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1.
Mol Pharm ; 21(2): 550-563, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38261609

RESUMO

4-Chlorokynurenine (4-Cl-KYN, AV-101) is a prodrug of a NMDA receptor antagonist and is in clinical development for potential CNS indications. We sought to further understand the distribution and metabolism of 4-Cl-KYN, as this information might provide a strategy to enhance the clinical development of this drug. We used excretion studies in rats, in vitro transporter assays, and pharmacogenetic analysis of clinical trial data to determine how 4-Cl-KYN and metabolites are distributed. Our data indicated that a novel acetylated metabolite (N-acetyl-4-Cl-KYN) did not affect the uptake of 4-Cl-KYN across the blood-brain barrier via LAT1. 4-Cl-KYN and its metabolites were found to be renally excreted in rodents. In addition, we found that N-acetyl-4-Cl-KYN inhibited renal and hepatic transporters involved in excretion. Thus, this metabolite has the potential to limit the excretion of a range of compounds. Our pharmacogenetic analysis found that a SNP in N-acetyltransferase 8 (NAT8, rs13538) was linked to levels of N-acetyl-4-Cl-KYN relative to 4-Cl-KYN found in the plasma and that a SNP in SLC7A5 (rs28582913) was associated with the plasma levels of the active metabolite, 7-Cl-KYNA. Thus, we have a pharmacogenetics-based association for plasma drug level that could aid in the drug development of 4-Cl-KYN and have investigated the interaction of a novel metabolite with drug transporters.


Assuntos
Ácido Cinurênico , Fármacos Neuroprotetores , Ratos , Animais , Cinurenina , Analgésicos , Fármacos Neuroprotetores/metabolismo
2.
Mol Pharm ; 18(1): 113-123, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33307708

RESUMO

Recent advances in the understanding of depression have led to increasing interest in ketamine and the role that N-methyl-d-aspartate (NMDA) receptor inhibition plays in depression. l-4-Chlorokynurenine (4-Cl-KYN, AV-101), a prodrug, has shown promise as an antidepressant in preclinical studies, but this promise has not been realized in recent clinical trials. We sought to determine if transporters in the CNS could be playing a role in this clinical response. We used radiolabeled uptake assays and microdialysis studies to determine how 4-Cl-KYN and its active metabolite, 7-chlorokynurenic acid (7-Cl-KYNA), cross the blood-brain barrier (BBB) to access the brain and its extracellular fluid compartment. Our data indicates that 4-Cl-KYN crosses the blood-brain barrier via the amino acid transporter LAT1 (SLC7A5) after which the 7-Cl-KYNA metabolite leaves the brain extracellular fluid via probenecid-sensitive organic anion transporters OAT1/3 (SLC22A6 and SLC22A8) and MRP4 (ABCC4). Microdialysis studies further validated our in vitro data, indicating that probenecid may be used to boost the bioavailability of 7-Cl-KYNA. Indeed, we found that coadministration of 4-Cl-KYN with probenecid caused a dose-dependent increase by as much as an 885-fold increase in 7-Cl-KYNA concentration in the prefrontal cortex. In summary, our data show that 4-Cl-KYN crosses the BBB using LAT1, while its active metabolite, 7-Cl-KYNA, is rapidly transported out of the brain via OAT1/3 and MRP4. We also identify a hitherto unreported mechanism by which the brain extracellular concentration of 7-Cl-KYNA may be increased to produce significant boosting of the drug concentration at its site of action that could potentially lead to an increased therapeutic effect.


Assuntos
Ácido Cinurênico/análogos & derivados , Cinurenina/análogos & derivados , Córtex Pré-Frontal/metabolismo , Probenecid/farmacologia , Pró-Fármacos/farmacologia , Animais , Ketamina/metabolismo , Ácido Cinurênico/metabolismo , Cinurenina/metabolismo , Masculino , Fármacos Neuroprotetores/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo
3.
Cell Mol Life Sci ; 76(5): 977-994, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30547226

