Varenicline is a potent partial agonist at α6β2* nicotinic acetylcholine receptors in rat and monkey striatum

Extensive evidence indicates that varenicline reduces nicotine craving and withdrawal symptoms by modulating dopaminergic function at α4β2* nicotinic acetylcholine receptors (nAChRs) (the asterisk indicates the possible presence of other nicotinic subunits in the receptor complex). More recent data suggest that α6β2* nAChRs also regulate dopamine release and mediate nicotine reinforcement. The present experiments were therefore done to test the effect of varenicline on α6β2* nAChRs and their function, because its interaction with this subtype is currently unclear. Receptor competition studies showed that varenicline inhibited α6β2* nAChR binding (K(i) = 0.12 nM) as potently as α4β2* nAChR binding (K(i) = 0.14 nM) in rat striatal sections and with ～20-fold greater affinity than nicotine. Functionally, varenicline was more potent in stimulating α6β2* versus α4β2* nAChR-mediated [(3)H]dopamine release from rat striatal synaptosomes with EC(50) values of 0.007 and 0.086 μM, respectively. However, it acted as a partial agonist on α6β2* and α4β2* nAChR-mediated [(3)H]dopamine release with maximal efficacies of 49 and 24%, respectively, compared with nicotine. We also evaluated varenicline's action in striatum of monkeys, a useful animal model for comparison with humans. Varenicline again potently inhibited monkey striatal α6β2* (K(i) = 0.13 nM) and α4β2* (K(i) = 0.19 nM) nAChRs in competition studies. Functionally, it potently stimulated both α6β2* (EC(50) = 0.014 μM) and α4β2* (EC(50) = 0.029 μM) nAChR-mediated [(3)H]dopamine release from monkey striatal synaptosomes, again acting as a partial agonist relative to nicotine at both subtypes. These data suggest that the ability of varenicline to interact at α6β2* nAChRs may contribute to its efficacy as a smoking cessation aid.     

Effects of the α7 nicotinic acetylcholine receptor agonist GTS-21 on the innate immune response in humans

The vagus nerve can reflexively attenuate the innate immune response via binding of the vagal neurotransmitter acetylcholine (ACh) to the α7 nicotinic ACh receptor (α7nAChR). We recently reported potent anti-inflammatory effects of the α7nAChR agonist GTS-21 in human leukocytes. In the present work, we investigated the anti-inflammatory effects of GTS-21 on the innate immune response during experimental human endotoxemia. We performed a double-blind placebo-controlled pilot study in 14 healthy nonsmoking male volunteers. Subjects received 150 mg GTS-21 (n = 7) or placebo (n = 7) orally three times per day during 3 days before endotoxin administration and on the day of the human endotoxemia experiment. This GTS-21 dosage scheme is the highest reported to be safe in humans. Subsequently, subjects were i.v. administered 2 ng/kg endotoxin (LPS derived from Escherichia coli O:113). Serial blood withdrawals were performed to determine GTS-21 plasma concentrations and inflammatory mediators. Plasma concentrations of GTS-21 and its active metabolite 4-OH-GTS-21 were highly variable between subjects. LPS administration resulted in a transient inflammatory response. There were no differences in the LPS-induced cytokine response between the GTS-21- and placebo-treated groups. However, within the GTS-21-treated group, higher GTS-21 plasma concentrations correlated with lower levels of TNF-α (r = -0.78, P = 0.03), IL-6 (r = -0.76, P = 0.04), and IL-1RA (r = -0.86, P = 0.01), but not IL-10 (r = -0.35, P = 0.25). In conclusion, although higher GTS-21 plasma concentrations significantly correlated with lower cytokine levels, the highest dose tested to be safe in humans did not result in significant differences in inflammatory mediators between the GTS-21- and placebo-treated groups.     

