Jun Yang

Opioids Activate Brain Analgesic Circuits Through Cytochrome P450/Epoxygenase Signaling

To assess the importance of brain cytochrome P450 (P450) activity in μ opioid analgesic action, we generated a mutant mouse with brain neuron–specific reductions in P450 activity; these mice showed highly attenuated morphine antinociception compared with controls. Pharmacological inhibition of brain P450 arachidonate epoxygenases also blocked morphine antinociception in mice and rats. Our findings indicate that a neuronal P450 epoxygenase mediates the pain-relieving properties of morphine.

Genetic Requirement for Pneumococcal Ear Infection

Background Ear infection or otitis media (OM) accounts for most bacterial respiratory infections in children in both developed and developing nations. Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis are the major OM pathogens. However, little is known about the genetic basis of bacterial OM largely due to practical difficulties in conducting research in ear infection models and genetically manipulating clinical isolates. Here, we report the first genome-scale in vivo screen for bacterial genes required for ear infection in a chinchilla model by signature tagged mutagenesis (STM), a high throughput mutant screen technique. Methodology/Principal Findings STM strains were constructed with a multi-drug resistant OM isolate ST556 (serotype 19F) and screened in a chinchilla OM model. Out of 5,280 mutants tested, 248 mutants were substantially underrepresented in the mutant pools recovered from the middle ear fluids of the infected chinchillas, indicating the impaired ability to survive and replicate in the middle ears due to genetic disruptions in the chromosome of strain ST556. Further DNA sequencing analysis mapped the mutations to 169 pneumococcal genes. Surprisingly, only 52 of these genes were required for pneumococcal nasopharyngeal colonization in a murine model. This infection site-specific gene requirement was verified by targeted mutagenesis in the selected genes. Conclusions/Significance These findings suggest that there are a subset of pneumococcal genes required for ear infection and that these may be distinct from those required for nasal colonization. Our data thus provide comprehensive gene targets for mechanistic understanding of pneumococcal ear infection. Finally, this study has also developed a model for future genome-scale search for virulence determinants in other pathogens associated with ear infections.

Leucine Stimulates Insulin Secretion via Down-regulation of Surface Expression of Adrenergic α2A Receptor through the mTOR (Mammalian Target of Rapamycin) Pathway IMPLICATION IN NEW-ONSET DIABETES IN RENAL TRANSPLANTATION

Background: Leucine can stimulate insulin release, but the mechanism has remained unclear. Results: Leucine regulates adrenergic α2 receptor trafficking. Rapamycin and clonidine together increase the risk of diabetes. Conclusion: mTOR activation by leucine elicits insulin release via adrenergic α2 receptors. Rapamycin and clonidine appear to synergistically facilitate new-onset diabetes. Significance: Our findings may have relevance in the clinical management of renal transplant patients. The amino acid leucine is a potent secretagogue, capable of inducing insulin secretion. It also plays an important role in the regulation of mTOR activity, therefore, providing impetus to investigate if a leucine-sensing mechanism in the mTOR pathway is involved in insulin secretion. We found that leucine-induced insulin secretion was inhibited by both the mTOR inhibitor rapamycin as well as the adrenergic α2 receptor agonist clonidine. We also demonstrated that leucine down-regulated the surface expression of adrenergic α2A receptor via activation of the mTOR pathway. The leucine stimulatory effect on insulin secretion was attenuated in diabetic Goto-Kakizaki rats that overexpress adrenergic α2A receptors, confirming the role of leucine in insulin secretion. Thus, our data demonstrate that leucine regulates insulin secretion by modulating adrenergic α2 receptors through the mTOR pathway. The role of the mTOR pathway in metabolic homeostasis led us to a second important finding in this study; retrospective analysis of clinical data showed that co-administration of rapamycin and clonidine was associated with an increased incidence of new-onset diabetes in renal transplantation patients over those receiving rapamycin alone. We believe that inhibition of mTOR by rapamycin along with activation of adrenergic α2 receptors by clonidine represents a double-hit to pancreatic islets that synergistically disturbs glucose homeostasis. This new insight may have important implications for the clinical management of renal transplant patients.

The capBCA Locus is Required for Intracellular Growth of Francisella tularensis LVS

Francisella tularensis is the causative agent of tularemia and a category A bioterrorism agent. The molecular basis for the extreme virulence of F. tularensis remains unclear. Our recent study found that capBCA, three neighboring genes, are necessary for the infection of F. tularensis live vaccine strain (LVS) in a respiratory infection mouse model. We here show that the capBCA genes are necessary for in vivo growth of F. tularensis LVS in the lungs, spleens, and livers of BALB/c mice. Unmarked deletion of capBCA in type A strain Schu S4 resulted in significant attenuation in virulence although the level of the attenuation in Schu S4 was much less profound than in LVS. We further demonstrated that CapB protein is produced at a low level under the in vitro culture conditions, and capB alone is necessary for in vivo growth of F. tularensis LVS in the lungs of BALB/c mice. Finally, deletional mutations in capB alone or capBCA significantly impaired intracellular growth of F. tularensis LVS in cultured macrophages, thus suggesting that the capBCA genes are necessary for intracellular adaptation of F. tularensis. The requirement of this gene locus in intracellular adaption at least in part explains the significant attenuation of F. tularensis capBCA mutants in virulence.

