yl substituent for the cytotoxic activity against the HCT-116 and MCF-7 lines. The studies regarding the mechanism of action of the active compounds included the assessment of the inhibition of MDM2-p53 interactions, cell cycle analysis and apoptosis induction examination. The results indicated that the studied compounds did not inhibit MDM2-p53 interactions but induced G0/G1 and G2/M cell cycle arrest in a p53-independent manner. Furthermore, the active compounds induced apoptosis in cells harboring wild-type and mutant p53. The compound design was conducted step by step and assisted by QSAR models that correlated the activity of the compounds against the HCT-116 cell line with molecular descriptors.

Fungal Keratitis: Diagnostic Characteristics of the Potassium Hydroxide Preparation With Calcofluor White in Northern California.

PURPOSE: The incidence of fungal keratitis demonstrates significant geographic and climatic variation. We report on the characteristics of the potassium hydroxide/calcofluor white (KOH-CFW) preparation observed at a tertiary center in Northern California, a region with a low incidence of fungal keratitis. METHODS: Culture-proven cases of microbial keratitis during a 5-year period were retrospectively reviewed. The sensitivity, specificity, and posttest probabilities were determined for the KOH-CFW assay. These results were compared with documented clinical impression and values reported in the literature. RESULTS: Three hundred three of 368 episodes of microbial keratitis during the study period documented the results of a fungal culture, KOH-CFW assay, and a clinical impression. Twenty-one (6.9%) of these cultures were positive for fungal organisms. The sensitivity and specificity of the KOH-CFW test were 29% and 93%, respectively. Clinicians' initial clinical impression based solely on patients' history and examination, without the aid of any histopathologic or biochemical test results, demonstrated a sensitivity and specificity of 33% and 89%, respectively. CONCLUSIONS: The observed sensitivity and specificity of the KOH-CFW preparation are significantly lower than many previously reported values. In regions with low incidence of fungal keratitis, the KOH-CFW preparation may have diagnostic performance similar to that of the clinical impression formed only on the basis of history and physical examination.

Branched alkyl of phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates as unique cytochrome P450 1A1-activated antimitotic prodrugs: Biological evaluation and mechanism of bioactivation.

We recently discovered a new family of prodrugs deriving from phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) bioactivatable by cytochrome P450 1A1 (CYP1A1) into potent antimitotics referred to as phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs). PAIB-SOs display significant selectivity toward human breast cancer cells based on the N-dealkylation of PAIB-SOs into their corresponding PIB-SOs by CYP1A1. In this study, we have evaluated the molecular mechanism of the bioactivation of PAIB-SOs into PIB-SOs by branching the linear alkyl chain on the imidazolidin-2-one (IMZ) moiety of PAIB-SOs by branched alkyl groups such as isopropyl, isobutyl and sec-butyl. Our results show that PAIB-SOs bearing an isobutyl group on the IMZ moiety and either a methoxy, a chloro or a bromo group at positions 3, 3,5 or 3,4,5 on the aromatic ring B exhibit antiproliferative activity ranging from 0.13 to 6.9 µM and selectivity toward MCF7 and MDA-MB-468 mammary cancer cells comparatively to other cell lines tested. Moreover, the most potent and selective PAIB-SOs bearing an isobutyl group and either a 3,5-Cl (44), 3,5-Br (45) or a 3,4,5-OMe (46) on the IMZ moiety exhibit antiproliferative activity in the sub-micromolar range and high selectivity ratios toward mammary cancer cells. They stop the cell cycle of MCF7 cells in the G2/M phase and disrupt their cytoskeleton. Furthermore, our studies evidenced that PAIB-SOs bearing either an isopropyl, a sec-butyl or an isobutyl group are hydroxylated on the carbon atom adjacent to the IMZ (Cα-OH) but only PAIB-SOs bearing an isobutyl group are bioactivated into PIB-SOs. Finally, PAIB-SOs 45 and 46 exhibit low toxicity toward normal cells and chick embryos and are thus promising antimitotic prodrugs highly selective toward CYP1A1-expressing breast cancer cells.

