Structure and properties of [(4,6-tBu2C6H2O)2Se]2An(THF)2, An = U, Np, and their reaction with p-benzoquinone.

The synthesis and characterization of U(iv) and Np(iv) selenium bis(phenolate) complexes are reported. The reaction of two equivalents of the U(iv) complex with p-benzoquinone results in the formation of a U(v)-U(v) species with a bridging reduced quinone. This represents a rare example of high-valent uranium chemistry as well as a rare example of a neptunium aryloxide complex.

Inhibition of native 5-HT3 receptor-evoked contractions in guinea pig and mouse ileum by antimalarial drugs.

Quinine, chloroquine and mefloquine are commonly used to treat malaria, however, with associated gastrointestinal (GI) side-effects. These drugs act as antagonists at recombinant 5-HT3 receptors and modulate gut peristalsis. These gastrointestinal side effects may be the result of antagonism at intestinal 5-HT3 receptors. Ileum from male C57BL/6 mice and guinea pigs was mounted longitudinally in organ baths. The concentration-response curves for 5-HT and the selective 5-HT3 agonist 2-Me-5-HT were obtained with 5-HT (pEC50 = 7.57 ± 0.33, 12) more potent (P = 0.004) than 2-Me-5-HT (pEC50 = 5.45 ± 0.58, n = 5) in mouse ileum. There was no difference in potency of 5-HT (pEC50 = 5.42 ± 0.15, n = 8) and 2-Me-5-HT (pIC50 = 5.01 ± 0.55, n = 11) in guinea pig ileum (P > 0.05). Quinine, chloroquine or mefloquine was applied for 10 min and inhibitions prior to submaximal agonist application. In mouse ileum, quinine, chloroquine and mefloquine antagonised 5-HT-induced contractions (pIC50 = 4.9 ± 0.17, n = 7; 4.76 ± 0.14, n = 5; 6.21 ± 0.2, n = 4, correspondingly) with mefloquine most potent (P < 0.05). Quinine, chloroquine and mefloquine antagonised 2-me-5-HT-induced contractions (pIC50 = 6.35 ± 0.11, n = 8; 4.64 ± 0.2, n = 7; 5.11 ± 0.22, n = 6, correspondingly) with quinine most potent (P < 0.05). In guinea-pig ileum, quinine, chloroquine and mefloquine antagonised 5-HT-induced contractions (pIC50 = 5.02 ± 0.15, n = 6; 4.54 ± 0.1, n = 7; 5.32 ± 0.13, n = 5) and 2-me-5-HT-induced contractions (pIC50 = 4.62 ± 0.25, n = 5; 4.56 ± 0.14, n = 6; 5.67 ± 0.12, n = 4) with chloroquine least potent against 5-HT and mefloquine most potent against 2-me-5-HT (P < 0.05). These results support previous studies identifying anti-malarial drugs as antagonists at recombinant 5-HT3 receptors and may also demonstrate the ability of these drugs to influence native 5-HT3 receptor-evoked contractile responses which may account for their associated GI side-effects.

Urinary ATP and visualization of intracellular bacteria: a superior diagnostic marker for recurrent UTI in renal transplant recipients?

Renal transplant recipients (RTR) are highly susceptible to urinary tract infections (UTIs) with over 50% of patients having at least one UTI within the first year. Yet it is generally acknowledged that there is considerable insensitivity and inaccuracy in routine urinalysis when screening for UTIs. Thus a large number of transplant patients with genuine urine infections may go undiagnosed and develop chronic recalcitrant infections, which can be associated with graft loss and morbidity. Given a recent study demonstrating ATP is released by urothelial cells in response to bacteria exposure, possibly acting at metabotropic P2Y receptors mediating a proinflammatory response, we have investigated alternative, and possibly more appropriate, urinalysis techniques in a cohort of RTRs. Mid-stream urine (MSU) samples were collected from 53 outpatient RTRs. Conventional leukocyte esterase and nitrite dipstick tests, and microscopic pyuria counts (in 1 µl), ATP concentration measurements, and identification of intracellular bacteria in shed urothelial cells, were performed on fresh unspun samples and compared to 'gold-standard' bacterial culture results. Of the 53 RTRs, 22% were deemed to have a UTI by 'gold-standard' conventional bacteria culture, whereas 87%, 8% and 4% showed evidence of UTIs according to leukocyte esterase dipstick, nitrite dipstick, and a combination of both dipsticks, respectively. Intracellular bacteria were visualized in shed urothelial cells of 44% of RTRs, however only 1 of the 23 RTRs (44%) was deemed to have a UTI by conventional bacteria culture. A significant association of the 'gold-standard' test with urinary ATP concentration combined with visualization of intracellular bacteria in shed urothelial cells was determined using the Fisher's exact test. It is apparent that standard bedside tests for UTIs give variable results and that seemingly quiescent bacteria in urothelial cells are very common in RTRs and may represent a focus of subclinical infection. Furthermore, our results suggest urinary ATP concentration combined with detection of intracellular bacteria in shed urinary epithelial cells may be a sensitive means by which to detect 'occult' infection in RTRs.