RESUMO

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated, apical anion channel that regulates ion and fluid transport in many epithelia including the airways. We have previously shown that cigarette smoke (CS) exposure to airway epithelia causes a reduction in plasma membrane CFTR expression which correlated with a decrease in airway surface hydration. The effect of CS on CFTR was dependent on an increase in cytosolic Ca2+. However, the underlying mechanism for this Ca2+-dependent, internalisation of CFTR is unknown. To gain a better understanding of the effect of Ca2+ on CFTR, we performed whole cell current recordings to study the temporal effect of raising cytosolic Ca2+ on CFTR function. We show that an increase in cytosolic Ca2+ induced a time-dependent reduction in whole cell CFTR conductance, which was paralleled by a loss of cell surface CFTR expression, as measured by confocal and widefield fluorescence microscopy. The decrease in CFTR conductance and cell surface expression were both dynamin-dependent. Single channel reconstitution studies showed that raising cytosolic Ca2+ per se had no direct effect on CFTR. In fact, the loss of CFTR plasma membrane activity correlated with activation of calcineurin, a Ca2+-dependent phosphatase, suggesting that dephosphorylation of CFTR was linked to the loss of surface expression. In support of this, the calcineurin inhibitor, cyclosporin A, prevented the Ca2+-induced decrease in cell surface CFTR. These results provide a hitherto unrecognised role for cytosolic Ca2+ in modulating the residency of CFTR at the plasma membrane through a dynamin- and calcineurin-dependent mechanism.


Assuntos
Calcineurina/fisiologia , Cálcio/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Citosol/metabolismo , Dinaminas/fisiologia , Brônquios/metabolismo , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Fosforilação
4.
J Physiol ; 594(6): 1643-61, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26574187

RESUMO

Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist-stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP-regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin-stimulated elevations in intracellular cAMP as well as both adenosine- and forskolin-stimulated increases in CFTR-dependent transepithelial short-circuit current, in polarised cultures of Calu-3 human airway cells. This CO2 -induced reduction in anion secretion was not due to a decrease in HCO3 (-) transport given that neither a change in CFTR-dependent HCO3 (-) efflux nor Na(+) /HCO3 (-) cotransporter-dependent HCO3 (-) influx were CO2 -sensitive. Hypercapnia also reduced the volume of forskolin-stimulated fluid secretion over 24 h, yet had no effect on the HCO3 (-) content of the secreted fluid. Our data reveal that hypercapnia reduces CFTR-dependent, electrogenic Cl(-) and fluid secretion, but not CFTR-dependent HCO3 (-) secretion, which highlights a differential sensitivity of Cl(-) and HCO3 (-) transporters to raised CO2 in Calu-3 cells. Hypercapnia also reduced forskolin-stimulated CFTR-dependent anion secretion in primary human airway epithelia. Based on current models of airways biology, a reduction in fluid secretion, associated with hypercapnia, would be predicted to have important consequences for airways hydration and the innate defence mechanisms of the lungs.


Assuntos
Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Cloretos/metabolismo , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Hipercapnia/metabolismo , Mucosa Respiratória/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Transporte de Íons , Transdução de Sinais , Sódio/metabolismo
5.
J Biol Chem ; 290(41): 24760-71, 2015 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-26294767

RESUMO

Evidence suggests that the plasma membrane Ca(2+)-ATPase (PMCA), which is critical for maintaining a low intracellular Ca(2+) concentration ([Ca(2+)]i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca(2+)]i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca(2+)]i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca(2+)]i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.


Assuntos
Trifosfato de Adenosina/metabolismo , Membrana Celular/enzimologia , Glicólise , Neoplasias Pancreáticas/patologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Adenocarcinoma/patologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Galactose/farmacologia , Glicólise/efeitos dos fármacos , Humanos , Ácido Iodoacético/farmacologia , Cetoácidos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores
6.
J Biol Chem ; 289(34): 23582-95, 2014 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-24993827

RESUMO

Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca(2+) ([Ca(2+)]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca(2+)]i are linked critically by the ATP-driven plasma membrane Ca(2+)-ATPase (PMCA) important for maintaining low resting [Ca(2+)]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca(2+)]i was measured by fura-2 imaging. An in situ [Ca(2+)]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mM) and POAEE (100 µM) induced a small but irreversible Ca(2+) overload response but had no significant effect on PMCA activity. POA (50-100 µM) induced a robust Ca(2+) overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca(2+) overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca(2+) overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.


Assuntos
Ácidos Graxos Monoinsaturados/farmacologia , Insulina/fisiologia , Pâncreas/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Morte Celular , Cromonas/farmacologia , Etanol/administração & dosagem , Etanol/metabolismo , Ácidos Graxos/metabolismo , Fluorescência , Morfolinas/farmacologia , Pâncreas/citologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Ratos , Ratos Sprague-Dawley
7.
Cells ; 11(22)2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36428960