A tale of two multi-focal therapies for glioblastoma: An antibody targeting ELTD1 and nitrone-based OKN-007.

Glioblastoma (GBM) is the most common primary malignant brain tumour in adults. Despite a multimodal treatment response, survival for GBM patients remains between 12 and 15 months. Anti-ELTD1 antibody therapy is effective in decreasing tumour volumes and increasing animal survival in an orthotopic GBM xenograft. OKN-007 is a promising chemotherapeutic agent that is effective in various GBM animal models and is currently in two clinical trials. In this study, we sought to compare anti-ELTD1 and OKN-007 therapies, as single agents and combined, against bevacizumab, a commonly used therapeutic agent against GBM, in a human G55 xenograft mouse model. MRI was used to monitor tumour growth, and immunohistochemistry (IHC) was used to assess tumour markers for angiogenesis, cell migration and proliferation in the various treatment groups. OKN and anti-ELTD1 treatments significantly increased animal survival, reduced tumour volumes and normalized the vasculature. Additionally, anti-ELTD1 was also shown to significantly affect other pro-angiogenic factors such as Notch1 and VEGFR2. Unlike bevacizumab, anti-ELTD1 and OKN treatments did not induce a pro-migratory phenotype within the tumours. Anti-ELTD1 treatment was shown to be as effective as OKN therapy. Both OKN and anti-ELTD1 therapies show promise as potential single-agent multi-focal therapies for GBM patients.

H2S Donors and Their Use in Medicinal Chemistry.

Hydrogen sulfide (H2S) is a ubiquitous gaseous signaling molecule that has an important role in many physiological and pathological processes in mammalian tissues, with the same importance as two others endogenous gasotransmitters such as NO (nitric oxide) and CO (carbon monoxide). Endogenous H2S is involved in a broad gamut of processes in mammalian tissues including inflammation, vascular tone, hypertension, gastric mucosal integrity, neuromodulation, and defense mechanisms against viral infections as well as SARS-CoV-2 infection. These results suggest that the modulation of H2S levels has a potential therapeutic value. Consequently, synthetic H2S-releasing agents represent not only important research tools, but also potent therapeutic agents. This review has been designed in order to summarize the currently available H2S donors; furthermore, herein we discuss their preparation, the H2S-releasing mechanisms, and their -biological applications.

Leptin promotes glycolytic metabolism to induce dendritic cells activation via STAT3-HK2 pathway.

Leptin is over-secreted in many autoimmune diseases, which can promote dendritic cells (DCs) maturation and up-regulate the expression of inflammatory cytokines, but the underlying mechanisms are not fully elucidated. Considering the major role of leptin in maintaining energy balance and the significant role of glycolysis in DCs activation, our study aims to investigate whether leptin promotes the activation of DCs via glycolysis and its underlying mechanisms. We demonstrated that leptin promoted the activation of DCs, including up-regulating the expression of co-stimulatory molecules and inflammatory cytokines, enhancing the proliferation and T helper 17 (Th17) cell ratio in peripheral blood mononuclear cells (PBMC) co-cultured with leptin-stimulated DCs. Leptin also enhanced DCs glycolysis with increased glucose consumption, lactate production, and the expression of hexokinase 2 (HK2). In addition, the activation of DCs stimulated by leptin could be inhibited by the glycolysis inhibitor 2-deoxy-d-glucose (2-DG). To explore the signaling pathways involved in leptin-induced HK2 expression, we observed that the inhibitors of STAT3 (NSC74859) could repress the enhancement of HK2 triggered by leptin stimulation. Therefore, our results indicated that leptin promoted glycolytic metabolism to induce DCs activation via STAT3-HK2 pathway.

Pharmacologic inhibiting STAT3 delays the progression of kidney fibrosis in hyperuricemia-induced chronic kidney disease.