Efficacy and safety of the α4β2 neuronal nicotinic receptor agonist ABT-894 in patients with diabetic peripheral neuropathic pain

Preclinical and clinical studies suggest that neuronal nicotinic receptor (NNR) agonists may be a novel and effective therapy for numerous painful conditions. Analgesic efficacy and safety of the highly selective α(4)β(2) NNR agonist ABT-894 was evaluated in 2 separate randomized, double-blind, multicenter, placebo-controlled clinical trials in patients with diabetic peripheral neuropathic pain (DPNP). Study 1 (280 patients randomized) tested 1, 2, and 4 mg ABT-894 twice daily compared with placebo and 60 mg duloxetine once per day over 8 weeks of treatment. Study 2 (124 patients randomized) tested 6 mg ABT-894 twice daily vs placebo for 8 weeks. The primary efficacy outcome measure in both studies was the weekly mean of the 24-hour average pain score recorded in each patient's diary. In both trials, none of the ABT-894 dose groups showed efficacy compared with placebo, whereas duloxetine achieved a statistically significant improvement over placebo in Study 1. All dose levels of ABT-894 were well tolerated, and no significant safety issues were identified. These results are in contrast to the outcome of a previously reported study of DPNP using the less selective α(4)β(2) NNR agonist ABT-594, which demonstrated efficacy compared with placebo, albeit with significant tolerability limitations. The failure of the highly selective α(4)β(2) NNR agonist ABT-894 indicates that it may not be possible to define a therapeutic index for this mechanism or that selectively targeting α(4)β(2) NNRs may not be a viable approach to treating neuropathic pain.     

S32212, a novel serotonin type 2C receptor inverse agonist/α2-adrenoceptor antagonist and potential antidepressant: II. A behavioral, neurochemical, and electrophysiological characterization

The present studies characterized the functional profile of N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-1,2-dihydro-3-H-benzo[e]indole-3-carboxamide) (S32212), a combined serotonin (5-HT)(2C) receptor inverse agonist and α(2)-adrenoceptor antagonist that also possesses 5-HT(2A) antagonist properties (J Pharmacol Exp Ther 340:750-764, 2012). Upon parenteral and/or oral administration, dose-dependent (0.63-40.0 mg/kg) actions were observed in diverse procedures. Both acute and subchronic administration of S32212 reduced immobility time in a forced-swim test in rats. Acutely, it also suppressed marble burying and aggressive behavior in mice. Long-term administration of S32212 was associated with rapid (1 week) and sustained (5 weeks) normalization of sucrose intake in rats exposed to chronic mild stress and with elevated levels of mRNA encoding brain-derived neurotrophic factor in hippocampus and amygdala (2 weeks). S32212 accelerated the firing rate of adrenergic perikarya in the locus coeruleus and elevated dialysis levels of noradrenaline in the frontal cortex and hippocampus of freely moving rats. S32212 also elevated the frontocortical levels of dopamine and acetylcholine, whereas 5-HT, amino acids, and histamine were unaffected. These neurochemical actions were paralleled by "promnemonic" properties: blockade of scopolamine-induced deficits in radial maze performance and social recognition and reversal of delay-induced impairments in social recognition, social novelty discrimination, and novel object recognition. It also showed anxiolytic actions in a Vogel conflict procedure. Furthermore, in an electroencephalographic study of sleep architecture, S32212 enhanced slow-wave and rapid eye movement sleep, while decreasing waking. Finally, chronic administration of S32212 neither elevated body weight nor perturbed sexual behavior in male rats. In conclusion, S32212 displays a functional profile consistent with improved mood and cognitive performance, together with satisfactory tolerance.     

S32212, a novel serotonin type 2C receptor inverse agonist/α2-adrenoceptor antagonist and potential antidepressant: I. A mechanistic characterization