RESUMO

N-methyl-D-aspartate (NMDA) receptors have been implicated in L-Dopa-induced dyskinesias (LID) in Parkinson's disease patients, but the use of antagonists that directly inhibit this receptor is associated with severe side effects. L-4-chlorokynurenine (4-Cl-KYN or AV-101) is a pro-drug of 7-chlorokynurenic acid (7-Cl-KYNA), a potent and specific antagonist of the glycine (GlyB) co-agonist site of NMDA receptors. The 7-Cl-KYNA has limited ability to cross the blood-brain barrier, whereas AV-101 readily accesses the brain. We investigated if AV-101 reduces LID in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys while maintaining the antiparkinsonian activity of L-Dopa. A first pilot study using three dyskinetic MPTP monkeys showed that acute AV-101 treatment (250 and 450 mg/kg) reduced LID and maintained the antiparkinsonian activity of L-Dopa. The main study using six additional dyskinetic MPTP monkeys showed that repeated AV-101 treatment (250 mg/kg, b.i.d. for 4 consecutive days) maintained their L-Dopa antiparkinsonian response. We measured significantly less LID when AV-101 was combined with L-Dopa treatment. AV-101 alone or with L-Dopa had no non-motor adverse effects in MPTP monkeys. Our study showed antidyskinetic activity of AV-101 in MPTP monkeys was comparable to amantadine tested previously in our laboratory in this model. We observed no adverse effects with AV-101, which is an improvement over amantadine, with its known side effects.


Assuntos
Discinesia Induzida por Medicamentos , Fármacos Neuroprotetores , Transtornos Parkinsonianos , Pró-Fármacos , Animais , Levodopa/efeitos adversos , Receptores de N-Metil-D-Aspartato , Discinesia Induzida por Medicamentos/tratamento farmacológico , Discinesia Induzida por Medicamentos/etiologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , Glicina/farmacologia , Glicina/uso terapêutico , Projetos Piloto , Transtornos Parkinsonianos/induzido quimicamente , Macaca fascicularis , Antiparkinsonianos/efeitos adversos , Fármacos Neuroprotetores/uso terapêutico , Amantadina/farmacologia , Amantadina/uso terapêutico
8.
Sci Rep ; 9(1): 13655, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541117

RESUMO

Chronic obstructive pulmonary disease (COPD), which is most commonly caused by cigarette smoke (CS) exposure, is the third leading cause of death worldwide. The cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane anion channel that is widely expressed in epithelia throughout the body. In the airways, CFTR plays an important role in fluid homeostasis and helps flush mucus and inhaled pathogens/toxicants out of the lung. Inhibition of CFTR leads to mucus stasis and severe airway disease. CS exposure also inhibits CFTR, leading to the decreased anion secretion/hydration seen in COPD patients. However, the underlying mechanism is poorly understood. Here, we report that CS causes CFTR to be internalized in a clathrin/dynamin-dependent fashion. This internalization is followed by retrograde trafficking of CFTR to the endoplasmic reticulum. Although this internalization pathway has been described for bacterial toxins and cargo machinery, it has never been reported for mammalian ion channels. Furthermore, the rapid internalization of CFTR is dependent on CFTR dephosphorylation by calcineurin, a protein phosphatase that is upregulated by CS. These results provide new insights into the mechanism of CFTR internalization, and may help in the development of new therapies for CFTR correction and lung rehydration in patients with debilitating airway diseases such as COPD.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumaça/efeitos adversos , Calcineurina/metabolismo , Linhagem Celular , Clatrina/metabolismo , Regulação para Baixo , Dinaminas/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Nicotiana
9.
Free Radic Biol Med ; 95: 333-48, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26944189

RESUMO

Reactive oxygen species, such as H2O2, can damage cells but also promote fundamental processes, including growth, differentiation and migration. The mechanisms allowing cells to differentially respond to toxic or signaling H2O2 levels are poorly defined. Here we reveal that increasing external H2O2 produces a bi-phasic response in intracellular H2O2. Peroxiredoxins (Prx) are abundant peroxidases which protect against genome instability, ageing and cancer. We have developed a dynamic model simulating in vivo changes in Prx oxidation. Remarkably, we show that the thioredoxin peroxidase activity of Prx does not provide any significant protection against external rises in H2O2. Instead, our model and experimental data are consistent with low levels of extracellular H2O2 being efficiently buffered by other thioredoxin-dependent activities, including H2O2-reactive cysteines in the thiol-proteome. We show that when extracellular H2O2 levels overwhelm this buffering capacity, the consequent rise in intracellular H2O2 triggers hyperoxidation of Prx to thioredoxin-resistant, peroxidase-inactive form/s. Accordingly, Prx hyperoxidation signals that H2O2 defenses are breached, diverting thioredoxin to repair damage.


Assuntos
Peróxido de Hidrogênio/química , Oxirredução , Peroxirredoxinas/química , Tiorredoxinas/química , Citoplasma/química , Citoplasma/metabolismo , Peróxido de Hidrogênio/metabolismo , Modelos Químicos , Peroxirredoxinas/genética , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tiorredoxinas/metabolismo
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