AIMS: Kidney fibrosis is a histological hallmark of chronic kidney disease (CKD), where hyperuricemia is a key independent risk factor. Considerable evidence indicated that STAT3 is one of the crucial signaling pathways in the progression of kidney fibrosis. Here, we investigated that pharmacological blockade of STAT3 delayed the progression of renal fibrosis in hyperuricemia-induced CKD. MAIN METHODS: In the study, we used the mixture of adenine and potassium oxonate to perform kidney injury and fibrosis in hyperuricemic mice, accompanied by STAT3 activation in tubular and interstitial cells. KEY FINDINGS: Treatment with STAT3 inhibitor S3I-201 improved renal dysfunction, reduced serum uric acid level, and delayed the progression of kidney fibrosis. Furthermore, S3I-201 could suppress fibrotic signaling pathway of TGF-ß/Smads, JAK/STAT and NF-κB, as well as inhibit the expression of multiple profibrogenic cytokines/chemokines in the kidneys of hyperuricemic mice. SIGNIFICANCE: These data suggested that STAT3 inhibition was a potent anti-fibrotic strategy in hyperuricemia-related CKD.

Targeting Purinergic Receptor P2RX1 Modulates Intestinal Microbiota and Alleviates Inflammation in Colitis.

Inflammatory bowel disease (IBD) remains one of the most prevalent gastrointestinal diseases worldwide. Purinergic signaling has emerged as a promising therapeutic target of inflammation-associated diseases. However, little is known about the specific roles of purinergic receptors in IBD. In the present study, expression profile of purinergic receptors was screened in the public Gene Expression Omnibus (GEO) datasets, and we found that expression of P2RX1 was significantly upregulated in inflamed colon tissues. Then, purinergic receptor P2RX1 was genetically ablated in the background of C57BL/6 mice, and dextran sulfate sodium (DSS) was used to induce mice colitis. RNA sequencing results of colon tissues showed that genetic knockout of P2RX1 suppressed the inflammation responses in DSS-induced mice colitis. Flow cytometry indicated that neutrophil infiltration was inhibited in P2RX1 ablated mice. 16S ribosomal DNA sequencing revealed major differences of intestinal microbiota between WT and P2RX1 ablated mice. Functional metagenomics prediction indicated that the indole alkaloid biogenesis pathway was upregulated in P2RX1 gene ablated mice. Further studies revealed that microbiota metabolites (indole alkaloid)-involved aryl hydrocarbon receptor (AhR)/IL-22 axis was associated with the beneficial effects of P2RX1 ablation. Finally, we found that a specific P2RX1 inhibitor succeeded to improve the therapeutic efficiency of anti-TNF-α therapy in DSS-induced mice colitis. Therefore, our study suggests that targeting purinergic receptor P2RX1 may provide novel therapeutic strategy for IBD.

LRG1 facilitates corneal fibrotic response by inducing neutrophil chemotaxis via Stat3 signaling in alkali-burned mouse corneas.

Leucine-rich α-2-glycoprotein-1 (LRG1) is a novel profibrotic factor that modulates transforming growth factor-ß (TGF-ß) signaling. However, its role in the corneal fibrotic response remains unknown. In the present study, we found that the LRG1 level increased in alkali-burned mouse corneas. In the LRG1-treated alkali-burned corneas, there were higher fibrogenic protein expression and neutrophil infiltration. LRG1 promoted neutrophil chemotaxis and CXCL-1 secretion. Conversely, LRG1-specific siRNA reduced fibrogenic protein expression and neutrophil infiltration in the alkali-burned corneas. The clearance of neutrophils effectively attenuated the LRG1-enhanced corneal fibrotic response, whereas the presence of neutrophils enhanced the effect of LRG1 on the fibrotic response in cultured TKE2 cells. In addition, the topical application of LRG1 elevated interleukin-6 (IL-6) and p-Stat3 levels in the corneal epithelium and in isolated neutrophils. The clearance of neutrophils inhibited the expression of p-Stat3 and IL-6 promoted by LRG1 in alkali-burned corneas. Moreover, neutrophils significantly increased the production of IL-6 and p-Stat3 promoted by LRG1 in TKE2 cells. Furthermore, the inhibition of Stat3 signaling by S3I-201 decreased neutrophil infiltration and alleviated the LRG1-enhanced corneal fibrotic response in the alkali-burned corneas. S3I-201 also reduced LRG1 or neutrophil-induced fibrotic response in TKE2 cells. In conclusion, LRG1 promotes the corneal fibrotic response by stimulating neutrophil infiltration via the modulation of the IL-6/Stat3 signaling pathway. Therefore, LRG1 could be targeted as a promising therapeutic strategy for patients with corneal fibrosis.