Although most antidepressants suppress serotonin (5-HT) and/or noradrenaline reuptake, blockade of 5-HT(2C) receptors and α(2)-adrenoceptors likewise enhances monoaminergic transmission. These sites are targeted by the urea derivative N- [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-1,2-dihydro-3-H-benzo[e]indole-3-carboxamide (S32212). S32212 was devoid of affinity for monoamine reuptake sites, yet displayed pronounced affinity (pK(i), 8.2) for constitutively active human 5-HT(2CINI) (h5-HT(2CINI)) receptors, behaving as an inverse agonist in reducing basal Gα(q) activation, [(3)H]inositol-phosphate production, and the spontaneous association of h5-HT(2CINI)-Renilla luciferase receptors with β-arrestin2-yellow fluorescent protein. Furthermore, upon 18-h pretreatment, S32212 enhanced the plasma membrane expression of h5-HT(2CINI) receptors as visualized by confocal microscopy and quantified by enzyme-linked immunosorbent assay. Its actions were prevented by the neutral antagonist 6-chloro-5-methyl-N-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl]indoline-1-carboxamide (SB242,084), which also impeded the induction by long-term exposure to S32212 of otherwise absent Ca(2+) mobilization in mouse cortical neurones. In vivo, S32212 blunted the inhibitory influence of the 5-HT(2C) agonist 2-(3-chlorobenzyloxy)-6-(1-piperazinyl)pyrazine (CP809,101) on ventrotegmental dopaminergic neurones. S32212 also blocked 5-HT-induced Gα(q) and phospholipase C activation at the h5-HT(2A) and, less potently, h5-HT(2B) receptors and suppressed the discriminative stimulus properties of the 5-HT(2A) agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane in rats. S32212 manifested marked affinity for human α(2A)- (pK(i) 7.2), α(2B)- (pK(i) 8.2), and α(2C)- (pK(i) 7.4) adrenoceptors, at which it abolished noradrenaline-induced recruitment of Gα(i3), Gα(o), adenylyl cyclase, and extracellular-regulated kinase1/2. Moreover, S32212 dose-dependently abolished the discriminative stimulus effects of the α(2)-adrenoceptor agonist (S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4,2'-(1',2',3',4'-tetrahydronaphthalene)] (S18616). Finally, S32212 displayed negligible affinity for α(1A)-adrenoceptors, histamine H(1) receptors, and muscarinic M(1) receptors. In conclusion, S32212 behaves as an inverse agonist at h5-HT(2C) receptors and as an antagonist at human α(2)-adrenoceptors (and h5-HT(2A) receptors). Its promising profile in preclinical models potentially relevant to the treatment of depression is described in J Pharmacol Exp Ther 340:765-780, 2012.     

Regulation of glutamate release by α7 nicotinic receptors: differential role in methamphetamine-induced damage to dopaminergic and serotonergic terminals

Regulation of glutamate release is an important underlying mechanism in mediating excitotoxic events such as damage to dopamine (DA) and serotonin (5-HT) neurons observed after exposure to methamphetamine (Meth). One way to regulate glutamate release may be through the modulation of α7 nicotinic acetylcholine (nACh) receptors. Meth administration is known to increase acetylcholine release; however, it is unknown whether Meth increases glutamate release and causes long-term damage to both DA and 5-HT terminals through the activation of α7 nACh receptors. To test this hypothesis, the α7 nACh receptor antagonist, methyllycaconitine (MLA), was administered before the administration of repeated doses of Meth while simultaneously monitoring extracellular striatal glutamate with in vivo microdialysis. In addition, the subsequent long-term decreases in markers of dopaminergic and serotonergic terminals, including DA reuptake transporter (DAT), serotonin reuptake transporter (SERT), vesicular monoamine transporter-2, vesicular DA, and vesicular 5-HT content in the rat striatum, were measured. The results show that MLA pretreatment prevented Meth-induced increases in striatal glutamate and protected against the subsequent long-term decreases in striatal DAT and vesicular DA content without affecting the hyperthermia produced by Meth. In contrast, the Meth-induced decreases in striatal SERT immunoreactivity and vesicular 5-HT content were not affected by MLA. This suggests that the α7 nACh receptor differentially mediates glutamate-dependent damage to DA but not 5-HT terminals in a manner that is independent of hyperthermia. Furthermore, antagonism of α7 nACh receptors may be a possible therapeutic strategy for decreasing extracellular glutamate and preventing the excitotoxic damage observed in other DA-related neurodegenerative disorders.     