Antagonism of neurosteroid modulation of native gamma-aminobutyric acid receptors by (3alpha,5alpha)-17-phenylandrost-16-en-3-ol.

Endogenous pregnane neurosteroids are allosteric modulators at gamma-aminobutyric acid type-A (GABAA) receptors at nanomolar concentrations. There is direct evidence for multiple distinct neurosteroid binding sites on GABAA receptors, dependent upon subunit composition and stoichiometry. This view is supported by the biphasic kinetics of various neuroactive steroids, enantioselectivity of some neurosteroids, selective mutation studies of recombinantly expressed receptors and the selectivity of the neurosteroid antagonist (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17PA) on 5alpha-pregnane steroid effects on recombinant GABAA receptors expressed in Xenopus oocytes and native receptors in dissociated neurons. However, it is unclear whether this antagonist action is present in a mature mammalian system. The present study evaluated the antagonist activity of 17PA on neurosteroid agonists both in vivo and in vitro by examining the effects of 17PA on 5alpha-pregnane-induced sedation in rats, native mature GABAA receptor ion channels utilizing the chloride flux assay and further studies in recombinant alpha1beta2gamma2 receptors. The data show that 17PA preferentially inhibits 3alpha,5alpha-THP vs. alphaxalone in vivo, preferentially inhibits 3alpha,5alpha-THDOC vs. alphaxalone potentiation of GABA-mediated Cl- uptake in adult cerebral cortical synaptoneurosomes, but shows no specificity for 3alpha,5alpha-THDOC vs. alphaxalone in recombinant alpha1beta2gamma2 receptors. These data provide further evidence of the specificity of 17PA and the heterogeneity of neurosteroid recognition sites on GABAA receptors in the CNS.

Acute ethanol administration rapidly increases phosphorylation of conventional protein kinase C in specific mammalian brain regions in vivo.

BACKGROUND: Protein kinase C (PKC) is a family of isoenzymes that regulate a variety of functions in the central nervous system including neurotransmitter release, ion channel activity, and cell differentiation. Growing evidence suggests that specific isoforms of PKC influence a variety of behavioral, biochemical, and physiological effects of ethanol in mammals. The purpose of this study was to determine whether acute ethanol exposure alters phosphorylation of conventional PKC isoforms at a threonine 674 (p-cPKC) site in the hydrophobic domain of the kinase, which is required for its catalytic activity. METHODS: Male rats were administered a dose range of ethanol (0, 0.5, 1, or 2 g/kg, intragastric) and brain tissue was removed 10 minutes later for evaluation of changes in p-cPKC expression using immunohistochemistry and Western blot methods. RESULTS: Immunohistochemical data show that the highest dose of ethanol (2 g/kg) rapidly increases p-cPKC immunoreactivity specifically in the nucleus accumbens (core and shell), lateral septum, and hippocampus (CA3 and dentate gyrus). Western blot analysis further showed that ethanol (2 g/kg) increased p-cPKC expression in the P2 membrane fraction of tissue from the nucleus accumbens and hippocampus. Although p-cPKC was expressed in numerous other brain regions, including the caudate nucleus, amygdala, and cortex, no changes were observed in response to acute ethanol. Total PKCgamma immunoreactivity was surveyed throughout the brain and showed no change following acute ethanol injection. CONCLUSIONS: These results suggest that ethanol rapidly promotes phosphorylation of cPKC in limbic brain regions, which may underlie effects of acute ethanol on the nervous system and behavior.

Common determinants of single channel conductance within the large cytoplasmic loop of 5-hydroxytryptamine type 3 and alpha4beta2 nicotinic acetylcholine receptors.

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (gamma) below the resolution of single channel recording (966 +/- 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range gamma values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4'), 436 (0'), and 440 (4') residues by 5-HT3B (-4'Gln, 0'Asp, and 4'Ala) residues increases gamma to 36.5 +/- 1.0 pS. The 0' residue makes the most substantial contribution to gamma of the 5-HT3AR. Replacement of 0'Arg by aspartate, glutamate (alpha7 nAChR subunit MA 0'), or glutamine (beta2 subunit MA 0') increases gamma to the resolvable range (>6 pS). By contrast, replacement of 0'Arg by phenylalanine (alpha4 subunit MA 0') reduced gamma to 416 +/- 107 fS. In reciprocal experiments with alpha4beta2 nAChRs (gamma = 31.3 +/- 0.8 pS), replacement of MA 0' residues by arginine in alpha4beta2(Q443R) and alpha4(F588R)beta2 reduced gamma slightly. By contrast, the gamma of double mutant alpha4(F588R)beta2(Q443R) was halved. The MA -4' and 4' residues also influenced gamma of 5-HT3ARs. Replacement of nAChR alpha4 or beta2 MA 4' residues by arginine made current density negligible. By contrast, replacement of both -4' residues by arginine produced functional nAChRs with substantially reduced gamma (11.4 +/- 0.5 pS). Homology models of the 5-HT3A and alpha4beta2 nAChRs against Torpedo nAChR revealed MA -4', 0', and 4' residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.

