Muscarinic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) gated by clozapine-N-oxide (CNO) allow selective G-protein cascade activation in genetically specified cell-types in ...vivo. Here we compare the pharmacokinetics, off-target effects and efficacy of CNO, clozapine (CLZ) and compound 21 (Cmpd-21) at the inhibitory DREADD human Gi-coupled M4 muscarinic receptor (hM4Di). The half maximal effective concentration (EC
) of CLZ was substantially lower (0.42 nM) than CNO (8.1 nM); Cmpd-21 was intermediate (2.95 nM). CNO was back-converted to CLZ in mice, and CLZ accumulated in brain tissue. However, CNO itself also entered the brain, and free cerebrospinal fluid (CSF) levels were within the range to activate hM4Di directly, while free (CSF) CLZ levels remained below the detection limit. Furthermore, directly injected CLZ was strongly converted to its pharmacologically active metabolite, norclozapine. Cmpd-21 showed a superior brain penetration and long-lasting presence. Although we identified a wide range of CNO and Cmpd-21 off-targets, there was hardly any nonspecific behavioural effects among the parameters assessed by the 5-choice-serial-reaction-time task. Our results suggest that CNO (3-5 mg/kg) and Cmpd-21 (0.4-1 mg/kg) are suitable DREADD agonists, effective at latest 15 min after intraperitoneal application, but both require between-subject controls for unspecific effects.
The blood-brain barrier is dysfunctional in epilepsy, thereby contributing to seizure genesis and resistance to antiseizure drugs. Previously, several groups reported that seizures increase brain ...glutamate levels, which leads to barrier dysfunction. One critical component of barrier dysfunction is brain capillary leakage. Based on our preliminary data, we hypothesized that glutamate released during seizures mediates an increase in matrix-metalloproteinase (MMP) expression and activity levels, thereby contributing to barrier leakage. To test this hypothesis, we exposed isolated brain capillaries from male Sprague Dawley rats to glutamate
and used an
/
approach of isolated brain capillaries from female Wistar rats that experienced status epilepticus as an acute seizure model. We found that exposing isolated rat brain capillaries to glutamate increased MMP-2 and MMP-9 protein and activity levels, and decreased tight junction protein levels, which resulted in barrier leakage. We confirmed these findings
in rats after status epilepticus and in brain capillaries from male mice lacking cytosolic phospholipase A
Together, our data support the hypothesis that glutamate released during seizures signals an increase in MMP-2 and MMP-9 protein expression and activity levels, resulting in blood-brain barrier leakage.
The mechanism leading to seizure-mediated blood-brain barrier dysfunction in epilepsy is poorly understood. In the present study, we focused on defining this mechanism in the brain capillary endothelium. We demonstrate that seizures trigger a pathway that involves glutamate signaling through cytosolic phospholipase A
, which increases MMP levels and decreases tight junction protein expression levels, resulting in barrier leakage. These findings may provide potential therapeutic avenues within the blood-brain barrier to limit barrier dysfunction in epilepsy and decrease seizure burden.
This study examined whether BI113823, a novel selective kinin B1 receptor antagonist can reverse established pulmonary arterial hypertension (PAH), prevent right heart failure and death, which is ...critical for clinical translation.
Left pneumonectomized male Wistar rats were injected with monocrotaline to induce PAH. Three weeks later, when PAH was well established, the rats received daily treatment of BI113823 or vehicle for 3 weeks.
Treatment with BI113823 from day 21 to day 42 after monocrotaline injection reversed established PAH as shown by normalized values of mean pulmonary arterial pressure (mPAP). BI113823 therapy reversed pulmonary vascular remodeling, pulmonary arterial neointimal formation, and heart and lung fibrosis, reduced right ventricular pressure, right heart hypertrophy, improved cardiac output, and prevented right heart failure and death. Treatment with BI113823 reduced TNF-α and IL-1β, and macrophages recruitment in bronchoalveolar lavage, reduced CD-68 positive macrophages and expression of proliferating cell nuclear antigen (PCNA) in the perivascular areas, and reduced expression of iNOS, B1 receptors, matrix metalloproteinase (MMP)-2 and MMP-9 proteins, and the phosphorylation of ERK1/2 and AKT in lung. Treatment with BI113823 reduced mRNA expression of ANP, BNP, βMHC, CGTF, collange-I and IV in right heart, compared to vehicle treated controls. In human monocytes cultures, BI113823 reduced LPS-induced TNF-α production, MMP-2 and MMP-9 expression, and reduced TNF-α-induced monocyte migration.