Pharmacological characterization of abediterol, a novel inhaled β(2)-adrenoceptor agonist with long duration of action and a favorable safety profile in preclinical models

Abediterol is a novel potent, long-acting inhaled β(2)-adrenoceptor agonist in development for the treatment of asthma and chronic obstructive pulmonary disease. Abediterol shows subnanomolar affinity for the human β(2)-adrenoceptor and a functional selectivity over β(1)-adrenoceptors higher than that of formoterol and indacaterol in both a cellular model with overexpressed human receptors and isolated guinea pig tissue. Abediterol is a full agonist at the human β(2)-adrenoceptor (E(max) = 91 ± 5% of the maximal effect of isoprenaline). The potency and onset of action that abediterol shows in isolated human bronchi (EC(50) = 1.9 ± 0.4 nM; t? onset = 7-10 min) is not significantly different from that of formoterol, but its duration of action (t? ～ 690 min) is similar to that of indacaterol. Nebulized abediterol inhibits acetylcholine-induced bronchoconstriction in guinea pigs in a concentration-dependent manner, with higher potency and longer duration of action (t? = 36 h) than salmeterol (t? = 6 h) and formoterol (t? = 4 h) and similar duration of action to indacaterol up to 48 h. In dogs, the bronchoprotective effect of abediterol is more sustained than that of salmeterol and indacaterol at doses without effects on heart rate, thus showing a greater safety margin (defined as the ratio of dose increasing heart rate by 5% and dose inhibiting bronchospasm by 50%) than salmeterol, formoterol, and indacaterol (5.6 versus 3.3, 2.2, and 0.3, respectively). In conclusion, our results suggest that abediterol has a preclinical profile for once-daily dosing in humans together with a fast onset of action and a favorable cardiovascular safety profile.     

Effects of ritobegron (KUC-7483), a novel selective β3-adrenoceptor agonist, on bladder function in cynomolgus monkey

We evaluated the pharmacological profile of ritobegron [KUC-7483; (-)-ethyl 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)-2,5-dimethylphenyloxy]acetate monohydrochloride] and its effects on the bladder in cynomolgus monkeys by in vitro and in vivo experiments. In vitro, ritobegron decreased the resting tension of the isolated bladder in a concentration-dependent manner (EC(50) 8.2 ± 2.3 × 10(-7) M; maximal relaxation 88.7 ± 3.7%). The β(3)-adrenoceptor (AR) antagonist 3-(2-allylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol hydrochloride (SR58894A) produced a rightward shift of this concentration-response curve without altering the maximal response (pK(B) value 6.56 ± 0.35). In isolated atria, ritobegron increased the atrial rate only at high concentrations (EC(50) 6.5 ± 1.2 × 10(-5) M). Ritobegron had no effect on tracheal contraction at concentrations from 10(-9) to 10(-4) M, and even at the highest concentration tested, 10(-3) M, the maximal relaxation it induced was only 26.7 ± 8.1%. Tests of the selectivity of ritobegron for the bladder gave values of 79.3- and 1200-fold higher versus atria and trachea, respectively. In the in vivo study ritobegron significantly decreased intravesical pressure (ED(50) 1.44 mg/kg) without affecting either mean blood pressure or heart rate. In conclusion, ritobegron displayed potent and selective β(3)-AR agonistic activity and relaxed the monkey isolated bladder, and in vivo it decreased intravesical pressure without affecting cardiovascular parameters. These results suggest that ritobegron may be a promising potential agent for the treatment of overactive bladder.     

Syndecan-1 regulates αvβ3 and αvβ5 integrin activation during angiogenesis and is blocked by synstatin,a novel peptide inhibitor

