Selective Alpha7 Nicotinic Acetylcholine Receptor Research Articles

GTS-21 attenuates ACE/ACE2 ratio and glycocalyx shedding in lipopolysaccharide-induced acute lung injury by targeting macrophage polarization derived ADAM-17

Acute lung injury (ALI) has received considerable attention in intensive care owing to its high mortality rate. It has been demonstrated that the selective alpha7 nicotinic acetylcholine receptor agonist Gainesville Tokushima scientists (GTS)-21 is promising for treating ALI caused by lipopolysaccharides (LPS). However, the precise underlying mechanism remains unknown. This study aimed to investigate the potential efficacy of GTS-21 in the treatment of ALI. We developed mouse models of ALI and alveolar epithelial type II cells (AT2s) injury following treatment with LPS and different polarized macrophage supernatants, respectively. Pathological changes, pulmonary edema, and lung compliance were assessed. Inflammatory cells count, protein content, and pro-inflammatory cytokine levels were analysed in the bronchoalveolar lavage fluid. The expression of angiotensin-converting enzyme (ACE), ACE2, syndecan-1 (SDC-1), heparan sulphate (HS), heparanase (HPA), exostosin (EXT)-1, and NF-κB were tested in lung tissues and cells. GTS-21-induced changes in macrophage polarization were verified in vivo and in vitro. Polarized macrophage supernatants with or without recombination a disintegrin and metalloproteinase-17 (ADAM-17) and small interfering (si)RNA ADAM-17 were used to verify the role of ADAM-17 in AT2 injury. By reducing pathological alterations, lung permeability, inflammatory response, ACE/ACE2 ratio, and glycocalyx shedding, as well as by downregulating the HPA and NF-κB pathways and upregulating EXT1 expression in vivo, GTS-21 significantly diminished LPS-induced ALI compared to that of the LPS group. GTS-21 significantly attenuated macrophage M1 polarization and augmented M2 polarization in vitro and in vivo. The destructive effects of M1 polarization supernatant can be inhibited by GTS-21 and siRNA ADAM-17. GTS-21 exerted a protective effect against LPS-induced ALI, which was reversed by recombinant ADAM-17. Collectively, GTS-21 alleviates LPS-induced ALI by attenuating AT2s ACE/ACE2 ratio and glycocalyx shedding through the inhibition of macrophage M1 polarization derived ADAM-17.

Quantification of Proliferating and Mitotically Active Retinal Cells in Mice by Flow Cytometry.

Adult mammals lack the ability to regenerate retinal neurons after injury. However, in previous studies from this lab, topical application of the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been associated with an increase in the number of retinal neurons in adult murine models both in the presence and absence of injury to the retina. Additionally, studies assaying mitotic markers have shown a substantial increase in the amount of mitotically active and proliferating cells with the topical application of the alpha7 nAChR agonist. However, these previous studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus limiting the number of antibodies that can be multiplexed. As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells. In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with Müller glia-derived progenitor cells that arise after treatment with PNU-282987. Key features • Neurogenesis in the adult mammalian retina. • Flow cytometry of retinal cells. • PNU-282987-induced mitotic activity in the retina. • Dissociation of the retina for flow cytometry analysis. Graphical overview Schematic demonstrating the protocol for preparation of retinal cells for flow cytometry analysis. (A) Adult mice (3-6 months) are subjected to topical PBS eyedrop treatment containing DMSO (control groups) or PNU-282987 (experimental groups). Both eyedrop treatments contain 1 mg/mL of BrdU to label proliferating cells. After treatment, mice are euthanized, and retinae are harvested for dissociation using papain. (B) Dissociated retina cells are fixed and permeabilized before aliquots are taken for cell counts on a hemocytometer. After determining the number of cells present, conjugated antibodies and unconjugated primary antibodies are added at the appropriate dilutions. Fluorescent secondary antibodies are added for markers that are unconjugated. Cells are then subjected to flow cytometric analysis using a BD LSRFortessa.