We conclude that BI113823 reverses preexisting severe experimental pulmonary hypertension via inhibition of macrophage infiltration, cytokine production, as well as down regulation of matrix metalloproteinase proteins.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A cure for epilepsy is currently not available, and seizure genesis, seizure recurrence, and resistance to antiseizure drugs remain serious clinical problems. Studies show that the blood–brain ...barrier is altered in animal models of epilepsy and in epileptic patients. In this regard, seizures increase expression of blood–brain barrier efflux transporters such as P-glycoprotein (P-gp), which is thought to reduce brain uptake of antiseizure drugs, and thus, contribute to antiseizure drug resistance. The goal of the current study was to assess the viability of combining in vivo and ex vivo preparations of isolated brain capillaries from animal models of seizures and epilepsy as well as from patients with epilepsy to study P-gp at the blood–brain barrier. Exposing isolated rat brain capillaries to glutamate ex vivo upregulated P-gp expression to levels that were similar to those in capillaries isolated from rats that had status epilepticus or chronic epilepsy. Moreover, the fold-increase in P-gp protein expression seen in animal models is consistent with the fold-increase in P-gp observed in human brain capillaries isolated from patients with epilepsy compared to age-matched control individuals. Overall, the in vivo/ex vivo approach presented here allows detailed analysis of the mechanisms underlying seizure-induced changes of P-gp expression and transport activity at the blood–brain barrier. This approach can be extended to other blood–brain barrier proteins that might contribute to drug-resistant epilepsy or other CNS disorders as well.
Behavioral strategies require gating of premature responses to optimize outcomes. Several brain areas control impulsive actions, but the neuronal basis of natural variation in impulsivity between ...individuals remains largely unknown. Here, by combining a Go/No-Go behavioral assay with resting-state (rs) functional MRI in mice, we identified the subthalamic nucleus (STN), a known gate for motor control in the basal ganglia, as a major hotspot for trait impulsivity. In vivo recorded STN neural activity encoded impulsive action as a separable state from basic motor control, characterized by decoupled STN/substantia nigra pars reticulata (SNr) mesoscale networks. Optogenetic modulation of STN activity bidirectionally controlled impulsive behavior. Pharmacological and genetic manipulations showed that these impulsive actions are modulated by metabotropic glutamate receptor 4 (mGlu4) function in STN and its coupling to SNr in a behavioral trait-dependent manner, and independently of general motor function. In conclusion, STN circuitry multiplexes motor control and trait impulsivity, which are molecularly dissociated by mGlu4. This provides a potential mechanism for the genetic modulation of impulsive behavior, a clinically relevant predictor for developing psychiatric disorders associated with impulsivity.
Abstract
Background
To examine the effects of BI 1029539 (GS-248), a novel selective human microsomal prostaglandin E synthase-1
(
mPGES-1) inhibitor, in experimental models of acute lung injury ...(ALI) and sepsis in transgenic mice constitutively expressing the mPGES1 (Ptges) humanized allele.
Methods
Series 1: Lipopolysaccharide (LPS)-induced ALI. Mice were randomized to receive vehicle, BI 1029539, or celecoxib. Series 2: Cecal ligation and puncture-induced sepsis. Mice were randomized to receive vehicle or BI 1029539.
Results
Series 1: BI 1029539 or celecoxib reduced LPS-induced lung injury, with reduction in neutrophil influx, protein content, TNF-ɑ, IL-1β and PGE
2
levels in bronchoalveolar lavage (BAL), myeloperoxidase activity, expression of mPGES-1, cyclooxygenase (COX)-2 and intracellular adhesion molecule in lung tissue compared with vehicle-treated mice. Notably, prostacyclin (PGI
2
) BAL concentration was only lowered in celecoxib-treated mice. Series 2: BI 1029539 significantly reduced sepsis-induced BAL inflammatory cell recruitment, lung injury score and lung expression of mPGES-1 and inducible nitric oxide synthase. Treatment with BI 1029539 also significantly prolonged survival of mice with severe sepsis. Anti-inflammatory and anti-migratory effect of BI 1029539 was confirmed in peripheral blood leukocytes from healthy volunteers.
Conclusions
BI 1029539 ameliorates leukocyte infiltration and lung injury resulting from both endotoxin-induced and sepsis-induced lung injury.