Syndecan-1 (Sdc1) is a matrix receptor shown to associate via its extracellular domain with the α_vβ₃ and α_vβ₅ integrins, potentially regulating cell adhesion, spreading, and invasion of cells expressing these integrins. Using Sdc1 deletion mutants expressed in human mammary carcinoma cells, we identified the active site within the Sdc1 core protein and derived a peptide inhibitor called synstatin (SSTN) that disrupts Sdc1''s interaction with these integrins. Because the α_vβ₃ and α_vβ₅ integrins are critical in angiogenesis, a process in which a role for Sdc1 has been uncertain, we used human vascular endothelial cells in vitro to show that the Sdc1 regulatory mechanism is also required for integrin activation on these cells. We found Sdc1 expressed in the vascular endothelium during microvessel outgrowth from aortic explants in vitro and in mouse mammary tumors in vivo. Moreover, we show that SSTN blocks angiogenesis in vitro or when delivered systemically in a mouse model of angiogenesis in vivo, and impairs mammary tumor growth in an orthotopic mouse tumor model. Thus, Sdc1 is a critical regulator of these two important integrins during angiogenesis and tumorigenesis, and is inhibited by the novel SSTN peptide.Angiogenesis, or the sprouting of new blood vessels from existing ones, occurs during development and in diseases such as diabetic retinopathy, endometriosis, psoriasis, rheumatoid arthritis, and tumor-induced angiogenesis (1). Vascular endothelial cells rely on signaling from multiple integrins during the angiogenic process (for review see reference 2), including the α_vβ₃ and α_vβ₅ integrins; signaling by the α_vβ₃ and α_vβ₅ integrin leads to endothelial cell proliferation, migration, matrix metalloprotease activation, and resistance to apoptosis (3).The α_vβ₃ and α_vβ₅ integrins are subject to regulation during angiogenesis. Fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), two potent angiogenic factors released by tumors, induce the expression of these two integrins that collaborate with the FGF and VEGF receptors in angiogenic signaling pathways (4); disrupting angiogenic signaling by inactivation of either integrin or growth factor receptor leads to endothelial cell apoptosis (5). The integrins are often up-regulated on metastatic tumors as well, leading to enhanced invasion, proliferation, and tumor survival (6–9) by largely the same mechanisms operative in endothelial cells. For these reasons, the integrins and their regulatory mechanisms are attractive targets for the development of therapeutic drugs. Drugs that are currently being tested range from inhibitory integrin antibodies (e.g., Vitaxin [10], based on the inhibitory antibody LM609 [11]), to cyclic RGD peptides that interfere with ligand binding (e.g., cRGDfV, cilengitide, and ST1646 [12–15]), to peptidomimetics based on the RGD sequence (e.g., S247 [16]). These inhibitors have all been shown to disrupt the growth of solid tumors as well as angiogenesis.We have recently identified a regulatory mechanism by which syndecan-1 (Sdc1), a cell-surface matrix receptor, regulates the activation of the α_vβ₃ and α_vβ₅ integrins on mammary carcinoma cells and fibroblasts (17–20). The syndecans are multifunctional extracellular matrix receptors on the surface of all adherent cells (21–23). They anchor to the matrix via heparan sulfate (HS) glycosaminoglycan chains attached near the distal tips of their core proteins; these chains recognize “heparin-binding” domains present in most matrix ligands, including fibronectin (FN), laminins, vitronectin (VN), thrombospondin, and the fibrillar collagens (21). In addition, mounting evidence suggests that they assemble with and control the signaling of other cell surface receptors, including integrins. McFall et al. first described a “cell-binding domain” in the extracellular domain of Sdc4 (24, 25); this site has recently been shown to regulate β1-containing integrins on mesenchymal cells, although the exact integrin target and regulatory mechanism remain unknown (26, 27). Recombinant Sdc2 extracellular domain alters adhesion mechanisms in colon carcinoma cells, suggesting that a regulatory site also exists in its extracellular domain (28, 29). More recently, we have shown that Sdc1 is necessary for activation of the α_vβ₃ integrin on mammary carcinoma cells (17, 20). Silencing Sdc1 expression, selective deletion of amino acids in its extracellular domain, or targeted competition with domain-specific antibodies or recombinant extracellular domain protein disrupts integrin activation and matrix recognition necessary for cell spreading and invasion. Similar activation of the α_vβ₅ integrin by Sdc1 occurs on B82L fibroblasts, which rely exclusively on this integrin for attachment to VN and FN (19). These extracellular syndecan-specific regulatory sites are readily accessible to therapeutic drugs and may hold promise as targets for combating tumorigenesis and other diseases in which their regulated mechanisms play a role.Given the importance of the α_vβ₃ and α_vβ₅ integrins in angiogenesis, we examined the possibility that Sdc1 regulates these integrins on vascular endothelial cells during tumor-induced angiogenesis. We found that Sdc1 is expressed by mouse and human endothelial cells in vitro, and is expressed during angiogenesis induced by FGF or VEGF in vitro and in vivo. We found that the α_vβ₃ and α_vβ₅ integrins associate with Sdc1 and that this association can be disrupted by a peptide called synstatin (SSTN) that is derived from the active site in the Sdc1 core protein. Furthermore, SSTN is an effective inhibitor of angiogenesis in vitro and in vivo, and of mammary carcinoma formation in nude mice. These results define the Sdc1 regulatory mechanism as a critical component of the angiogenic and tumorigenic process.     