GTS-21, a selective alpha7 nicotinic acetylcholine receptor agonist, ameliorates diabetic nephropathy in Leprdb/db mice

Diabetic nephropathy (DN) is a serious complicating factor in human type 2 diabetes mellitus (T2DM), and it commonly results in end-stage renal disease (ESRD) that requires kidney dialysis. Here, we report that the α7 nicotinic acetylcholine receptor (α7nAChR) agonist GTS-21 exerts a novel anti-inflammatory action to ameliorate DN, as studied using an inbred strain of Leprdb/db mice in which hyperglycemia and obesity co-exist owing to defective leptin receptor (Lepr) signaling. For this analysis, GTS-21 was administered to 10–12 week-old male and female mice as a 4 mg/kg intraperitoneal injection, twice-a-day, for 8 weeks. Kidney function and injury owing to DN were monitored by determination of plasma levels of BUN, creatinine, KIM-1 and NGAL. Histologic analysis of glomerular hypertrophy and mesangial matrix expansion were also used to assess DN in these mice. Concurrently, renal inflammation was assessed by measuring IL-6 and HMGB1, while also quantifying renal cell apoptosis, and apoptotic signaling pathways. We found that Leprdb/db mice exhibited increased markers of BUN, creatinine, NGAL, KIM-1, IL-6, cytochrome C, and HMGB-1. These abnormalities were also accompanied by histologic kidney injury (mesangial matrix expansion and apoptosis). Remarkably, all such pathologies were significantly reduced by GTS-21. Collectively, our results provide new evidence that the α7nAChR agonist GTS-21 has the ability to attenuate diabetes-induced kidney injury. Additional studies are warranted to further investigate the involvement of the vagal cholinergic anti-inflammatory reflex pathway (CAP) in ameliorating diabetic nephropathy.

Transcriptome Changes in Retinal Pigment Epithelium Post-PNU-282987 Treatment Associated with Adult Retinal Neurogenesis in Mice.

PNU-282987, a selective alpha7 nicotinic acetylcholine receptor agonist, has previously been shown to have both neurogenic and broad regenerative effects in the adult murine retina. The objective of this study was to assay the molecular mechanism by which PNU-282987 promotes the production of Muller-derived progenitor cells through signaling via the resident retinal pigment epithelium. These Muller-derived progenitor cells generate a myriad of differentiated neurons throughout the retina that have previously been characterized by morphology. Herein, we demonstrate that topical application of PNU-282987 stimulates production of functional neurons as measured by electroretinograms. Further, we examine the mechanism of how this phenomenon occurs through activation of this atypical receptor using a transcriptomic approach isolated retinal pigment epithelium activated by PNU-282987 and in whole retina. We provide evidence that PNU-282987 causes a bi-modal signaling event in which early activation primes the retina with an inflammatory response and developmental signaling cues, followed by an inhibition of gliotic mechanisms and a decrease in the immune response, ending with upregulation of genes associated with specific retinal neuron generation. Taken together, these data provide evidence that PNU-282987 activates the retinal pigment epithelium to signal to Muller glia to produce Muller-derived progenitor cells, which can differentiate into new, functional neurons in adult mice. These data not only increase our understanding of how adult mammalian retinal regeneration can occur, but also provide therapeutic promise for treating functional vision loss.

Cholinergic anti-inflammatory pathway and COVID-19.

The cholinergic anti-inflammatory pathway (CAP) first described by Wang et al, 2003 has contemporary interest arising from the COVID-19 pandemic. While tobacco smoking has been considered an aggravating factor in the severity of COVID-19 infections, it has been suggested by some that the nicotine derived from tobacco could lessen the severity of COVID-19 infections. This spotlight briefly describes the CAP and its potential role as a therapeutic target for the treatment of COVID-19 infections using vagus nerve stimulation or selective alpha7 nicotinic acetylcholine receptor agonists.

Potentiation of cognitive enhancer effects of Alzheimer’s disease medication memantine by alpha7 nicotinic acetylcholine receptor agonist PHA-543613 in the Morris water maze task

RationaleThere are controversial pieces of evidence whether combination therapies using memantine and cholinesterase inhibitors are beneficial over their monotreatments. However, results of preclinical studies are promising when memantine is combined with agonists and allosteric modulators of the alpha7 nicotinic acetylcholine receptor (nAChR).ObjectivesHere, we tested the hypothesis that cognitive enhancer effects of memantine can be potentiated through modulating alpha7 nAChRs in a scopolamine-induced amnesia model.MethodsMonotreatments, as well as co-administrations of selective alpha7 nicotinic acetylcholine receptor agonist PHA-543613 and memantine were tested in the Morris water maze task in rats. The efficacy of the co-administration treatment was observed on different domains of spatial episodic memory.ResultsLow dose of memantine (0.1 mg/kg) and PHA-543613 (0.3 mg/kg) successfully reversed scopolamine-induced short-term memory deficits both in monotreatments and in co-administration. When recall of information from long-term memory was tested, pharmacological effects caused by co-administration of subeffective doses of memantine and PHA-543613 exceeded that of their monotreatments.ConclusionOur results further support the evidence of beneficial interactions between memantine and alpha7 nAChR ligands and suggest a prominent role of alpha7 nAChRs in the procognitive effects of memantine.