The cause of antiseizure drug (ASD) resistance in epilepsy is poorly understood. Here, we focus on the transporter P‐glycoprotein (P‐gp) that is partly responsible for limited ASD brain uptake, which ...is thought to contribute to ASD resistance. We previously demonstrated that cyclooxygenase‐2 (COX‐2) and the prostaglandin E receptor, prostanoid E receptor subtype 1, are involved in seizure‐mediated P‐gp up‐regulation. Thus, we hypothesized that inhibiting microsomal prostaglandin E2 (PGE2) synthase‐1 (mPGES‐1), the enzyme generating PGE2, prevents blood‐brain barrier P‐gp up‐regulation after status epilepticus (SE). To test our hypothesis, we exposed isolated brain capillaries to glutamate ex vivo and used a combined in vivo‐ex vivo approach by isolating brain capillaries from humanized mPGES‐1 mice to study P‐gp levels. We demonstrate that glutamate signaling through the NMDA receptor, cytosolic phospholipase A2, COX‐2, and mPGES‐1 increases P‐gp protein expression and transport activity levels. We show that mPGES‐1 is expressed in human, rat, and mouse brain capillaries. We show that BI1029539, an mPGES‐1 inhibitor, prevented up‐regulation of P‐gp expression and transport activity in capillaries exposed to glutamate and in capillaries from humanized mPGES‐1 mice after SE. Our data provide key signaling steps underlying seizure‐induced P‐gp up‐regulation and suggest that mPGES‐1 inhibitors could potentially prevent P‐gp up‐regulation in epilepsy.—Soldner, E. L. B., Hartz, A. M. S., Akanuma, S.‐I., Pekcec, A., Doods, H., Kryscio, R. J., Hosoya, K.‐I., Bauer, B. Inhibition of human microsomal PGE2 synthase‐1 reduces seizure‐induced increases of P‐glycoprotein expression and activity at the blood‐brain barrier. FASEB J. 33, 13966‐13981 (2019). www.fasebj.org
Myeloid-derived growth factor (MYDGF) is a paracrine-acting protein that is produced by bone marrow-derived monocytes and macrophages to protect and repair the heart after myocardial infarction (MI). ...This effect can be used for the development of protein-based therapies for ischemic tissue repair, also beyond the sole application in heart tissue. Here, we report the X-ray structure of MYDGF and identify its functionally relevant receptor binding epitope. MYDGF consists of a 10-stranded β-sandwich with a folding topology showing no similarities to other cytokines or growth factors. By characterizing the epitope of a neutralizing antibody and utilizing functional assays to study the activity of surface patch-mutations, we were able to localize the receptor interaction interface to a region around two surface tyrosine residues 71 and 73 and an adjacent prominent loop structure of residues 97-101. These findings enable structure-guided protein engineering to develop modified MYDGF variants with potentially improved properties for clinical use.
Kinin B1 receptors (B1Rs) are implicated in the pathogenesis of fibrosis. This study examined the anti-fibrotic effects of B1R blockade with BI 113823 in two established mouse models of hepatic ...fibrosis induced by intraperitoneal carbon tetrachloride (CCl
) injection or bile duct ligation (BDL). The mechanisms underlying the protection afforded by B1R inhibition were examined using human peripheral blood cells and LX2 human hepatic stellate cells (HSCs).
Fibrotic liver diseases were induced in mice by intraperitoneal carbon tetrachloride (CCl
) injection for 6 weeks, and by bile duct ligation (BDL) for 3 weeks, respectively. Mice received daily treatment of vehicle or BI 113823 (B1R antagonist) from onset of the experiment until the end of the study.
B1Rs were strongly induced in fibrotic mouse liver. BI 113823 significantly attenuated liver fibrosis and portal hypertension (PH), and improved survival in both CCl
and BDL mice. BI 113823 significantly reduced the expression of fibrotic proteins α-SMA, collagens 1, 3, 4, and profibrotic growth factors PDGF, TGFβ, CTGF, VEGF, proliferating cell nuclear antigen; and reduced hepatic Akt phosphorylation in CCl
- and BDL-induced liver fibrosis. BI 113823 also reduced expression of Cytokines IL-1, IL-6; chemokines MCP-1, MCP-3 and infiltration of inflammatory cells; and inhibited human monocyte and neutrophil activation, transmigration, TNF-α & MPO production in vitro. BI 113823 inhibited TGF-β and B1R agonist-stimulated human-HSC activation, contraction, proliferation, migration and fibrosis protein expression, and inhibited activation of PI3K/Akt signalling pathway.
B1Rs merits consideration as a novel therapeutic target for chronic liver fibrosis and PH.
Increased expression of drug efflux transporters at the blood-brain barrier accompanies epileptic seizures and complicates
therapy with antiepileptic drugs. This study is concerned with identifying ...mechanistic links that connect seizure activity
to increased P-glycoprotein expression at the blood-brain barrier. In this regard, we tested the hypothesis that seizures
increase brain extracellular glutamate, which signals through an N -methyl- d -aspartate (NMDA) receptor and cyclooxygenase-2 (COX-2) in brain capillaries to increase blood-brain barrier P-glycoprotein
expression. Consistent with this hypothesis, exposing isolated rat or mouse brain capillaries to glutamate for 15 to 30 min
increased P-glycoprotein expression and transport activity hours later. These increases were blocked by 5 H -dibenzo a , d cyclohepten-5,10-imine (dizocilpine maleate) (MK-801), an NMDA receptor antagonist, and by celecoxib, a selective COX-2 inhibitor;
no such glutamate-induced increases were seen in brain capillaries from COX-2-null mice. In rats, intracerebral microinjection
of glutamate caused locally increased P-glycoprotein expression in brain capillaries. Moreover, using a pilocarpine status
epilepticus rat model, we observed seizure-induced increases in capillary P-glycoprotein expression that were attenuated by
administration of indomethacin, a COX inhibitor. Our findings suggest that brain uptake of some antiepileptic drugs can be
enhanced through COX-2 inhibition. Moreover, they provide insight into one mechanism that underlies drug resistance in epilepsy
and possibly other central nervous system disorders.
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