Lipopolysaccharide upregulates α7 acetylcholine receptors: stimulation with GTS-21 mitigates growth arrest of macrophages and improves survival in burned mice

Nicotinic stimulation of the α7 acetylcholine receptors (α7AChRs) mitigates the lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF-α) and other cytokines release in macrophages. This effect is blocked by α7AChR antagonist, α-bungarotoxin (BTX). We tested and confirmed the hypotheses that LPS upregulates α7AChRs, and the prototypical α7AChR antagonists, vecuronium and BTX, do not block the effects of GTS-21, a specific α7AChR agonist, on TNF-α release. With the knockdown of α7AChR expression by short interference RNA, GTS-21 effects on inhibition of TNF-α release were not demonstrable. In addition, GTS-21 mitigated the LPS-induced growth arrest of macrophages in vitro in J774A.1 cells and ex vivo in peritoneal macrophages obtained from mice at 3 days after burn. Moreover, GTS-21 reduced mortality after burn injury in mice. These results indicate that (i) LPS upregulates α7AChRs; (ii) the therapeutic beneficial effects of GTS-21 on cytokine release are specifically mediated via α7AChRs and are preserved even when cotreated with prototypical antagonist, BTX, or clinically used muscle nicotinic antagonist, vecuronium; (iii) activation of α7AChRs by GTS-21 partially reverses the LPS-induced proliferation arrest; and (iv) GTS-21 reduces mortality in mice with burn injury. The in vivo beneficial effects of GTS-21 in burn injury warrant further studies.     

Tamoxifen regulation of bone growth and endocrine function in the ovariectomized rat: discrimination of responses involving estrogen receptor α/estrogen receptor β, G protein-coupled estrogen receptor, or estrogen-related receptor γ using fulvestrant (ICI 182780)

Tamoxifen is a selective estrogen receptor (ER) modulator, but it is also a deactivating ligand for estrogen-related receptor-γ (ERRγ) and a full agonist for the G protein-coupled estrogen receptor (GPER). Fulvestrant is a selective ER down-regulator that lacks agonist effects on ERα/ERβ, is inactive on ERRγ, but acts as a full agonist on GPER. Fulvestrant effects on tamoxifen actions on uterine and somatic growth, bone, the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis, and pituitary prolactin were analyzed to pharmacologically discriminate tamoxifen effects that may be mediated by ERα/ERβ versus ERRγ versus GPER. Ovariectomized rats received tamoxifen (0.6 mg/kg/daily) plus fulvestrant at 0, 3, 6, or 12 mg/kg/daily for 5 weeks; controls received vehicle or 6 mg/kg fulvestrant daily. Tamoxifen effects to increase uterine weight, decrease serum IGF-I, increase pituitary prolactin, and increase bone mineral density could be fully blocked by fulvestrant, indicating mediation by ERα/ERβ. Tamoxifen effects to decrease pituitary GH, tibia length, and body weight were only partially blocked by fulvestrant, indicating involvement of mechanisms unrelated to ERα/ERβ. Fulvestrant did not inhibit tamoxifen actions to reduce total pituitary protein, again indicating effects not mediated by ERα/ERβ. Tamoxifen actions to reduce serum GH were mimicked rather than inhibited by fulvestrant, pharmacological features consistent with GPER involvement. However, fulvestrant alone increased IGF-I and also blocked tamoxifen-evoked IGF-I decreases; thus fulvestrant effects on serum GH might reflect increased IGF-I feedback inhibition. Fulvestrant alone had no effect on the other parameters. The findings indicate that mechanisms unrelated to ERα/ERβ contribute to tamoxifen effects on body weight, bone growth, and pituitary function.     