The selective alpha7 nicotinic acetylcholine receptor agonist AR-R17779 does not affect ischemia-reperfusion brain injury in mice.

Inflammation plays a central role in stroke-induced brain injury. The alpha7 nicotinic acetylcholine receptor (α7nAChR) can modulate immune responses in both the periphery and the brain. The aims of the present study were to investigate α7nAChR expression in different brain regions and evaluate the potential effect of the selective α7nAChR agonist AR-R17779 on ischemia–reperfusion brain injury in mice. Droplet digital PCR (ddPCR) was used to evaluate the absolute expression of the gene encoding α7nAChR (Chrna7) in hippocampus, striatum, thalamus and cortex in adult, naïve mice. Mice subjected to transient middle cerebral artery occlusion (tMCAO) or sham surgery were treated with α7nAChR agonist AR-R17779 (12 mg/kg) or saline once daily for 5 days. Infarct size and microglial activation 7 days after tMCAO were analyzed using immunohistochemistry. Chrna7 expression was found in all analyzed brain regions in naïve mice with the highest expression in cortex and hippocampus. At sacrifice, white blood cell count was significantly decreased in AR-R17779 treated mice compared with saline controls in the sham groups, although, no effect was seen in the tMCAO groups. Brain injury and microglial activation were evident 7 days after tMCAO. However, no difference was found between mice treated with saline or AR-R17779. In conclusion, α7nAChR expression varies in different brain regions and, despite a decrease in white blood cells in sham mice receiving AR-R17779, this compound does not affect stroke-induced brain injury.

Dexmedetomidine attenuates inflammation and pancreatic injury in a rat model of experimental severe acute pancreatitis via cholinergic anti-inflammatory pathway.

BackgroundExcessive inflammatory responses play a critical role in the development of severe acute pancreatitis (SAP), and controlling such inflammation is vital for managing this often fatal disease. Dexmedetomidine has been reported to possess protective properties in inflammatory diseases. Therefore, this study aimed to investigate whether dexmedetomidine pre-treatment exerts an anti-inflammatory effect in rats with SAP induced by sodium taurocholate, and if so, to determine the potential mechanism.MethodsSAP was induced with sodium taurocholate. Rats received an intraperitoneal injection of dexmedetomidine 30 min before sodium taurocholate administration. α-bungarotoxin, a selective alpha-7 nicotinic acetylcholine receptor (α7nAchR) antagonist, was injected intra-peritoneally 30 min before dexmedetomidine administration. The role of the vagus nerve was evaluated by performing unilateral cervical vagotomy before the administration of dexmedetomidine. Efferent discharge of the vagal nerve was recorded by the BL-420F Data Acquisition & Analysis System. Six hours after onset, serum pro-inflammatory cytokine (tumor necrosis factor α [TNF-α] and interleukin 6 [IL-6]) levels and amylase levels were determined using an enzyme-linked immunosorbent assay and an automated biochemical analyzer, respectively. Histopathological changes in the pancreas were observed after hematoxylin and eosin staining and scored according to Schmidt criteria.ResultsPre-treatment with dexmedetomidine significantly decreased serum levels of TNF-α, IL-6, and amylase, strongly alleviating pathological pancreatic injury in the rat model of SAP (TNF-α: 174.2 ± 30.2 vs. 256.1±42.4 pg/ml; IL-6: 293.3 ± 46.8 vs. 421.7 ± 48.3 pg/ml; amylase: 2102.3 ± 165.3 vs. 3186.4 ± 245.2 U/L). However, the anti-inflammatory and pancreatic protective effects were abolished after vagotomy or pre-administration of α-bungarotoxin. Dexmedetomidine also significantly increased the discharge frequency and amplitude of the cervical vagus nerve in the SAP rat model (discharge frequency: 456.8 ± 50.3 vs. 332.4 ± 25.1 Hz; discharge amplitude: 33.4 ± 5.3 vs. 20.5 ± 2.9 μV).ConclusionsDexmedetomidine administration attenuated the systemic inflammatory response and local pancreatic injury caused by SAP in rats through the cholinergic anti-inflammatory pathway involving vagus- and α7nAChR-dependent mechanisms.