5-HT(3A) receptor subunit is required for 5-HT3 antagonist-induced reductions in alcohol drinking.

The ionotropic serotonin subtype-3 (5-HT3) receptor has emerged as a potential therapeutic target in the treatment of alcohol abuse and alcoholism because selective pharmacological antagonists reduce alcohol consumption in preclinical and clinical models. 5-HT binds to the extracellular N-terminus of the 5-HT(3A) receptor subunit but receptor activation is also enhanced by distinct allosteric sites, which indicates the presence of other receptor subunits. It is not known if specific molecular subunits of the 5-HT3 receptor modulate alcohol drinking. To address this issue, we characterized acute locomotor response to alcohol and alcohol consumption in a two-bottle home-cage procedure by congenic C57BL/6J mice with a targeted deletion of the 5-HT(3A) receptor subunit gene. 5-HT(3A)-null mice did not differ from wild-type littermate controls on measures of spontaneous locomotor activity, habituation to a novel environment, or locomotor response to ethanol (0, 0.5, 1, or 2 g/kg). Moreover, null mice did not differ from controls on measures of ethanol (2-10%) intake and preference during or after a two-bottle home-cage sucrose fading procedure. Systemic administration of the 5-HT3 antagonist LY-278,584 (0-10 mg/kg) decreased intake of both sweetened (2% sucrose+10% ethanol) and unsweetened (10% ethanol) ethanol in wild-type mice only. These findings indicate that reduction of alcohol drinking produced by 5-HT3 antagonism is dependent on the presence of 5-HT(3A)-containing receptors.

A cytoplasmic region determines single-channel conductance in 5-HT3 receptors.

5-hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the 'HA-stretch') within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.

Targeted gene deletion of the 5-HT3A receptor subunit produces an anxiolytic phenotype in mice.

Anxiety disorders are the most common psychiatric disorders. Typical medications used to treat patients are benzodiazepines or antidepressants that target serotonin (5-HT) activity. The ionotropic 5-HT(3) receptor has emerged as a potential therapeutic target because selective antagonist compounds reduce anxiety in rodents, primates, and humans. 5-HT binds to the extracellular N-terminus of the 5-HT(3A) receptor subunit, but receptor activation is also enhanced by distinct allosteric sites. It is not known if specific molecular subunits of the 5-HT(3) receptor modulate anxiety. To address this issue, we characterized anxiety-like behavior of mice with a targeted deletion of the 5-HT(3A) receptor subunit gene in the light/dark box, elevated plus maze, and novelty interaction animal models of anxiety. 5-HT(3A) null mice exhibited an anxiolytic behavioral phenotype that was highly correlated across behavioral measures. This evidence indicates that the 5-HT(3A) molecular subunit influences anxiety-like behavior. Pharmacotherapy that targets specifically the 5-HT(3A) receptor subunit may provide a novel treatment for anxiety disorders.

The 5-HT3 antagonist Y-25130 blocks cocaine-induced lowering of ICSS reward thresholds in the rat.

Serotonin-3 (5-HT(3)) receptor antagonists have been shown to attenuate drug-induced increases in mesolimbic dopamine (DA), locomotor activation, and drug self-administration. In the present study, we tested whether the selective 5-HT(3) antagonist Y-25130 would attenuate cocaine-induced lowering of intracranial self-stimulation (ICSS) reward thresholds. Rats (n=6) were surgically prepared with bipolar stimulation electrodes and trained to self-administer electrical stimulation delivered to the medial forebrain bundle-lateral hypothalamus (MFB-LH). A discrete-trial, rate-free threshold determination procedure was used to detect pharmacologically induced changes from baseline reward thresholds. Four doses of Y-25130 (0.0, 0.03, 0.3, and 3.0 mg/kg ip) were given alone and in combination with cocaine (4.0 mg/kg ip). Y-25130 did not significantly alter reward thresholds or response latencies when given alone as compared to baseline measures. While there were no significant effects at lower doses, the middle and highest doses of Y-25130 (0.3 and 3.0 mg/kg) did attenuate the threshold-lowering effect of cocaine. These findings suggest that the rewarding effects of cocaine are mediated through 5-HT(3) receptor activity.