Design, synthesis and biological activity of bis-sulfonyl-BODIPY probes for tumor cell imaging.

In recent years, BODIPY derivatives have become one of the research hotspots in the field of bioprobes, but most of them have the problems of poor hydrophilicity, low biocompatibility and no targeting. In this paper, novel ethylenediamine bridging bis-sulfonyl-BODIPY fluorescent probes were successfully designed and synthesized to solve these problems; What's more, the cytotoxicity analysis, cell imaging, in vivo imaging and apoptosis experiments were carried out. Ethylenediamine bridges and oxygen-rich sulfonyl groups made such probes had certain hydrophilicity, so they could be dissolved in dimethylsulfoxide and methanol. The IC50 value of compound 9 in HCT-116 cells was 93.12 ± 6.33 µM, and in HeLa cells was 89.09 ± 11.84 µM, which indicating that the probe had certain inhibitory effect on cancer cells. The excellent biocompatibility and potential tumor targeting properties of the compound were clearly observed in cell and mice imaging. This study is of great significance for the rational design of novel targeted BODIPY probes with good hydrophilicity and biocompatibility.

Development of succinimide-based inhibitors for the mitochondrial rhomboid protease PARL.

While the biochemistry of rhomboid proteases has been extensively studied since their discovery two decades ago, efforts to define the physiological roles of these enzymes are ongoing and would benefit from chemical probes that can be used to manipulate the functions of these proteins in their native settings. Here, we describe the use of activity-based protein profiling (ABPP) technology to conduct a targeted screen for small-molecule inhibitors of the mitochondrial rhomboid protease PARL, which plays a critical role in regulating mitophagy and cell death. We synthesized a series of succinimide-containing sulfonyl esters and sulfonamides and discovered that these compounds serve as inhibitors of PARL with the most potent sulfonamides having submicromolar affinity for the enzyme. A counterscreen against the bacterial rhomboid protease GlpG demonstrates that several of these compounds display selectivity for PARL over GlpG by as much as two orders of magnitude. Both the sulfonyl ester and sulfonamide scaffolds exhibit reversible binding and are able to engage PARL in mammalian cells. Collectively, our findings provide encouraging precedent for the development of PARL-selective inhibitors and establish N-[(arylsulfonyl)oxy]succinimides and N-arylsulfonylsuccinimides as new molecular scaffolds for inhibiting members of the rhomboid protease family.

Feed-forward activation of STAT3 signaling limits the efficacy of c-Met inhibitors in esophageal squamous cell carcinoma (ESCC) treatment.

c-Hepatocyte growth factor receptor (Met) inhibitors have demonstrated clinical benefits in some types of solid tumors. However, the efficacy of c-Met inhibitors in esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we discovered that c-Met inhibitors induced "Signal Transducer and Activator of Transcription (STAT3)-addiction" in ESCC cells, and the feedback activation of STAT3 in ESCC cells limits the tumor response to c-Met inhibition. Mechanistically, c-Met inhibition increased the autocrine of several cytokines, including CCL2, interleukin 8, or leukemia inhibitory factor, and facilitated the interactions between the receptors of these cytokines and Janus Kinase1/2 (JAK1/2) to resultantly activate JAKs/STAT3 signaling. Pharmacological inhibition of c-Met together with cytokines/JAKs/STAT3 axis enhanced cancer cells regression in vitro. Importantly, combined c-Met and STAT3 inhibitors synergistically suppressed tumor growth and promoted the apoptosis of tumor cells without producing systematic toxicity. These findings suggest that inhibition of the STAT3 feedback loop may augment the response to c-Met inhibitors via the STAT3-mediated oncogene addiction in ESCC cells.

Cardiopreventive capacity of a novel (E)-N'-(1-(7-methoxy-2-oxo-2H-chromen-3-yl) ethylidene)-4-methylbenzenesulfonohydrazide against isoproterenol-induced myocardial infarction by moderating biochemical, oxidative stress, and histological parameters.