ICH3, a selective alpha7 nicotinic acetylcholine receptor agonist, modulates adipocyte inflammation associated with obesity.

The alpha7 nicotinic acetylcholine receptor (α7nAChR), involved in the modulation of inflammation and insulin sensitivity, is downregulated in white adipose tissue (WAT) of obese patients. This study aims to test the ability of a selective synthetic α7nAChR agonist, the spirocyclic Δ2-isoxazoline derivative (R)-(-)-ICH3 (ICH3), to counteract acute inflammation and obesity-associated modifications in WAT. We employed the LPS-septic shock murine model, human primary adipocytes and diet-induced obese (DIO) mice. Inflammatory factor expression was assessed by ELISA and quantitative real-time PCR. Flow cytometry was employed to define WAT inflammatory infiltrate. Insulin signaling was monitored by quantification of AKT phosphorylation. In the septic shock model, ICH3 revealed antipyretic action and reduced the surge of circulating cytokines. In vitro, ICH3 stimulation (10µM) preserved viability of human adipocytes, decreased IL-6 mRNA (P < 0.05) and blunted LPS-induced peak of TNFα (P < 0.05) and IL-6 (P < 0.01). Chronic administration of ICH3 to DIO mice was associated with lower numbers of CD8+ T cells (P < 0.05) and to changed WAT expression of inflammatory factors (Hp, P < 0.05; CD301/MGL1, P < 0.01; Arg-1, P < 0.05). As compared to untreated, ICH3 DIO mice exhibited improved insulin signaling in the skeletal muscle (P < 0.01) mirrored by an improved response to glucose load (ipGTT: P < 0.05 at 120min). We proved that ICH3 is an anti-inflammatory drug, able to reduce inflammatory cytokines in human adipocytes and to blunt the effects of obesity on WAT inflammatory profile, on glucose tolerance and on tissue insulin sensitivity.

EFFECT OF GTS-21, AN ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR AGONIST, ON CLP-INDUCED INFLAMMATORY, GASTROINTESTINAL MOTILITY, AND COLONIC PERMEABILITY CHANGES IN MICE

During abdominal sepsis, the inhibition of gastrointestinal (GI) motility together with mucosal barrier dysfunction will lead to increased bacterial translocation and maintenance of sepsis. The activation of the vagal anti-inflammatory pathway remains an appealing therapeutic strategy in sepsis. In this respect, selective alpha7 nicotinic acetylcholine receptor (α7nAChR) agonists have shown anti-inflammatory properties in several animal models of inflammation. Sepsis was induced in OF-1 mice by cecal ligation and puncture (CLP). GI transit was quantified, and cytokine levels were determined in serum and colon. Colonic permeability was assessed by means of Evans blue injection. We studied the effect of GTS-21, an α7nAChR agonist, on the aforementioned parameters. Splenectomized animals as well as α7nAChR-knock-out animals (Chrna7) were included to study the role of splenic macrophages and the α7nAChR during polymicrobial abdominal sepsis. In septic animals, GTS-21 significantly ameliorated GI motility, lowered systemic and colonic levels of IL-6, decreased colonic permeability, and decreased the number of positive cultures obtained from blood and mesenteric lymph nodes. Splenectomy prevented animals from developing sepsis-induced ileus. Chrna7 mice displayed a more severe septic phenotype, whereas GTS-21 remarkably was also beneficial in these animals. Our results show that peripheral targeting of the vagal anti-inflammatory pathway proves beneficial in an animal model of polymicrobial abdominal sepsis. A major role is allocated to splenic immune cells in the development of sepsis, as preventive splenectomy was protective for the development of sepsis. Data on the Chrna7 mice suggest that the beneficial effects mediated by GTS-21 on inflammation and motility might be related to activation of other receptors besides the α7nAChR.

Modulatory effects of α7 nAChRs on the immune system and its relevance for CNS disorders.