Preventive effect of GGsTop, a novel and selective γ-glutamyl transpeptidase inhibitor, on ischemia/reperfusion-induced renal injury in rats

GGsTop [2-amino-4-{[3-(carboxymethyl)phenyl](methyl)phosphono}butanoic acid], is a novel, highly selective, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor with no inhibitory activity on glutamine amidotransferases. In this study, we investigated the effects of treatment with GGsTop on ischemia/reperfusion-induced renal injury in uninephrectomized rats. Ischemic acute kidney injury (AKI) was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion 2 weeks after contralateral nephrectomy. Renal function in vehicle-treated AKI rats markedly decreased at 1 day after reperfusion. Treatment with GGsTop (1 and 10 mg/kg i.v.) 5 min before ischemia attenuated the ischemia/reperfusion-induced renal dysfunction in a dose-dependent manner. Histopathological examination of the kidney of AKI rats revealed severe renal damage, which was significantly suppressed by the GGsTop treatment. In renal tissues exposed to ischemia/reperfusion, GGT activity was markedly increased immediately after reperfusion, whereas renal superoxide production and malondialdehyde level were significantly increased 6 h after reperfusion. These alterations were abolished by the treatment with GGsTop. In addition, renal glutathione content was decreased by the 45-min ischemia, but its level was markedly elevated by the GGsTop treatment. Our results demonstrate that the novel and highly selective GGT inhibitor GGsTop prevents ischemia/reperfusion-induced AKI. The renoprotective effect of GGsTop seems to be attributed to the suppression of oxidative stress by inhibiting GGT activation, thereby preventing the degradation of glutathione.     

Characterization of two mutations, M287L and Q266I, in the α1 glycine receptor subunit that modify sensitivity to alcohols

Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels. Ethanol potentiates glycine activation of the GlyR, and putative binding sites for alcohol are located in the transmembrane (TM) domains between and within subunits. To alter alcohol sensitivity of GlyR, we introduced two mutations in the GlyR α1 subunit, M287L (TM3) and Q266I (TM2). After expression in Xenopus laevis oocytes, both mutants showed a reduction in glycine sensitivity and glycine-induced maximal currents. Activation by taurine, another endogenous agonist, was almost abolished in the M287L GlyR. The ethanol potentiation of glycine currents was reduced in the M287L GlyR and eliminated in Q266I. Physiological levels of zinc (100 nM) potentiate glycine responses in wild-type GlyR and also enhance the ethanol potentiation of glycine responses. Although zinc potentiation of glycine responses was unchanged in both mutants, zinc enhancement of ethanol potentiation of glycine responses was absent in M287L GlyRs. The Q266I mutation decreased conductance but increased mean open time (effects not seen in M287L). Two lines of knockin mice bearing these mutations were developed. Survival of homozygous knockin mice was impaired, probably as a consequence of impaired glycinergic transmission. Glycine showed a decreased capacity for displacing strychnine binding in heterozygous knockin mice. Electrophysiology in isolated neurons of brain stem showed decreased glycine-mediated currents and decreased ethanol potentiation in homozygous knockin mice. Molecular models of the wild-type and mutant GlyRs show a smaller water-filled cavity within the TM domains of the Q266I α1 subunit. The behavioral characterization of these knockin mice is presented in a companion article (J Pharmacol Exp Ther 340:317-329, 2012).     

Genetic and biochemical characterization of a novel metallo-β-lactamase, TMB-1, from an Achromobacter xylosoxidans strain isolated in Tripoli, Libya

An Achromobacter xylosoxidans strain from the Tripoli central hospital produced a unique metallo-β-lactamase, designated TMB-1, which is related to DIM-1 (62%) and GIM-1 (51%). bla(TMB-1) was embedded in a class 1 integron and located on the chromosome. The TMB-1 β-lactamase has lower k(cat) values than both DIM-1 and GIM-1 with cephalosporins and carbapenems. The K(m) values were more similar to those of GIM-1 than those of DIM-1, with the overall k(cat)/K(m) values being lower than those for GIM-1 and DIM-1.     