Brain P450 epoxygenase activity is required for the antinociceptive effects of improgan, a nonopioid analgesic

&NA; The search for the mechanism of action of improgan (a nonopioid analgesic) led to the recent discovery of CC12, a compound that blocks improgan antinociception. Because CC12 is a cytochrome P450 inhibitor, and brain P450 mechanisms were recently shown to be required in opioid analgesic signaling, pharmacological and transgenic studies were performed in rodents to test the hypothesis that improgan antinociception requires brain P450 epoxygenase activity. Intracerebroventricular (ICV) administration of the P450 inhibitors miconazole and fluconazole, and the arachidonic acid (AA) epoxygenase inhibitor N‐methylsulfonyl‐6‐(2‐propargyloxyphenyl)hexanamide (MS‐PPOH) potently inhibited improgan antinociception in rats at doses that were inactive alone. MW06‐25, a new P450 inhibitor that combines chemical features of CC12 and miconazole, also potently blocked improgan antinociception. Although miconazole and CC12 were weakly active at opioid and histamine H3 receptors, MW06‐25 showed no activity at these sites, yet retained potent P450‐inhibiting properties. The P450 hypothesis was also tested in Cprlow mice, a viable knock‐in model with dramatically reduced brain P450 activity. Improgan (145 nmol, ICV) antinociception was reduced by 37% to 59% in Cprlow mice, as compared with control mice. Moreover, CC12 pretreatment (200 nmol, ICV) abolished improgan action (70% to 91%) in control mice, but had no significant effect in Cprlow mice. Thus, improgan’s activation of bulbospinal nonopioid analgesic circuits requires brain P450 epoxygenase activity. A model is proposed in which (1) improgan activates an unknown receptor to trigger downstream P450 activity, and (2) brainstem epoxygenase activity is a point of convergence for opioid and nonopioid analgesic signaling. The present study found that the nonopioid analgesic drug improgan utilizes cytochrome P450 epoxygenase enzymes in the brain to produce its pain‐relieving actions.

Rapamycin Inhibition of mTOR Reduces Levels of the Na+/H+ Exchanger 3 in Intestines of Mice and Humans, Leading to Diarrhea

BACKGROUND & AIMS The immunosuppressant rapamycin frequently causes noninfectious diarrhea in organ transplant recipients. We investigated the mechanisms of this process. METHODS We performed a retrospective analysis of renal transplant recipients treated with rapamycin from 2003 through 2010 at Albany Medical College, collecting data on serum levels of rapamycin. Levels of the Na+/H+ exchanger 3 (NHE3) were measured in human ileal biopsy specimens from patients who did and did not receive rapamycin (controls), in ileum tissues from rats or mice given rapamycin, and in mice with intestine-specific disruption of mammalian target of rapamycin (Mtor) (mTOR(f/f):Villin-cre mice) or Atg7 (Atg7(flox/flox); Villin-Cre). Exchange activity and intestinal water absorption were measured using a pH-sensitive dye and small intestine perfusion, respectively. RESULTS Episodes of noninfectious diarrhea occurred in organ recipients after increases in serum levels of rapamycin. The expression of NHE3 was reduced in the ileal brush border of patients with diarrhea. In rats and mice, continuous administration of low doses of rapamycin reduced levels of NHE3 in intestinal tissues; this effect was not observed in mice with intestinal deletion of ATG7, indicating that autophagy is required for the reduction. Administration of single high doses of rapamycin to mice, to model the spikes in rapamycin levels that occur in patients with severe diarrheal episodes, resulted in reduced phosphorylation of S6 and AKT in ileal tissues, indicating inhibition of the mTOR complex (mTORC1 and mTORC2). The intestines of mice with intestine-specific deletion of mTOR were dilated and contained large amounts of liquid stools; they also had reduced levels of total NHE3 and NHERF1 compared with control mice. We observed a significant reduction in Na(+)/H(+) exchange activity in ileum tissues from these mice. CONCLUSIONS Rapamycin inhibition of mTOR reduces levels of NHE3 and Na(+)/H(+) exchange activity in intestinal tissues of patients and rodents. This process appears to require the autophagic activity mediated by ATG7. Loss of mTOR regulation of NHE3 could mediate the development of diarrhea in patients undergoing rapamycin therapy.