The clinical development of selective alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonists has hitherto been focused on disorders characterized by cognitive deficits (e.g., Alzheimer’s disease, schizophrenia). However, α7 nAChRs are also widely expressed by cells of the immune system and by cells with a secondary role in pathogen defense. Activation of α7 nAChRs leads to an anti-inflammatory effect. Since sterile inflammation is a frequently observed phenomenon in both psychiatric disorders (e.g., schizophrenia, melancholic and bipolar depression) and neurological disorders (e.g., Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis), α7 nAChR agonists might show beneficial effects in these central nervous system disorders. In the current review, we summarize information on receptor expression, the intracellular signaling pathways they modulate and reasons for receptor dysfunction. Information from tobacco smoking, vagus nerve stimulation, and cholinesterase inhibition is used to evaluate the therapeutic potential of selective α7 nAChR agonists in these inflammation-related disorders.

750 GTS-21, a Selective Alpha7 Nicotinic Acetylcholine Receptor Agonist, Ameliorates Impaired Mucosal Barrier Function and Inhibits Bacterial Translocation in a Mouse Cecal Ligation and Puncture Model

750 GTS-21, a Selective Alpha7 Nicotinic Acetylcholine Receptor Agonist, Ameliorates Impaired Mucosal Barrier Function and Inhibits Bacterial Translocation in a Mouse Cecal Ligation and Puncture Model

Effects of splenectomy and GTS-21, a selective α7 nicotinic acetylcholine receptor agonist, on the development of septic ileus in mice

Sepsis remains a leading cause of mortality in our ICUs. Ileus, defined as the inhibition of the propulsive motility in the gastrointestinal (GI) tract, together with mucosal barrier dysfunction will maintain sepsis by the translocation of intestinal bacteria. Preliminary data in our group showed that the administration of GTS-21, a selective alpha7 nicotinic acetylcholine receptor (α7nAChR) agonist, ameliorates inflammation and GI motility disturbances during sepsis [1]. Activation of the α7nAChR is the final step in the vagal anti-inflammatory pathway, a spleen-dependent and macrophage-dependent pathway that dampens inflammation. We aimed to study the effects of splenectomy (SPLX) combined with GTS-21 on GI motility and on local colonic and systemic inflammation, as the role of the spleen in the vagal anti-inflammatory pathway is currently under debate.

Acetylcholine inhibits hypoxia‐induced tumor necrosis factor‐α production via regulation of MAPKs phosphorylation in cardiomyocytes

Recent findings have reported that up-regulation of tumor necrosis factor-alpha (TNF-α) induced by myocardial hypoxia aggravates cardiomyocyte injury. Acetylcholine (ACh), the principle vagal neurotransmitter, protects cardiomyocytes against hypoxia by inhibiting apoptosis. However, it is still unclear whether ACh regulates TNF-α production in cardiomyocytes after hypoxia. The concentration of extracellular TNF-α was increased in a time-dependent manner during hypoxia. Furthermore, ACh treatment also inhibited hypoxia-induced TNF-α mRNA and protein expression, caspase-3 activation, cell death and the production of reactive oxygen species (ROS) in cardiomyocytes. ACh treatment prevented the hypoxia-induced increase in p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) phosphorylation. Co-treatment with atropine, a non-selective muscarinic acetylcholine receptor antagonist, or methoctramine, a selective type-2 muscarinic acetylcholine (M(2) ) receptor antagonist, abrogated the effects of ACh treatment in hypoxic cardiomyocytes. Co-treatment with hexamethonium, a non-selective nicotinic receptor antagonist, and methyllycaconitine, a selective alpha7-nicotinic acetylcholine receptor antagonist, had no effect on ACh-treated hypoxic cardiomyocytes. In conclusion, these results demonstrate that ACh activates the M(2) receptor, leading to regulation of MAPKs phosphorylation and, subsequently, down-regulation of TNF-α production. We have identified a novel pathway by which ACh mediates cardioprotection against hypoxic injury in cardiomyocytes.

Preclinical and the first clinical studies on [11C]CHIBA-1001 for mapping α7 nicotinic receptors by positron emission tomography