This study is carried out to assess the cardiopreventive effect of (E)-N'-(1-(7-methoxy-2-oxo-2H-chromen-3-yl) ethylidene)-4-methylbenzenesulfonohydrazide or SHC, a novel synthesized coumarin, against myocardial infarction induced by isoproterenol (ISO). The SHC compound was identified and characterized by spectral methods (infrared, 1 H NMR [nuclear magnetic resonance], 13 C NMR, Nuclear Overhauser Effect Spectroscopy, and high-resolution mass spectroscopy). Male Wistar rats were divided into four groups: Control, ISO (rats were injected subcutaneously by 85 mg/kg body weight [BW] of isoproterenol at Days 6 and 7 of the experience), ISO + SHC (150 µg/kg BW, orally for 7 days) and ISO + acenocoumarol (150 µg/kg BW, orally for 7 days). Results showed that ISO induced a remarkable alteration of electrocardiogram (ECG) pattern and increases of plasma cardiac troponin T, creatine kinase-MB, total cholesterol, triglycerides, low-density lipoprotein-cholesterol, lactate dehydrogenase, aspartate transaminase, and malondialdehyde. In addition, ISO reduced the high-density lipoprotein-cholesterol content and the activities of superoxide dismutase and glutathione peroxidase, with the induction of myocardial necrosis. However, SHC administration revealed a significant decrease in cardiac dysfunction markers, restored normal ECG pattern, as well as improving lipids parameters. Moreover, SHC treatment remarkably alleviated the cardiac oxidative stress and the myocardial remodeling process. Overall, the SHC offers good protection from acute myocardial infarction through the antioxidant capacity.

Mesenchymal stem cell-derived interleukin-28 drives the selection of apoptosis resistant bone metastatic prostate cancer.

Bone metastatic prostate cancer (PCa) promotes mesenchymal stem cell (MSC) recruitment and their differentiation into osteoblasts. However, the effects of bone-marrow derived MSCs on PCa cells are less explored. Here, we report MSC-derived interleukin-28 (IL-28) triggers prostate cancer cell apoptosis via IL-28 receptor alpha (IL-28Rα)-STAT1 signaling. However, chronic exposure to MSCs drives the selection of prostate cancer cells that are resistant to IL-28-induced apoptosis and therapeutics such as docetaxel. Further, MSC-selected/IL-28-resistant prostate cancer cells grow at accelerated rates in bone. Acquired resistance to apoptosis is PCa cell intrinsic, and is associated with a shift in IL-28Rα signaling via STAT1 to STAT3. Notably, STAT3 ablation or inhibition impairs MSC-selected prostate cancer cell growth and survival. Thus, bone marrow MSCs drive the emergence of therapy-resistant bone metastatic prostate cancer yet this can be disabled by targeting STAT3.

Phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonates and phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonamides as new antimicrotubule agents targeting the colchicine-binding site.

We recently designed and prepared new families of potent antimicrotubule agents designated as N-phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) and phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamides (PIB-SAs). Our previous structure-activity relationship studies (SAR) focused on the aromatic ring B of PIB-SOs and PIB-SAs leaving the impact of the phenylimidazolidin-2-one moiety (ring A) on the binding to the colchicine-binding site (C-BS) poorly studied. Therefore, the aim of the present study was to evaluate the effect of replacing the imidazolidin-2-one (IMZ) group by a pyrrolidin-2-one moiety. To that end, 15 new phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonate (PYB-SO) and 15 phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonamide (PYB-SA) derivatives were designed, prepared, chemically characterised and biologically evaluated. PYB-SOs and PYB-SAs exhibit antiproliferative activity in the low nanomolar to low micromolar range (0.0087-8.6 µM and 0.056-21 µM, respectively) on human HT-1080, HT-29, M21 and MCF7 cancer cell lines. Moreover, they block cell cycle progression in G2/M phase. Immunofluorescence, tubulin affinity and tubulin polymerisation assays show that they cause microtubule depolymerisation by docking the C-BS. In addition, docking assays with the most potent derivatives show binding affinity toward the C-BS and they also exhibit weak or no toxicity toward chick embryos. Finally, physicochemical properties calculated using the SwissADME algorithm show that PYB-SOs and PYB-SAs are promising new families of antimicrotubule agents.