Complete Genome Sequence of Streptococcus pneumoniae Strain ST556, a Multidrug-Resistant Isolate from an Otitis Media Patient

Streptococcus pneumoniae is a major pathogen causing bacterial infection in the middle ear of humans. We previously used S. pneumoniae strain ST556, a low-passage 19F isolate from an otitis media patient, to perform a whole-genome screen for ear infection-associated genes in a chinchilla model. This report presents the complete genome sequence of ST556. The genome sequence will provide information complementary to the experimental data from our genetic study of this strain.

High-affinity binding of [3H]cimetidine to a heme-containing protein in rat brain.

[3H]Cimetidine (3HCIM) specifically binds to an unidentified site in the rat brain. Because recently described ligands for this site have pharmacological activity, 3HCIM binding was characterized. 3HCIM binding was saturable, heat-labile, and distinct from the histamine H2 receptor. To test the hypothesis that 3HCIM binds to a cytochrome P450 (P450), the effects of nonselective and isoform-selective P450 inhibitors were studied. The heme inhibitor KCN and the nonselective P450 inhibitor metyrapone both produced complete, concentration-dependent inhibition of 3HCIM binding (Ki = 1.3 mM and 11.9 μM, respectively). Binding was largely unaffected by inhibitors of CYP1A2, 2B6, 2C8, 2C9, 2D6, 2E1, and 19A1 but was eliminated by inhibitors of CYP2C19 (tranylcypromine) and CYP3A4 (ketoconazole). Synthesis and testing of CC11 [4(5)-(benzylthiomethyl)-1H-imidazole] and CC12 [4(5)-((4-iodobenzyl)-thiomethyl)-1H-imidazole] confirmed both drugs to be high-affinity inhibitors of 3HCIM binding. On recombinant human P450s, CC12 was a potent inhibitor of CYP2B6 (IC50 = 11.7 nM), CYP2C19 (51.4 nM), and CYP19A1 (140.7 nM) and had a range of activities (100–494 nM) on nine other isoforms. Although the 3HCIM binding site pharmacologically resembles some P450s, eight recombinant human P450s and three recombinant rat P450s did not exhibit 3HCIM binding. Inhibition by KCN and metyrapone suggests that 3HCIM binds to a heme-containing brain protein (possibly a P450). However, results with selective P450 inhibitors, recombinant P450 isoforms, and a P450 antibody did not identify a 3HCIM-binding P450 isoform. Finally, CC12 is a new, potent inhibitor of CYP2B6 and CYP2C19 that may be a valuable tool for P450 research.

Leucine as a Stimulant of Insulin

Leucine is one of the most potent insulin secretogues among the branched-chain amino acids that promote glucose-induced insulin release from pancreatic β-cells. Leucine exerts its secretagogue effects via multiple routes. Leucine can either serve as a fuel source for ATP production or it may be converted to a metabolic intermediate α-ketoisocaproate (KIC) to inhibit KATP channel activity, leading to membrane depolarization and triggering insulin secretion. It also regulates insulin release by acting on glutamate dehydrogenase, a key enzyme that fuels amino acids into the tricarboxylic acid cycle. Leucine also triggers calcium oscillations in pancreatic β-cells and regulates the expression of some key genes that are critical for insulin secretion in pancreatic islets. Recent studies revealed that leucine is a potent activator of mTOR. Leucine appears to regulate insulin release by influencing adrenergic activity via the mTOR pathway. This route has been implicated in new-onset diabetes in renal transplant patients.

Tu1642 Inhibition of mTOR Mediates Alleviation of Intestinal Fibrosis via Suppression of Myofibroblast Proliferation

a porcine model of stress-induced gut injury. Nine-week-old pigs that were previously subjected to early weaning stress, co-mingled with unfamiliar (non-littermate) pigs to induce social stress. Six-hours following social stress, animals were treated daily with either lubiprostone (μ0.3μg/ kg) or placebo once daily gavage PO for 3-days. Following the treatment period, colonic tissues were harvested and mounted in Ussing chambers. The transepithelial electrical resistance (TER) in the colon of stressed pigs was found to be reduced by 30% compared to control non-stressed pig colon (p,0.01). Stressed pigs treated with lubiprostone showed significantly increased TER compared to placebo treated stressed pigs. Consistent with the changes in TER, stressed placebo-treated pigs had increased paracellular flux of 3H-mannitol, and treatment with lubiprostone prevented the increase in paracellular flux of mannitol caused by stress. Initial histopathological evaluation of tissues revealed no differences between groups in epithelial appearance, which appeared normal and continuous. However, further evaluation of the mucosa and sub mucosa is required. Thus, data demonstrate a beneficial influence of lubiprostone on intestinal barrier properties in a porcine model of psychosocial stress. * (Amitiza) -R-Tech-Ueno, Tokyo, Japan