4-[(11)C]methylphenyl 2,5-diazabicyclo[3.2.2]nonane-2-carboxylate ([(11)C]CHIBA-1001), a 4-methyl-substituted derivative of the selective alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) partial agonist 4-bromophenyl 1,4 diazabicyclo[3.2.2]nonane-4-carboxylate (SSR180711), is a potential radioligand for mapping alpha7 nAChRs in the brain by positron emission tomography (PET). In this study, we performed preclinical and first clinical PET studies using [(11)C]CHIBA-1001 for imaging alpha7 nAChRs in the human brain. [(11)C]CHIBA-1001 was synthesized by methylation of the tributylstannyl precursor with [(11)C]CH(3)I in a palladium-promoted Stille cross-coupling reaction. The radiation absorbed-dose of [(11)C]CHIBA-1001 in humans was calculated from distribution data in mice. The acute toxicity of CHIBA-1001 at a dose of 3.20 mg/kg body weight, which is more than 41,000-fold the clinical equivalent dose of [(11)C]CHIBA-1001, was evaluated. The mutagenicity of CHIBA-1001 was studied by a reverse mutation test in Salmonella typhimurium (Ames test). Metabolite analysis in the mouse brain was carried out by high-performance liquid chromatography. The first clinical PET imaging of alpha7 nAChRs with [(11)C]CHIBA-1001 in a normal volunteer was also performed. A suitable preparation method for [(11)C]CHIBA-1001 injection was established. The radiation absorbed-dose by [(11)C]CHIBA-1001 in humans was low enough for clinical use, and no acute toxicity or mutagenicity of CHIBA-1001 was found. Most radioactivity in the mouse brain was detected as an unchanged form, although peripherally [(11)C]CHIBA-1001 was degraded. We successfully performed brain imaging by PET with [(11)C]CHIBA-1001 in a normal volunteer. A 90-min dynamic scan showed a rapid accumulation and gradual washout of radioactivity in the brain. The highest distribution volume of [(11)C]CHIBA-1001 was found in the thalamus; however, regional differences in brain radioactivity were small. Peripherally, [(11)C]CHIBA-1001 was stable in humans: >80% of the radioactivity in plasma was detected as the unchanged form for 60 min. These results demonstrate that [(11)C]CHIBA-1001 is a suitable radioligand to use in clinical trials for imaging alpha7 nAChRs in the human brain, providing acceptable dosimetry and pharmacological safety at the dose required for adequate PET imaging.

Neuropeptide Modulators of High Mobility Group Box 1 Secretion as Potential Therapeutic Agents for Severe Sepsis

This Commentary describes a study suggesting that the endogenous neuropeptides vasoactive intestinal peptide (VIP) and urocortin can improve survival in an animal model of lethal sepsis, possibly through their ability to decrease circulating levels of the cytokine-like protein high mobility group box 1 (HMGB1).

Selective α7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis*

Tumor necrosis factor and high mobility group box 1 are critical cytokine mediators of inflammation. The efferent vagus nerve inhibits cytokine release through alpha7-nicotinic acetylcholine receptor-mediated cholinergic signaling. Here we studied whether GTS-21, a selective alpha7-nicotinic acetylcholine receptor agonist, inhibits proinflammatory cytokines in vitro and in vivo and improves survival in murine endotoxemia and severe sepsis. Randomized and controlled in vitro and in vivo study. Research laboratory and animal facility rooms. RAW 264.7 cells and BALB/c mice treated with endotoxin or subjected to cecal ligation and puncture (CLP). RAW 264.7 cells were exposed to endotoxin (4 ng/mL or 10 ng/mL) in the presence or absence of GTS-21 (1-100 muM), and tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation were analyzed. Mice were treated with GTS-21 (0.4 mg/kg or 4 mg/kg, intraperitoneally) or saline 30 mins before endotoxin (6 mg/kg, intraperitoneally), and serum tumor necrosis factor was analyzed 1.5 hrs after the onset of endotoxemia. In survival experiments, mice were treated with GTS-21 (0.4 or 4.0 mg/kg, intraperitoneally) or saline 30 mins before and 6 hrs after endotoxin and then twice daily for 3 days. Severe sepsis was induced by CLP. Mice were treated with GTS-21 (4 mg/kg) or saline immediately and 6 hrs and 24 hrs after CLP, and serum high mobility group box 1 was analyzed 30 hrs after CLP. In survival experiments, GTS-21 (0.4 or 4 mg/kg) treatment was initiated 24 hrs after CLP and continued twice daily for 3 days. GTS-21 dose-dependently inhibited tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation in vitro. GTS-21 (4 mg/kg) significantly inhibited serum tumor necrosis factor during endotoxemia and improved survival (p < .0001). GTS-21 (4 mg/kg) significantly inhibited serum high mobility group box 1 levels in CLP mice and improved survival (p < .0006). These findings are of interest for the development of alpha7-nicotinic acetylcholine receptor agonists as a new class of anti-inflammatory therapeutics.