Soluble transferrin receptor in urine,a new biomarker for IgA nephropathy and Henoch–Schönlein purpura nephritis

ObjectivesIgA nephropathy (IgAN) and Henoch–Schönlein purpura nephritis (HSPN) might represent different ends of a continuous spectrum of glomerular disease. In both conditions, upregulated soluble transferrin receptor (sTfR) might be excreted in urine, which could be a potential biomarker to monitor disease activity and therapeutic response.MethodsIn this pilot study, 132 Caucasian patients consulting the Nephrology Department at the Ghent University Hospital because of a glomerulopathy and 50 normal controls were included. Urinary sTfR concentrations were determined in concentrated urine using a newly developed latex-enhanced immunonephelometric assay.ResultsMedian urinary sTfR concentration was higher in patients with a primary glomerulopathy than in healthy subjects (p < 0.0001). More importantly, absolute median levels of urinary sTfR were markedly higher in patients with active IgAN or HSPN [10 μg/L, 95% confidence interval (CI): 6–18 μg/L] in comparison with those with other morphological types of glomerulopathy (2 μg/L, 95%CI: 1–4 μg/L) (p < 0.0001). A statistically significant difference in urinary sTfR concentration was observed between patients with active IgAN or HSPN and patients who had achieved partial or complete remission (p < 0.0001). Multiple regression analysis with urinary sTfR as dependent variable revealed that proteinuria was the main predictor of urinary sTfR concentration (r² = 0.52, p < 0.001).ConclusionDetermination of sTfR in urine is a new and sensitive method for a potential biomarker of IgAN and HSPN.     

5-Aminoimidazole-4-carboxyamide-ribonucleoside (AICAR)-stimulated hepatic expression of Cyp4a10, Cyp4a14, Cyp4a31, and other peroxisome proliferator-activated receptor α-responsive mouse genes is AICAR 5'-monophosphate-dependent and AMP-activated protein kinase-independent

5-Aminoimidazole-4-carboxyamide-ribonucleoside (AICAR), a prodrug activator of AMP-activated protein kinase (AMPK), increased hepatic expression of cytochrome P450 4a10, 4a14, and 4a31 mRNAs 2-, 3-, and 4-fold, respectively, and liver microsomal lauric acid ω-hydroxylation increased 2.8-fold. Likewise, mRNA levels of the peroxisome proliferator-activated receptor α (PPARα)-responsive genes, Acox1, Acadm, Cpt1a, and Fabp1, were also increased by AICAR treatment. AICAR did not elicit these changes in PPARα null mice. In isolated murine hepatocytes, AICAR and adenosine produced similar effects, and these responses were blocked by the PPARα antagonist [(2S)-2-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl]amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester (GW6471). Inhibition of AMPK using compound C (dorsomorphin or 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine) did not block the induction of the PPARα-responsive genes by AICAR or adenosine, and 6,7-dihydro-4-hydroxy-3-(2'-hydroxy[1,1'-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662), a non-nucleoside, direct activator of AMPK, did not increase expression of PPARα-responsive genes. An inhibitor of adenosine kinase, 5-iodotubercidin, blocked these responses, suggesting that the phosphorylation of AICAR and adenosine to AICAR 5'-monophosphate (ZMP) or AMP, respectively, was required. Concentrations of ZMP and AMP were elevated and ATP levels diminished at 24 h. The PPARα-dependent responses were associated with increased concentrations of oleic acid, a potent PPARα agonist, and diminished levels of oleoyl-CoA. Oleoyl-CoA synthase activity was inhibited by ZMP and AMP with IC(50) values of 0.28 and 0.41 mM, respectively. These results suggest that PPARα is activated by increased concentrations of free fatty acids that may arise from impaired fatty acid metabolism caused by altered levels of ATP, AMP, and ZMP after AICAR or adenosine treatment.