Electrophysiological assessment and pharmacological treatment of blast-induced tinnitus.

Tinnitus, the phantom perception of sound, often occurs as a clinical sequela of auditory traumas. In an effort to develop an objective test and therapeutic approach for tinnitus, the present study was performed in blast-exposed rats and focused on measurements of auditory brainstem responses (ABRs), prepulse inhibition of the acoustic startle response, and presynaptic ribbon densities on cochlear inner hair cells (IHCs). Although the exact mechanism is unknown, the "central gain theory" posits that tinnitus is a perceptual indicator of abnormal increases in the gain (or neural amplification) of the central auditory system to compensate for peripheral loss of sensory input from the cochlea. Our data from vehicle-treated rats supports this rationale; namely, blast-induced cochlear synaptopathy correlated with imbalanced elevations in the ratio of centrally-derived ABR wave V amplitudes to peripherally-derived wave I amplitudes, resulting in behavioral evidence of tinnitus. Logistic regression modeling demonstrated that the ABR wave V/I amplitude ratio served as a reliable metric for objectively identifying tinnitus. Furthermore, histopathological examinations in blast-exposed rats revealed tinnitus-related changes in the expression patterns of key plasticity factors in the central auditory pathway, including chronic loss of Arc/Arg3.1 mobilization. Using a formulation of N-acetylcysteine (NAC) and disodium 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07) as a therapeutic for addressing blast-induced neurodegeneration, we measured a significant treatment effect on preservation or restoration of IHC ribbon synapses, normalization of ABR wave V/I amplitude ratios, and reduced behavioral evidence of tinnitus in blast-exposed rats, all of which accorded with mitigated histopathological evidence of tinnitus-related neuropathy and maladaptive neuroplasticity.

Neuropeptide Y facilitates P2X1 receptor-dependent vasoconstriction via Y1 receptor activation in small mesenteric arteries during sympathetic neurogenic responses.

ATP, norepinephrine and NPY are co-released by sympathetic nerves innervating arteries. ATP elicits vasoconstriction via activation of smooth muscle P2X receptors. The functional interaction between neuropeptide Y (NPY) and P2X receptors in arteries is not known. In this study we investigate the effect of NPY on P2X1-dependent vasoconstriction in mouse mesenteric arteries. Suramin or P2X1 antagonist NF449 abolished α,ß-meATP evoked vasoconstrictions. NPY lacked any direct vasoconstrictor effect but facilitated the vasoconstrictive response to α,ß-meATP. Mesenteric arteries expressed Y1 and Y4 receptors, but not Y2 or Y5. Y1 receptor inhibition (BIBO3304) reversed NPY facilitation of the α,ß-meATP-evoked vasoconstriction. L-type Ca2+ channel antagonism (nifedipine) had no effect on α,ß-meATP-evoked vasoconstrictions, but completely reversed NPY facilitation. Electrical field stimulation evoked sympathetic neurogenic vasoconstriction. Neurogenic responses were dependent upon dual α1-adrenergic (prazosin) and P2X1 (NF449) receptor activation. Y1 receptor antagonism partially reduced neurogenic vasoconstriction. Isolation of the P2X1 component by α1-adrenergic blockade allowed faciliatory effects of Y1 receptor activation to be explored. Y1 receptor antagonism reduced the P2X1 receptor component during neurogenic vasoconstriction. α1-adrenergic and P2X1 receptors are post-junctional receptors during sympathetic neurogenic vasoconstriction in mesenteric arteries. In conclusion, we have identified that NPY lacks a direct vasoconstrictor effect in mesenteric arteries but can facilitate vasoconstriction by enhancing the activity of P2X1, following activation by exogenous agonists or during sympathetic nerve stimulation. The mechanism of P2X1 facilitation by NPY involved activation of the NPY Y1 receptor and the L-type Ca2+ channel.